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POLYMER SCIENCE AND TECHNOLOGY
ENCYCLOPEDIA OF POLYMER RESEARCH (2 VOLUME SET)
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POLYMER SCIENCE AND TECHNOLOGY
ENCYCLOPEDIA OF POLYMER RESEARCH (2 VOLUME SET)
CAROLINE E. JONES EDITOR
Nova Science Publishers, Inc. New York
Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Encyclopedia of polymer research / Editor, Caroline E. Jones. p. cm. ISBN 978-1-61122-209-8 (eBook) 1. Polymers--Encyclopedias. I. Jones, Caroline E. TP1110.E46 20 668.9--dc22 2010037853
Published by Nova Science Publishers, Inc. † New York
CONTENTS Volume 1 xi
Preface Chapter 1
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Research and Review and Studies Induced Self-Assembly of Diblock Copolymers Eri Yoshida
1
A Novel Thermosensitive Composite Hydrogel Based on Poly(Ethylene Glycol)-Poly(Ε-Caprolactone)-Poly(Ethylene Glycol) (PECE) Copolymer and Pluronic F127 ChangYang Gong, Shuai Shi, PengWei Dong, MaLing Gou, XingYi Li, YuQuan Wei and ZhiYong Qian
29
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer for Catalytic Enantioselective Reactions Christine Saluzzo and Stéphane Guillarme
45
Small Molecule Stabilization: A Novel Concept for the Stabilization of Small Inorganic Nanoparticles Georg Garnweitner
173
Molecular Implications in the Solubilization of the Antibacterial Agent Triclocarban by Means of Branched Poly (Ethylene Oxide)Poly (Propylene Oxide) Polymeric Micelles Diego A. Chiappetta, José Degrossi, Ruth A. Lizarazo, Deisy L. Salinas, Fleming Martínez and Alejandro Sosnik
197
213
Chapter 6
Siloxane-Containing Compounds as Polymer Stabilizers Carmen Racles, Thierry Hamaide and Etienne Fleury
Chapter 7
Amphiphilic Block Copolymers: Potent Efflux Pump Inhibitors for Drug Delivery and Cancer Therapy Martin Werle and Hirofumi Takeuchi
235
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers Z. Yang
243
Chapter 8
vi
Contents
Chapter 9
Current Developments in Double Hydrophilic Block Copolymers G. Mountrichas and S. Pispas
Chapter 10
Thermo-Oxidation Stability of Poly(Butylene Terephthalate) and Catalyst Composition Antonio Massa, Valeria Bugatti, Arrigo Scettri and Socrate Contessa
Chapter 11
Chapter 12
Chapter 13
Chapter 14
Chapter 15
Chapter 16
Chapter 17
Chapter 18
Chapter 19
Chapter 20
Hindered Amine Stabilizers as Sources of Markers of the Heterogeneous Photooxidation / Photostabilization of Carbon Chain Polymers J. Pilař and J. Pospíšil A Review of the Hazardous Effects of Polycyclic Aromatic Hydrocarbons on Living Organisms Maria Aparecida Marin-Morales; Daniela Morais Leme and Dânia Elisa Christofoletti Mazzeo
291
327
343
359
Polycyclic Aromatic Hydrocarbons Toxicity in Animals and Humans Pierina Visciano and Monia Perugini
407
Polycyclic Aromatic Hydrocarbons and Carcinogenic Risk Assessment in Developing Countries Mahmoud A. Hassanien
423
Adipose Tissue as a key Contributor to Polycyclic Aromatic Hydrocarbon-Induced Carcinogenesis P. Irigaray and D. Belpomme
453
Chlorinate Polycyclic Aromatic Hydrocarbons: A New Class of Environmental Toxin? Takeshi Ohura and Hiroyuki Sakakibara
463
Recent Trends in Analytical Methods to Chemically Monitor PAH Pollution Verónica Pino
469
New Tendencies in Liquid Chromatography for Controlling PAHs Pollution R. Herráez-Hernández, P. Campíns-Falcó, J. Verdú-Andrés, A. Sevillano-Cabeza and L. Ferrando-Climent
557
Pahs in Sediments Associated with Coal and Coal-Derived Particles - Occurrence, Mobility and Risk Assessment Yi Yang and Thilo Hofmann
577
Analysis of Polycyclic Aromatic Hydrocarbons Adsorbed on Soot Surface by Laser Desorption, Laser Ionization and Mass Spectrometry Michael Ziskind, Alessandro Faccinetto, Pascale Desgroux, Eric Therssen and Cristian Focsa
587
Contents
vii 605
Chapter 21
Toxicity and Toxicology of Benzo[a]pyrene M. S. Yang and W. S. To
Chapter 22
Chromatographic Analysis of Polycyclic Aromatic Hydrocarbons by Π-Electron Containing Polymeric Organic Phase-Grafted on Silica M. Mizanur Rahman, Makoto Takafuji, Ashequl A. Rana, Abul K. Mallik, Usha G. Gautam and Hirotaka Ihara
621
Determination of Polycyclic Aromatic Hydrocarbons in SolidMatrix Foods Using a Non-Destructive Method Diana Martin and Jorge Ruiz
651
Chapter 23
Chapter 24
Chapter 25
PAH Removal by Two Native Tropical Plants Cultured on Model Contaminated Soil E. Escalante-Espinosa, L. Rodríguez-García and M. Gutiérrez-Rojas In Vitro Phytoremediation Mechanisms of PAH Removal by Two Plant Species L. Reynoso-Cuevas, F. Cruz-Sosa and M. Gutiérrez-Rojas
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677 691
Chapter 26
Fibers Spun from Polysaccharides Lingyan Kong, Gregory R. Ziegler and Rajesh Bhosale
Chapter 27
Development and Characterization of Starch Based Nano-Composites Xiaozhi Tang and Sajid Alavi
735
Characterization and Mechanism of Chitosan Hydrolysis by Non-specific Enzymes Wenshui Xia and Ping Liu
775
Volume 2
Chapter 28
Chapter 29
Use of Cyclodextrin Polymers in Separation of Organic Species Cezary A. Kozlowski and Wanda Sliwa
Chapter 30
Properties, Applications and Current Development of Edible Polysaccharide Films and Coatings Pau Talens, María José Fabra and Amparo Chiralt
Chapter 31
Carbohydrate Binding Modules: Functions and Applications Susana Moreira and Miguel Gama
Chapter 32
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification Barbara Simončič, Brigita Tomšič, Boris Orel and Ivan Jerman
817
857 891
927
viii Chapter 33
Contents Pathways to Bioactive Oligosaccharides: Biological Functions and Potential Applications Ellen C. Giese, Aneli M. Barbosa and Robert F. H. Dekker
969
Chapter 34
Amidation of Cellulose Materials Nadege Follain
1001
Chapter 35
D/L-Hybrid Galactans from Red Seaweeds: Do they Really Exist? Marina Ciancia and Alberto S. Cerezo
1045
Chapter 36
Development of Polysaccharide Networks for L-(+)-Ascorbic Acid Stabilization into Antioxidant/Nutritional Supplier-Edible Films Ana M. Rojas, Paula G. León, Silvia K. Flores, Carolina D. Pérez and María D. De’Nobili
Chapter 37
Chapter 38
Chapter 39
Chapter 40
Chapter 41
Chapter 42
Employing Polymer-Polymer Incompatibility for Oil Microencapsulation by Coacervation Jaroslav M. Katona, Verica J. Sovilj, Lidija B. Petrović and Jadranka L. Milanović Main Characteristics of Some Gelling and Thickening Polysaccharides: Relation between the Molecular Structure, the Physical Properties and the Applications Marguerite Rinaudo
1083
1109
1145
Development and Applications of Nutraceuticals from Marine Carbohydrates Y. Dominic Ravichandran and Se-Kwon Kim
1169
Carbohydrate Polymers as Versatile Materials in Controlled Release Technologies Anamika Roy, J. Bajpai and A.K. Bajpai
1195
Structural Organization and Biological Activity of Chitin Nanofibrils Anyarat Watthanaphanit and Ratana Rujiravanit
1225
Development of Carbohydrate Polymer Based New Hydrogel Materials Derived from Seaweed Polysaccharides A. K. Siddhanta, Ramavatar Meena, Gayatri Prasad, Mahesh U Chhatbar, Gaurav K Mehta, Mihir D Oza, Sanjay Kumar and Kamalesh Prasad
1245
Chapter 43
Chitosans: New Vectors for Gene Therapy Riccardo A.A. Muzzarelli
1273
Chapter 44
Extremophiles as Sources of Exopolysaccharides Hande Kazak, Ebru Toksoy Öner and Robert F. H. Dekker
1295
Chapter 45
Alginate: A Promising Polysaccharide for Delivery of Essential Oils Pranee Lertsutthiwong, Ian S. Haworth and Pornchai Rojsitthisak
1311
Contents Chapter 46
Chapter 47
Obtention of Products Enriched in Carbohydrate Polymers from the Food Industry Leftovers Lía N.Gerschenson, Ana M. Rojas, Marina de Escalada Pla, Eliana Fissore and María E. Latorre Recent Developments in the Preparation and Applications of Chitosan-Based Microspheres Dongfeng Wang, Haiyan Li, Binjie Liu and Xingyu Wang
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1329
1345
Chapter 48
Chemoenzymatic Synthesis of Amylose-Grafted Polymers Yoshiro Kaneko and Jun-ichi Kadokawa
1361
Chapter 49
Polysaccharides and their Derivatives for Biomedical Applications Liqun Yang and Li-Ming Zhang
1383
Chapter 50
Feruloylated Arabinoxylans Recovered from Low-Value Maize by-Products Guillermo Niño-Medina, Elizabeth Carvajal-Millan, Jaime Lizardi, Agustín Rascón-Chu, Alfonso A. Gardea
Chapter 51
Chapter 52
Chapter 53
1401
Effect of Visible Light on Physical and Enzymatic Transformations of Polysaccharides Maciej Fiedorowicz and Piotr Tomasik
1417
The Topology Morphology Comparison of Nano-structural Units between Heating and High-pressure Gelatinization of Starch Granules Liu Zhongdong, Guo Yanyan, Gao li, Liu Peng, Gao Xinlou, Chen Zhaotan, Dong Caiwen and Liu Boxiang
1431
Partial Characterization of a New Acidic Heteropolysaccharide Produced by a Native Isolate of Lactobacillus sp. CFR-2180 R. Mohan and S. V. N. Vijayendra
1447
Chapter 54
“Polysaccharide-based Intelligent Delivery” Calls Cancer to Stop Hong Wu, Li Fan and Fei Li
1457
Chapter 55
Study on the Physical Properties of Waxy Wheat Starch Chen Jianhui, Liu Zhongdong, Liu Peng, Zhang Zesheng, Lv Xiaoling, Sun Ping and Liu Boxiang
1469
Chapter 56
Studies on the Morphology of Waxy Maize Zein Film Liu Zhongdong, An Hongjie, Sun Youning, Guo Yunchang, Liu Peng and Li Junchao
1483
Chapter 57
Carbohydrate Polymers from Mushrooms: Recent Development and Applications as Functional Foods Peter C. K. Cheung
1489
x Chapter 58
Index
Contents Application of Macrocyclic Polymers for Metal Ions Separation Cezary Kozlowski, Wladyslaw Walkowiak, Joanna Konczyk and Anna Nowik-Zajac
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PREFACE This book presents important research advances in the study of polymer research. Topics discussed herein include small molecule stabilization; amphiphilic block copolymers; effects of polycyclic aromatic hydrocarbons in living organisms; PAHs pollution; cyclodextrin polymers; polymer-polymer incompatability; carbohydrate polymers in controlled release technologies; and macrocyclic polymers for metal ions separation. Chapter 1 - The molecular self-assembly is induced by variation in the surroundings, such as temperature, pressure, pH, salt formation, and noncovalent bond cross-linking. The block copolymers are molecularly converted in situ from the nonamphiphilic copolymers completely dissolved in a solvent to amphiphilic copolymers due to these stimuli. Therefore, the association and dissociation of the isolated copolymers are reversibly controlled by such stimuli. The induced self-assembly has advantages over direct self-assembly of amphiphilic copolymers in molecular designing. There is no dependence on the balance of solvophilic and solvophobic moieties when designing the copolymers. Thus, a better selection of the driving force can be provided. The advantages also include the fact that a variety of amphiphilic copolymers can be created from one nonamphiphilic copolymer in situ by selecting the stimuli. Chapter 2 - A novel kind of biodegradable thermosensitive composite hydrogel was successfully prepared in this work, which was a flowing sol at ambient temperature and became a non-flowing gel at body temperature. The composite hydrogel was composed of poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) and Pluronic F127 copolymer. By varying the composition of above two copolymers, in vivo degradation rate and in vitro drug release behavior could be controlled. Histopathological study of tissue at injection site showed no significant inflammatory reaction and toxicity, which means that the composite hydrogel might serve as a safe candidate as in situ gelforming controlled drug delivery system. Chapter 3 - This paper reviews the recent progress made in insoluble polymer supported amino alcohols, amino thiols, oxazolines, salens, sulphonamides, oxazaborolidines and diamines ligands. This paper deals also with various approaches of stabilization of the catalytic system by immobilization of the chiral catalyst onto the polymer by the way of immobilization of the chiral ligand. Different types of ligand immobilization are presented: pendant ligands anchored on a polymer prepared by a polymer reaction, ligands on the backbone prepared by copolymerization and molecular imprinting technique. Examples of their use, performance and recyclability in a variety of enantioselective reactions such as alkylation and reductions of C=O bonds (hydrogenation, hydrogen transfer reduction)
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reduction of C=N bonds, C-O bond formations (epoxidation, dihydroxylation), C-C bond formations (Diels Alder, cyclopropanation, aldolisation, allylic substitution) and oxidation … are presented. Chapter 4 - In the last 20 years, the synthesis of nanoparticles with defined size and shape has been studied with strongly growing interest, leading to a multitude of synthetic approaches and strategies. Whereas the synthesis of the nanocrystals has been studied in great detail, far less effort has been directed towards the stabilization of the obtained materials against agglomeration. This is surprising as the stabilization determines their dispersibility in various solvents, which is a crucial parameter for most applications. For conventional colloids, the classical theories of electrostatic, steric and electrosteric stabilization are well established, but application of these theories to the stabilization of small nanomaterials leads to some peculiarities and at the same time has some limitations, which is known from experimental experience but has not been studied in a systematic fashion yet. One important conclusion from the theories is that short organic molecules sufficiently serve to provide steric stabilization of nanoparticles less than about 50 nm in size, without a need for long-chain polymeric stabilizers. This concept has been successfully applied using commercial metal oxide nanoparticles in the 50 nm size range, and it is even possible to tailor nanoparticle dispersions with respect to their rheological properties by adjustment of the stabilizer size. Through proper choice of the stabilizer, nanoparticle slurries with high solids content but at the same time low viscosity can be realized, which is highly advantageous for applications especially in the field of ceramic processing. For ultrasmall nanoparticles in the sub-10-nm regime, the picture is somewhat different. On the one hand, the dispersions of such particles in a stabilized state show very special properties on the verge to molecular solutions, rendering them highly relevant for applications and thus their preparation highly important. On the other hand, due to the lack of suitable model materials, the fundamentals of interaction and stabilization of such small nanoparticles remains largely in the dark. Only a small number of reports were specifically directed to adress these problems and systematically investigate the effects of stabilizer chemistry and structure as well as solvent influence. A brief overview of these studies is provided to show that first concepts have been presented, but the general applicability of these concepts still remains to be seen, and to demonstrate the substantial need for further research in this field in order to develop concepts for the rational stabilization and preparation of dispersions with tailored nanoparticle interactions and thus tailored properties. Chapter 5 - Aiming to gain further insight into the complexity of drug/polymeric micelle interaction phenomena, the present chapter investigated the incorporation of the poorly watersoluble topical antibacterial agent triclocarban (TCC) into polymeric micelles of the branched pH/temperature-responsive poly(ethylene oxide)-poly(propylene oxide) block copolymers Tetronic® 1107 (MW = 15 kDa, 70 wt% PEO) and 1307 (MW = 15 kDa, 70 wt% PEO). Solubility extents showed a sharp increase of up to 4 orders of magnitude. Due to the pHdependent character of both the carrier and the drug, studies were performed under different pH conditions. Due to a more efficient poloxamine aggregation at higher pH-values, a clear increase in the solubilization capacity was apparent under these conditions. However, ionization of TCC at pH 12.7 constrained the formation of hydrogen bonds between the urea moieties and the polyether chain, leading to a decrease in solubility above this pH. The size and size distribution of drug-loaded micelles was evaluated by Dynamic Light Scattering (DLS). Findings indicated the increase in the size of the aggregates with the incorporation of
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the drug. The morphology of the nanostructures was visualized by transmission electron microscopy (TEM). The stability of the systems over time was also evaluated. Finally, the antibacterial activity of different TCC/poloxamine complexes was assayed on different bacteria collections. For example, while a poloxamine-free TCC aqueous solution (pH 7.4) was not effective on Staphylococcus aureus, a 10% drug-containing T1307 system inhibited the bacterial growth to some extent. These results supported the release of the drug from the polymeric reservoir. However, as opposed to previous reports, overall findings indicated the limited intrinsic activity of TCC against the investigated pathogens. Chapter 6 – Generally, surfactants are used as stabilizers of interfaces or particles and their applications are very wide, from foams or adhesion modifiers to the orientation of chemical reactions. Siloxane surfactants are known for their ability to decrease the surface tension of liquids in such extent that is comparable only with some fluorinated compounds, which are thought to exhibit potential toxicological problems. On the other hand, polysiloxanes are unique by their set of properties, like for example low glass transition temperature, hydrophobic behavior, transparency to visible and UV light, high permeability to various gases (especially oxygen), physiological inertness, excellent blood compatibility (low interaction with plasma proteins). In addition, their chemistry is very versatile, and as a result, a very broad range of siloxane-organic compounds can be synthesized, including amphiphilic macromers or polymers. The most commonly known siloxane surfactants are the so called „silicone polyethers”, but other nonionic, as well as ionic surface active agents have been prepared and used over the years in cosmetics, textile conditioning, foam stabilization, coatings or agriculture. Recent developments in this research field and especially our experimental results on the synthesis, properties and applications of siloxane-containing surfactants will be reviewed. Our main interest is to propose new surfactants or alternative synthetic procedures, and new stabilization systems for polymeric nanoparticles. Carbohydrate modified (poly)siloxanes with different architectures have particularily been studied and tested, due to their biocompatibility and bioavailability. Chapter 7 - The ability of amphiphilic block copolymers to modulate multi drug resistance related processes has been demonstrated the first time more than 10 years ago. Nowadays, the efflux pump inhibitory activity of amphiphilic block copolymers is used in two main areas. First, to improve the transport of efflux pump substrates across the blood brain barrier (BBB) and second, in cancer therapy. It has been shown that in the presence of amphiphilic block copolymers higher concentrations of certain anticancer drugs, which are known as efflux pump substrates, can be found in the brain. Within the current chapter, recent developments in the field of amphiphilic block copolymer mediated efflux pump inhibition are discussed. Besides presenting data from in vitro and vivo studies, also the mechanisms involved in efflux pump inhibition are addressed. In addition, the influence of hydrophilicity/lipophilicity of various amphiphilic block copolymers as well as factors such as micelle formation on the efflux pump inhibitory activity are explained. Chapter 8 - The effects of physical aging near the surface region of glassy polymers are studied via the relaxations of (1) surface topographic features created by rubbing, and (2) the rubbing induced birefringence (RIB). Extensive experimental results are presented to show that physical aging processes that would have drastic effects on the relaxations of bulk polymers have little effects on the relaxations of rubbed surfaces. The authors also found that
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surface topographic features, such as ditches and ridges created by rubbing, relax at temperatures at about 20 °C below the bulk glass transition temperature of the polystyrene for the molecular weight of 442 kg/mol, even though the Laplace Pressure driving the relaxation is 1/500 of the yield limit. The relaxation of RIB in polystyrene (PS), its derivatives with modified side group, and polycarbonate (PC), involves only the length scale of the order of an individual segment. A phenomenological model based on individual birefringence elements is proposed for the RIB relaxation. The relaxation times (RT’s) of the elements are found to be independent of the thermal or stress history of the samples, either before or after the formation of the birefringence. The RT’s are also independent of the molecular weight, rubbing conditions, and film thickness, while the RT’s distribution function does depend on the molecular weight and rubbing conditions. The model provides quantitative interpretations that agree very well with all the reported experimental results, and sheds important light to the novel behaviors of the RIB relaxation. The absence of physical aging effects is probably due to the combined effects of small length scale of the RIB relaxation, and the accelerated aging speed in the near surface region. This is consistent with the mobility enhancement in the surface layer previously reported in the literature. Chapter 9 - Double hydrophilic block copolymers (DHBCs) constitute a novel class of water-soluble macromolecules with potential utilization in a wide range of applications. The exceptional combination of features, coming from their block copolymer structure and their ability to be stimuli responsive, establishes this class of copolymers as a core of intense research interest, aiming at elucidating aspects regarding their targeted synthesis, solution behavior and application possibilities. In this chapter, the current developments in the field of double hydrophilic block copolymers are discussed. In particular, synthetic strategies leading to the preparation of DHBCs are described. Moreover, their aqueous solution behavior is examined in respect to their ability to self assemble, due to changes in the solution temperature, and/or pH, as well as due to complexation. Additionally, the potential applications of DHBCs in mineralization processes, nanomedicine, nanotechnology and so on are mentioned. Finally, future perspectives in the field of DHBCs regarding general polymer science and nanotechnology issues, as well as open scientific questions, on synthesis and solution behavior of this class of materials, are also discussed. Chapter 10 - Polyesters are heterochain macromolecular substances characterized by the presence of carboxylate ester groups in the repeating units of their chains. Predominant in terms of volume and products value are those based on poly(ethylene terephthalate) (PET), long established as basis of fibers, films, molding plastics and containers for liquids, and poly(butylene terephthalate) (PBT) largely used to produce fibers as well as for special applications in motor and electric industry. Chapter 11 - Application of Hindered Amine Stabilizers (HAS) is the state-of-the-art approach to protection of carbon-chain polymers such as polyolefins and polystyrene or blends containing these against weathering. During outdoor exposure, the polymers loose their material properties due to solar radiation-triggered photooxidation. The complex mechanism of the stabilization involving cyclic oxidation-triggered transformation of HAS is outlined. Monitoring of the formation of the HAS-developed key transformation products, HAS-related nitroxides, responsible within the regenerative mechanism for the effective stabilization was used to confirm the heterogeneous character of photooxidation of two carbon-chain polymers, polypropylene and a specific polyethylene copolymer. Depth profiles of nitroxides were monitored in a long-term photooxidation regime using Electron Spin
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Resonance Imaging (ESRI) technique. The shape of concentration profiles of the nitroxides accumulated in the equilibrium state upon filtered Xenon lamp-equipped Weather-Ometer exposure was interpreted in terms of the oxygen diffusion limited oxidation and radiation penetration in oxidation-stressed polymer surfaces. The data indicate differences in the character of the heterogeneous process in dependence on the polymer matrix and on the used stabilizer system based on secondary HAS and O-alkylhydroxylamine HAS and/or HAS combination with UV absorbers. Imaging of nitroxides is a precise tool for marking heterogeneous oxidation of polyolefins. Chapter 12 - Polycyclic aromatic hydrocarbons (PAHs) are one of the most important, abundant and dangerous class of persistent organic contaminants found in air, water, food and waste sites. The great concern with this class of pollutants is due to the fact that these chemicals cause various deleterious effects, which could jeopardize the genome of exposed organisms or even lead them to death. Many studies have been conducted in several laboratories distributed around the world and with different organisms to quantify the impact of those pollutants and to diagnose their action mechanisms. Thus, there is a real necessity to compile the data already described in the literature, obtained from tests in situ, in vivo and in vitro, derived from simulation experiments, occupational exposure or environmental disasters, in order to produce a report with the main effects already described for that class of pollutant. This review article intends to address the following issues: toxicity, cytotoxicity, genotoxicity, mutagenicity, teratogenicity and carcinogenicity of PAHs, besides information about estrogenicity and metabolism of these substances. In this context, it will be also presented the test systems that better responded to the assessments of the PAHs effects, in an attempt to show the most sensitive and efficient organisms to detect the contamination of these agents. Chapter 13 - Polycyclic aromatic hydrocarbons (PAHs) are a group of about 100 different chemicals that are formed during incomplete combustion or heat-induced decomposition of organic matter. Humans can be exposed to PAHs through different routes. For non-smokers, the major route of exposure is from food with a minor contribution from inhaled air. Food can be contaminated from environmental sources, industrial food processing and from home food preparation. The European regulator has found that there is a low risk to consumers from PAHs in foods based on average dietary exposure but risk management action may be required for high dietary intake. Several PAHs have been shown to be genotoxic carcinogens. In 2002, the Scientific Committee on Food (SCF) reviewed PAH toxicity. For 15 compounds it concluded that there was clear evidence for their toxicity. In 2005, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) performed a risk assessment on PAHs, and basically agreed with the SCF selection. Also the US Environmental Protection Agency has classified some PAHs as priority-pollutants based on toxicity, potential for human exposure, frequency of occurrence and the extent of information available. Seven of these PAHs (benzo(a)anthracene, chrysene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, dibenzo(a,h)anthracene and indeno(1,2,3-cd)pyrene) are considered as probable human carcinogens, but benzo(a)pyrene is the most studied and used as a marker for PAHs in ambient air and food. PAHs are themselves chemically inert but undergo metabolic activation in mammalian cells to diol-epoxides that bind covalently to cellular macromolecules, including DNA, causing errors in DNA replication and mutations that initiate the carcinogenic process. Other reactive intermediates can be generated by one-electron oxidation process that can result in chemically unstable alkylation of DNA, which leads to depurination,
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a potentially mutagenic event. In this chapter the most important effects of PAHs on animals and humans health are reported and discussed to define new possible preventive and regulatory programs for these pollutants. Chapter 14 - Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds composed of fused aromatic rings. Some high molecular weight PAHs are considered to be carcinogenic, mutagenic, teratogenic even at low concentrations and have received more environmental concern. PAHs are resistant to biodegradation and can bioaccumulate in the environment due to their hydrophobic nature and low solubility. PAHs are ubiquitous environmental contaminants found in air, soil and water. The main sources of PAHs in the environment and for general population exposure are natural and intentional combustion of organic matter and petroleum manipulation. Atmospheric emissions will arise from the processing of coal and petroleum products and incomplete combustion during industrial processes. In the developing countries, present information concerning the sources and inputs of PAHs in these countries is rather limited. PAHs concentrations have been reported in a number of urban areas as well as in remote places and are consistently higher in winter than in summer, and in urban than in rural areas. The higher values are found in particular places like stack gases, tunnels of motorways, open burning sites, etc. There are no ambient air quality standards and no specific guideline value can be recommended for PAHs in air. PAHs in air are attached to particles, which may also play a role in their carcinogenicity. Although food is thought to be the major source of human exposure to PAHs, part of this contamination may arise from air pollution with PAHs. Exposure to PAHs mainly occurs from atmospheric pollution through inhalation, ingestion and dermal contact. PAHs half-lives in humans are in fact in the range of days/hours, and metabolism is responsible of the formation of carcinogenic metabolites. Risk assessments and potency assessments of various individual and complex mixtures of PAHs have been attempted. BaP is the only PAH for which a database is available, allowing a quantitative risk assessment. PAH potencies are used to determine quantitative health risks posed by PAH exposure. The risks posed by a mixture of PAHs are based on an assumption of additively of the individual risks posed by the PAHs. The objectives of this chapter are to: 1) report PAH concentrations in the environmental media in developing countries; 2) collate published and unpublished PAH information from environmental agencies and write a report on the state of knowledge of PAHs; 3) characterize the priority PAHs, carcinogenic risk assessment and potential health impacts. Chapter 15 – The authors have previously shown that benzo[a]pyrene used at extremely low dose can cause weight gain in mice and that the increase in adipose tissue mass is due to inhibition of β-adrenergic stimulation of lipolysis. Moreover they have suggested that adipose tissue is a reservoir for many carcinogens including Polycyclic Aromatic Hydrocarbons (PAHs).In this chapter, the authors demonstrate that the two C4 PAHs pyrene and phenanthrene can accumulate into adipocytes, but at the difference of benzo[a]pyrene have no impact on lipolysis.Since benzo[a]pyrene has been proved to be mutagenic while C4 PAHs are tumor promoters, they consider that a mixture of the three PAHs may act as a complete carcinogen. Furthermore, because adipose tissue can be induced by benzo[a]pyrene, is a reservoir for many PAHs and other carcinogens and can stimulate proliferation of normal or cancer cells, through the secretion of adipokines by adipocytes, they therefore propose that
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adipose tissue is a key contributor to chemical carcinogenesis by acting not only upon initiation and promotion but also upon progression. Chapter 16 - A review has provided the environmental sources and behavior of chlorinated 3- to 5-ring polycyclic aromatic hydrocarbons (ClPAHs). ClPAHs have been known to be ubiquitous contaminants, which can be found in urban air, vehicle exhaust gas, snow, tap water and sediments. The concentrations in the each environment are generally higher than those of chlorinated dioxins, although which are significantly lower than the concentrations of parent compounds, PAHs. Based on the environmental data and emission sources analysis for the ClPAHs, the dominant production process is by incineration of organic materials including chlorine like as the case of dioxins. On the other hand, treatment of PAHs with chlorine in water also produced ClPAHs. Indeed, such secondary reaction for ClPAHs generation could occur in the gaseous phase. For the biological effects, the toxicities such as mutagenicity and aryl hydrocarbon receptor activity of certain ClPAHs are emphasized than those of parent PAH, in which the toxicities of exposure to ClPAHs based on AhR activity were estimated to be approximately 30-50 times higher than that of dioxins. To investigate the sources and environmental behavior of ClPAHs is of great importance to assess human health risks. Chapter 17 - Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic compounds with two or more fused aromatic rings. They have received increased attention in recent years in pollution studies because some of these compounds are highly carcinogenic or mutagenic. Some, while not carcinogenic, may act as synergists. These compounds are widespread in the environment. Their occurrence is largely a result of anthropogenic and natural emissions such as fossil fuel burning, motor vehicles, waste incinerators, oil refining, coke and asphalt production, aluminum production, etc. PAHs have relatively low solubility in water but are highly lipophilic. Most of the PAHs with low vapor pressure in the air are adsorbed on particles. When dissolved in water or adsorbed on particulate matter, PAHs can undergo photodecomposition when exposed to UV light. In the atmosphere, PAHs can react with many other pollutants such as ozone, nitrogen oxides and sulfur dioxide, yielding diones, nitro- and dinitro-PAHs, and sulfonic acids, respectively. PAHs may also be degraded by some microorganisms in soils. PAHs in soils/sediments can be taken up by biota, and some bio-magnification of these toxic compounds may occur as they pass through the food chain. The US Environmental Protection Agency (EPA) has identified 16 un-substituted PAHs as priority pollutants. The European Food Security Authority (EFSA) has classified 15 PAHs by their food toxicity. Thus, potential exposure to PAHs must be assessed by testing contaminated soils, sediments, air, water and foods, and estimating the degree of intake of each of these media into the human body, as well as the overall toxicity.There are several main problems associated with the determination of PAHs in environmental and food samples: their low concentrations, the complexity of the samples, and the necessity for fast and reliable analytical methods. To achieve successful results, many extraction-separation techniques have been developed depending on the nature of the sample. An in-depth description of the most commonly-used techniques, together with recent trends in analytical methods to monitor PAHs levels in different kind of samples, will be the main focus of this chapter. Among those that will be cited are Soxhlet extraction, liquid-liquid extraction (LLE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), pressurized fluid extraction (PFE), supercritical fluid extraction (SFE), solid-phase extraction (SPE), and solid-phase microextraction (SPME), mainly in combination with chromatographic
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techniques (HPLC and GC). Moreover, the utilization of active or passive samplers in pollution monitoring, as well as the increased utilization of semi-permeable membrane passive samplers (SPMDs), will be described. Chapter 18 - Different configurations were studied for the screening analysis of common polycyclic aromatic hydrocarbons (PAHs) in environmental waters by liquid chromatography (LC). Analyte enrichment on a traping microcolumn connected by column switching to the analytical column, and in-valve in-tube solid-phase microextraction (IT-SPME) were tested as alternatives to conventional solid-phase extraction (SPE) with disposable cartridges. For separation conventional LC, capillary LC and LC with a monolithic column were tested and compared, whereas UV-diode array detection (DAD) or fluorescence detection were used for signal monitoring. The compounds assayed were naphthalene, anthracene, fluoranthene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene and benzo[ghi]perylene. On the basis of the results obtained, new methods are presented for the on-line screening analysis of PAHs in several water matrices. The main advantages of the proposed procedures are: (i) the minimum off-line sample manipulation as the samples only need to be filtered; (ii) the rapidity, as the total analysis time can be reduced to less than 20 min, (iii) the improved detection limits, especially for the in-valve IT-SPME configurations, as they are compatible with the injection of very large sample volumes (up to 4.0 mL), and (iv) the total elimination of organic solvents in the sample treatment. Therefore, the proposed methods are environmentally friendly and costeffective alternatives for the routine monitoring of PAHs in waters. The occurrence of the tested PAHs in waters of The Comunidad Valenciana (Spain) was also tested. Chapter 19 - Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous hydrophobic organic contaminants in the environment. They tend to be associated with particles and are widely transported by flooding and atmospheric pathways, resulting in elevated concentrations in sediments/soils. Coal and coal-derived particles in natural sediments/soils can act not only as strong sinks for the PAHs, but also as very important sources of PAHs in sediments/soils. The understanding of the mobility of these contaminants from the sediments/soils, especially the sequestration of PAHs by coal and coal-derived particles is very important, because they can control the transportation, bioavailability, degradation and hence the potential risk of these contaminants in the environment. Chapter 20 - Polycyclic aromatic hydrocarbons (PAHs) are notably formed and emitted by incomplete combustion of coal, oil, petrol, wood, tobacco. In addition, they participate to soot inception. The remaining gaseous PAHs are then adsorbed onto the porous soot matrix so formed, conditioning physicochemical properties of these particles and consequently their impact on human health and environment. This has motivated various studies. However, an extensive analysis of the adsorbed PAHs onto soot particles is still challenging. The authors are developing an experimental set-up, based on the combined use of 3 techniques - laser desorption, multi-photon ionization and time-of-flight mass spectrometry, dedicated to this study with particular focus on the dependence of the adsorbed species on the stage of combustion and the nature of the fuel. The complexity of the first obtained mass spectra led us to successively investigate the laser desorption of pure PAHs then "synthetic" soot samples- obtained by adsorbing PAHs on various carbonaous substrates- to characterize the processes involved in our method, and finally combustion soot collected from various diffusion flames. They present an overview of these studies together with their first practical implications, obtained on soot collected from Diesel and Diesel surrogate flames.
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Chapter 21 - Benzo[a]pyrene (BaP) is one of the most well studied PAHs. It is a product of incomplete combustion of fossil fuel. BaP can be deposited into food, soil and water. The major route of human exposure to BaP is via food consumption. BaP can interact with DNA to form bulky adducts, or biotransformed to produce free radicals. Both processes may lead to mutation and cancer. Despite the clarity of its actions, it is still difficult to establish a clear dose-response relation between BaP and cancer in human. Currently, BaP is considered class 2A carcinogen by the International Agency for Research in Cancer (IARC), a chemical with well documented evidence as an animal carcinogen, but limited evidence as human carcinogen. It is believed that only by understanding the basic difference between animal and human biochemistry will it be possible to address the toxicity of BaP in the various biological systems. In the present chapter, experiments pertaining to studying the response to BaP in human and animal cells are discussed. Chapter 22 - Polycyclic aromatic hydrocarbons (PAHs) are well-known organic compounds derived from incomplete combustion of organic matters. PAHs have been widely studied in a broad field such as material science, industrial technologies and pharmaceutical sciences, as well known environmental pollutants, and an increasing particular concern has been paid to their adverse harmful effects to human due to the carcinogenic and mutagenic properties of many PAH species. Clearly, the concerns over adverse health effects of PAHs in human and wildlife provide the impetus for research on their sources, environmental fate and food chain transfer. High performance liquid chromatography (HPLC) is useful and conventional method to monitor PAHs. More than 60% of all HPLC separations are carried out under reversed-phase (RP) conditions and alkylated-silicas were successfully employed for the separation of mixtures of PAHs. In RP-HPLC chromatography, not only long alkyl chains but also aromatic compounds such as benzene, pyrene, and hetero aromatic selector such as vinylpyridine have been investigated for organic phase of stationary phase so far. However, development of new stationary phases has been required for separation of similar compounds such as structural isomers, positional isomers and optical isomers. In this chapter, the authors focus on the chromatographic analysis of PAHs by π-electron containing ordered and disordered polymeric organic phase-grafted silica. Polymeric phase-grafted silica stationary phases exhibits unexpected enhancement of separation for PAHs by a multipleinteraction mechanism, even if the polymer does not form any specific secondary structure. Poly(octadecylacrylate) and poly(4-vinylpyridine)-grafted mesoporous silica particles (SilODAn and Sil-VPn, respectively) will be described as typical examples of such stationary phases. Effects of grafting method and grafting-density of polymeric phases on the separation of PAHs in HPLC will be also discussed. Extensive chromatographic studies showed that selectivity for PAHs can be enhanced by multiple π-π interactions between the π-electron containing polymeric phases and the guest PAHs molecules. The presence of aligned carbonyl groups in a crystalline state of poly(octadecylacrylate) on silica significantly enhanced the selectivity for PAHs by π-π interaction mechanism. However, Sil-VPn recognizes molecular shape (planar and disk-like) of PAHs through π-π interactions between pyridyl groups of grafted-polymer and PAHs, along with electrostatic interactions involving a inductive effects and /or a quadrupolar effect in the normal phase HPLC. Comparison of the HPLC separations for PAHs with conventional octadecylsilylated silica (ODS) demonstrates the efficiencies and effectiveness of these π-electron containing polymeric organic phases grafted silica particles.
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Chapter 23 - The use of a direct extraction device (DED) for coupling to solid phase microextraction (SPME) is described. Such a device enables the introduction of the SPME fibre in the core of solid matrixes, allowing the analysis of volatile compounds, such as polycyclic aromatic hydrocarbons (PAHs), with no or little deterioration of the solid product. The feasibility of using SPME-DED for extraction of PAHs from solid matrixes mimicking solid foodstuffs (gelatine alone, gelatine containing fat and salt, or cooked chicken patties) and from commercial smoked meat products (smoked tenderloin, smoked jowl, smoked dry cured sausage and smoked potato sausage) was tested. Moreover, the diffusion of PAHs in solid matrixes by SPME-DED was also evaluated. SPME-DED appeared as a rapid, nondestructive technique for primary screening of low molecular weight (MW) PAHs in all solid matrixes assayed, as well as for the study of the diffusion process of PAHs within solid systems, without sampling and any laboratorial manipulation. Chapter 24 - Cyperus laxus and Cyperus ligularis are native plants growing at swamp highly contaminated with hydrocarbons. Anthracene and pyrene phytoremediation from a hydrocarbon mixture (hexadecane 50%; pristane 14%; anthracene 18% and pyrene 18%) by both species were evaluated. The aim of this chapter was to demonstrate the ability of two native plants to remove PAH from contaminated model soils under greenhouse controlled conditions. Experiments were carried out in a model system using perlite to simulate soil conditions. Initial hydrocarbon mixture concentration was 4 000 mg kg-1 of dry perlite for C. laxus and 1 000 mg kg-1 of dry perlite for C. ligularis. Plants were inoculated with hydrocarbon-degrader microorganisms. Root dry biomass, total microbial viable counts, hydrocarbon removal and hydrocarbon sorption by roots were determined during 150 days of culture. Root dry biomass from inoculated plants was 1.3 times higher than those from non inoculated plants. At the end of culture, microbial viable counts for all treatments were around 109 CFU g-1 of dry perlite for bacteria and 107 CFU g-1 of dry perlite for fungi. PAH and aliphatics were completely removed by C. ligularis during first 60 days of culture. C. laxus removed pyrene totally until 150 days of culture while anthracene only 75%. Hydrocarbons from C. ligularis roots were negligible. PAH sorption by roots was detected for C. laxus since 75 days of culture, 125 and 215 mg kg-1 of dry roots for pyrene and anthracene, respectively. At the end of culture, pyrene sorption was reduced (60 mg kg-1 of dry roots) possibly due to plant metabolism. For species, hydrocarbon removal and accumulation in roots, at 150 days of culture, was independent on the microbial inoculum added. C. laxus and C. ligularis were capable to remove efficiently anthracene and pyrene, these plants can be useful for phytoremediation of PAH contaminated soils. Chapter 25 - Polycyclic aromatic hydrocarbons (PAH) are widespread environmental pollutants of natural and anthropogenic origins. Despite their poor water solubility, they can be taken up and bioaccumulated by plants. The effect of PAH on selected plant (Festuca arundinacea and Bouteloua curtipendula) at early grown stage was evaluated under in vitro conditions. Plant species were cultured on Murashige and Skoog (MS) medium with three different PAH: phenanthrene (PHE), pyrene (PYR), and benzo[a]pyrene (BaP). PAH plant accumulation and distribution were evaluated with GC and HPLC. To date, number phytoremediation mechanisms of pollutants removal are known, including: (i) degradation (phyto- and rhizodegradation), (ii) removal (phytoextraction, phytovolatilization) and (iii) immobilization (phytoestabilization). The objective of this study was to identify the phytoremediation mechanisms of selected plants use to control and/or remove PAH from MS medium. To accomplish the task, plant samples, after 90 d culture, were collected and
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analyzed periodically to determine the residual PAH content in MS medium, roots and plants stems. F. arundinacea was able to transform 40.40% of initial PHE, accumulated 6.99% in stems and almost three times in roots (20.66%). With B. curtipendula, total removal was 66.14% and PHE was found in roots (14.38%) and stems (6.45%). Both species were able to remove and transform PYR, higher concentrations were found in roots than in stems. BaP outlined in a different pattern: both species were able to remove BaP from MS medium, B. curtipendula was more efficient than F. arundinacea 57.41% and 38.93%, respectively; bioaccumulation of BaP was the higher while transformation the lower. The dominant mechanisms observed for PHE and PYR in both plant species were phytodegradation and phytoextraction. B. curtipendula employed phytoextraction for BaP; it was able to bioaccumulate 45.05% in roots, 1.8 times more than F. arundinacea (24.99%). Several studies investigated the effect of plant-microbe interactions on the degradation of organic contaminants. In our study, plants were able to remove and transform PAH under sterile in vitro conditions, i.e. without microorganisms. Chapter 26 - Polysaccharides are complex carbohydrates made of chains of monosaccharide units linked by glycosidic bonds. Some polysaccharides, e.g. cellulose and chitin, are naturally occurring fibrous materials developed during cellular growth, while other polysaccharides are not capable of forming elongated structures in vivo because of their molecular structure or physicochemical environments. A variety of polysaccharides have been artificially spun into fibers, for instance, cellulose and its derivatives, chitin and chitosan, alginate, hyaluronic acid, pullulan, and dextran, as well as polysaccharide blends or blends with other materials. Ideal substitutes for synthetic fibers derived from fossil fuels, polysaccharides are renewable and exhibit biodegradability and biocompatibility, and often preferred to protein fibers (silks) in terms of their abundance and economics. This chapter reviews the literature on fibers spun from several polysaccharides and fiber spinning techniques, including both conventional fiber spinning methods and electrospinning. Resulting fiber properties and prospective applications are discussed. Chapter 27 - Starch-based biodegradable materials are considered one of the most promising candidates to replace certain types of conventional plastics. Starch is relatively cheap and available from a broad range of plants. However, the use of starch for packaging materials is restricted because of its sensitivity to water and inferior mechanical properties. The unique properties of nano-structured substances have opened windows of opportunity for the creation of high performance materials with a critical impact on starch-based materials. This chapter presents a review of ongoing research and development activities in our lab and in the wider scientific community related to starch-clay nanocomposites. As a valuable way to further enhance properties of these nanocomposites, blends involving biodegradable polymers such as poly (vinyl alcohol) are also discussed. Melt intercalation, using extrusion processing, has proved to be an efficient way for preparation of starch-based nanocomposites with improved thermal, mechanical, and barrier properties. The chapter also elaborates the mechanisms of clay exfoliation and dispersion in the starch matrix, which are based on both chemistry and processing effects. Improved interfacial compatibility of the multiphase system (starch, biodegradable polymer, plasticizers, nanofiller, etc.), as well optimized processing parameters, can lead to the design of nanocomposites with enhanced properties. A special emphasis is also given to an overview of mathematic models for some critical nanocomposite properties, such as barrier and mechanical properties. The establishment of a mathematic
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model provides guidelines for tailoring clay modification, clay dispersion and processing parameters for fabricating starch-based nanocomposites with unique properties. Chapter 28 - Chitosan, the only alkaline polysaccharide of β-1,4 linked N-acetylglucosamine and glucosamine,could be hydrolyzed by many non-specific enzymes such as cellulase, protease, and lipase, especially cellulase, which show high activity on chitosan. The hydrolytic mechanisms of these non-specific enzymes have been received growing attentions. The focus of this chapter was the characterizations and hydrolyzing mechanism of the non-specific enzymes toward chitosan choosing the three typical non- specific enzymes: cellulase, lipase and papain as objects. The enzymatic characteristics, purification, product analysis, glycoside bond cleavage, active sites and gene cloning of these enzymes to expatiate their non-specific hydrolysis mechanism were studied. From these, the authors obtained two bifunctional enzymes with chitosanolytic activity from commercial cellulase and lipase,respectively,and one chitosanase from papain.The three purified enzymes were the main reasons for the non-specific chitosanolytic hydrolysis of cellulase,lipase and papain, respectively. Moreover, It is identified that the bifunctional enzyme with chitosanolytic and cellulolytic activity(CCBE) from cellulase (T.viride) is identified as a cellobiohydrolase I with exo-β-D-glucosaminidase activity belonging to glycosyl hydrolysase 7 family.The enzmye with chitinase and chitosanase activity (CNBE)from lipase (A. oryzae) is the exo-β-D -glucosaminidase with N-acetyl-chitobiosidase activity belonging to glycosyl hydrolysase 18 family. Both of the two enzymes are novel and first reported in chitosanase families. Besides, the active sites and gene expression analysis of CCBE indicated that their dual activities originated from two distinct catalytic domains; while the two active sites overlapped partially. Chapter 29 - In the first part the preparation of CD polymers is described and examples of CD polymers useful as drug carriers are shown. It is pointed out that CD polymer nanoparticles are promising for application in various biomedical and biotechnological applications, e.g. in drug delivery or enzyme immobilization. A special attention was paid to removal of organic dyes from industrial wastewaters by β-CD polymers, having in view the wide use of dyes in textile and cosmetic plants. The preparation of β-CD copolymers with carbon nanotubes is described in the aspect of their application for removal of p-nitrophenol and trichloroethylene from water. It was pointed out that ceramic membranes impregnated with crosslinked silylated CD polymers are promising for removal of polycyclic aromatic hydrocarbons from water. In recent years the importance of imprinted CD polymers is growing; as an example of their application the recognition of oligopeptide structures is presented. Membranes based on CD polymers are often used today in separation processes; an increasing attention is paid now especially to chiral separation processes. In this aspect the use of chitosan/CD composite membranes in chiral separation of tryptophan was described. In the final part the selected examples of supramolecular architectures based on CD polymers are presented having in view the rapid development of supramolecular chemistry. Chapter 30 - Moisture, oxygen, carbon dioxide, lipid, flavor and/or aroma transfer between food components or between foods and their surrounding environment can provoke deterioration of food texture, flavour, color, aroma or nutritional values which results in food quality loss. Regulating the mass transfer in food systems by edible films and coatings can increase food-product shelf life and food quality. Besides their barrier properties, edible films and coatings can act as carriers for functional food additives, antioxidants, antimicrobial
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agents and nutrients; and due to their biodegradability nature, could have an impact on overall packaging requirements. Edible films and coatings are produced from edible biopolymers and food-grade additives. Film-forming biopolymers can be proteins, polysaccharides (carbohydrates and gums) or lipids. Plasticizers and other additives are combined with the film-forming biopolymers to modify the physical properties or functionality of films. The composition of the film must be chosen according to specific food applications, the type of food products and the major mechanisms of quality deterioration. Polysaccharide films and coatings are used to extend the shelf life of fruits, vegetables, seafood, meats and confectionary products by preventing dehydration, oxidation rancidity, surface browning and oil diffusion; and in some specific cases can improve the physicochemical, nutritional and sensorial properties of the products. The common poly saccharides used for edible films are: starches and their derivatives; cellulose and its derivatives; seaweed extracts; gums; pectins and chitosan. The objectives of this chapter are to (a) review research on polysaccharide film-formation and characteristics, (b) analyze mechanical and barrier properties (water vapour permeability, gas permeabilities and volatile permeability) of polysaccharide-based films, (c) summarize applications of polysaccharide films in food products, and (d) make conclusions as to the status of polysaccharide films and their future developmental direction. Chapter 31 - Carbohydrate-active enzymes (CAZymes) are associated to the synthesis and breakdown of complex carbohydrates and glycoconjugates. CAZymes, beside the catalytic domain (CD), usually present a substrate-binding module named carbohydratebinding module (CBM), which has independent fold and function. Nearly 7% of the CAZymes contain at least one CBM module. Presently, 58 CBMs families are described in the CAZy database (http://www.cazy.org/), presenting considerable heterogeneity in binding specificity, towards crystalline, amorphous and soluble polysaccharides, both between and within the families. CBMs are known to potentiate the activity of many enzymes, by targeting and promoting a prolonged interaction with the substrate. Since CBMs are functional and structurally independent of the other protein modules, several applications have been described using CBMs obtained by enzyme proteolysis or by DNA recombinant technology. The present revision focus on recent developments on CBMs applications in the biomedical, biological and biotechnological fields. Chapter 32 - Natural cellulose fibers are high-molecular polymers that are strongly susceptible to microbial degradation. Fungal and bacterial attack on the fibers cause depolymerization of cellulose macromolecules, which is reflected in decreased molecular weight and strength, increased solubility and a changed crystallinity. Although biodegradable textile fibers are classified as environmentally-friendly materials, the biodegradation process that occurs when the textile product is still in use could cause serious functional, aesthetic and hygienic problems because of textile deterioration, staining, discoloration and odor. To protect the cellulose against biodegradation, chemical modification is of great importance. In this research, therefore, fibers were chemically modified by the use of an antimicrobial finish on the basis of AgCl (AG) in combination with a reactive organic-inorganic binder (RB), water and oil repellent finishes on the basis of fluoroalkylfunctional siloxane (FAS), perfluorooctyltriethoxysilane (PFOTES) in combination with di-ureapropyltriethoxysilane [bis(aminopropyl)terminated-polydimethylsiloxane (1000) (PDMSU) and aminopropylperfluoroisooctyl polyhedral oligomeric silsesquioxane (AP2PF2IO4 POSS), as well as with an
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easy-care and durable press finish on the basis of imidasolidinone (DMeDHEU). Moreover, it was expected that these finishes would provide the active or passive antimicrobial properties of the modified fibers. Biodegradation of the finished cellulose fibers was carried out by the soil burial test according to SIST EN ISO 11721-2:2003. The chemical and functional properties of the fibers were determined by means of DP, SEM, AFM, FT-IR, XPS, ICP-MS, appropriate microbiological tests and static contact angle measurements of different liquids on the fiber surface. It can be seen from the study that AgCl embedded in RB imparts active antibacterial and antifungal properties to the coated fibers resulting in excellent microbial reduction and, consequently, strong inhibition of biodegradation. The presence of FAS, PFOTES-PDMSU and AP2PF2IO4 POSS coatings, which cause an increase in fiber hydrophobicity, as well as a high drop in surface free energy, results in decreased adhesion of microorganisms and impaired conditions of their growth. In the modification process of cellulose fibers by DMeDHEU, the formation of covalent bonds between the finish and the cellulose macromolecules strengthens the less ordered amorphous regions, resulting in a decrease of fiber swelling. This inhibits the penetration of microorganisms into the fibers, where biodegradation takes place. By applying a combination of FAS and DMeDHEU finishes, a synergistic action of the components is obtained in the coating. In this case, the protective properties of the coating against biodegradation are much higher than those obtained by single component finishing. Chapter 33 - Oligosaccharides present specific physicochemical and biological properties that can be exploited for specific applications in foods and pharmacology. They can be produced through a number of different physical, chemical and enzymatic catalysed reactions from their parent polysaccharides as well as through transglycosylation reactions. This chapter examines the pathways leading to the production of bioactive oligosaccharides that have biotechnological applications. These carbohydrate oligomers constitute a nutritional type of “fiber” that benefits the growth of bifidobacteria and lactobacilli in the colon promoting human health and well-being. The use of oligosaccharides to modify biological responses was recently reported, and this has included their effects as anti-inflammatory and anti-cholesterolaemic stimulating compounds. An overview of nutraceutical and biological functions of these carbohydrate fragments mainly for human health is also reported. Chapter 34 - Native cellulose is a structural material that is biosynthesized as microfibrils by a number of living organisms ranging from higher and lower plants, to some ameobae, sea animals, bacteria and fungi. Depending on their origin, individual cellulose microfibrils have diameters from 2 to 20 nm, while their length can reach several tens of microns. The chemical modification of cellulose microfibrils is investigated for preparing new bio-based materials with end-use properties in the fields of adhesion, textile, detergent, paint, cosmetic, medicine, food, etc. Among the possible chemical modifications, the selective oxidation of the primary alcohol group of polysaccharides has been studied for more than a half-century. Recently, a method for selectively oxidizing primary alcohol groups of polysaccharides has been described in literature without degradation of products. The technique is based on a reaction catalyzed by 2,2,6,6-tetramethyl-1-piperidine oxoammonium radical (TEMPO) in presence of NaOBr, generated in situ by NaOCl and NaBr, the catalyst being regenerated during the reaction. The chemical modification is a way to modify and introduce specific functionalities leading to the development of new biopolymers in macromolecular prodrug carrier, bio-based composites, nanocomposites, for example. The polymer must be biodegradable and / or biocompatible and must contain appropriate functional sites for chemical conjugation.
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Despites on large interest on natural and synthetic biodegradable polymers investigated, important efforts are continuing to search for new systems, notably on cellulose. In this report, the amidation of cellulose materials previously modified by carboxylation reaction is realized from the selection of some amines (cyclic and linear structures). The carboxylation is resulted from the TEMPO mediated oxidation of cellulose, leading to partially or totally oxidized cellulose, in presence of carbodiimide which is known to increase the reactivity of carboxyl groups toward amidation but used rarely for polysaccharides holding carboxyl moieties. Few reports were found in the literature on the use of carbodiimide in the preparation of cellulose conjugates with amines in order to develop new modified cellulose materials. The goals of this report are to develop ways of preparing the cellulose conjugates which can be water-soluble materials or water-insoluble materials, to identify linkage with carboxylated cellulose materials through amide bonds and to understand the obtained results following by FT-IR, NMR spectroscopies (at liquid and solid states) and electron paramagnetic resonance spectroscopy. The carboxyl groups content of oxidized cellulose materials after carboxylation and after amidation reactions are equally determined by titration curves of conductimetry and elemental analysis. Chapter 35 - Several points concerning the presence in red seaweeds of galactans with cooccurrence of carrageenan and agaran structures (DL-hybrid galactans) are addressed: a.
Analysis of D- and L-galactose enantiomers and their methylated and cyclic derivatives. Spectroscopic, enzymatic and chemical methodologies. b. Systems of galactans, in which the presence of D- and L-galactose has been determined (DL-galactan systems). c. The possible deviant structures of carrageenans and agarans in DL-hybrid galactans. d. The isolation of “pure” carrageenans and agarans from DL-galactan systems. e. The formation of molecular complexes between carrageenans, agarans and carrageenans/agarans. f. DL-hybrid galactans or molecular complexes: Evidences in favor of each hypothesis.
Chapter 36 - In the past half century, synthetic petroleum-based polymers have been widely used in a variety of packaging materials but have become a major source of waste disposal problems due to their poor biodegradability. With the increasing demand by consumers for high-quality foods and concerns about limited natural resources and the environment, the use of renewable resources to produce edible and biodegradable packaging materials that can improve product quality and reduce waste disposal problems are being explored. Biopolymer-based edible films and coatings are intended to function as barriers against moisture, oxygen, flavor, aroma and oil, as well as carriers of additives thereby improving food quality and enhancing the shelf life of food products. Water-soluble polysaccharides are commonly used in food related applications as thickeners due to their increase in viscosity when hydrated. Polysaccharides can also constitute edible films, an important quality since these polymers are of natural origin and come from renewable sources and are biodegradable as well. Though it is recognized that polysaccharide edible films are not good barriers against water vapour, they can find interesting applications as food interfaces, as carriers of active compounds or preservatives, constituting delivery systems with local activity.
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The present chapter deals with the study of edible films based on deacylated and/or acylated forms of gellan gum to support L-(+)-ascorbic acid (AA) in view of natural antioxidant protection of foods, by leveraging its activity as a vitamin in the human metabolism. Kinetics of AA-destruction and subsequent non enzymatic browning development were studied in the films stored at constant temperature (25ºC) and relative humidity (33.3, 57.7 or 75.2%) and their relationship with the microstructure, at the macromolecular and molecular levels, was also analyzed with the purpose of ensuring a better AA retention as well as lower browning rate as a consequence of controlled water mobility in the polymeric networks. Chapter 37 - Microencapsulation by coacervation is a common method for microcapsules production. It can be achieved by employing different methods, where the most common one is formation of an insoluble complex of two oppositely charged polymers and its subsequent deposition at surface of dispersed particles (e.g. emulsified oil droplets). In this way, microcapsules with coacervate shell are formed. Composition and microstructure of the coacervate shell are key to determine properties and application of microcapsules. In this chapter, novel method for microencapsulation by coacervation is presented. The method employs polymer-polymer incompatibility taking place in a ternary system composed of sodium carboxymethyl cellulose (NaCMC), hydroxypropylmethyl cellulose (HPMC), and sodium dodecylsulfate (SDS). In the ternary system, various interactions between HPMCNaCMC, HPMC-SDS and NaCMC-(HPMC-SDS) take place. The interactions were investigated by carrying out detailed conductometric, tensiometric, turbidimetric, viscosimetric, and rheological study. The interactions may result in coacervate formation as a result of incompatibility between NaCMC molecules and HPMC/SDS complex, where the ternary system phase separates in HPMC/SDS complex rich coacervate and NaCMC rich equilibrium solution. By tuning the interactions in the ternary system coacervate of controlled rheological properties was obtained. Thus obtained coacervate was deposited at the surface of dispersed oil droplets in emulsion, and oil-content microcapsules with a coacervate shell of different properties were obtained. Formation mechanism and stability of the coacervate shell, as well as stability of emulsions depend on HPMC-NaCMC-SDS interaction. Emulsions stabilized with coacervate of different properties were spray dried and powder of microcapsules was obtained. Dispersion properties of microcapsules, and microencapsulation efficiency were investigated and found to depend on both properties of deposited coacervate and the encapsulated oil type. Chapter 38 - Natural polysaccharides are produced by animals, plants or microorganisms; a large variety of chemical structures is found and as a consequence a large variety of physical properties is available. This chapter focuses on water soluble polysaccharides from different origins; their chemical structures are given as well as their macromolecular characteristics. The selected polysaccharides presented in the following are: alginates from seaweeds, gellan and xanthan from bacteria, galactomannans from seeds, chitin and chitosan from crustaceous shells. Galactomannans, xanthan and chitosans are thickening linear polysaccharides; the first one is neutral, xanthan is an anionic polysaccharide and chitosan is a cationic polymer in acidic conditions. The main physical properties of these different polysaccharides in dependence with the experimental conditions (temperature, ionic concentrations, nature of counterions…) are described. Considering alginates and gellan, a specific behaviour is
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observed: physical gelation for which the mechanisms of gelation are discussed in relation with thermodynamic conditions. The main applications of these different polysaccharides are described in different domains. At the end, mixtures of two of these polymers are considered: polyelectrolyte complexes formed between alginate and chitosan; cooperative interaction between galactomannans and xanthan. These mixtures open the way to new applications. Chapter 39 - Nutraceuticals are food or food ingredient that provides medical or health benefits. Carbohydrates are one of the most abundant bioactive substances in the marine organisms. Many marine organisms produce carbohydrates with diverse applications due to biofunctional properties. A great deal of research has been conducted in order to assess the possible use of these marine carbohydrates. The marine carbohydrates including glucosamine glycon, chitin, chitosan, fucoidan, carageenan and alginic acid have a host of bioactivities such as antioxidative, antibacterial, antiviral, antitumor, immunostimulatory and anticoagulant. Moreover, these carbohydrates have many beneficial effects and hence could be developed into potential nutraceuticals. This review describes the current progress in the development of marine carbohydrates into nutraceuticals and their applications. Chapter 40 - The present chapter intends to focus mainly on applications of various carbohydrate polymers such as aloe vera, chitosan, guar gum, starch etc. to design controlled delivery formulations for release of a variety of bioactive agents such as low and high molecular weights drugs and pharmaceutical compounds, agrochemicals such as pesticides, insecticides etc. The chapter also presents a critical review of various studies carried out over recent past particularly in the fields of controlled release technologies using carbohydrate polymers as carriers. A mention of the current state-of-the-art of carbohydrate polymers in biomedical, pharmaceutical and agricultural fields has also been presented. Chapter 41 - Chitin, the second most abundant structural biopolymer, has a complicated, multi-level supermolecular construction. All chitins are developed from superfine fibrils having diameters in the nano to micro scales, and each nanofibril contains ordered nanocrystallites inserted into low-ordered non crystalline domains. Acid hydrolysis can be used to dissolve away regions of the marginal domains so that the water-insoluble, highly crystalline residue can be converted into a stable suspension by vigorous mechanical shearing action. Highly crystalline chitin nanofibrils, otherwise called ‘whiskers’, exhibit enormous surface improvement that allows them to impart strength to several materials such as poly(caprolactone), soy proteins, natural rubber, poly(vinyl alcohol), chitosan, and silk fibroin. Apart from their ability to improve mechanical integrity, chitin nanofibrils may interact well with enzymes, platelets, growth factors, and other cell compounds existing in living tissues. Thus, the wound recovery ability and the ability to induce the formation of granulation tissues are obtained so that the applicability of chitin nanofibrils in medical areas is supported. In this chapter, the structural building principles and nanofibril structure of chitin from various sources and the methods of preparation of nanofibrillated chitin are discussed. Biological activities, as well as attempts to modify chitin nanofibril, are subsequently described. Chapter 42 - In the course of the work on value addition of Indian seaweeds in the laboratory, modifications of seaweed polysaccharides by various methods such as grafting of these carbohydrate polymers with different substrates, blending and cross linking were embarked upon. This has resulted in hydrogel materials exhibiting improved properties and
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functions. In this article, our earlier works on modification of seaweeds using grafting as well as cross linking with non-toxic genipin, the naturally occurring cross linker, will be discussed including those recently carried out with the polysaccharides of the seaweed species Chetomorpha antennina, Chamaedoris auriculata, Grateloupia indica and Sargassum wightii of Indian waters. Galactans, arabinogalatan, rhamno-galacto-ribo-arabinan, polyuronates and other heteropolymers as well as polysaccharide blends were cross linked with genipin in a water-based eco-friendly process yielding new polymeric hydrogel materials. Galactanpolyuronic acid blend was grafted with a polymeric substrate to yield a robust hydrogel. It appears that the genipin cross linked polymeric products with improved properties are best prepared with homopolymers, the heteropolymers are comparatively less amicable to this modification strategy, which produces remarkably improved functional effects. Polysaccharide blends are also a good starting material of choice for the modification work. The cross linking reaction brings about a significant makeover in the chiroptical properties of the polysaccharides. The modified products exhibited considerable thermal stability and pHresponsiveness in aqueous media. All these new properties predispose these modified products to potential applications in various domains including ingestible and non-ingestible ones. Chapter 43 - During the last quinquennium, chitosan has suddenly gained importance as a plasmid vector to be used in gene therapy in alternative to unsafe viral vectors. The main characteristics that qualify chitosan for this role are its spontaneous reactivity with DNA and oligonucleotides in general, the protection of DNA against enzymatic destruction by nucleases, the slow release of DNA, the capacity to penetrate into tissues by opening tight junctions of cells, besides its full biocompatibility with human cells and its safe assimilation in the body. The chitosan chemistry has been refined in order to impart to chitosan better solubility in physiological media, by acting on its cationicity and hydrophilicity / hydro phobicity, or combining it with poly(ethyleneimine), polyethylene glycol and derivatives, or conjugating it with cell-specific ligands. The transfection efficacy of said modified chitosans was greatly improved compared to plain chitosan. In order to understand the fate of internalized vector-DNA complexes within cells, fluorophores including the green fluorescent protein have been conjugated with DNA or with chitosan. After the exceptionally good performances of chitosans as functional wound dressing materials and drug carriers, it appears that the involvement of chitins and chitosans in gene therapy will develop into an additional exceptional achievement for this class of polysaccharides. Chapter 44 - Extreme environments, generally characterized by atypical temperatures, pH, pressure, salinity, toxicity and radiation levels, are inhabited by various microorganisms specifically adapted to these particular conditions. These microorganisms, called extre mophiles, are of significant biotechnological importance as their enzymes (extremozymes) and biopolymers possess unique properties that offer insights into their biology and evolution. The enthusiastic search for novel extremophiles has largely been stimulated by the uniqueness of their survival mechanisms. This uniqueness can be transformed into valuable applications ranging from wastewater treatment to the diagnosis of infectious and genetic diseases. One adaptation strategy of particular importance to extremophiles is the production of extracellular polymeric substances (EPSs) that envelop the cell as a barrier protecting them against environmental extremes such as desiccation, temperature, pressure, salinity, acidity, heavy metals, and radiation. Due to their many interesting physicochemical and rheological properties, these biopolymers possess novel functionality that is generally superior to
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petrochemical-derived polymers in aspects that embrace biodegradability, and environmental and human compatibility. Consequently, biopolymers of extremophiles are widely used in foods, cosmetics, pharmaceutical products, textiles, detergents, adhesives, oil-recovery from wells, brewing and waste treatment processes. This chapter presents a brief overview of life under extreme environmental conditions. This is followed by a discussion of extremophilic microorganisms and their adaptation mechanisms, and specifically focuses on the production of EPSs and their ecological and physiological functions. The application areas of industrially important EPSs from various extremophilic producer strains are also mentioned. Chapter 45 - Naturally occurring polysaccharides are of interest in pharmaceutical and cosmetic applications due to their biodegradability, biocompatibility and non-toxicity. Among these polysaccharides, alginate, a linear polysaccharide that is extracted from marine brown algae, is widely used as a particulate carrier for encapsulation, controlled release and delivery of a variety of bioactive compounds to target organs. Due to its bioadhesive, anionic and colloidal properties, alginate can interact with divalent cations or cationic polymers to form hydrogel micro- or nanoparticles that have specificity for mucosal tissues. A particular area of interest is the use of alginate as a shell or matrix material for encapsulation of essential oils. These oils have therapeutic properties that include antibacterial, antifungal, antiviral, antioxidant, and insect-repellent effects, but are insoluble in water, volatile and unstable in certain environments. This has limited development of new formulations and encapsulation of the oil in carriers is required to overcome these problems. Size reduction of the carriers to the nanometer scale greatly improves the efficacy of the delivery system and may allow targeting of the pharmacological activity of the oil to specific organs. This review focuses on an overview of alginate and essential oils, the preparation of alginate matrices from large to nanometer scale, and encapsulation of essential oils in these matrices. The prospects for development of improved alginate nanoparticles as delivery systems for essential oils are discussed at the end of the chapter. Chapter 46 - Wastes from the food processing industry have some common characteristics such as large amounts of organic materials: proteins, carbohydrates and lipids. The great production of processed vegetable products is generating an increase of the amount of wastes which represent a valuable source of by products such as carbohydrate polymers. Utilization of by-products may contribute to the efficiency of the processing and also to the sustainability of the environment. Fruits and vegetables are important sources of dietary fiber although their content is lower than the one of cereals. The cell wall matrix is the major fiber source in this type of foods and these fibers are rich in pectins. The carbohydrate composition, molecular weight and physiological properties of these fibers might be influenced by conditions of extraction as well as sources, location and many other environmental factors. The object of this research was the study of products enriched in carbohydrate polymers and obtained from quince (Cydonia oblonga Miller) or red beet (Beta vulgaris L. var. conditiva). Different methods of obtention which involved dehydration, acid and/or ethanol treatment were assayed and yield, chemical composition, molecular weight, physiological and technological properties of isolated products were stated. Chapter 47 - In recent years, interesting works have been focused on the amino polysaccharide chitosan-based microspheres. The chitosan microspheres have been applied in diversified fields, such as food processing, water treatment and biological fields. To bring new or improved properties, the chemical modification of chitosan-based microspheres is of
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rapidly growing interest. After chemical modification, chitosan-based microspheres can be used as functional materials with modified properties, such as adsorption resins, magnetic resins and nanocomposites, for specific end use in food industry. This paper presents a review of the developments in the preparation and applications of chitosan-based microspheres. The main objective of this review is to provide recent information and to show the development trend about chitosan-based microspheres. Chapter 48 – This chapter reviews the synthesis of amylose-grafted polymeric materials with well-defined structures by chemoenzymatic method, which is the combination of following chemical with enzymatic reactions. As a chemical reaction for the present chemoenzymatic method, introduction of a maltooligosaccharide as a primer, which is an initiating point for the amylose-forming polymerization, to polymer backbones or polymerizable groups is carried out to produce maltooligosaccharide-grafted polymeric materials or macromonomers having maltooligosaccharide components. As an enzymatic reaction, formation of amylose is performed by phosphorylase-catalyzed enzymatic poly merization using α-D-glucose-1-phosphate as a monomer. This enzymatic polymerization is initiated from a meltooligosaccharide as a primer and the propagation proceeds by the reversible reaction to produce amylose. The main-chain backbones of the amylose-grafted polymeric materials describing in this chapter are polystyrene and polyacetylene as organic synthetic polymers, polydimethylsiloxane and silica gel as inorganic materials, and chitin, chitosan, cellulose, and polypeptide as biopolymers. Chapter 49 - Polysaccharides are one of essential biomacromolecules in nature, which play important roles in life activities. Some of them exhibit various biological activities or have special functions. Moreover, polysaccharides contain reactive groups, such as hydroxyl, amino and carboxylic acid groups. Thus, they may react with some compounds to produce polysaccharide derivatives. Amphiphilic polysaccharide derivatives can self-assemble into stable micelles in aqueous solution, promising as a new carrier for the controlled release of drugs. In order to overcome various barriers which hinder drug delivery, the drug molecules may be conjugated with polysaccharide chains through chemical bonds to form prodrugs. The parent drug can be released through an enzymatic or chemical transformation during the metabolizing process. This article reviews the investigations of bioactive and functional polysaccharides, amphiphlic polysaccharide derivatives and polysaccharide-based prodrugs for biomedical applications. Chapter 50 - Maize bran and nejayote (a maize processing waste water) generated from tortilla-making industries in Mexico were investigated as source of water soluble feruloylated arabinoxylans. The tortilla industry is important in Mexico since half of the total volume of consumed food is maize. Therefore, maize residues are potential source of these added-value biomolecules as hydrocolloids for the food industry, considering the volume they represent. Both by-products were treated for these polysaccharides extraction. Because of their different nature, the polymers extracted from each byproduct showed different composition, physicochemical and functional properties. Feruloylated arabinoxylans from maize bran (FAXMB) presented an arabinoxylan content of 74% (w/w), a ferulic acid content of 0.34 μg/mg FAXMB, an arabinose to xylose ratio (A/X) of 0.85, an intrinsic viscosity [η] of 208 mL/g and a molecular weight (Mw) of 190 kDa. Gels were obtained from this FAXMB by laccase covalent cross-linking of ferulic acid leading to the formation of diferulic (di-FA) and triferulic (tri-FA) acid. Gels elasticity increased from 9 to 14 Pa as the FAXMB concentration changed from 2.5 to 3.5 % (w/v), while the di-FA and tri-FA contents remained
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constant (0.030 and 0.015 μg/mg FAXMB, respectively). On the other hand, feruloylated arabinoxylans extracted from nejayote (FAXN) showed a ferulic acid content of 0.23 μg/mg, an A/X ratio of 0.65, an [η] of 183 mL/g and a Mw of 60 kDa. Gels were obtained from 4% (w/v) FAXN solution, while no gelation was observed at lower concentrations. Gels elasticity increased from 2 to 4 Pa, as the FAXN concentration increased from 4 to 8 % (w/v), while no change in the di-FA and tri-FA contents was registered (0.020 and 0.010 μg/mg FAXN, respectively). The entrapment of insulin or β-lactoglobulin at 0.1% (w/v) in gels at 3.5 % (w/v) in FAXMB was investigated as well. Insulin and β-lacto-globulin did not modify either the gel elasticity or the cross-links content. Protein release rate from gels was dependent upon their molecular weight. The apparent diffusion coefficient was 0.95 × 10-7 and 0.74 × 10-7 cm2/s for insulin (5 kDa) and β-lactoglobulin (18 kDa), respectively. The results suggest that FAXMB gels can be potential candidates for the controlled release of proteins. Furthermore, recovery of these gums from low-value maize by-products could represent a commercial advantage in comparison to other gums commonly used. Chapter 51 - There are a number of reports on the use of polarized light to cure severe burns, heal wounds, and treat eczema psoriasis. Illumination with polarized light increases immunological response in peripheral blood. Amazing applications of polarized light have been recently extended to technically important transformations of polysaccharides. Visible light, particularly linearly polarized visible light (LPVL), has been found useful in transformations of polysaccharides, starches of various botanical origin, such as xylan, chitin, chitosan, and cellulose. There are two features of the application of that source of energy. Thus, on illumination of aqueous suspension of polysaccharides with LPVL the side chains of branched polysaccharides have initially been split, however, on prolonged illumination repolymerization of the split side chains into linear polysaccharide took place. Response of the polysaccharide to the illumination with LPVL depended on crystallinity of the polysaccharides. The effect of the illumination has been stronger in highly crystalline polysaccharide networks. Illumination of polysaccharides with red and green LPVL showed certain differences in the action of those wavelengths. Green light stimulated depolymerization of the polysaccharide to a higher degree with no effect on repolymerization of split fragments whereas red light was slightly more efficient in repolymerization than in depolymerization. LPVL has been capable of stimulation of hydrolases and cyclases providing acceleration of the enzymatic hydrolysis of amylopectin, chitin, chitosan, xylan and cellulose with relevant enzymes and higher yield of the products of hydrolysis. Depending on polysaccharide and relevant enzyme used, effect of the stimulation with white non-polarized light could be observed but effect of such illumination was always much weaker compared to the effect of LPVL. Illumination of cyclase with LPVL resulted in production α-, β- and γ-cyclodextrins with higher yield and different proportion of three products compared to these available from the process run without stimulation of the enzyme with LPVL. Stimulation of enzymes with LPVL presents an essential potential in practical applications. It is beneficial to perform such 1 -2 hour stimulation in a small reaction vessel followed by the application of the enzyme into bioreactor without any further illumination. Chapter 52 - It is directly observed a variety of morphologies of the cereal starch nanostructural units after the heating gelatinization by using atomic force microscope in the atmosphere. According to the observation results, nano-structural units of starch grains can be
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classified into spherical, ball chain, branched, cyclic, rod-shaped form and so on. On this basis, the authors observed the starch nano-structural units of high-pressure gelatinization, and compared the topology structure differences of cereal starch nano-structural units under different conditions of gelatinization in nano-scaled. The results showed: cereal starch nano-structural units is intermediates which are composed of natural starch granules, and their topology morphology is not the simple "chain" connections of glucose 1-4 and 1-6 glycosidic bond speculated by classic study, they have more complex composition ways, structural level. Cereal starch of High-pressure gelatinization compared with that heating gelatinization, have different nano-structure patterns; and the same germplasm starch also have different nano-structure patterns after different pressure treatment; therefore, it can be made different gelatinization characteristics of starch nano-materials by different gelatinization methods. Chapter 53 - Polysaccharides are being used in food and allied industries as texture improving agents, stabilizers, for the preparation of the edible films etc. Plant and seaweed derived polysaccharides suffer lack of assured supply and variations in quality. Microbial exopolysaccharides (EPS) provide a valid alternative. An EPS producing lactic acid bacterium was isolated from cabbage and identified as Lactobacillus sp. CFR-2180. Production of 22 g/L of EPS in 24 h at 30°C was noticed. The EPS had 65% total carbohydrates, 0.7% protein, 10% uronic acid and 2.0% moisture. Analysis by gas chromatography revealed that the EPS is a heteropolysaccharide with the presence of mannose, galactose and glucose in a ratio of 1:7:5, respectively. Gel permeation chromatography and HPLC analysis of the EPS indicated presence of multiple peaks with molecular weight ranging from 1.8 x 104 to 2.5 x 106 Da., confirming the heterogeneity of the EPS. The results of the preliminary characterization of the EPS indicated that it is a new EPS that has not been reported earlier. Chapter 54 - The article presents an overview of the latest advances in investigations about the development of polysaccharide-based intelligent antitumor drug delivery. Biodegradable, non-toxic and stimuli sensitivity nature of many kinds of polysaccharide encourages its potential use as a carrier for drug delivery system. Besides, it is reported to possess many biological activities, such as antiviral, antitumor, antimicrobial and anticoagulant activities. The innovative idea of incorporating polysaccharide to “intelligent delivery” had motivated the development of nano drug carriers with a triggered release in response to specific external or internal stimuli. Especially, acidic changes are characteristic signals for treating solid-tumors, since the relatively acidic extracellular pH is a distinguishing phenotype of solid tumors from surrounding normal tissues, and more acidic conditions are also encountered in endosomes once the micelle or nanoparticle enters the cells via endocytosis pathways. Base on this idea, quite a few intelligent polysaccharides based polymeric nanocarriers have been exploited to date. This paper summarized innovative ideas polysaccharide-based intelligent antitumor drug delivery, such as chitosan based pH-sensitive micelle systems loaded Paclitaxel, chitosan-alginate multilayer microcapsules as drug delivery vehicle, cholesteryl-bearing pullulans nanogels trapping hydrophobic molecules (antitumor drugs), etc. The paper also contained its developments in the past few decades. Meanwhile, it highlights recent progress of pH sensitive nanosystems developed in our research group mainly about preparation, characterization and biological evaluating of chitosan and dextran based super pH sensitive nanoparticles loading antitumor drugs and introduced previous misdirections and the existing research directions. This article also
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pointed out prior errors and problems facing in the experiments, one step further investigated the trends in this field. Chapter 55 - At present, with the rapid development of starch industries, there is a need to expand the breeds of starch, to look for excellent starch resources to meet application requirements in different fields. The mesoscopic scale and form of starch directly effect food quality, color and biological titer. For example, the amylopectin starch structure of cereal starch determines the nature of the material, and also the strength, flexibility and scope of use of starch environmental protection material. Research on all kinds of topology structures such as the column, chain, and ring of starch nano-structural units, obtain the relationship between cereal food quality and the form of starch nano-structural units, provide micro-scale reference for selecting high-quality breeding; in this indicator, successfully developing specialty corn and wheat germplasm, it was found that high-branched-chain corn and wheat products triggered a sense of unique food, having a quality specificity mechanism such as food rheology and so on. Waxy wheat, a new breed of wheat varieties, is about to realize commercial cultivation. Its starch can be applied to industrial production, and industrial application must understand the physical properties. Compared with traditional wheat, the research on waxy wheat is just in the initial stage. Only part of the nature of waxy wheat starch is studied, but there is no further research on the structure and application of starch. The study, through system and indepth researching on a variety of physical and chemical properties and nano-structure units, comparing the similarities and differences with common wheat starch, waxy corn starch and other commonly used industrial starch, provides a theoretical foundation for waxy wheat starch applications in industry. The results show that in the physical properties, the waxy wheat starch has low gelatinization onset temperature, a large gelatinization range and low gelatinization endothermic enthalpy change; there are a high peak viscosity, a moderate low viscosity and cold plastic viscosity, the larger collapse of the value and high viscosity of the anti-aging properties in gelatinization processing; the gel surface formatted is brittle, easily broken, the smaller internal hardness, the smaller adhesion and good flexibility. If played by external force, it is easier to restore to the original shape. After the gelatinization of its starch, it has higher transparency, water-holding capacity and moderate apparent viscosity, and antifreeze-thaw stability, viscosity shear stability and viscosity stability is the very best. In general, a variety of physical and chemical characteristics of the waxy wheat starch are similar to that of potato starch. The research on Waxy starch is helpful to the huge macromolecular level, providing references for the application and selection of food ingredients and starch materials, guiding the effective application of starch resources, resolving the scientific questions, for example, whether this reflects the starch grain varieties characteristics, genetic characteristics and nutritional characteristics, material characteristics and so on. Chapter 56 - Zein is the major storage protein of maize, which is widely used environmentally for packing-materials in food engineering. In this paper, nano-structures of waxy maize zeins were investigated in virtue of atomic force microscopy (AFM), and morphological properties of nano-particles composing waxy maize zein film were analyzed. Chapter 57 - Mushrooms along with other fungi belong to the Myceteae Kingdom. Mushrooms have a very diverse structural morphology that they exist as aerial fleshy fruiting bodies, vegetative filamentous mycelia and underground nonfleshy sclerotia. The differentiation of all these structures involve very complicated fungal morphogenesis and
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metamorphosis that are related to cell wall synthesis and metabolism (Bartnicki-Garcia, 1999; De Groot et al., 2005). With such unique characteristic, mushrooms become a rich source of carbohydrate polymers that are derived from fungal cell wall formed at different morphological stages (Zhang et al., 2007). Unlike carbohydrate polymers obtained from cell wall of higher plants, mushroom carbohydrate polymers are mainly non-starch polysaccharides with beta-glucan-chitin complex and mannoproteins (Bartnicki-Garcia, 1970). Very large structural variations and content of these mushroom carbohydrate polymers exist among the different developmental stages of the mushrooms (Zhang et al., 2007). Mushroom carbohydrate polymers have been known for their bioactive functions such as immunopotentiating, antitumor, antioxidant activities of which their mechanisms are not fully understood (Wasser, 2002; Cheung, 2008; Ooi, 2008). Recently, there is a growing popularity of developing mushroom carbohydrate polymers as functional foods (Chang, 2008). The success of such application requires active research. Chapter 58 - In this chapter selected examples of metal ion separations with polymeric macrocycles such as crown ethers, calixarenes, resorcinarenes, calixcrowns and cyclodextrins, reported in recent literature, are presented. Particularly, the use of those polymers in separation processes such as ion flotation, solvent extraction as well as transport across liquid and polymer membranes is shown. First, selected examples of crown ether polymers variety cross-linked as metal ion carriers are described, then selectivity species by donor sites bonding and coordination are characterized. Selected calixarene and calixcrown polymers as ligands of metal ions are described showing their possible applications, especially in the aspect of the separation of toxic metals ions. Other macrocyclic compounds such as cyclodextrins (CD) can be also polymerized and then used as ion carriers for transport of metal ions from aqueous solutions. The hydrophobic CD polymers have been used as ion carriers for separation of transition metal cations from dilute aqueous solutions by transport across polymer inclusion membranes. Recent developments of the CD polymers application in sorption processes of heavy metal ion from aqueous solution will be also presented in this chapter. It is also shown that hydrophilic, water soluble CD polymers obtained by cross-linking of CD with anhydrides are useful in the removal of transition metal ions from diluted aqueous solutions by ion flotation process.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 1
RESEARCH AND REVIEW AND STUDIES INDUCED SELF-ASSEMBLY OF DIBLOCK COPOLYMERS Eri Yoshida Department of Materials Science, Toyohashi University of Technology, Hibarigaoka, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
1. INDUCED SELF-ASSEMBLY BY ELECTRON TRANSFER The molecular self-assembly is induced by variation in the surroundings, such as temperature [1-4], pressure [5-9], pH [10-14], salt formation [13-18], and noncovalent bond cross-linking [1921]. The block copolymers are molecularly converted in situ from the nonamphiphilic copolymers completely dissolved in a solvent to amphiphilic copolymers due to these stimuli. Therefore, the association and dissociation of the isolated copolymers are reversibly controlled by such stimuli. The induced self-assembly has advantages over direct self-assembly of amphiphilic copolymers in molecular designing. There is no dependence on the balance of solvophilic and solvophobic moieties when designing the copolymers. Thus, a better selection of the driving force can be provided. The advantages also include the fact that a variety of amphiphilic copolymers can be created from one nonamphiphilic copolymer in situ by selecting the stimuli. Electron transport systems perform important functions concerning respiration and energy metabolism in eucaryotes [22, 23]. The electron transport reactions occur at the mitochondria inner membrane formed by electron transport proteins [24] and the lipid bilayer built up by the self-assembly of phospholipids as vital surfactants [25, 26]. The electron transport proteins include redox catalysts such as nicotinamide, iron [27, 28], and quinones [29].
Figure 1-1-1. Redox system of TEMPO
The electrons produced by these redox reactions transfer through the lipid bilayer. While the relationship between the electron transport mechanisms and the molecular self-assembly
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in vivo has been clarified, control of the self-assembly by electron transport has been applied for an artificial polymeric surfactant.
Figure 1-1-2. The PVTEMPO-b-PSt diblock copolymer.
1.1. Oxidation-Induced Micellization Oxidation-induced micellization of a diblock copolymer was determined for a diblock copolymer containing 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) on the side chains [30]. TEMPO is a stable nitroxyl radical known as a spin trapping reagent [31], a spin label reagent [32], and a mediator for living radical polymerization [33, 34]. TEMPO forms a redox system in which the radical is converted into the oxoaminium cation (OA) by one-electron oxidation and is converted into the aminoxy anion (AA) by one-electron reduction [35] (Figure 1-1-1). The oxidation of TEMPO into the OA is caused by chlorine [36], bromine [37], copper (II), and iron (III) [38], while the reduction into AA is brought about by hydrazobenzene [39], quinones [40], and ascorbic acid [41, 42]. The OA salt serves as a oneelectron oxidizing agent for amines [35, 36], sulfides [35, 43], and organometallic compounds [44] to produce their radical cation salts or radical intermediates. The OA salt also acts as a two-electron oxidizing agent for converting an alcohol into an aldehyde or ketone [45]. The salts such as the OA chloride, nitrate, trifluoroborate, and hexafluroantimonate are easily prepared by disproportionation of TEMPO in ether by the acids [46]. The oxidation-induced micellization was attained using poly(4-vinylbenzyloxy-TEMPO)block-polystyrene (PVTEMPO-b-PSt) diblock copolymer obtained by the reaction of 4hydroxy-TEMPO and PVBC-b-PSt (Figure 1-1-2). The molecular weight of copolymer was Mn(PVTEMPO-b-PSt) = 31,200-b-49,400. The PVTEMPO-b-PSt diblock copolymer showed no self-assembly in carbon tetrachloride, a nonselective solvent. Dynamic light scattering demonstrated that the copolymer self-assembled into micelles when chlorine gas was added to the copolymer solution. An excess of chlorine (1.94 equivalents relative to the TEMPO) was added in order to complete the reaction with the TEMPO when it was taken into consideration that part of the chlorine gas would escape. The hydrodynamic diameter (DH) of the micelles was estimated to be 49.5 nm by cumulant analysis, while that of the isolated copolymer was 15.6 nm. Figure 1-1-3 shows the scattering intensity distribution vs the hydrodynamic diameter of the copolymer before and after the reaction. The scattering intensity distribution was obtained by the Marquadt analysis [47]. The scattering intensity distribution of the micelles completely took the place of the unimer distribution by the reaction with chlorine.
Research and Review and Studies Induced Self-Assembly of Diblock Copolymers
Figure 1-1-3. Scattering intensity distribution of the hydrodynamic diameter for before and after the reaction with the chlorine. [PVTEMPO-b-PSt] = 1.71 X 10-3 g/mL.
Figure 1-1-4. ESR spectra of PVTEMPO-b-PSt in CCl4 before (a) and after (b) the reaction with the chlorine, and after the reaction with TMPD (c), and of Wursters’ blue chloride separately prepared (d).
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Figure 1-1-5. Micellization of PVTEMPO-b-PSt by the chlorine.
ESR studies verified that the radical concentration of the TEMPO in the copolymer decreased due to the reaction with chlorine. Figure 1-1-4 shows the ESR spectra of the copolymer before and after the reaction. Before the reaction with chlorine, a broad signal was observed due to the random orientation, probably caused by the restriction of the mobility of the TEMPO supported on the side chains. They should undergo a strong interaction with each other. After the reaction, the broad signal changed to a characteristic triplet attributed to the isotropy along with a decrease in the signal intensity. The g values of the radicals before and after the reaction were 2.0066 and 2.0064, respectively. This negligible difference in the g values indicates that they are identical radicals originating from the TEMPO. The initial concentration of the TEMPO radical was estimated to be 2.30 mM based on the molar ratio of the VTEMPO unit to the St (VTEMPO/St = 0.186/0.814). The radical concentration after the reaction with 1.94 equivalents of chlorine was estimated to be 6.76×10-2 mM on the basis of the integral curves obtained from the differential curves of the radicals. Ninety-seven percent of the TEMPO was consumed by 1.94 equivalents of the chlorine and only 3% of the TEMPO remained unreacted.
Figure 1-1-6. Variation in the UV absorbance of the PVTEMPO-b-PSt copolymer during the micellization. The chlorine to the VTEMPO unit was 0, 0.13, 0.28, 0.58, 1.11, and 1.94 equivalents from the bottom.
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Figure 1-1-7. The variation in UV absorbance at 360 nm, relative scattering intensity (I/I0),and hydrodynamic diameter (DH) of PVTEMPO-b-PSt vs amount of the chlorine.
UV analysis revealed that as the TEMPO were oxidized into the OA chloride (OAC), the block copolymer became amphiphilic in nature, and hence the polymers underwent micellization (Figure 1-1-5). OA salts are insoluble in carbon tetrachloride; however, in good solvents, such as acetonitrile, the salts show absorption at 360 nm. As can be seen in Figure 1-1-6, the absorption at 360 nm increased as a result of increasing the chlorine. The increase in the absorption at 360 nm indicates an increase in the OAC. Figure 1-1-7 shows the plots of the absorbance at this wavelength, the relative scattering intensity (I/I0), and the hydrodynamic diameter of the copolymer vs the amount of chlorine. The absorbance increased with an increase in the amount of chlorine, while the scattering intensity and hydrodynamic diameter remained almost constant over 1.11 equivalents of chlorine. It was assumed that no reaction except for the oxidation of the TEMPO by chlorine to the OAC occurred, and the degrees of oxidation of the TEMPO to the OAC were estimated at each
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amount of chlorine. The oxidation degrees were determined based on the UV absorbance and the conversion at 1.94 equivalents by the ESR analysis. Figure 1-1-8 shows the variation in the scattering intensity and hydrodynamic diameter of the copolymer vs the oxidation degree. The hydrodynamic diameter rapidly increased at a 16% oxidation degree. Only 16 % of the OAC induced the micellization. The scattering intensity also rapidly increased at the 16% oxidation degree; however, it increased almost proportionally with an increase in the oxidation degree. The continuous increase in the scattering intensity over 16% should be based on increases in the aggregation number or the number of micelles. This consequence was supported by the results for the dependence of the scattering intensity on the copolymer concentration. Figure 1-1-9 shows the plots of the scattering intensity and hydrodynamic diameter of the micelles vs the copolymer concentration. Whereas the micellar size was almost independent of the copolymer concentration, the scattering intensity increased with increasing copolymer concentration. The number of micelles increased as a result of increasing copolymer concentration, causing an increase in the scattering intensity. TEM observations confirmed that the POAC-b-PSt copolymer self-assembled into spherical micelles (Figure 1-1-10). The size of the micelles was almost equal to that estimated by the dynamic light scattering. In common cases, some micelles show a smaller size in the TEM image than in light scattering due to swelling of the micelles in solution. The POAC-b-PSt micelles may have difficulty swelling in carbon tetrachloride, because the micelles have the salts with low affinity for the solvent in the micellar cores, resulting in a slight difference in micellar size between that in light scattering and that in TEM.
Figure 1-1-8. The plots of relative scattering intensity and hydrodynamic diameter of PVTEMPO-b-PSt vs the degree of oxidation.
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The POAC-b-PSt copolymer seemed not to be very thermally stable, because the orange color of the OAC gradually faded out over room temperature, although the micellar structure was maintained even after the color disappeared. However, below 0°C the micellar solution retained the orange color for several hours.
Figure 1-1-9. The plots of the relative scattering intensity and hydrodynamic diameter of PVTEMPO-bPSt vs copolymer concentration.
Figure 1-1-10. A TEM image of the POAC-b-PSt micelles.
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Figure 1-1-11. A 1H NMR spectrum of the POAC-b-PSt micelles after the reaction with benzyl alcohol. Solvent: CCl4 with benzene-d6 as the lock solvent and diethyl ether as the standard to estimate the conversion.
The micelles served as an oxidizing agent for converting benzyl alcohol into benzaldehyde. When 1 equivalent of benzyl alcohol relative to the VTEMPO unit was added to the POAC-b-PSt micellar solution in carbon tetrachloride, the orange solution became colorless. 1H NMR demonstrated the quantitative formation of benzaldehyde. Figure 1-1-11 shows the 1H NMR spectrum of the reaction mixture. Signals originating from benzaldehyde are observed at 7.72, 8.00, and 10.13 ppm. The signals at 1.30 and 3.54 ppm are attributed to diethyl ether added as a standard to estimate the conversion into benzaldehyde. The conversions were determined from the ratio of the signal intensity at 10.13 ppm to that at 3.54 ppm, with the results after 20 and 45 min being 91 and 97%, respectively. The 97% conversion of benzyl alcohol into benzaldehyde confirms that the VTEMPO units were almost quantitatively converted to the OAC by the chlorine. The signals based on the blocks containing the pendant groups were not observed even after the oxidation of benzylalcohol, indicating that the copolymer maintains the micellar structure after the reaction. The light scattering revealed that no changes occurred in the micellar size and in the relative scattering intensity after the reaction. The OAC served as a two-electron oxidizing agent for benzyl alcohol, converting to the insoluble hydroxylamine-hydrochloride salt. Consequently, no dissociation of the micelles occurred due to the oxidation. It can be deduced that the micelles oxidized benzyl alcohol in the cores and released soluble benzaldehyde from the cores maintaining the micellar structure (Figure 1-1-12).
Figure 1-1-12. Oxidation of benzyl alcohol into benzaldehyde by the POAC-b-PSt micelles.
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Figure 1-1-13. The UV spectrum of Wurster’s blue chloride produced through the oxidation of TMPD by the POAC-b-PSt micelles.
The POAC-b-PSt micelles also oxidized N,N,N’,N’-tetramethyl-1,4-phenylenediamine (TMPD) to produce Wurster’s blue chloride. As 1 equivalent of TMPD relative to the VTEMPO unit was added to the micellar solution prepared by 1.94 equivalents of chlorine, the solution with orange colored micelles immediately turned purple. Figure 1-1-13 shows the UV spectrum of the micellar solution after the reaction. The characteristic absorption of Wurster’s blue [48] was confirmed at 536, 574, and 624 nm. It was suggested that the Wurster’s blue chloride was generated in the micellar cores by a one-electron transfer from TMPD to the OAC, because the insoluble Wurster’s blue chloride was dissolved into carbon tetrachloride.
Figure 1-1-14. Scattering intensity distribution, weight exchange distribution, and number exchange distribution of the hydrodynamic diameter of the copolymer after the reaction with TMPD.
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The one-electron transfer mechanism from TMPD to the oxoaminium salt was supported by the ESR analysis. As can be seen in Figure 1-1-4c, the signal intensity of the TEMPO increased due to the reaction with TMPD. The g value of the signal was 2.0063, showing good agreement with that before the reaction (g = 2.0064). In the triplet signal, another sharp signal was discerned. This singlet signal had a g value of 2.0034. We separately prepared Wurster’s blue chloride in carbon tetrachloride by the reaction of TMPD with chlorine. Wurster’s blue chloride was obtained as an insoluble black precipitate by the direct oxidation of TMPD by chlorine in carbon tetrachloride; however, the radical salt was unstable by itself and was rapidly decomposed thus losing its radical nature. Figure 1-1-4d shows the ESR spectrum of Wurster’s blue chloride. Wurster’s blue chloride alone showed a singlet signal with g = 2.0034 in carbon tetrachloride. The identification of g values verified that Wurster’s blue chloride was produced from the reaction of TMPD and the POAC-b-PSt micelles and was solubilized within the micellar cores. The Marquadt analysis also revealed that the POAC-b-PSt micelles were dissociated into the PVTEMPO-b-PSt copolymer by the reaction with TMPD. Figure 1-1-14 shows three different distributions of the hydrodynamic diameter of the copolymer; i.e., the scattering intensity distribution, weight exchange distribution, and number exchange distribution. The scattering intensity distribution showed the formation of huge particles over 500 nm, in addition to particles with a size similar to that of the POAC-b-PSt micelles. The huge particles should be attributed to the insoluble Wurster’s blue dropped from the micelles, because the resulting solution gradually became a white suspension, thus losing the purple color. However, there were not many huge particles, because the distribution of the huge particles was not seen in the weight exchange distribution. On the other hand, the unimer distribution slightly discerned in the scattering intensity distribution was clearly observed in the weight exchange distribution. The number exchange distribution showed only the unimer distribution, suggesting that most of the micelles were dissociated into unimers by the reaction with TMPD. TEM observations showed that the POAC-b-PSt micelles reverted into PVTEMPO-b-PSt unimers. Figure 1-1-15 shows a TEM image of the copolymer after the reaction with TMPD. It is observed that larger particles with cores and smaller particles almost without cores co-exist. The larger particles were expected to originate from the micelles including Wurster’s blue. The larger particles are still bigger than the POAC-b-PSt micelles and have a somewhat distorted shape compared with the micelles. The distortion of the shape should be caused by the copolymer associating through a weak force. This weak association of the copolymer is also reflected in the fact that Wurster’s blue chloride gradually dropped out of the micelles. The many small particles were considered to be the isolated copolymers, because the average size of the particles was 17.0 nm, almost the same size as the unimer determined by light scattering. Furthermore, unimers separating from the large particles were also observed. (Figure 1-1-16). It was deduced that the POAC-b-PSt micelles oxidized TMPD to the Wurster’s blue chloride, reverting into the PVTEMPO-b-PSt copolymers (Figure 1-1-17). Most of the copolymers reverted into the isolated copolymers, while some of them still surrounded the Wurster’s blue particles to solubilize them.
1.2. Reduction-Induced Micellization While the oxidation-induced micellization was based on the OAC/TEMPO system using chlorine as the oxidizing agent, the reduction-induced was attained through the TEMPO/HA system using phenylhydrazine as the reducing agent [49].
Research and Review and Studies Induced Self-Assembly of Diblock Copolymers
Figure 1-1-15. A TEM image of the POAC-b-PSt copolymer after the reaction with TMPD.
Figure 1-1-16. TEM images of PVTEMPO-b-PSt separating from the micelles.
Figure 1-1-17. The reaction of TMPD by the POAC-b-PSt micelles.
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Figure 1-2-1. Variation in the UV absorbance as phenylhydrazine was added to the copolymer solution in benzene. The PH/TEMPO ratios were 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 from the top.
Figure 1-2-2. Plots of the UV absorbance at 500 nm (a), relative scattering intensity (b) and hydrodynamic diameter (c) vs the PH/TEMPO ratio.
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TEMPO is red in color and has a UV absorption around 470 nm. This radical is reduced by phenylhydrazine to the colorless hydroxylamine [50]. The PVTEMPO-b-PSt diblock copolymer showed an absorption with a λmax value at 467 nm based on the TEMPO radical. It was found that the red color of the copolymer solution in benzene faded out as phenylhydrazine was added to the solution. Figure 1-2-1 shows the variation in the UV absorption of the copolymer with the addition of the phenylhydrazine. The absorbance based on TEMPO decreased with an increase in the molar ratio of the phenylhydrazine to the VTEMPO unit (PH/TEMPO). The absorbance plotted at 500 nm versus the PH/TEMPO ratio is shown in Figure 1-2-2a. The absorbance continued to decrease up to 0.8 and was almost constant above it. The absorbance did not reach zero even at 1.0 due to the long foot of the large absorption peak observed at 240-390 nm. The formation of the hydroxylamine was also confirmed by the 1H NMR measurement of the copolymer in CDCl3 in the presence of phenylhydrazine. Signals based on the tetramethyl protons of the hydroxylamine derivative of TEMPO were observed at 1.18 and 1.26 ppm. Light scattering studies demonstrated that the scattering intensity of the copolymer solution was inversely correlative to the variability in the UV absorbance. Figure 1-2-2b shows the variation in the relative scattering intensity versus the PH/TEMPO ratio. The scattering intensity increased with an increase in the ratio and became almost constant over 0.8. The increase in the scattering intensity suggests the formation of micelles. The hydrodynamic diameter of the copolymer also increased with the increase in the PH/TEMPO ratio (Figure 1-2-2c). The diameter of the copolymer rapidly increased with the addition of a small amount of phenylhydrazine and became steady over 0.2. Whereas the scattering intensity became constant over the PH/VTEMPO ratio of 0.8, the hydrodynamic diameter was constant over 0.2. This difference in the variability between the hydrodynamic diameter and scattering intensity can be accounted for by the fact that the increase in the hydrodynamic diameter indicates the formation of micelles, while the increase in the scattering intensity exactly means an increase in the number of the micelles. The formation of the micelles by the addition of only a 0.2 ratio of the PH/TEMPO suggests that the micellization occurred before all the TEMPO radicals were converted into the hydroxylamine. The hydrogen bonding among the hydroxyl groups should have effectively caused the micellization in the nonpolar solvent. The Marquadte analysis of the scattering intensity distribution of the copolymer also revealed the micelle formation. The PVTEMPO-b-PSt copolymer showed no self-assembly in benzene, because both the blocks of PVTEMPO and PSt were solvophilic to benzene. Therefore, PVTEMPO-b-PSt existed as isolated copolymers, that is, unimers in the absence of phenylhydrazine. The hydrodynamic diameter of the unimers was estimated to be 19.2 nm based on the Marquadte analysis. Figure 1-2-3 shows the scattering intensity distributions obtained at the PH/VTEMPO ratios of 0.1 and 1.0. The scattering intensity distribution in the absence of phenylhydrazine could not be obtained due to the very low scattering intensity. It is obvious that the distribution of the unimers was shifted to the higher side of the hydrodynamic diameter at the 1.0 ratio, although part of the distributions overlapped. The micellar size was estimated to be 55.6 nm. It has been reported that hydroxylamine is oxidized to TEMPO by oxygen [34]. To the micellar solution containing the hydroxylamine was added oxygen by bubbling after the hydroxylamine was converted into the aminoxy anions by sodium hydride in order to facilitate the oxidation by oxygen. As oxygen was added to the micellar solution, the UV absorbance due
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to the TEMPO radicals increased. The hydroxylamine in the copolymer was converted into TEMPO by the oxygen, although the radicals immediately reverted to the hydroxylamine again due to the presence of phenylhydrazine in the solution. In addition, this experiment was performed at the PH/TEMPO ratio of 0.5 in order to minimize the influence of the hydrazine.
Figure 1-2-3. Scattering intensity distribution of the hydrodynamic diameter of the copolymer at the 0.1 and 1.0 PH/TEMPO ratios.
1.3. Disproportionation-Induced Micellization TEMPO is disproportionated into the OA and the AA by the acids [46]. The disproportionation of TEMPO also promoted the micellization of the PVTEMPO-b-PSt copolymer [51]. The series of micellizations using the TEMPO redox systems indicate that the electron transport becomes a trigger that causes self-assembly of molecules, in addition to external triggers such as temperature, pressure, pH, salt formation, and noncovalent bond cross-linking. PVTEMPO-b-PSt shows no self-assembly in 1,4-dioxane because both blocks are solvophilic to the solvent. Dynamic light scattering studies demonstrated that the copolymer formed micelles in this solvent by the addition of hydrochloric acid. Figure 1-3-1 shows variation in the hydrodynamic diameter and scattering intensity of the copolymer as the hydrochloric acid was added to the copolymer solution. The hydrodynamic diameter rapidly increased at a 0.8 molar ratio of hydrochloric acid to the VTEMPO unit (HCl/VTEMPO). This suggested that the micellization started at 0.8. The hydrodynamic diameter gradually continued to increase over 0.8 and almost became a constant at 1.6. At the complete micellization, the copolymer formed micelles with the hydrodynamic diameter of 53.8 nm, estimated by the cumulant analysis. The scattering intensity also started increasing at 0.8. The disagreement of the variation in the scattering intensity with that of the hydrodinamic diameter was based on the fact that the scattering intensity was attributed to the aggregation number of the micelles. Figure 1-3-2 shows the scattering intensity distribution obtained by
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the Marquadt analysis for the hydrodynamic diameter of the copolymer before and after the addition of the hydrochloric acid. The Marquadt method is much better than the cumulant in analyzing the intensity distribution of the hydrodynamic diameter for polymers with comparatively narrow molecular weight distributions. The distribution of the hydrodynamic diameter for the isolated copolymers, that is unimers, was observed around 30 nm. The distribution of the micelles at the HCl/VTEMPO ratio of 2.0 was observed around ca. 55 nm. The unimer distribution was completely shifted to the micellar distribution, indicating that all the unimers were engaged in forming the micelles.
Figure 1-3-1. Variation in the hydrodynamic diameter and scattering intensity of the PVTEMPO-b-PSt copolymer vs. HCl/VTEMPO. [PVTEMPO-b-PSt] = 1.71 g/L.
Figure 1-3-2. Scattering intensity distribution of the hydrodynamic diameter for PVTEMPO-b-PSt before and after the reaction with HCl at HCl/VTEMPO of 2.0. [PVTEMPO-b-PSt] = 1.71 g/L.
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Figure 1-3-3. Variation in the UV absorbance of the PVTEMPO-b-PSt copolymer during the micellization. HCl/VTEMPO from the bottom = 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0. [PVTEMPO-b-PSt] = 1.71 g/L.
Figure 1-3-4. Micellization of PVTEMPO-b-PSt by the disproportionation of TEMPO.
A UV analysis confirmed that the oxoaminium chloride was formed in the micellar cores via the disproportionation using hydrochloric acid. Figure 1-3-3 shows the variability in the absorbance of the copolymer solution during the micellization. The oxoaminium chloride derived from 4-methoxy-TEMPO was insoluble in 1,4-dioxane, however, in a good solvent such as acetonitrile, the oxoaminium chloride showed an absorption at 360 nm. The hydroxylamine is soluble in the solvent, but had no UV absorption. The absorbance at 360 nm increased with an increase in the amount of HCl. This observation implies that the insoluble oxoaminium chloride was dissolved in the micelles by being supported on the core blocks. The increase in the absorbance indicates an increase in the amount of the oxoaminium chloride in the micellar cores. The disproportionation of TEMPO proceeded into the oxoaminium chloride and the hydroxylamine as the amount of the hydrochloric acid increased (Figure 1-3-4). ESR studies demonstrated that the radical concentration of the TEMPO in the copolymer decreased with the increasing HCl. The ESR spectra of the copolymer during the micellization are shown in Figure 1-3-5. The copolymer showed broad signals before the addition of HCl. The broad signals are based on the random orientation caused by the restriction of the mobility of the TEMPO supported on the polymer side chains. The broad signals gradually changed to the typical triplet of TEMPO as the HCl increased. It is considered that the number of TEMPO molecules decreased as the disproportionation proceeded, resulting in the fact that the interaction among the TEMPO molecules was
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reduced and showed the original triplet. In fact, the signal intensity decreased with this change in the signal, although the g values were almost constant throughout the micellization. The radical concentrations were estimated on the basis of the integral curves obtained from these differential curves in the ESR. Figure 1-3-6 shows the variation in the TEMPO concentration during the micellization. The radical concentration decreased as the HCl increased and became constant over a 1.6 HCl/VTEMPO ratio. It was found that 40 mol% of the TEMPO radicals remained unreacted. This result suggests that the micellization hindered the TEMPO disproportionation due to the fact that the remaining TEMPO was isolated in each of the micellar cores. However, the disproportionation continued to proceed unless the micellization was completed, because the TEMPO concentration continued to decrease even over the HCl/TEMPO ratio of 0.8 at which the micellization started. TEM observations demonstrated that spherical micelles were formed by the disproportionation-induced micellization. The TEM image of the micelles is shown in Figure 1-3-7. The micelles did not have completely spherical outlines, suggesting that the micelles were constructed with a weak association force. This weak association should be caused by non-quantitative disproportionation and by the presence of the soluble hydroxylamine in the cores. The diameter of the micelles was ca. 72.7 nm in the TEM image. This micellar size was larger than that estimated by the dynamic light scattering. This difference may be accounted for by the fact that the micelles expanded by an increase in the repulsion among the OA cations when the solvent was removed.
Figure 1-3-5. ESR spectra of PVTEMPO-b-PSt. [PVTEMPO-b-PSt] = 1.71 g/L.
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Figure 1-3-6. Plots of the radical concentration of the TEMPO vs. HCl/VTEMPO.
Figure 1-3-7. A TEM image of the micelles obtained by the disproportionation.
REFERENCES [1] [2] [3] [4] [5] [6]
Alexandre A., Reynafarje B., Lehninger AL (1978). Proc Natl Acad Sci, 75, 5296. Arotcarena M., Heise B., Ishaya S., Laschewsky A (2002). J Am Chem Soc, 124, 3787. Bar RS., Deamer DW., Cornwall DG (1966). Science, 153, 1010. Brik ME (1990). Synth Commun, 20, 3283. Brontein LM., Sidorov SN., Valetsky PM (1999). Langmuir, 15, 6256. Buhler E., Dobrynin AV., DeSimone JM., Rubinstein M (1998). Macromolecules, 31, 7347. [7] Capaldi RA (1982). Biochim Biophys Acta, 695, 291. [8] Celso L., Triolo A., Triolo F., Donato DI., Steinhart M., Kriechbaum M., Amenitsch H., Triolo R (2002). Eur Phys J Soft Matter, 8, 311. [9] Chan SI., Li PM (1990). Biochemistry, 29, 1. [10] Erecinska M., Wilson DF (1982). J Membr Biol, 70, 1.
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[11] Georges MK., Veregin RPN., Kazmaier PM., Hamer GK (1993). Macromolecules, 26, 2987. [12] Gohy JF., Lohmeijer GG., Varshney SK., Decamps B., Leroy E., Boileau S., Schubert US (2002). Macromolecules, 35, 9748. [13] Gohy JF., Varshney SK., Jerome R (2001). Macromolecules, 34, 3361. [14] Golubev VA., Rozantsev EG., Neiman MB (1965). Bull Acad Sci USSR, 1898. [15] Golubev VA., Rozantsev EG., Neiman MB (1965). Izv Akad Nauk SSSR Ser Khim, 1927. [16] Griffiths DE (1965). Essays Biochem, 1, 120. [17] Guranova VV., Kovarsksaya BM., Krinitzkaya LA., Neiman MB., Rozantsev EG (1965). Vysokomol Soedin, 7, 1515. [18] Hageman HJ., Overeem T (1981). Makromol Chem Rapid Commun, 2, 719. [19] Hu Y., Kramer MC., Boudreaux CJ., McCormick CL (1995). Macromolecules, 28, 7100. [20] Hubbel WL., Metcalfe JC., Metcalfe SM., McConnell HM (1970). Biochim Biophys Acta., 219, 415. [21] Koga T., Zhou S., Chu B (2001). Appl Optics, 40, 4170. [22] Lee AS., Butun V., Vamvakaki M., Armes S., Pople JA., Gast AP (2002). Macromolecules, 35, 8540. [23] Lehninger., AL Reynafarje B., Alexandre A., Villalobo A (1980). Ann NY Acad Sci, 341, 585. [24] Liu S., Weaver JVM., Tang Y., Billingham NC., Armes SP (2002). Macromolecules, 35, 6121. [25] Liu S., Zhang G., Jiang M (1999). Polymer, 40, 5449. [26] Liu YC., Wu LM., Chen P (1985). Tetrahedron Lett, 26, 4201. [27] Lowe AB., Billingham NC., Armes SP (1997). Chem Commun, 1035. [28] Marquardt DW (1963). J Soc Indust Appl Math, 11, 431. [29] Martin TJ., Prochazka K., Munk P., Webber SE (1996). Macromolecules, 29, 6071. [30] McClain JB., Canelas DA., Samulski ET., DeSimone JM., Londono JD., Cochran HD., Wignall GD., Chillura-Martino GD., Triolo R (1996). Science, 274, 2049. [31] Miyazawa T., Endo T., Shiihashi S., Ogawara M (1985). J Org Chem, 50,1332. [32] Neradovic, D., Nostrum, C. F., Hennink, W. E. (2001). Macromolecules, 34, 7589. [33] Paleos CM., Dais P (1977). Chem Commun, 10, 345. [34] Paper I., Bobbitt JM., Cecile M., Flores L (1988). Heterocycles, 27, 509. [35] Rozantsev, E. G. & Golubev, V. A. (1966). Izv Akad Nauk SSSR Ser Khim, 891. [36] Rozenberg VI., Piotrovskii VK., Golubev VA., Gvon KI., Nikanorov VA., Bundel YG., Reutov OA (1975). Bull Acad Sci USSR Chem Ser, 24, 2508. [37] Slater EC (1983). Trends Biochem Sci, 8, 239. [38] Wang RL., Tam KY., Compton RG (1997). J Electroanal Chem, 434, 105. [39] Weaver JVM., Armes SP., Butun V (2002). Chem Commun, 2122. [40] Yoshida E., Kunugi S (2002). Macromolecules, 35, 6665. [41] Yoshida, E. & Ogawa, H. (2007). J Oleo Sci, 56, 297. [42] Yoshida E., Tanaka M., Takata., T (2005). Collid Polym Sci, 284, 51. [43] Yoshida E., Tanaka T (2006). Colloid Polym Sci, 285, 135. [44] Yoshida, E. & Tanaka, T. (2008). Colloid Polym Sci, 286, 827. [45] Yoshida E., Takata T., Endo T (1992). Macromolecules, 25, 7282.
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[46] Yoshida E., Takata T., Endo T., Ishizone T., Hirao A., Nakahama S (1994). Chem Lett, 1827. [47] Yoshida E., Terada Y (2005). Collid Polym Sci, 283, 1190. [48] Yoshida E., Sugita A (1996). Macromolecules, 29, 6422. [49] Zhao H., Douglas EP (2002). Mater Res Soc Symp Proc, 43. [50] Zhdanov RI., Golubev VA., Gida VM., Rozantsev EG (1971). Dokl Akad Nauk SSSR, 196, 856. [51] Zhou S., Chu B (1998). Macromolecules, 31, 5300.
2. INDUCED SELF-ASSEMBLY BY PHOTOLYSIS Light is a handy, easily available, and environmentally clean stimulant to cause selfassembly. In vivo, the photoreceptor proteins in animal eye cells change their highdimensional structure by receiving photons [1], while artificial polymers responsive to light contain photochromic compounds such as azobenzene [2-4], spiropyran [5,6], stilbene [7-9], cinnamate [10], and triphenylmethane leuco residues [11]. The polymers reversibly change their structure through the cis-trans isomerization, dimerization, and conformational changes of the photochromic compounds. This reversible behavior is manipulated by UV wavelength of the compounds or sometimes temperature. Compared to these reversible reactions required as a function of the on-off switches, irreversible reactions are convenient to fix the spatial structure changed by photo irradiation. The structure change effects by the photo irreversible reaction have been investigated on the photolysis of diazosulfonates [12-14], 1iminopyridinium ylides [15], [4(4'-alkoxybenzoyl)phenylmethyl]phosphonic acids [16], and didecyl-2-methoxy-5-nitrophenyl phosphate [17]. The former three kinds of surfactants loose their surface-active ability by photolysis, resulting in the destruction of the micelles and vesicles. On the other hand, didecyl-2-methoxy-5-nitrophenyl phosphate formed vesicles by the photolysis. The self-assembly induced by the photolysis was determined for a poly(4-tert-butoxystyrene)block-polystyrene diblock copolymer (PBSt-b-PSt) [18] (Figure 2-1). In this photolysis-induced self-assembly, a diblock copolymer produced by the photolysis formed micelles. This new way of molecular self-assembly induced by photoirradiation has the potential to produce new applications for optical memory materials and optical devices using the photoirreversible reaction.
Figure 2-1. The PBSt-b-PSt diblock copolymer.
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The PBSt-b-PSt diblock copolymer shows no self-assembly in dichloromethane since the PBSt and PSt blocks are solvophilic to it. Light scattering studies have demonstrated that the copolymer is self-assembled into micelles in dichloromethane by irradiation in the presence of a photoacid generator. Figure 2-2 shows the variation in the hydrodynamic diameter (DH) and the relative scattering intensity (I/I0) of the copolymer with the molecular weight of Mn(PBSt-b-PSt) = 15,000-b-97,000 during the irradiation using bis(alkylphenyl)iodonium hexafluorophosphate (BAI) as a photoacid generator. The molar ratio of BAI to the BSt unit was 0.38. The hydrodynamic diameter and scattering intensity showed a good correlation. They increased at 4.5 h and became constant over 5 h, indicating that the micellization was completed over 5 h. The hydrodynamic diameter of the micelles averaged 63.0 nm, while that of the isolated copolymer, which is a unimer, was 16.6 nm based on the cumulant analysis. The observation of the jump and the constant state within the short time period suggests the rapid micellization due to the strong aggregation force.
Figure 2-2. The variation in the hydrodynamic diameter (DH), relative scattering intensity (I/I0), and conversion of the copolymer during the irradiation using BAI. Mn(PBSt-b-PSt) = 15,000-b-97,000, [copolymer]0 = 3.30 g/L.
The variation in the scattering intensity distribution of the hydrodynamic diameter also supported the formation of the micelles by the rapid association. Figure 2-3 shows the
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scattering intensity distribution obtained by the Marquadt analysis. The distribution was shifted to the higher side of the hydrodynamic diameter over time by the irradiation. The slight shift in the distribution at 3.5 h implies that aggregates with a lower aggregation number were formed during the first stage and those associated into micelles, rather than that the unimers inserted step by step into the micelles.
Figure 2-3. Scattering intensity distributions of the hydrodynamic diameter of the copolymer. [copolymer]0 = 3.30 g/L
Figure 2-4. A TEM image of the micelles. Mn(PBSt-b-PSt) = 15,000-b-97,000,
TEM observation confirmed the formation of spherical micelles through the irradiation. The TEM image of the micelles is shown in Figure 2-4. The diameter of the micelles was
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estimated to average 40.6 nm based on the TEM. Compared to the micellar size determined by the cumulant analysis, the TEM exhibited a smaller diameter of the micelles than the dynamic light scattering. The estimation of the micelles as the smaller size can be accounted for by the fact that the micelles in the solution contracted when isolated in air. The irradiation of the copolymer in the absence of BAI and the dark reaction in its presence produced no changes in the hydrodynamic diameter and scattering intensity. These two control experiments suggest that the structure of PBSt-b-PSt was changed by the irradiation on BAI. The 1H NMR confirmed that the micellization was caused by the elimination of the tert-butyl groups in the copolymer. Figure 2-5 shows the 1H NMR spectra of the copolymer before and after the irradiation. The 1H NMR measurements were performed in 1,4-dioxane-d8. Signals at 1.29 ppm based on the tert-butyl groups were hardly observed after the irradiation. The disappearance of the signals implies that the tert-butyl groups were eliminated from the copolymer. PBSt-b-PSt should have been converted into poly(4-vinyl phenol)-block-PSt (PVPh-b-PSt) by the hydrolysis of the tert-butoxy groups with the photoacid generator as a catalyst (Figure 2-6), based on the mechanism of the hydrolysis of poly(4-tert-butoxystyrene) [19]. A signal based on the hydroxyl groups of the PVPh blocks could not be discerned due to the fact that it overlapped with the signals of the aromatic protons and had too low an intensity. In addition, it is clear that the disappearance of the butyl proton signals and no observation of the hydroxyl signal were not based on the self-assembly of the copolymer into micelles. This is because PVPh-b-PSt showed no selfassembly in 1,4-dioxane-d8 and existed as unimers. The conversion of the BSt units into the VPh units was estimated based on the signal intensity of the tert-butyl protons to that of the aromatic protons at 6.3-7.7 ppm. The time-conversion plots are shown in Figure 2-2. The conversion started increasing at an earlier stage than the scattering intensity. The scattering intensity jumped when the conversion reached 50%, indicating that the micellization was dependent on the degree of the VPh unit formation.
Figure 2-5. 1H NMR spectra of the copolymer before (bottom) and after the irradiation (upper, the irradiation time = 5.5 h). Solvent: 1,4-dioxane-d8.
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Figure 2-6. The micellization induced by photolysis of PBSt-b-PSt.
Figure 2-7. The variation in the hydrodynamic diameter, scattering intensity, and conversion of the copolymer during the irradiation in the presence of BAI (o), DPI (z), and TPS (▲). [copolymer]0 = 3.30 g/L. Photoacid generator/BSt unit = 0.38.
Research and Review and Studies Induced Self-Assembly of Diblock Copolymers
25
The study of the micellization using different kinds of photoacid generators demonstrated that the micellization, coupled with the conversion, were dependent on the ability of the photoacid generator. The micellization by the irradiation was evaluated using diphenyliodonium hexaflurophosphate (DPI) and triphenylsulfonium triflate (TPS). Figure 27 shows the variation in the hydrodynamic diameter, scattering intensity, and conversion during the irradiation by BAI, DPI, and TPS. The conversion for DPI started increasing slightly earlier than that for BAI, although there is a negligible difference in the transition of the scattering intensity and hydrodynamic diameter. On the other hand, TPS needed a longer irradiation time to promote the micellization as compared to BAI and DPI. This difference in promoting the micellization was clarified on the basis of the UV analysis of the photoacid generators. Figure 2-8 shows the UV spectra of the photoacid generators and the PBSt-b-PSt copolymer, coupled with the illumination intensity of the irradiation versus the wavelength for the high-pressure mercury lamp. It is considered that the irradiation reaction of the photoacid generators occurred around 290 nm, because at this wavelength, the absorption of the photoacid generator overlapped at a highest proportion with the illumination intensity of the lamp without any obstruction by the copolymer. The absorbance of the photoacid generators decreased in the order of BAI > DPI > TPS. In particular, TPS had a slight absorption at 290 nm. It can be deduced that the difference in the absorption intensity among the photoacid generators was reflected in the irradiation time needed to initiate the micellization.
Figure 2-8. UV spectra of BAI (a), DPI (b), TPS (c), and PBSt-b-PSt (d) with the illumination intensity of the irradiation of the high-pressure mercury lamp (e). Solvent: dichloromethane. Mn(PBSt-b-PSt) = 15,000-b-97,000.
The efficiency of the micellization was also dependent on the concentration of the photoacid generator. Figure 2-9 shows the variation in the hydrodynamic diameter, scattering intensity, and conversion during the irradiation in the presence of DPI at the DPI/BSt molar ratios of 0.38 and 1.00. More sharply and earlier jumping was observed at 1.00, indicating that the micellization was promoted more effectively at 1.00. Consequently, the irradiation time needed for the micellization was manipulated by the concentration of the photoacid generator. The block length of the copolymer had an effect not only on the micellar size and scattering intensity, but also on the conversion. For the identical PBSt block length (Mn = 15,000), the effect of the PSt block length on the micellization was explored. Figure 2-10 shows the variation in the hydrodynamic diameter, scattering intensity, and conversion during the irradiation using copolymers with the different PSt block lengths: Mn = 63,000 and
26
Eri Yoshida
170,000. Regarding the hydrodynamic diameter and the scattering intensity, it was observed that the copolymer with the shorter PSt block showed a transition at an earlier stage than that with the longer PSt. The copolymer with the shorter PSt more easily aggregated into micelles. The shorter PSt sample produced smaller micelles with a higher aggregation number due to the shorter length of the PSt blocks. The conversion of the shorter PSt sample also started increasing slightly earlier than that for the longer sample. The reason why the shorter PSt sample initiated the micellization more earlier is also based on the faster conversion.
Figure 2-9. The variation in the hydrodynamic diameter, scattering intensity, and conversion during the irradiation at 0.38 (O) and 1.00 (z) of DPI/BSt. [copolymer]0 = 3.30 g/L, Mn(PBSt-b-PSt) = 15,000-b97,000.
Research and Review and Studies Induced Self-Assembly of Diblock Copolymers
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Figure 2-10. The variation in the hydrodynamic diameter, scattering intensity, and conversion during the irradiation using copolymers with Mn(PSt block) = 63,000 (z) and 170,000 (O). Mn(PBSt block) = 15,000, [copolymer]0 = 3.30 g/L, DPI/BSt = 0.38.
REFERENCES [1]
[2] [3] [4] [5] [6]
Palczewski, K., Kumasaka, T., Hori, T., Behnke, C., Motoshima, B., Fox, A., Le Trong, I., Teller, T., Okada, T., Stenkamp, R. E., Yamamoto, M. & Miyano, M. (2000). Science, 289, 739 Lovrien, R. (1967). Proc Natl Acad Sci USA, 57, 236 Matejka, L., Dusek, K. (1998). Makromol Chem, 182, 3223 Pieroni, O. & Fissi, A. (1992). J Photochem Photobiol B Biol, 12, 125 Menju, A., Hayashi, K. & Irie M. (1981). Macromolecules, 14. 755 Taguchi, M., Li, G., Gu, Z., Sato, O. & Einaga, Y. (2003). Chem Mater, 15, 4756
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Eri Yoshida Meier, H. (1992). Angew Chem Int Ed Engl, 31, 1399 Szczubialka, K. & Nowakoaska, M. (2003). Polymer, 44, 5269 Eastoe, J., Sanchez, M., Dominguez, S., Wyatt, P., Beeby, A. & Heenan, R. (2002). Langmuir, 18, 7837. Szczubialka, K. & Nowakoaska, M. (2003). Polymer, 44, 5269 Irie, M. & Hosoda, M. (1985). Makromol Chem Rapid Commun, 6, 533 Dunkin, I. R., Gittinger, A., Sherrington, D. C. & Whittaker, P. (1994). J Chem Soc Chem Commun, 2245. Mezger, T., Nuyken, O., Meindl, K. & Wokaun, A. (1996). Prog Org Coatings, 29, 147. Nuyken, O. & Voit, B. (1997). Macromol Chem Phys, 198, 2337. Haubs, M. & Ringsdorf, H. (1987). New J Chem, 11, 151. Okamoto, Y., Yoshida, H. & Takamuku, S. (1988) Chem Lett, 569. Veronese, A., Berclaz, N. & Luisi, P. L. (1998). J Phys Chem B, 102, 7078. Yoshida, E., Kuwayama, S. (2007). Colloid Polym Sci, 285, 1287. Conlon, D. A., Crivello, J. V., Lee, JL. & O'Brien, M. J. (1989). Macromolecules, 22, 509.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 2
A NOVEL THERMOSENSITIVE COMPOSITE HYDROGEL BASED ON POLY(ETHYLENE GLYCOL)POLY(Ε-CAPROLACTONE)-POLY(ETHYLENE GLYCOL) (PECE) COPOLYMER AND PLURONIC F127 ++
Chang Yang Gong, Shuai Shi, Peng Wei Dong, Ma Ling Gou, Xing Yi Li, Yu Quan Wei and Zhi Yong Qian* State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, and School of Life Sciences, Sichuan University, Chengdu, 610041, China.
ABSTRACT A novel kind of biodegradable thermosensitive composite hydrogel was successfully prepared in this work, which was a flowing sol at ambient temperature and became a nonflowing gel at body temperature. The composite hydrogel was composed of poly(ethylene glycol)-poly(ε-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG, PECE) and Pluronic F127 copolymer. By varying the composition of above two copolymers, in vivo degradation rate and in vitro drug release behavior could be controlled. Histopathological study of tissue at injection site showed no significant inflammatory reaction and toxicity, which means that the composite hydrogel might serve as a safe candidate as in situ gelforming controlled drug delivery system.
++
This work was financially supported by National Natural Science Foundation (NSFC20704027), National 863 Project (2007AA021900), Sichuan Prominent Young Talents Program (07ZQ026-033), and Sichuan Key Project of Science and Technology (2007SGY019), and Chinese Key Basic Research Program (2004CB518807). * Corresponding author: Tel: 86-28-85164063, Fax: 86-28-85164060, E-mail:
[email protected].
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1. INTRODUCTION Cancer is one of the most severe diseases and causes millions of death each year in the world. More than 1 million new cancer cases and 500,000 deaths from cancer are projected to occur in 2008 in USA [1]. Chemotherapy, as conventional cancer treatment, was widely used in clinical. However, conventional chemotherapy had severe side effects, which are very painful for patients to tolerate. Novel controlled drug delivery systems (DDS) are intended to deliver drugs at predetermined rates for predefined periods of time, which might overcome the shortcomings of conventional drug formulations, therefore could diminish the side effects and improve the life quality of the patients [2-3]. Thus, a suitable controlled drug delivery system is extremely important for chemotherapy. An optimal controlled drug delivery system should sustained release drugs in an extended period, and should be biodegradable, biocompatible, and non-toxic. Hydrogels are a special series of materials that could absorb a considerable amount of water while maintaining their three-dimension integrity in water. Over the past decades, the stimuli-sensitive copolymer hydrogel has attracted increasing attention owing to their smart responsibility to the environmental stimulus including chemical substances and changes in temperature, pH, or electric field [4-10]. Especially, the biodegradable thermosensitive physical crosslinked hydrogels consisted of hydrophobic and hydrophilic blocks have been extensively studied owing to their smart responsibility to the environmental stimulus, biodegradability, and biocompatibility. Therefore, biodegradable thermosensitive hydrogels, which might have potential biomedical applications, have been investigated as in situ gelforming system, such as controlled drug delivery, tissue repair, and cell encapsulation [1123]. In previous contributions, many biodegradable thermosensitive hydrogels have been studied, such as poly(ethylene glycol)-poly(D,L-lactic acid-co-glycolic acid) (PEG-PLGA) copolymers [24-27], poly(ethylene glycol)-poly(L-lactic acid) (PEG-PLLA) copolymers [10, 28], chitosan derivatives [29-32], methylcellulose, polyphosphazene, and etc. Poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) triblock copolymer (PEG-PPG-PEG), known as Pluronic or Poloxamer, has been extensively studied as a potential drug delivery vehicle due to their excellent biocompatibility and thermosensitivity [33-34]. These copolymers have been widely used as emulsifiers, wetting agents, and solubilizers [35]. However, due to weak hydrophobicity of PPG block and high PPG content, the Pluronic copolymer forms a fast-eroding gel and can not persist longer than a few hours in vivo, which greatly restricted its application as in situ gel-forming systems. In our previous study, we prepared a new kind of biodegradable and injectable thermosensitive poly(ethylene glycol)–poly(ε-caprolactone)–poly(ethylene glycol) (PEGPCL-PEG, PECE) hydrogel controlled drug delivery system that undergoes sol-gel-sol transition. In vitro drug release behavior and in vivo gel formation and degradation test upon subcutaneous injection of PECE hydrogel in the mice model were also conducted [36]. PEG and PCL are materials that are biocompatible and have benn used in several FDA-approved products [37-42]. And PCL is lack of toxicity and has great permeability [43]. Due to combination of great advantages of PEG and PCL, the PECE copolymer might have great potential application in biomedical field. In this study, we prepared a new kind of biodegradable and injectable composite hydrogel based on PECE and Pluronic F127 copolymer. The composite hydrogels are free-flowing sol
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at room temperature and becomes gel at body temperature, and the influence of composition of the composite hydrogel on the sol-gel-sol transition was investigated. We wonder whether composite hydrogel with different composition would result in different drug release rate and in vivo gel degradation rate. Therefore, in vitro drug release behavior and in vivo gel formation and degradation test were conducted in this paper. The result showed that by altering the composition of composite hydrogel, in vitro drug release rate and in vivo gel degradation rate could be controlled, which was very important for their further application as injectable in situ gel-forming controlled drug delivery system.
2. MATERIALS AND METHODS 2.1. Materials Poly(ethylene glycol) methyl ether (MPEG, Mn=550, Aldrich, USA), ε-caprolactone (εCL, Alfa Aesar, USA), Pluronic F127 (Sigma, USA), hexamethylene diisocyanate (HMDI, Aldrich, USA), stannous octoate (Sn(Oct)2, Sigma, USA), bovine serum albumin (BSA, BR, BoAo Co. Ltd, China), and VB12 (Sigma, USA) were used without any further purification. All the materials used in this article were analytic reagent (AR) grade and used as received, expect BSA. BALB/c mice, at weight of 20±2g, were used for in vivo gel formation and degradation test. The animals were purchased from the Laboratory Animal Center of Sichuan University. The animals were housed at controlled temperature of 20-22oC, relative humidity of 50-60% and 12h light-dark cycles. Free access to food and water was allowed. All the animals would be in quarantine for a week before treatment. All animal care and experimental procedures were conducted according to Institutional Animal Care and Use guidelines.
2.2. Synthesis and Characterization of PECE Copolymer PECE copolymer was synthesized as described in our previous work according to Figure 1. Briefly, PEG-PCL diblock copolymer was prepared by ring opening polymerization of ε-CL initiated by MPEG using stannous octoate as catalyst; PEG-PCL-PEG triblock copolymer was synthesized by coupling PEG-PCL diblock copolymer using HMDI as the coupling agent [18, 36]. The just-obtained PECE copolymer was first dissolved in AR grade dichloromethane, and reprecipitated from the filtrate using AR grade excess cold petroleum ether. Then the mixture was filtered and vacuum dried to constant weight at room temperature. The purified copolymer was kept in air-tight bags before further use. The obtained PECE copolymer was characterized by FTIR (NICOLET 200SXV, Nicolet, USA), 1H-NMR (Varian 400 spectrometer, Varian, USA), and GPC (Agilent 110 HPLC, USA).
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2.3. Preparation of Composite Hydrogel Preparation of composite hydrogels was described in Figure 2. Different amount of Pluronic F127 copolymer were dissolved in icy cold deionized water to a transparent solution, and PECE solution at different concentration were prepared by dissolution of PECE copolymer in deionized water at a designated temperature then cooled to 4oC. Subsequently, the two prepared solutions were mixed together under mild agitation to obtain a homogeneous liquid solution. The final solution contained a given concentration and composition of the two copolymers to form different composite hydrogel samples. The composite hydrogel prepared in this work were listed in Table 1. Table 1. The composite hydrogel prepared in this work Code S1 S2 S3 S4 S5
PECE: Pluronic F127 (w/w) 100:0 70:30 50:50 30:70 0:100
Phase Transition behavior Sol-gel-sol Sol-gel-sol Sol-gel-sol Sol-gel-sol Sol-gel-sol
The concentration region with phase transition behavior (Wt%) 15% to 35% 25% to 35% 20% to 35% 20% to 35% 15% to 35% O
O
H3 C
x
H O
O
Y
(MPEG)
(epsilon-CL)
130oC
Sn(Oct)2
O O
O
H
O
H 3C
OCN(CH2 )6NCO
X
Y
(HMDI)
(MPEG-PCL)
130oC
Sn(Oct)2 O
O O H3C
OY
O
O
O OCHN(CH2)6 NHCO
X
PEG Figure 1. Synthesis scheme of PECE copolymer
PCL
XO
Y
PEG
CH3
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33
Figure 2. Preparation of composite hydrogel.
2.4. Sol-Gel-Sol Phase Transition Behavior Study Sol-gel-sol phase transition diagrams of composite hydrogel were recorded using test tube-inverting method [18, 36]. The sol-gel-sol transition was visually observed by inverting the vials, and conditions of sol and gel were defined as “flow liquid sol” and “no flow solid gel” in one minute respectively.
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In this study, the volume of the composite hydrogel solution was kept 0.5 mL in total regardless of the concentration. After incubated in water bath at 0oC for 20 minutes, the hydrous samples were slowly heated at a rate of 0.5oC/min, from 0oC to the temperature when precipitation occurred.
2.5. Scanning Electron Microscopy (SEM) of Composite Hydrogel SEM was employed to investigate morphology of composite hydrogel. The composite hydrogels were frozen in liquid nitrogen and lyophilized for 72 h. The hydrogels were sputtered with gold before observation. In this study, morphology of prepared composite hydrogels was examined on JEOL SEM (JSM-5900LV, JEOL, Japan).
2.6. In vitro Drug Release Behavior Freshly prepared VB12 loaded composite hydrogel were used to assay in vitro release behavior of hydrophilic drugs. In detail, 200µl of prepared VB12 loaded composite hydrogel containing 1mg VB12 were placed into 4 mL EP tubes and allowed to gel in an incubator at 37oC for 12 h. Then, the gels were immersed in 1 mL of PBS (pH=7.4) and were shaken at 100rpm at 37oC. At specific time intervals, all of the release media were removed and replaced by fresh release media. After centrifugation at 13000rpm for 10min, the supernatant of the removed release media were collected and stored at -20oC until analysis. The collected supernatants were detected on UV spectrophotometer at 362 nm to determine the concentration of VB12. BSA was used as model protein drug to determine the release behavior of protein or peptide from protein loaded composite hydrogel in vitro. The detail procedure was similar to the study of VB12 release, but the initial drug loading amount were 4mg. The amount of BSA present in the supernatant was determined by bicinchoninic acid (BCA) assay and BCATM Protein Assay Kit (PIERCE, USA) was used. The SDS-polyacrylamide gel electrophoretic (PAGE) analysis was used to assay the stability of BSA during the release period.
2.7. In vivo Gel Formation and Degradation Study In vivo gel formation and degradation tests were performed in BALB/c mice (20±2g). The composite hydrogel (S1, S2, S3, S4 and S5) at concentration of 30wt% were prepared. Then, each animal was subcutaneously injected with 0.5 mL of composite hydrogel solutions by a syringe with a 25 gauge needle. At predetermined time (2h, 6h, 1d, 3d, 7d, 10d, and 14d), the animals were sacrificed by cervical dislocation. And then the injection site was carefully cut open and the in situ formed gel was taken photo.
2.8. Histopathologic Study Tissue samples of injection site were obtained after taken photo at each time point (2h, 6h, 1d, 3d, 7d, 10d, and 14d), which described in section 2.7. Control group was only given
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similar volume of normal saline at the same time and same way. All the tissue samples were preserved in 10% buffered formaldehyde and were subsequently embedded in paraffin. Then, paraffin sections were stained with haematoxylin-eosin (HE) for histopathologic examination. In order to investigate the inflammatory reaction and toxicity of composite hydrogel at injection site, the histopathological changes of tissue at injection site were observed on light microscope. At day 14, composite hydrogels would be physiologically metabolized and removed from injection site. Therefore, we save the picture at day 14 as an illustration.
3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PECE Copolymer The synthesis of PECE triblock copolymer has been described in our previous work [18, 36]. Briefly, ring-opening copolymerization of ε-CL onto MPEG was performed to synthesize PEG-PCL diblock copolymer using stannous octoate as catalyst. PEG-PCL diblock copolymer was then coupled using HMDI as coupling agent to produce the biodegradable PEG-PCL-PEG triblock copolymer. FTIR, 1H-NMR and GPC were used to characterize the chemical structure of PECE copolymer (data not shown). The results of FTIR, 1H-NMR and GPC indicated that the PECE triblock copolymer designed by controlling the feed composition was synthesized successfully. The number-average molecular weight (Mn) and the PEG/PCL block ratios of PECE triblock copolymer synthesized in this work were calculated from 1H-NMR spectra. The Mn of PECE triblock copolymer was 3408 and the PEG/PCL block ratios was 960/2448, which was consistent with theoretical value calculated from feed ratio (Mn=3300, PEG/PCL= 1100/2200). Mn and polydispersity (PDI, Mw/Mn) of PECE copolymer determined by GPC were 4391 and 1.30 respectively.
3.2. Temperature-Dependent Sol-Gel-Sol Transition Behavior The composite hydrogel prepared in this work were composed of PECE and Pluronic F127 hydrogel, which individually presented sol-gel-sol transition. Due to combination of PECE hydrogel and Pluronic F127 hydrogel, the composite hydrogel also showed sol-gel-sol transition. As presented in Table 1, composite hydrogel based on PECE and Pluronic F127 copolymers from S1 to S5 showed temperature-dependent reversible sol-gel-sol phase transition. The composite hydrogels flowed freely at lower temperature, but became gelation at body temperature about 37oC (Figure 3). Figure 4 presented the sol-gel-sol phase transition diagram of prepared composite hydrogel. When the copolymer concentrations are above the critical gelation concentration (CGC), composite hydrogel changed from “sol” phase to “gel” phase with increase in temperature to the lower critical gelation temperature (LCGT). With further increase of temperature to upper critical gelation temperature (UCGT), the sol phase occurs.
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o
Temperature ( C)
Figure 3. Photograph of composite hydrogel (30wt%). S1 at 10oC (A) and 37oC (B); S3 at 10oC (C) and 37oC (D); S5 at 10oC (E) and 37oC (F);
120 110 100 90 80 70 60 50 40 30 20 10 0
S1 (ECE:100%, F127:0%) S2 (ECE:70%, F127:30%) S3 (ECE:50%, F127:50%) S4 (ECE:30%, F127:70%) S5 (ECE:0%, F127:100%)
Sol Gel Sol 0
5
10
15 20 25 30 35 Concentration (wt%)
40
Figure 4. Sol-gel-sol transition phase diagram of composite hydrogel .
According to Figure 4, pure PECE hydrogel (S1) and pure Pluronic F127 hydrogel (S5) both have a CGC of approximately 15wt%, but S5 have a much wider gelation window than that of S1. The UCGT of S5 at the concentration of 30wt% and 35wt% was not detected in the temperature range of 0oC to 100oC. The CGC of S2, S3, and S4 were 25wt%, 20wt%, and 20wt% respectively, which were much higher than that of two pure hydrogels. By mixing the two hydrogel together, the CGC of the composite hydrogel increased accordingly. CGC of S2 increased approximately 10wt% than that of S1, whereas CGC of S4 increased approximately 5wt% compared with that of S5. This phenomenon indicated that with regard to CGC the influence of Pluronic F127 hydrogel on PECE hydrogel was more dramatic than that of PECE hydrogel on Pluronic F127 hydrogel.
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As shown in Figure 4, with increase in ECE hydrogel content in composite hydrogel, the UCGT decreased significantly, whereas the LCGT increased slightly. The UCGT of S5 hydrogel at concentration of 30wt% and 35wt% could not detected in the range of 0oC to 100oC, but the UCGT at concentration of 30wt% and 35wt% were detected in S4 and S3 hydrogel respectively due to the increase content of PECE hydrogel. Therefore, it was obvious that sol-gel-sol transition behavior of composite hydrogel was depended on the composition of the PECE and Pluronic F127 hydrogel. In fact, by altering the composition of composite hydrogel, the temperature range of sol-gel-sol phase transition could be broadened to a certain extent, which might be very useful for their further application as injectable in situ gel-forming drug delivery system in different situation.
3.3. Morphology of Composite Hydrogel Interior morphology of composite hydrogel was investigated by SEM. The composite hydrogels were frozen in liquid nitrogen and lyophilized for 72 h before the test. Figure 5-A, B and C showed the morphology of S1, S3 and S5 hydrogel respectively. All the hydrogel samples showed porous three-dimension structure, but the shape and mesh size of pores in the hydrogel were different. S1 showed approximately spherical pore with small mesh size, whereas S5 showed cylindrical-like shape with relatively larger mesh size compared to S1. S3 hydrogel, composed of 50% PECE hydrogel and 50% Pluronic F127 hydrogel, also showed spherical pore, but have a larger mesh size compared to pure PECE hydrogel (S1). The morphology of S3 hydrogel suggested that two compositions of S3 hydrogel both have great influence on S3 hydrogel interior structure.
Figure 5. SEM photograph of composite hydrogel (×500). A: S1 hydrogel (30wt%); B: S3 hydrogel (30wt%); B: S5 hydrogel (30wt%)
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3.4. In vitro Drug Release Profile of Composite Hydrogel In vitro release profiles of VB12 from VB12 loaded composite hydrogel in PBS solution were investigated, and the results were shown in Figure 6. According to Figure 6, VB12 could release from composite hydrogel in an extended period and the composition of composite hydrogel had great effect on VB12 release profile. S5 hydrogel disappeared completely in PBS solution in 12h, whereas S1 and S3 hydrogel could maintain their integrity in the whole release period. VB12 released faster and reached higher cumulative release rate (94.2%) from S5 hydrogel compared to S1 and S3 hydrogel. In S1 hydrogel, an initial burst release of 20.2% of loaded VB12 occurred in the first one hour, followed by release of 82.9% in 7 days, whereas, in S3 hydrogel, the cumulative release rate of one hour and 7 days were 22.6% and 87.2% respectively. In this study, BSA was used as a model drug to investigate in vitro release behavior of protein or peptide drugs from composite hydrogel, and the data were summarized in Figure 7. As presented in Figure 7-A, higher PECE hydrogel content in composite hydrogel resulted in slower release rate of BSA from composite hydrogel. The cumulative release rate of BSA from S1, S3, and S5 hydrogel were 32.7%, 46.1%, and 90.7% respectively. SDS-PAGE was performed to evaluate the stability of BSA in the in vitro release period. According to Figure 7-B, the major band for BSA appeared at about 67KD (lane2 to lane10) according to the protein marker, which means that BSA was stable in all the composite hydrogel and the whole release period. Thus, composition of composite hydrogel substantially affected the drug release behavior of composite hydrogel, where higher Pluronic F127 content resulted in higher cumulative release rate and higher initial burst release rate. By altering the composition of PECE and Pluronic F127 copolymers in composite hydrogel, drug release rate could be varied and controlled.
3.5. In vivo Gel Formation and Degradation Behavior The application of composite hydrogel as in situ gel-forming drug delivery system was tested by subcutaneous injection with 0.5 mL of hydrogel solution (from S1 hydrogel to S5 hydrogel, 30wt%) into BALB/c mice. In Figure 8, from A to E showed the photograph took at 1st day, 3rd day, 7th day, 10th day and 14th day after composite hydrogel injection respectively ( From left to right: S4, S3, S2 and S1 hydrogel). F and G presented the photograph took at 2nd hour and 6th hour after S5 hydrogel injection. The injectable sol state of composite hydrogel became gel in mice just in a few seconds and maintained its integrity in vivo in an extended period. The formed gel of S1 and S2 hydrogel were spherical shaped rather than spread to form sheet shaped. In the process of time after composite hydrogel injection, the in vivo formed gel decreased in size and finally disappeared due to the degradation. Hydrogels of S5, S4, S3, and S2 were disappeared in vivo at 6th hour, 7th day, 10th day, and 14th day after injection respectively. At 14th day after injection (Figure8-E), only a small mount of S1 hydrogel was found in vivo, which indicated that S1 hydrogel could sustain longer than 14days.
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Cumulative release (%)
100 80 60 40
S1 (ECE:F127=100:0) S3 (ECE:F127=50:50) S5 (ECE:F127=0:100)
20 0
0
24
48
72 96 120 144 168 Time (h)
Figure 6. In vitro release behavior of VB12 from composite hydrogel.
Cumulative release (%)
100 S1 (ECE:F127=100:0) S3 (ECE:F127=50:50) S5 (ECE:F127=0:100)
80 60 40 20 0
A 0
48
96 144 192 240 288 336 384 Time (h)
Figure 7. In vitro release behavior of BSA from composite hydrogel (A) and SDS-PAGE results of BSA in vitro release profile (B) (Lane 1: marker; Lane 2: BSA standard; Lane 3: S1 at 24th hour; Lane 4: S1 at 168th hour; Lane 5: S1 at 360th hour; Lane 6: S5 at 1st hour; Lane 7: S5 at 12th hour; Lane 8: S3 at 24th hour; Lane 9: S3 at 168th hour; Lane 10: S3 at 360th hour;).
According to Figure 8, we could find that the sustained time of the composite hydrogel in vivo could be controlled by altering the composition of above two hydrogel, which might be very useful for its potential application in injectable in situ gel-forming drug delivery system.
3.6. Histopathological Study All samples were histopathological observed by light microscope, and no significant histopathological changes were observed due to composite hydrogels (Figure 9). As shown in Figure 9, the morphology of the composite hydrogel-treated tissue (Figure 9-B, C) did not show significant difference when compared with control group (Figure 9-A). Histopathological study of tissue at injection site showed no significant inflammatory reaction and histopathological changes, which mean the composite hydrogel were nontoxicity and might serve as a safe candidate as in situ gel-forming drug delivery system.
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Figure 8. In situ gel formation and degradation behavior of composite hydrogels. From A to E showed the photograph took at 1d, 3d, 7d, 10d and 14d after injection respectively ( From left to right: S4, S3, S2 and S1). F and G presented the photograph took at 2h and 6h after Pluronic F127 hydrogel injection.
Figure 9. Photograph of tissue samples from injection site after composite hydrogel injection for 14d ( ×400). A: Control group; B: S3 hydrogel injection group; C: S1 hydrogel injection group.
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CONCLUSION A series of novel biodegradable and injectable thermosensitive composite hydrogel were successfully prepared. The obtained composite hydrogel underwent sol-gel-sol transition as temperature increased, which was a flowing sol at ambient temperature and became a nonflowing gel at physiological temperature, and the sol-gel-sol phase transition behavior of the copolymers aqueous solutions was determined using the test tube inverting method. By varying the composition of PECE and Pluronic F127 copolymers, the in vivo degradation rate and in vitro drug release profile of composite hydrogel could be controlled, which was very useful for its potential applications as an in situ gel-forming controlled drug delivery system. Histopathological study suggested the composite hydrogel is a safe candidate for its applicantion in biomedicine fields.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
[13]
Jemal, A; Siegel, E; Ward, E; Hao, Y; Xu, J; Murray, T; Thun, MJ. Cancer statistics, CA Cancer J Clin, 2008, 58, 71-96. Qiu, Y; Park, K. Environment-sensitive hydorgels for drug delivery. Adv Drug Deliver Rev, 2001, 51, 321-339. Hatefi, A; Amsden, B. Biodegradable indectable in situ forming drug delivery systems. J Control Rel, 2002, 80, 9-28. Suzuki, A; Tanaka, T. Phase transition in polymer gels induced by visible-light. Nature, 1990, 346, 345-347. Galaev, IY; Mattiasson, B. ‘Smart’ polymers and what they could do in biotechnology and medicine. Trends in Biotechnol, 1999, 17, 335-340. Nanjawade, BK; Manvi, FV; Manjappa, AS. In situ-forming hydrogels for sustained ophthalmic drug delivery. J Control Rel, 2007, 122, 119-134. Miyta, T; Asami, N; Uragami, T. A reversibly antigen-responsive hydrogel. Nature, 1999, 399, 766-769. Lee, KY; Mooney, Dj. Hydrogels for tissue engineering. Chem Rev, 2001, 101, 18691879. Kamath, KR; Park, K. Biodegradable hydrogels in drug delivery. Adv Drug Deliv Rev, 1993, 11, 59-84. Jeong, B; Bae, YH; Lee, DS; Kim, SW. Biodegradable block copolymers as injectable drug-delivery systems. Nature 1997, 388, 860-862. Jeong B; Kim SW; Bae YH. Thermosensitive sol-gel reversible hydrogels. Adv Drug Deliver Rev, 2002, 54, 37-51. Kissel, T; Li, Y; Unger, F. ABA-triblock copolymers from biodegradable polyerster Ablocks and bydrophilic poly(ethylene oxide) B-blocks as a candidate for in situ forming hydrogel delivery systems for proteins. Adv Drug Deliver Rev, 2002, 54, 99-134. Gariépy, ER; Leroux, JC. In situ-forming hydrogels—review of temperature-sensitive systems. Eur J Pharm Biopharm, 2004, 58, 409-426.
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[14] Dimitrov, I; Trzebicka, B; Muller, AH; Dworak, A; Tsvetamov, CB. Thermosensitive water-soluble copolymers with doubly responsive reversibly interacting entities. Progress Polym Sci, 2007, 32, 1275-1343. [15] Song, MJ; Lee, DS; Ahn, JH; Kim, DJ; Kim, SW. Thermosensitive sol-gel transition behaviors of poly(ethylene oxide)/aliphatic polyester/poly(ethylene oxide) aqueous solutions. J Polym Sci, Part A, Polym Chem, 2004, 42, 772-784. [16] Liu, CB; Gong, CY; Huang, MJ; Wang, JW; Pan, YF; Zhang, YD; et al. Thermoreversible Gel-Sol Behavior of Biodegradable PCL-PEG-PCL Triblock Copolymer in Aqueous Solutions. J Biomed Mater Res B, 2008, 84, 165-175. [17] Choi, SW; Choi, SY; Jeong, B; Kim, SW; Lee, DS. Thermoreversible gelation of poly(ethylene oxide) biodegradable polyester block copolymers.II. J Polym Sci, Part A, Polym Chem, 1999, 37, 2207-2218. [18] Gong, CY; Qian, ZY; Liu, CB; Huang, MJ; Gu, YC; Wen, YJ; et al. A Thermosensitive Hydrogel Based on Biodegradable Amphiphilic Poly(ethylene glycol)polycaprolactone-poly(ethylene glycol) block Copolymers. Smart Mater Struct, 2007, 16, 927-933. [19] Liu, CB; Gong, CY; Pan, YF; Zhang, YD; Wang, JW; Huang, MJ; et al. Synthesis and Characterization of a Thermosensitive Hydrogel Based on Biodegradable Amphiphilic PCL-Pluronic(L35)-PCL block Copolymers. Colloids Surfaces A, 2007, 302, 430-438. [20] Bromberg, LE; Ron, ES. Temperature-responsive gels and thermogelling polymer matrices for protein and peptide delivery. Adv Drug Deliver Rev, 1998, 31, 197–221. [21] Li, J; Li, X; Ni, X; Leong, KW. Synthesis and Characterization of New Biodegradable Amphiphilic Poly(ethylene oxide)-b-Poly[(R)-3-hydroxybutyrate]-b-Poly(ethylene oxide) Triblock Copolymers. Macromolecules, 2003, 36, 2661–2667. [22] Lee, JW; Hua, FJ; Lee, DS. Thermoreversible gelation of biodegradable poly(εcaprolactone) and poly(ethylene glycol) multiblock copolymers in aqueous solutions. J Control Rel, 2001, 73, 315-327. [23] Li, J; Ni, X; Leong, KW. Injectable Drug-Delivery Systems Based on Supramolecular Hydrogels Formed by Poly(ethylene oxide)s and Cyclodextrin. J Biomed Mater Res, Part A, 2003, 65, 196-202. [24] Jeong, B; Bae, YH; Kim, SW. Biodegradable thermosensitive micelles of PEG-PLGAPEG triblock copolymers. Colloid Surface B, 1999, 16, 185-193. [25] Lee, DS; Shim MS; Kim SW; Lee H; Park I; Chang T. Novel thermoreversible gelation of biodegradable PLGA-block-PEO-block-PLGA triblock copolymers in aqueous solution. Macromol Rapid Commun, 2001, 22, 587-592. [26] Chung, YM; Simmons, KL; Gutowska, A; Jeong, B. Sol-gel transition temperature of PLGA-g-PEG aqueous solutions. Biomacromolecules, 2002, 3, 511-516. [27] Jeong, B; Bae, YH; Kim, SW. Thermoreversible Gelation of PEG-PLGA-PEG Triblock Copolymer Aqueous Solutions. Macromolecules, 1999, 32, 7064-7069. [28] Choi, SW; Choi, SY; Jeong, B; Kim, SW; Lee DS. Thermoreversible gelation of poly(ethylene oxide) biodegradable polyester block copolymers.II. J Polym Sci, Part A, Polym Chem, 1999, 37, 2207-2218. [29] Schuetz, YB; Gurny, R; Jordan, O. A Novel Thermoresponsive Hydrogel Based on Chitosan. Eur J Pharm Biopharm, 2008, 68, 19-25.
A Novel Thermosensitive Composite Hydrogel Based on…
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[30] Bhattarai, N; Ramay, HR; Gunn, J; Matsen, FA; Zhang, M. PEG-grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release. J Control Release, 2005, 10, 609-624. [31] Berger, J; Reist, M; Mayer, JM; Felt, O; Peppas, NA; Gurny, R. Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications. Eur J Pharm Biopharm, 2004, 57, 19–34. [32] Chenite, A; Chaput, C; Wang, D; Combes, C; Buschmann, MD; Hoemann, CD; et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials, 2000, 21, 2155-2161. [33] Gilbert, JC; Richardson, JL; Davies, MC; Palin, KJ; Hadgraft. The Effect of Solutes and Polymers on The Gelation Properties of Pluronic F-127 Solutions for Controlled Drug Delivery. J Controlled Release, 1987, 5, 113-118. [34] Xiong, XY; Tam, KC; Gan, LH. Synthesis and aggregation behavior of Pluronic F127/Poly(lactic acid) block copolymers in aqueous solutions. Macromolecules, 2003, 36, 9979-9985. [35] Rangelov, S; Dimitrov, P; Tsvetanov, CB. Mixed block copolymer aggregates with tunable temperature behavior. J Phys Chem B, 2005, 109, 1162-1167. [36] Gong, CY; Shi, S; Dong, PW; Kan, B; Gou, ML; Wang, XH; et al. Synthesis and Characterization of PEG-PCL-PEG Thermosensitive Hydrogel. Int J Pharm, 2009, 365, 89-99. [37] Chung, YM; Simmons, KL; Gutowska, A; etc. Sol-gel transition temperature of PLGAg-PEG aqueous solutions. Biomacromolecules, 2002, 3, 511-516. [38] Chen, X; Qian, ZY; Gou, ML; Chao, GT; Zhang, YD; Gu, YC; Huang, MJ; Wang, JW; Pan, YF; Wei, YQ; Chen, JP, Tu, MJ. Acute Oral Toxicity Evaluation of Biodegradable and pH-sensitive Hydrogel based on Polycaprolactionem, Poly(ethylene glycol) and Methylacrylic acid (MAA). J Biomed Mater Res, Part A, 2008, 84, 589-597. [39] Bea, SJ; Suh, JM; Sohn, YS; Bae, YH; Kim, SW; Jeong, B. Thermogelling Poly(caprolactone-b-ethylene glycol-bcaprolactone) Aqueous Solutions. Macromolecules, 2005, 38, 5260-5265. [40] Li, J; Li, X; Ni, X; Wang, X; Li, H; Leong, KW. Self-assembled supramolecular hydrogels formed by biodegradable PEO-PHB-PEO triblock copolymers and εcyclodextrin for controlled drug delivery. Biomaterials, 2006, 27, 4132-4140. [41] Li, Z; Ning, WWJ; etc. Controlled gene delivery system based on thermosensitive biodegradable hydrogel. Pharmacol Res, 2003, 20, 884-888. [42] Iza, M; Stoianovici, G; Viora, L ; etc. Hydrogels of poly(ethylene glycol): mechanical characterization and release of a model drug. J Control Rel, 1998, 52, 41-51. [43] Zhou, SB; Deng, XM; Yang, H. Biodegradable poly(ε-caprolactone)-poly(ethylene glycol) block copolymers: characterization and their use as drug carriers for a controlled delivery system. Biomaterials, 2003, 24, 3563-3570.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 3
NITROGEN-CONTAINING LIGANDS ANCHORED ONTO POLYMERS AS CATALYST STABILIZER FOR CATALYTIC ENANTIOSELECTIVE REACTIONS Christine Saluzzo *and Stéphane Guillarme Université du Maine UCO2M UMR-CNRS 6011, Av. O. Messiaen, 72085 Le Mans Cedex 9, France
ABSTRACT This paper reviews the recent progress made in insoluble polymer supported amino alcohols, amino thiols, oxazolines, salens, sulphonamides, oxazaborolidines and diamines ligands. This paper deals also with various approaches of stabilization of the catalytic system by immobilization of the chiral catalyst onto the polymer by the way of immobilization of the chiral ligand. Different types of ligand immobilization are presented: pendant ligands anchored on a polymer prepared by a polymer reaction, ligands on the backbone prepared by copolymerization and molecular imprinting technique. Examples of their use, performance and recyclability in a variety of enantioselective reactions such as alkylation and reductions of C=O bonds (hydrogenation, hydrogen transfer reduction) reduction of C=N bonds, C-O bond formations (epoxidation, dihydroxylation), C-C bond formations (Diels Alder, cyclopropanation, aldolisation, allylic substitution) and oxidation … are presented.
1. INTRODUCTION The need to develop effective methods for enantioselective synthesis is becoming ever more important as only a single enantiomer of a racemic bioactive compound is generally required for pharmaceuticals [1], agrochemicals [1,2], flavour [1,3] or fragrance [1,3]. Homogeneous asymmetric catalysis has been used to perform a variety of transformations *
Corresponding author: e-mail :
[email protected] 46
Christine Saluzzo and Stéphane Guillarme
under mild conditions with high enantioselectivity. As these fine and specialty chemicals are manufactured to meet high and well defined standards of purity compatible with the desired performance, chemical industry had to adopt efficient and clean technology [4]. From an industrial point of view, homogeneous methods remain unpratical, particularly due to the high cost of the chiral metal catalysts (range of the chiral catalyst loadings from 1 to 10% and in some cases up to 30%) and the difficulty of their recovery and reuse. Thus, from an economic, environmental and technical point of view, homogeneous supported catalysis is preferable to homogeneous catalysis because of the handling, separation and recycling abilities [5]. Many approaches have been employed to immobilize the homogeneous catalyst. [6]. In the classical immobilization with organic polymer, the chiral ligand units are anchored onto polymers (type 1, Scheme 1). The incorporation of chiral ligands on the main chain of the polymers is another approach (type 2, Scheme 1) and is done by copolymerization of the chiral ligand with a linker. The last but less known approach consists in the use of an imprinted polymer (type 3, Scheme 1), an extensively used methodology to form artificial antibodies. The imprinted polymer is based on a copolymerization and a crosslinking of a polymer around a molecule used as a printed molecule (PM). This latter included into the polymer by means of an interaction with functional groups is then removed, thus leaving its imprint in the polymeric matrix. If a chiral PM is used, the formed chiral cavity is able to act as a center of molecular recognition [7]. L GP
type 1
L M
PM
L
Ligand Ligand L
M M type 2 Ligand
L M Ligand
M M PG: polymerizable group M: metal PM: printed molecule
PM
L L L M L
type 3
Scheme 1.
The aim of this review is to discuss the contribution of soluble polymer-supported ligands and insoluble polymer-supported ligands to asymmetric catalysis in the field of reduction of C=O bonds, cyclopropanation, Diels-Alder, alkylation, allylation, dihydroxylations, epoxydation reactions, kinetic resolution of terminal epoxide ….. by means of nitrogen containing ligands complexed with metal as asymmetric catalyst. Each type of asymmetric homogeneous supported catalyst will be examined successively from the perspective of using phosphine-free nitrogen-containing ligands complexed with a metal. Results were analyzed in comparison to the results in the literature for similar systems.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
47
2. REDUCTION OF C=O BOND The three major catalytic reduction procedures which have emerged are enantioselective hydride reduction, hydrogenation and hydrogenation transfer reduction (HTR) close to the hydrosilylation which represent only few examples in asymmetric polymer-supported catalyst.
2.1. Hydrogenation In homogeneous catalysis, various phosphines have been developped for asymmetric hydrogenation [8]. However, for hydrogenation of unfunctionalized ketone, a remarkably high reactivity emerges when the Ru compounds are further complexed with a 1,2 diamine ligand. Thus, the precatalyst RuCl2(phosphine)2(1,2-diamine) combined with an inorganic base in isopropanol is considered as one of the most powerful systems for the hydrogenation of ketones (Scheme 2, i-PrOH as solvent) [9, 10].
O Ar
1,2-diamine-(S)-BINAP-RuCl2
R1
+ H2 (1 MPa) t-BuOK, i-PrOH or i-PrOH-DMF
OH H R Ar 1
Scheme 2.
Close to polymer bound-BINAP/diamine ruthenium precatalyst [11], complexes of BINAP or their derivatives in the presence of polymer bound diamine have been employed. These catalytic systems have been mainly studied by Itsuno with immobilized diphenylethylenediamine (DPEN). As the use of N-substituted 1,2-diamine ligands decreased the catalytic activity of hydrogenation of simple ketones contrary to free diamine ligands, they have been less employed. However, N-substituted 1,2-diamine ligands are rather effective in the hydrogenation of α-substituted ketone, such as α-amido ketones. Thus, two types of immobilized DPEN have been performed. Functionalized DPEN for subsequent grafting onto polymer or copolymerization are presented (Scheme 3). If free primary amines were maintained, grafting was realized by the modification of phenyl groups into phenolic ones [12, 13] and copolymerization by using a phenolic ether group containing a vinylphenyl functionality as monomer. ((S,S)-2, (R,R)-3 and (S,S)-4, -5, 6; (R,R)-5, -6, -7, -9 respectively (Scheme 3) [13, 14, 15]. The first type of polymers was formed by reaction of the phenoxide derived from DPEN with 1% crosslinked chloromethylated polystyrene (Schemes 4 and 5). It has been observed that in the cases of higher content of chloromethyl group or a higher degree of crosslinking, some unreacted chloromethyl groups remain.
48
Christine Saluzzo and Stéphane Guillarme Homogeneous ligands Functionalized DPEN for subsequent grafting or copolymerization OH
HO
H2 N
O
OH
NH2 H2N NHBoc (R,R)-3
H2N NH2 (S,S)-2
(S,S)-DPEN (S,S)-1
CH2
CH2 NH2 H2 N (R,R)-DPEN (R,R)-1
O
OMe
H2C
O
MeO
MeO
O
CH2
n
O
n
OMe H2C
CH2
BocHN NHBoc n = 1, (R,R)-5; (S,S)-5 n = 4, (R,R)-6; (S,S)-6 O OR
O
O
n
BocHN NHBoc (S,S)-4 H2N NHBoc (R,R)-7a, n = 1, R = Me (R,R)-7b, n = 1, R = Bn (R,R)-7c, n = 4, R = Me (R,R)-7d, n = 4, R = Bn H2N
NH Bn
H2N
NH
(R,R)- N-benzyl DPEN (R,R)-8
O
CH2
(R,R)-9
Scheme 3.
x
y
CH2Cl +
z
CH2 O
NaH, DMF
H2N H2N
O CH2
(
(S,S)-2
z
)
x
y
(S,S)-10a: x = 0.01, y = 0.2, z = 0.79 (S,S)-10b: x = 0.05, y = 0.2, z = 0.75
Scheme 4.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
x
y
x
z
cross linkage 'CH2)nX
y
49
z
cross linkage
1) NaH, DMF 2) HCl, THF 3) Et3N, THF
'CH2)n O
+ (R,R)-3
O H2N crosslinkage: (R,R)-11a: x = 0.10, y = 0.01 (R,R)-11b: x = 0.10, y = 0.05 crosslinkage: (R,R)-12a: x = 0.10, y = 0.05 (R,R)-12b: x = 0.10, y = 0.10
NH2
CH2(OCH2CH2)2OCH2
Scheme 5.
To perform the catalytic homogeneous-supported hydrogenation of acetophenone with precatalyst ((S,S)-10a, (S,S)-10b, (R,R)-11a, (R,R)-11b, (R,R)-12a or (R,R)-12b)-(R)-BINAPRuCl2, it has been shown that the suitable solvent is a mixture of i-PrOH/DMF/ (1/1, v/v). If i-PrOH is employed alone, hydrogenation does not take place due to little swelling of the polymer catalyst (Scheme 2, Ar = Ph). Moreover, a lower degree of crosslinking gave better reactivity; higher ees and conversion are obtained (Table 1). Table 1. Enantioselective hydrogenation of acetophenone with diamine polymersupported-BINAP-RuCl2 Ligand-BINAP-RuCl2 10aa 10ba 11ab 11bb 12ab 12bb
Yield (%) 100% 39% 91% 46% >99% 70%
Ee (%) 73% 68% 70% 67% 23% 65%
Ref. [12] [12] [13] [13] [13] [13]
a
Diamine/BINAP/RuCl2/ketone/t-BuOK : 1/1/1/200/20 Diamine/BINAP/RuCl2/ketone/t-BuOK : 2/1/1/1000/20
b
The best results are observed if xylBINAP is used instead of BINAP (ie 93% ee, 100% yield in the presence of 10a). The immobilized (S,S)-10a-(R)-BINAP-RuCl2 can be reused at least four times without loss of activity and selectivity (from the first to the fourth reuse ee is about 73-74% with 100% yield) [12]. Enantioselectivities obtained with cocatalyst DPEN modified by a vinylphenyl group copolymerized under radical conditions (polymers 13 [14] and 14, 15 (Schemes 6 and 7) [15] and 16 (Scheme 8) [13] also exhibited almost the same level as those obtained from the low-
50
Christine Saluzzo and Stéphane Guillarme
molecular-weight counterpart in solution system (73-80% and 80% ee respectively with conversions always >99% for polymer containing less than 5% DVB).
x
y
CH2 O
x
OMe
MeO
y
H2C O
MeO
OMe CH2
+ (S,S)-2
H2C
O
O
H2N
NH2
13a: x = 0.05, y = 0.95 13b: x = 0.10, y = 0.90 Scheme 6.
)
R2
( R1 CH2 O
n
H2C O
n
R2
CH2 O
+ R1
n
O H2C
n
O
O
a: MMA: R1 = OMe R2 = Me BocHN NHBoc b: HEMA: R1 = O(CH2)2OH H N NH2 2 R2 = Me 5: n = 1 14: n = 1 c: BMA: R1 = OBu 6: n = 4 15: n = 4 R2 = Me Scheme 7.
Catalytic systems derived from 13 and 14, 15 and 16a have successfully been recycled from four (for 13 [14]), 14 [15] and 15 [15]) to sixteen times (for 16a [13]) without any change of activity and selectivity. They were also tested with other aromatic ketones and high yields and enantioselectivities were observed (some examples are presented in Table 2).
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
x
+
1) AIBN 2 mol% THF 70°C 2) HCl, THF 3) Et3N, THF
y
51
z
) (
(CH2)n O
(R,R)-7 OR H2N NH2 16a: R = Me, n = 1, x = 0.10, z = 0.05 16b: R = Bu, n = 1, x = 0.10, z = 0.10
Scheme 8.
Table 2. Hydrogenation of several ketones with polymeric diamine/BINAP/RuCl2 catalyst Ketone
Diamine
Time (h)
acetophenone
13aa or 13ba 14ab 15cb
propiophenone
13aa 14ab 15cb 16a 13aa 14ab 15cb 16ac
1’-acetonaphtone
Ee (%) (config.) 77 (R) 78 (R) 79 (R)
Ref.
18 5 5
Conv. (%) 100 >99 >99
4 12 12 24 18 12 12 24
100 >99 >99 99 100 >99 >99 >99
82 (R) 84 (R) 83 (R) 82 96 (R) 96 (R) 96 (R) 97
[14] [15] [15] [13] [14] [15] [15] [13]
[14] [15] [15]
a
Diamine/BINAP/RuCl2/ketone/t-BuOK : 2/1/1/1000/20 Diamine/BINAP/RuCl2/ketone/t-BuOK : 2/1/1/200/20 c Diamine/BINAP/RuCl2/ketone/t-BuOK : 1/1/1/200/20 b
For the hydrogenation of the racemic α-(N-benzoyl-N-methylamino)propiophenone, (Scheme 9) polymers containing N-substituted DPEN were prepared by radical copolymerization of (R,R)-9 (Scheme 3) with various crosslinking agents (Scheme 10) [16]. O
OH
Ph
+ H2 (1 MPa) Ph
N O
Scheme 9.
1,2-diamine-(R)-BINAP-RuCl2 t-BuOK, i-PrOH-DMF
Ph N
Ph O
52
Christine Saluzzo and Stéphane Guillarme R1 0.10
R2
R3 0.88
R4
0.02
R3 O HN H2N 17a: R1 = R3 = H, R2 = Ph, R4 = C6H4 17b: R1= H, R2 = Ph, R3 = Me, R4 = CO2CH2CH2OCO 17c: R1= Me, R2 = CO2CH2CH2OH, R3 = Me, R4 = CO2CH2CH2OCO 17d: R1= Me, R2 = CO2CH2CH2OH, R3 = H, R4 = C6H4 17e: R1 = R3 = Me, R2 = CO2Me, R4 = CO2CH2CH2OCO 17f: R1 = H, R2 = CONHi-PrMe, R3 = Me, R4 = CO2CH2CH2OCO 17g: R1 = H, R2 = C6H4CH2OH, R3 = Me, R4 = CO2CH2CH2OCO
Scheme 10.
For the catalytic reduction, the solvent of choice was the mixture 1/1 (v/v) of i-PrOH/DMF (conditions for good swelling of the polymeric ligands 17). The polarity of the crosslinking agent has a direct influence on the results of the catalysis. The precatalyst obtained with 17a produced a low conversion (7%) and an excellent diastereoselection (>99%) for an enantiomeric excess of 61%. A slight improvement was observed for 17b formed with EGDMA a polar crosslinker (conversion: 10%, ee: 88%). Compared to them 17e present a better conversion (89%) with >99% of de and ee. The precatalysts prepared from 17c, 17d, 17f, 17g from better hydrophilic polymer support have exhibited excellent catalytic performance for this dynamic kinetic resolution of racemic α-amide ketone via hydrogenation to yield the corresponding syn β-amide alcohol (100% conversion, de >99% and ee ranging from 90 to >99%). In addition, the polymer-supported catalyst could be reused several times without loss of catalytic activity.
2.2. Hydrogenation Transfer Reduction Hydrogenation transfer reduction (HTR) is defined as a reduction of mainly C=O and C=N bonds employing an hydrogen donor (DH2) such as cyclohexene, cyclohexadiene, alcohols, formic acid or hydrazine, in the presence of a catalyst, avoiding all the risks inherent to molecular hydrogen (Scheme 11). For this reason and for a procedural simplicity, HTR can be regarded as an alternative for the asymmetric hydrogenation [17]. For HTR, the first heterogeneous catalytic systems were formed with homogeneous-supported phosphines, which were also oxygen sensitive contrary to amine ligands. Moreover, nitrogen-containing ligands could be easily polymerized. O R1 Scheme 11.
OH
cat*
R2
+ DH2
OH +
R1
R2
R1
R2
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
53
2.2.1. 1,2-diamine and derivatives As for hydrogenation, most of the chiral ligands involved in heterogeneous HTR have been prepared starting from functionalized DPEN (Scheme 12). In 1993, Lemaire found that N,N-dimethyl-1,2-diphenylethylenediamine (18) complexed with Rh could be employed as ligand for asymmetric HTR, leading to 67% ee and 100% for the reduction of acetophenone and methyl phenyl glyoxylate respectively [18]. Due to the presence of the amino groups, this C2 diamine could be easily transformed into diurea and dithiourea derivatives.
HN
HN
(S,S)-18
H2N
NH2
NH SO2
NH2
(S,S)-19
(R,R)-20
BocHN
NH SO2
COOH (R,R)-21
BocHN
NH SO2
O ( ) COOH n (R,R)-22
Scheme 12.
As good results for the asymmetric HTR of acetophenone were obtained (conversion 100% and 91% ee) with diurea [19], not only copolymerization of diamine 18 has been performed but 1,2-cyclohexyldiamine 19 was also used. Thus pseudo-C2 polyamide 23, polyureas 24, 24 and 26 or polythioureas 27 were prepared by polycondensation with diacid chloride, diisocyanate [20] or dithioisocyanate respectively (Scheme 12) [21]. With rhodium complexes the conversions varied from 22% to 100% and ee from 0% to 60% for HTR with acetophenone at 70°C, in the presence of [Rh(cod)Cl]2 with diamine polymer unit (23-26)/Rh ratio of 10, 2-propanol and KOH. Polyamide 23 proved to be useless (only 22% conversion and 28% ee) contrary to polyurea 25 which presents similar ee to those observed with diamine 18 when (Rh(cod)Cl)2 was employed as the catalytic precursor for HTR (ie 100% conversion for both of them and 55% and 59% ee respectively). Polyureas 24a, 24b, 25 and crosslinked 26 led to better conversions, 80, 50, 97 and 100% with respectively 0, 13, 39 and 60% ee. Moreover, the chiral crosslinked polyurea 26 presented a slight increase in enantioselectivity over the monomer analog 18 (55% ee and 94% conversion under similar conditions) and the reaction rate appeared to be even higher than in the homogeneous phase. Catalytic system from 25 showed a capacity to recycle (Scheme 13) [20]. For thioureas, it has already been shown that under homogeneous conditions, they are better ligands than ureas, when using ruthenium complexes as precursors [22]. Thus, polythioureas were good candidates for the HTR reaction [21]. For polythioureas 27, studies of the DP were performed and it had been showed that DP depends on the ratio of the reagents. During the polymerization, a guanidine moiety could be formed, stopping the chain growing. To avoid its formation, dithioisocyanate must be used in excess.
54
Christine Saluzzo and Stéphane Guillarme R1
R1
R2 NH HN R2 ClOC Ph
XCN (R)
COCl
Ph
R1
N N C O (R) O C R2 R2 O O n
NCX
R1
N N C R2 R2 X
NH (R) NH C X n
23: R1 = Ph, R2 = Me
24: R1 = -(CH2)4-, R2 = H, X = O Me Me
R=
a: R = 25: R1 = Ph, R2 = Me, X = O R=
b: R =
26: crosslinked 25 formed with 70/30 mixture of di and triisocyanatodiphenylmethane
27: R1 = Ph, R2 = H, X = S a: R = -(CH2)4b: R = Me Me c: R = d: R = Me Me
Scheme 13.
O Ph
cat* 5% i-PrOH/KOH 70°C
cat*: 24a (Rh(cod)Cl)2 1st use conv. 100% 60% ee 2nd use conv. 100% 59% ee 3rd use conv. 100% 60% ee
OH Ph cat*: 27c (Ru(benzene)Cl2)2 1st use conv. 92%, 70% ee 2nd use conv. 98%, 67% ee 3rd use conv. 99%, 66% ee 4th use conv. 99%, 63% ee 5th use conv. 98%, 61% ee
Scheme 14.
The HTR of various ketones was then performed in isopropanol with a substrate/ligand/tBuOK/Ru ratio of 20/1.5/4/1. The chiral induction depended on the flexibility of the linker. The more flexible the linker, the less enantioselective the reaction: 31%, 70%, 70% and 65% ee for respectively 27a, 27b, 27c, 27d ruthenium complexes with conversion up to 90% for 27a, 27b, 27c and only 47% for 27d. For this latter ligand which was fully crosslinked, the decrease in conversion was due to the low accessibility of the catalytic site although a ligand/metal ratio of 8 had to be used. The recovery and reusing of the 27c polymeric catalyst has been performed with practically no loss of either catalytic activity or enantioselectivity (Scheme 14). These results could be
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
55
explained by the rigidity of the active site which is crucial for the selectivity and the stability of the catalytic system. Compared to the polyureas most of the polythioureas tested were more efficient. Modification of 1,2-diphenylethylenediamine into N-p-toluenesulfonyl-1,2-diphenylethylenediamine (TsDPEN) led to one of the most selective ligand for the homogeneous HTR of ketones (97% ee for acetophenone) [23]. The heterogeneization of this ligand was first carried out with N-(vinylbenzene-psulfonyl)-1,2-diphenylethylenediamine (20), styrene and divinylbenzene as crosslinker (Scheme 15).
+ NH2
AIBN CH2Cl2
+
NH
O S NH O
NH2
SO2
20
1 1
/ /
10 10
/ /
0 0.5
AIBN CH2Cl2
28 77% yield 29 71% yield
Scheme 15.
When tested as ligands for the IrI and RuII catalyzed HTR [24] of acetophenone, in presence of 2.5% of metal precursor and 2-propanol/base, at 70°C, Ir complexes led to higher ee (28: 92% and for the crosslinked 29: 94%) compared to the homogeneous analog for which only 75% of conversion and 89% ee were obtained. Nevertheless, the Ir catalyst was more efficient than ruthenium (Ir: 92% ee, Ru: 64% ee crosslinked Ir: 94% ee, crosslinked Ru: 84% ee), but for Ru the conversions were of about 20% in both cases. On the other hand the homogeneous Ir complex analog led to only 75% conversion at 89% ee. This difference could be explained by the formation of chiral microenvironments upon polymerization and stabilization of the reactive Ir complex. Attempts to reuse the catalysts showed that the one containing Ru which is less selective is more stable upon reuse than the Ir derivative. Itsuno [25] has also shown that polymer-supported DPEN monosulfonamides containing sulfonated pendent group (Scheme 16) are able to catalyze the HTR reduction of ketones in water with sodium formiate as hydrogen donor (S/C = 100). However, TsDPEN immobilized on polystyrene crosslinked or not, polymer 30 and 31 respectively, shrank in water. Sodium p-styrene sulfonate was copolymerized with chiral N-(vinylbenzene-p-sulfonyl)-DPEN (20) under radical polymerization conditions with or without DVB leading respectively to ligand 32 and 33. Control of the balance hydrophilicity/hydrophobicity of the polymer support is carried out by changing the salt from Na+ to quaternary ammonium. All of these polymers swelled in water, and their respective ruthenium, rhodium or iridium complexes were prepared. Compared to sodium salt polymer-supported catalyst from 32a and 33a, ammonium
56
Christine Saluzzo and Stéphane Guillarme
supported catalyst from 32b and 33b have produced the best results in terms of activity and selectivity for the reaction in aqueous media. Moreover, almost the same activity was observed for the reduction of acetophenone with the ammonium non crosslinked catalyst 32b prepared from [RhCl2Cp]2 and [RuCl2(p-cymene)]2 (100% and 99% conversion; 98% and 98% ee respectively) while the catalyst from [IrCl2Cp]2 decreased both reactivity and enantioselectivity (77% conversion, 89% ee).
0.1
Ph
NH SO2
Ph
NH2
(R,R)-30
0.1
Ph Ph
0.1
0.9
NH SO2 NH2
Ph
NH SO2
Ph
NH2
0.1
(R,R)-31
0.1
0.9
Ph
SO3- X+
Ph
(R,R)-32 +
0.8
0.1
0.8
SO3-X+
NH SO2 (R,R)-33
NH2
+
a: X = Na ; b: + N
Scheme 16.
Enantioselectivities of 97-98% were obtained in five recycle runs by using (R,R)-33b derived catalyst. Earlier, Bayston [26] has synthesized the polymer 34 (Scheme 17) starting from the aminostyrene Merrifield-type polymer. By means of ruthenium precursor, 88% conversion with 91% ee was obtained for reduction of acetophenone. Compared with the preceding ligand 29 complexed with ruthenium, better conversion and enantioselectivities are shown; these latter results were comparable with the Ir catalyst. O
O2S NH NH2 34
Scheme 17.
N H
PS
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
57
A similar heterogeneization of TsDPEN was made by Wang [27] with N-(carboxy, carbomethoxy or carboethoxy) benzene -p-sulfonyl)-1,2-diphenylethylenediamine (21 and 22 scheme 12). The acid function permitted the immobilization of the ligand to an aminomethylated polystyrene via an amide function (Scheme 18). O O2S NH
1) aminomethylated polystyrene, DCC, DMAP pentafluorophenol 2) TFA, CH2Cl2
(R,R)-20
CONH n
31 n = 1 32 n = 2 NH2
aminomethylpolystyrene: 1.07 mmol/g; DVB 1% CONH O2 S NH 33 NH2 O Ph
R
L* (RuCl2(p-cymene)2), 30°C HCOOH/Et3N, CH2Cl2 (1/1) Ketone/L*/Ru: 100/1.2/1
OH Ph *
R
R = CO2Et, CONHCH3, CN Scheme 18.
Ruthenium complexes of 31, 32 and 33 using the azeotrope triethylamine/formic acid as hydrogen donnor permitted the reduction of β-keto ester, amide and nitrile with high conversion (> 95%) and ee (> 90%) (Scheme 18). The electronic character and the spacer length between the polystyrene part and the benzene ring had very little effect on the reduction outcome, for a same substrate conversion and ee are similar. Here also, it has been shown that the catalyst formed with ligand 32 could be reused at least three times without loss of activity and enantioselectivity. Xiao proposed a poly(ethylene glycol)-supported ligand (34) in order to perform the HTR of aromatic ketones in water, using as hydrogen donor, the azeotrope triethylamine/formic acid [28] or sodium formate/water (Scheme 19) [29]. These ligands were formed by immobilization of TsDPEN by functionalization of the phenyl group by a hydroxy group in the meta position followed by a nucleophilic reaction with a monomethylether mesylate PEG (Mn 2000) (Scheme 19). The Ru-catalyst formed was soluble under the conditions of the reaction. It could be isolated from the reaction mixture by precipitation, though addition of a low polarity solvent,
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Christine Saluzzo and Stéphane Guillarme
allowing recycling. Up to 80% ee and up to 85% ee were achieved with all of the ketones tested using the azeotrope triethylamine/formic acid or sodium formate/water systems respectively. Nevertheless, convertion depended on the structure of the ketone and mainly on the position of the withdrawing group of the aromatic group (Cl, OMe, CF3).
OH
HO
H2N
HN Ts
1) MeO PEG OMs CsCO3, DM F, 50°C
O
O
2) CF 3 COOH, CH 2 Cl2 , H 2 O, r.t.
H2N
L* (RuCl2 (p-cym ene) 2), 50°C HCOOH/Et 3 N/H 2 O (1/1/1 v/v/v), or NaCOOH/H 2 O S/C: 100/1
O Ar
HN Ts 34 OH
Ph *
Ar: p-Cl, o-Cl, p-Me, o-Me, m-OM e, m-Br, p-CF 3 Phenyl; 1-naphtyl, 2-naphtyl
Scheme 19.
For the sodium formate/water reduction system [29], several parameters were studied. A 20°C decrease of the temperature led to a slightly higher ee at a longer time. The lower the formate concentration, the slower the reduction is and the lower ee. Moreover, the reaction was not affected by the presence of a surfactant. The ligand itself probably acted as a phase transfer catalyst. The presence of an organic solvent, inducing a biphasic system, led to a slower reaction and a decrease in ee. Concerning the catalytic system stability, it appeared that in sodium formate/water medium, a possible decomposition occurred because catalytic recycle led quickly to loss of catalytic activity and ee, which was not the case when formate/water system was used: more than ten recycles with no loss of activity and enantioselectivity. The same approach was involved in 2008 by Chan [30]. As an alternative method for attaching a PEG chain onto TsDPEN ligand, a PEG-750 (ie Mn = 750) was anchored at the para-position of an aryl sulphate group leading to polymer 35 in 51% overall yields starting from chiral DPEN (Scheme 20). Polymer 35 was examined in the Ru-catalyzed asymmetric HTR of acetophenone in water using HCOONa as the hydrogen donor at room temperature. Best ee (96%) was obtained with 5 equivalents of HCOONa and the high ratio of substrate to catalyst (S/C = 1000) led to slower reaction with a slight decrease in ee (90% ee). Other aryl ketones have been reduced. Compared with the precedent catalytic system formed with polymer 34, similar results were obtained in term of enantioselectivity. Once again, the catalyst could be easily recovered and reused at least eight times. Ph
Ph
Ph
HN HN Boc
SO2
1) MeO PEG OTs CsCO3, acetone
HN
SO2
2) CF3COOH, CH2Cl2
OH
Scheme 20.
H2N
Ph
35
O
O
O
16
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
59
2.2.2. Imprinting technique for hydrogen transfer reduction with 1,2-diamines In 1995, Lemaire [32] reported the immobilized transfer hydrogenation catalyst using a tetramine-rhodium complex 35 in order to study the effect of molecular imprinting. The upper rhodium complex was formed by copolymerization with a diisocyanide in the presence of sodium (S)-phenylethanolate (PM) leading to the imprinted polymers 36a and 36b (Scheme 21). The PM is then removed by adding 2-propanol. The resulting polymer as well as the nontemplated polymer were tested in the transfer hydrogenation of acetophenone and phenylethylketone in the presence of 5 mol % of Rh-polymer with KOH/[Rh] ratio of 4 at 60°C. Results are presented in Table 3.
Ph HN Me
H NaO Ph + NH Me
Me OCN
R
Me
NCO
CH2Cl2, r.t. Ar
N
HO
Rh N
Rh(cod)Cl 2 Me 36a R =
OH
36
HO H Me
particle size : 80-120 μm
Ph
Me N
36b R = HO
Me
HO
Rh N
R
O
R Ph OH
Ph O
R N
HO
Rh N
Scheme 21.
Table 3. Molecular imprinting effect in reduction by using Rh catalyst with templated and non-templated polymers 35a and 35b. Substrate acetophenone acetophenone acetophenone acetophenone phenylethylketone phenylethylketone
Chiral polymer-Rh catalyst 36a polymerized 36a (S) templated 36b polymerized 36b (R) templated 36b polymerized 36b (R) templated
Inductor configuration (R,R) (R,R)
Conv. (%) 44 42
Time (days) 1 1
Ee (%) (config.) 33 (S) 43 (S)
(S,S) (S,S)
98 98
1 1
25 (R) 43 (R)
(S,S) (S,S)
96 91
6 9
47 (R) 67 (R)
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Christine Saluzzo and Stéphane Guillarme
In homogeneous catalysis using chiral diamine 18 complexed with Rh, the acetophenone was reduced quantitatively with 55% ee, in 7 days. In the case of polymerized complex 36a, acetophenone reduction leads to 33% ee and with its templated analog 43% ee. With 36b, an increase of about 20% ee is observed between polymerized and templated ligand. These increases in ee were ascribed to a favourable molecular imprinting effect of the PM, creating chiral pockets within the polymer network. Nevertheless, in the case of sterically demanding substrates little or no reduction occured, indicating a substrate stereospecificity different from that observed in homogeneous catalysis or when using catalyst precursor belonging to the polymer backbone [32]. For the reduction of acetophenone, crosslinked templated polymers were studied. Optimization of the crosslinking ratio led to the best compromise between activity and selectivity (70% ee for a crosslinking ratio of 50/50 of triisocyanate/diisocyanate) [33, 34]. This selectivity could be explained by a certain rigidity of the cavity, permitting good accessibility to the reaction sites. This molecular imprinting technique was also used by Severin [35] [36] who proposed the synthesis of phosphonato complexes 37 (Scheme 22) in order to mimic the six membered cyclic transition structure suggested for HTR. This immobilized catalyst was prepared in a three step reaction (Scheme 22). In order to compare the effect of such polymeric organometallic transition state analog (TSA 38), the polymer 40 was prepared (Scheme 22) by copolymerization of the non phosphonato complexed catalyst with ethylene glycol dimethacrylate (EGDMA) (Ru/EGDMA: 1/99). The ability of such polymers to catalyse the reduction of benzophenone was tested, using the azeotrope formic acid/Et3N as hydrogen donor.
R R N Ru O NH2 R1 P R O 37
1
N Ru O NH2 R1 P R1 O EGDMA, CH2Cl2 V-70, 35°-65°C
AgO2PPh2 CH2Cl2, r.t.
TSA 38 [BnNEt3]Cl, MeOH
R 1) EGDMA, CH2Cl2 V-70, 35°-65°C 2) [BnNEt3]Cl, MeOH
N Ru Cl NH2 R1 R1 R = SO2C6H4CH=CH2
R N Ru Cl NH2 R1 R1
non-imprinted polymer 40
a: R1 = H b: R1 = -(CH2)4Scheme 22.
39
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
61
The molecular imprinted polymer 39a was significantly more active than polymer 40a (TOF (40a) = 51.4 h-1, TOF (39a) = 16.5 h-1). With this enhancement of the rate of the reaction using the imprinting polymer, substancially higher specificity for benzophenone was observed when completion of HTR of an equimolar amont of benzophenone and a ketonic cosubstrate was performed. This behaviour was slightly larger for aliphatic ketones as compared to aromatic ketones. These results were consistent with the activity and selectivity using organometallic TSA as template with an imprinting technique. A similar study was made with the polymer including rhodium complexes coordinated with phosphinato ligands (polymer 38b) and the imprinted polymer 39b. After 6 hours, the reduction of acetophenone led to 81% yields and 95% ee, and ruthenium complexes gave up to 70% ee for the reduction of the ethyl phenyl ketone [37]. Due to the structurally defined transition state analogs, included in a polymer, a shapeselective cavity close to the catalytic active center was formed. This cavity is highly selective and an enhancement of the activity was observed Furthermore, it has also been shown that activity and selectivity depended on how the metal complex was attached to the polymer backbone: a rigid connection by two styrene side chains is superior [36].
2.2.3. Aminoalcohol and aminothiol as ligand Chiral β-amino alcohols can also be used for homogeneous HTR of ketones and are known to be effective for reduction of aromatic ketones [38] but, only few examples of organic polymer-supported ligands have been reported. Recently, poly((S)-glycidylmethacrylate-co-ethyleneglycol dimethacrylate) (41) (poly((S)-GMA-co-EGDMA)), [39][40] (poly((S)-PhGMA-co-EGDMA)) (42), [41] poly((S)glycidylmethacrylate-co-divinylbenzene (poly((S)-GMA-co-DVB)) (43) [41] [42] and ((S)thiiranylmethylmethacrylate-co-ethyleneglycol dimethacrylate) (44) [42] different precursor polymers were synthesized. They were formed by radical suspension polymerisation of enantiopure (S)-GMA (30 wt % for 41a or 70 wt % for 41b) with EGDMA or DVB (70 wt % for 43), or (S)-PhGMA (40 wt % for 42) with EGDMA or (S)-TMA (30 wt % for 45) with EGDMA in presence of AIBN, with a mixture of cyclohexanol/dodecanol (91/9 wt/wt) and 2% of aqueous polyvinylpyrrolidone (PVP) as stabilizer (Scheme 23). Chiral beads of 41 and 43 presented respectively a specific surface area of 82 and 275 m2/g, and in both the cases, 2.11 mmol/g of epoxyde function. A specific area of 50 m2/g with 4.92 mmol/g of epoxyde function, a specific area of 35 m2/g with 1.83 mmol/g of epoxyde function and a specific area of 92 m2/g with 1.72 mmol/g of epoxyde function were respectively observed with 42, 44 and 45. The subsequent epoxide or episufide ring opening with diverse amines led to the corresponding polyaminoalcohols and polyaminothiols. Their ruthenium complexes were applied to the HTR of benzophenone (Scheme 23). It is noteworthy that the nature of the amine of the aminoalcohol played a crucial role. The best results in terms of conversion and enantioselection are obtained with polyaminoalcohols and polyaminothiols derived from methylamine and benzylamine (Table 4) [39]. It appeared that the efficiency of the catalyst depends on the nature and the proportion of the crosslinking agent (catalyst presenting 70% of EGDMA was more efficient than this with 30% of EGDMA and for DVB a decrease in conversion and ee was observed). It depended also on the specific surface, the nature of the ligand (aminoalcohol or aminothiol) and on the
62
Christine Saluzzo and Stéphane Guillarme
steric hindrance (aminoalcohol and ligands from GMA compared with PhGMA were more efficient) (Table 4) [41]. Moreover, attempts to recycle ruthenium complexe of 46a led to a marked decrease in activity from 94 to 27% and lowered selectivity from 70 to 54%. O
R1 +
O X X = O: GMA X = S: TMA
R2
O
R1 * H O X R4 41 - 45 R3HN R5 (3 eq)
AIBN, PVP dodecanol/cyclohexanol
R R2
DMF 100°C, 22 h
X = O 41, 42, 46 and 47: X = S 45 and 48:
O
O
O
O
R1 * N O HX R3 R5 46 - 60 H R4 O
R1 = H, R2 = Me;
R=
and R1 = H, R2 = H
X = O 43 and 60: R = O
O
O
O
C-X configuration: S if X = O; R if X = S C-R1 configuration: S if R1 = Ph
R1 = Ph, R2 = Me;
X = O 44 and 49: R = O Ph
Me
i-PrOH, t-BuOK, 80°C [RuCl2(p-cymene)]/46-60
OH H Me Ph
acetophenone/Ru/aminoalcohol polymer unit/t-BuOK: 20/1/4/5
Scheme 23.
Table 4. HTR of acetophenone using several aminoalcohols and aminothiols (L*/Ru/Ketone/t-BuOH: 4/1/20/5, 80°C) R1 * O HX HN CH2R5 O
Phenylethanol
X (C-X config.) O (S)
R1 (C-R1 config.) H
R5
Ligand
Functionality (mmol/g)
Ph
46a
O(S)
H
H
O (S)
H
S (R) S (R) O (S) O (S) O (S)
Time (h)
Conv. (%)
Ee (%) (config.)
1,46
3
94
71 (R)
46b
0,85
1
95
65 (R)
Ph
47a
2.90
72
51
57 (R)
H
Ph
48a
1.11
22
55
50 (S)
H Ph (S) Ph (S) H
H Ph H Ph
48b 49a 49b 60a
0,90 0.65 0.51 1.28
22 22 72 22
50 61 95 > 95
Ee (%) 85 84 62 99 63 98 95 44 94 96
Recycling (ee %) 3 (58) 1 (78) 16 8
Ref.
[56] [56] [57] [57] [58] [58] [59] [59]
In general, the crosslinked CBS (64, 64D or 66) afforded enantioselectivities almost identical to those of the CBS in homogeneous conditions (58a and 58b) and superior to the pendent-linker equivalent ligand (ie respectively 65, 65D and 67). These results could be ascribed to the superior swelling characteristics of the crosslinked catalyst in the solvent of the reaction which permits a better diffusion of the reagents into the catalyst [56-58]. For polymer 67, the weaker coordination with borane, due to the steric hindrance on the nitrogen atom, may be responsible of the slower catalysis. With 68 and 69 the results of the reaction were almost similar. After the 20th reuse, analysis of the polymeric mixture 68 showed that the overall weigh fell to 57% of its original value with an average loss per reaction of 98.5%. The recycling of the chiral supported-sulfonamide in the reduction of the meso precursor of d-biotin could be involved at least 5 times with non change in activity and enantioselectivity. Ph O
O S
Ph OH
N (0.15 eq)
O
O
70
NH H
NH H
BH3.SMe2 THF reflux 6h
O
N Bn
O
NH NH H H H O HO N Bn
Scheme 33.
Recently, Wang [64] prepared by radical copolymerization a cinchona alkaloid copolymer: the methyl acrylate-co-quinine (PMA-QN (71)) (Scheme 34). Complexed with palladium(II), its catalytic activity in the heterogeneous catalytic reduction of aromatic ketones by sodium borohydride was studied. High yields in their corresponding alcohols are obtained but it is found that the efficiency of the catalyst depended on the nature of the solvent and the ketone which related to the accessibility of the catalytic active site. The optical yields in methanol and ethanol 95% were lower than in ethanol. This ability was attributed to a bad coordination between PMA-QN-PdCl2 and sodium borohydride and a reaction rate which was very rapid. The stability of the chiral copolymer catalyst was studied
70
Christine Saluzzo and Stéphane Guillarme
via the study of the recycling efficiency. Only 2 wt% loss of metal was observed after repeated use (at least 5 times) with only a little decrease in enantioselectivity.
n
m
COOMe
N OH
H
N
MeO
OH
H N
MeO
QN
PMA-QN
N 71 Scheme 34.
2.4. Hydrosilylation with Ligands as Pendent Group Asymmetric hydrosilylation of ketone had been studied in homogeneous supported catalyst with nitrogen containing ligands and ony few examples have been reported. In 1998, Enders reported immobilized triazolium salts as precursors to chiral carbene-rhodiumcatalyzed asymmetric hydrosilylation (Scheme 35). This Rh complex gave 24% ee compared to 17% ee for the homogeneous reaction (Scheme 34). The recycling of the solid supported catalyst 71 was successful with only slowly decreasing yields which were comparable to the yields of the homogeneous reactions with catalyst 72 [65].
N N
O
O
+
ClO4 H
N
ClO4 H
N N N
p-TSA CH2Cl2
HO ( ) t-Bu n
O
O
O ( ) t-Bu n
[CODRhCl]2 THF, Et3N
N N
Cl N N
Rh
N O
O
Ph Scheme 35.
Rh
N
O ( ) t-Bu n 72 O
Cl
1) Ph2SiH2 (1eq) THF 72 or 73 2) MeOH, p-TSA
BnO
t-Bu 73
OH Ph *
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
71
3. REDUCTION OF C=N BOND Although highly effective asymmetric reduction of carbonyl compounds has been extensively investigated, enantioselective reduction of imine derivatives to amines has been less studied, mainly in homogeneous supported catalysis [66]. With polymer containing nitrogen (2-amino-3-(p-hydroxyphenyl)-1,1-diphenylpropan-1ol [67], 2-piperazinemethanol), Itsuno [68] has shown that in stoechiometric conditions, oxime ethers could be reduced with borane with excellent ee (up to 99%). More recently, in organocatalysis, Kočovsky [69] has reported asymmetric reduction of imine with trichlorosilane catalyzed by an (N-methylvaline)-derived formamide anchored to a polymer. Under the best conditions, with 15 mol% of the catalyst enantiomeric excesses were about 85% and the catalyst could be reuse. Then, in 2009, Itsuno [70] utilized for HTR of N-benzylimine the polymer-immobilized chiral ligands 31, 33a and 33b (Scheme 16) as their RuII complexes. These complexes were already used to catalyze the HTR reduction of ketones in water [25]. The HTR was carried out in organic solvent (dichloromethane) and in water using as hydrogen donor respectively the azeotrope HCOOH/Et3N and HCOONa. Catalytic system with ligand 31 was effective for the HTR of N-benzyl imines in organic solvent. The corresponding amines were formed in good yields and ee (92-96% yields and 84-88% ee). In contrast, RuII complexes obtained from amphiphilic polymer 33a and 33b were found to be effective for the HTR of cyclic imines in water (50-95% yields 86-94% ee). The catalytic activity, in water, seemed to be controlled by the hydrophilic-hydrophobic balance in a polymer-supported catalyst.
4. CYCLOPROPANATION Highly functionalized cyclopropanes are important building blocks for obtaining numerous natural compounds (terpenes, pheromones ….), and compounds which are of high value in biological (ie: agrochemical active species) and medicinal chemistry. A wide number of methods have emerged in order to produce optically active cyclopropanes [71]. From all of them, highly enantioselective cyclopropanes are formed by asymmetric catalytic insertion of carbene to prochiral olefins. This method, first employed by Noyori in 1966 [72] consists in the metal-catalyzed decomposition of substituted diazo compounds in the presence of various prochiral alkenes. Under homogeneous conditions [73], various chiral Schiff base-Cu(II or I) or Ru(II) complexes have been used as chiral catalyst precursors and from all of them the most efficient systems are formed with oxazolines and bis-oxazolines. Thus after their first use by Masamune [74], oxazoline-derived ligand have been extensively employed in homogeneous enantioselective catalytic reaction particularly in cyclopropanation [75]. Thus nitrogenanchoring ligands on organic polymers are mainly formed by immobilized oxazolines.
72
Christine Saluzzo and Stéphane Guillarme
4.1. Oxazolines Ligands Bis-oxazolines (Box), azabis-oxazolines (azaBox) and pyridine bis-oxazoline (PyBox) with a C2 symmetry form the great majority of various oxazoline-derived ligands which are used in immobilized catalytic cyclopropanation reactions (Scheme 36). For these reactions, immobilized ruthenium and copper complexes which are able to form carbene intermediates have been tested. Most of the time, the reaction involved styrene and ethyl diazoacetate (EDA). However other alkenes (diphenyl ethylene …) and other diazo compounds (i.e. tertiobutyl and ethyl 2-phenyl diazoacetate) have also been investigated (Scheme 36).
O
O N
N
N R
R
H N
O
R1
+
N2 C
R PyBox 76
a: R = Ph b: R = t-Bu c: R = Bn
a: R = i-Pr
R2 R2
R4 COOR3
CuL*
N
R R AzaBox 75
a: R = Ph b: R = t-Bu c: R = Bn d: R = Et e: R = i-Pr
O
N N
N
R
Box 74
O
O
R1 R2 R1
R4 A
COOR3
R2 +
COOR3 C R4
COOR3
R1 B
R4
R1 R2
R4
D
COOR3
Scheme 36.
First, heterogeneous catalytic systems involved N-anchoring ligands on inorganic supports such as zeolithes [76], clays [77] …. Immobilization of the catalyst on organic support leading to insoluble or soluble polymers was made by grafting or copolymerization of the chiral ligand. In all cases, the immobilization is performed via alkylation of the methylene bridge or of the nitrogen of the bridge (AzaBox, PyBox).
4.1.1. Insoluble supported-polymer Box as ligands In 2000, Mayoral [78] described the first immobilization of Box on insoluble polymer. For a same ligand, grafting and several methods of polymerization (Scheme 37) were reported in order to compare the catalytic efficiency of these supported ligands after their complexation with Cu. The study was based on immobilization of bis-(oxazoline) 74a-c (Scheme 36) and their corresponding Cu-complexes were tested in the cyclopropanation of styrene with EDA, a well known reaction in homogeneous catalysis [79].
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
73
The Box methylene bridge of 74a, [78, 80] 74b [78, 80, 81, 82] or 74c [78, 80] was first functionalized with two p-vinylbenzyl groups in order to perform homo-polymerization [78, 80] (polymer 79), or co-polymerization with styrene [78, 80, 81] (polymer 78), DVB [78, 80, 81, 82] (polymer 80α) divinylbenzyl polyethylene glycol [80, 81, 82] (polymer 80β) and divinylbenzyl resorcinol [80, 81] (polymer 80γ) or dendrimers 80δ (Scheme 2). The best results were observed with the homopolymer 79b (R = t-Bu) [78, 80]. This homopolymer 79 prepared by thermal polymerization [78] was able to coordinate a large amount of Cu and presented a better activity than the same homopolymer prepared in presence of AIBN (respectively 51% and 34% yield, 35/65 and 39/61 trans/cis ratio, 75% ee and 77% ee for the trans cyclopropane, 70% ee and 73% ee for the cis cyclopropane) [78]. For all the copolymers, the low efficiency of the copper complexes (cyclopropanation of styrene: yield up to 40%) may be explained by the non accessible core of polymer by Cu probably due to the high degree of crosslinking [78, 80]. But moderate to high ee were observed (up to 78% ee) and contrary to homopolymers, the cis cyclopropane is mainly formed. Attempts to use copolymers from monofunctionalized Box (Scheme 38) as ligand did not improve the efficiency (yield up to 16%); the diastereoselectivity is in favour of the trans stereoisomer. As for copolymer 80α, yields obtained with 80β−δ, do not exceed 35% and the diastereoselection gave mainly the trans cyclopropane. With 80β the formation of trans isomer is up to 67% [81, 83]. The presence of oxygen atom in the crosslinking agents could induced more coordination with the metal. This effect is detrimental for the catalytic properties for the resulting Cu-loaded resin [81]. The homopolymer 79b could be reused up to five times without loss of ee but with a slight decrease in activity [80] In order to avoid the non accessibility to the core of polymer by the metal due to excessive crosslinking, a monofunctionalization of the Box leading to Box pendant ligand 81 was performed [80] (Scheme 38). Unfortunately, the incorporation of copper into polymers 81 was not found to be improved (Cu loading: 0.07-0.08 mmol/g). Furthermore, a higher loading was observed with the less crosslinked polymer 81a. It is noteworthy that if the ligand 81c is transformed into a polymer with bisoxazoline moieties by treatment with MeLi and MeI, the results obtained for the cyclopopanation reaction were slightly worse that those obtained with 81c itself (yields 16% vs 16%; trans/cis ratio 60/40 vs 57/43, ee trans 23% vs 29%; ee cis 22% vs 34%). Salvadori [84] showed that a copolymer structure presenting a single spacer, but flexible one linking the ligand moiety to the inert polystyrene backbone was effective with solutionlike behaviour (Scheme 39). For the cyclopropanation of styrene with EDA, Cu-polymerized 82 complexes afforded similar results than the non supported Box. In the first case 61% and 60% yield, 67/33 and 71/29 dr in favour of the trans, 93% and 94% ee were respectively observed. The catalytic system could be reused without loss of its catalytic properties. Moreover, it is noteworthy that the immobilization method did not influence the catalytic behaviour: catalytic systems formed with copolymerized (route A) [84] or graft methodology (route B) [85] were substantially equivalent (variation of about 1 to 2% of the yield, ee or dr). In 2004, Yifei reported Merrifield (3.8 mmol/g) polymer-supported Box 74c-e [85] (Schemes 36 and 40) for the cyclopropanation of 1,1-diphenylethene with EDA.
74
Christine Saluzzo and Stéphane Guillarme
The highest enantioselectivity of 85% ee with 82% yield was achieved by using 83d showing as in homogeneous conditions that the steric hindrance of the R group of the oxazoline had a great influence on the ee and the yield. Under recycling, the catalytic system led to an important decrease in enantioselectivity. In order to restrict the conformational mobility of the groups on the bridge of the Box, a 1,3-dioxane group was introduced, then the Box was grafted onto a Wang resin (0.7 mmol Br/g) (Scheme 41) [86]. In that condition, only 0.26 mmol/g has been grafted onto the active site and the copper content was 0.018 mmol/g. The ligand 84 was evaluated in the cyclopropanation of styrene with EDA. The trans cyclopropane was obtained in 65% ee. The high ee was due to less steric hindrance between the two oxazoline groups, as the consequence of the chair conformation of the dioxane ring. AzaBox as well as PyBox have the advantage towards Box to present a donor nitrogen atom in the bridge allowing to a higher coordinating ability. Thus a better stabilization and a better recoverability, with regard to their analogous Box ligand are observed. Additionally, AzaBox and PyBox ligands have only one link point in the bridge which makes grafting easier.
4.1.2. Insoluble supported-polymer AzaBOX as ligands Recently, AzaBox ligands were attached to a TentaGel (polymer with a polystyrene backbone and PEG periphery) (87) or to polystyrene (88 and 89) by direct grafting of the ligand onto the polymer or by copolymerization of the ligand functionalized with a styryl group (Scheme 42) [87]. The corresponding catalyst presented a copper content of 0.2-0.3 mmol/g. Cyclopropanation of styrene and 1,1-diphenylstyrene with EDA was studied with 11.5 mol% of the catalyst. In these experiments, catalyst formed with copolymerized ligand 89 did not show any advantage over grafting ones. Moreover, Merrifield resin was a better support than TentaGel. With 87a the yield was up to 51% but its diastereoselection (64/34) and and its ee (up to 62%) are lower than those obtained with 88 or 89 (yield up to 32%, dr 70/30, ee up to 88%) for the cyclopropanation of styrene. 4.1.3. Insoluble supported-polymer PyBox as ligands The first immobilization of PyBox was made by Mayoral [88] by means of radical copolymerization of 4-vinylPyBox with styrene and DVB in presence of AIBN and a porogen (toluene or a mixture toluene/dodecanol) (ligand 90 Scheme 43). PyBox was incorporated in the polymer with high yield from 75 to 95%. The corresponding Ru-complex was formed at about 50-60% showing that all the ligands were not accessible. The nature of the porogen solvent was important. A polymeric ligand prepared in presence of dodecanol gave a lower Ru functionalization and was detrimental to the trans/cis selectivity (77/23). The best catalytic system, high trans/cis selectivity (85/15) and ee (trans: 85%), was formed by the polymer prepared in toluene. However, it has been observed that with a lower crosslinking degree, dodecanol has less importance. The best catalysts were reused twice, but a marked decrease in both selectivities and activities were observed for the second recycle.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
O
O N
1) MeLi
N 2)
R
R
Cl
74a-c
1) MeLi
O
O N
77 [11]
N R
R 2) n
m
78 [9, 11]
Cl
AIBN
O
O N
N
N R
R
O
O
a: m = 20, n = 80 b: m = 50, n = 50 c: m = 80, n = 20 d: m = 90, n = 10
N
n AIBN or heat
R
R AIBN
O
O N
crosslinker
R 79 [78, 80]
R x
z
y
crosslinker : α:
x=20, y=70, z=10 [78, 80, 81]
β:
80
O O 7.7O x=20, y=70, z=10 [78, 80, 81]
O
O N
N γ:
R
R
O
O R1O
O x=20, y=70, z=10 [78, 80, 81]
OR1 δ:
O
O
O
x=10, y=85, z=5
OR1
[80, 81]
R1 = R1O
N
O
O OR1
R1 =
O
O O
OR1
Scheme 37.
[81, 82]
75
76
Christine Saluzzo and Stéphane Guillarme
y
x
AIBN
H N
Styrene
H
DVB
O
O
81 a: x = 0, y = 90 b: x = 40, y = 50 c: x= 70, y = 20
10
O
O
N
N t-Bu
t-Bu
N t-Bu
t-Bu
Scheme 38. (
)(
) ( y
x
)
z
Ph ) (
O (
O +
+
route A ref. [84]
O
O N
82
AIBN
)n
O N
N
N
t-Bu t-Bu route A n = 1, x/y/z ~4/43/53 route B n = 2, x/y/z ~6/92/2
t-Bu
t-Bu
O
route B ref. [85] 1) 9-BBN, THF 2) H2O2, NaOH 3) KH, THF 4) Merrifield resin cat. KI and C[18-6]
O(
)2 O
O N t-Bu
N t-Bu
Scheme 39.
R
O O
O
Cl
N R
N
R 74 c: R = Bn d: R = Et e: R = i-Pr
Scheme 40.
Merrifield's resin (3.8 mmol Cl/g)
N C H2
83
N O
R
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
77
O
OH O
O
1) NaH
O
O
2) Br-Wang
O
O N i-Pr
O
O
O O
O
N
O
O i-Pr
N
N
N
N i-Pr
i-Pr i-Pr
i-Pr 84
85
Scheme 41.
Subsequent studies involving not only copolymerized ligands but also polymers grafted onto a Merrifield resin (Scheme 43) were performed [89]. For the latter methodology, functionalization of the PyBox was required, thus 4-aminophenoxyPyBox and 4mercaptoPyBox were synthesized, leading to the corresponding ligands supported polymers 91-93. For the Ru catalysts, the introduction of a spacer is important. The longer the spacer is, the higher Ru functionalization. This is consistent with the better accessibility of the grafted ligands contrary to copolymerized ones. Concerning their evaluation for the cyclopropanation of styrene with EDA it has been found that to obtain high catalytic performance and good recyclability, the heterogeneous catalyst must have a gel type organic support with a low cross linking (2% DVB) and a support with no electron-donating effect on the pyridine ring (C-C bond between the support and the ligand). Thus taking into account these parameters, a highly active (40-65% yield) and enantioselective (87-91% ee) catalyst was obtained. Reused at least 3 times of the catalytic system with the same performance was possible. Mayoral [90] also demonstrated that the PyBox-Ru supported complexes on macroporous monolithic polymers were efficient catalysts for the cyclopropanation reaction. PyBox monolithic minireactors were prepared in a stainless column by radical copolymerization of 4-vinyl or 4-styryl PyBox in presence of styrene and DVB using 10% toluene/50% dodecanol as the precipitating porogeneous mixture (polymers 94 and 95 Scheme 44). To form the Ru complex, a solution of [RuCl2(p-cymene)]2 was passed through the column at low flow then washed with dichloromethane to remove the non complexed Ru. For the cyclopropanation of styrene with EDA, several parameters have been studied: flow rate, morphology of the catalyst and solvent. An increase of the flow rate induced a decrease of the cyclopropanation and a dimerization of EDA which is one of the poisons of the catalyst. Besides both region and enantioselectivities gave good values: 80/20 trans/cis, cis isomer 48 ± 5% ee, trans isomer 75 ± 5% ee. The catalyst could be used for several runs (4-6 times) without change in catalytic performance. Concerning the morphology of the catalyst, a slight decrease in catalytic
78
Christine Saluzzo and Stéphane Guillarme
activity was found with ligand 95 containing an aryl spacer. If the crosslinking degree was higher (51% DVB instead of 20%), the activity and chemoselectivity were improved. The reaction performed without solvent provided an increase in both efficiency and chemoselectivity for a mixture styrene/EDA: 1/7 and minimize the dimerization of EDA. With scCO2 as solvent, yields were about 25% but chemoselectivities and ee of both diastereoisomers rise. Moreover, the total number of turnover (TON) is greatly increased. O n-BuLi, -78°C-0°C THF
O Tenta- O Gel
TentaO Gel N
O Br O
H N
N
O
O N
poly styrene
O
87 R a: R = i-Pr b: R = t-Bu loading 0.1-0.12 mmol/g poly styrene
N R
R X
N
R
N N R 75a R 75b
H N
O
N
O
75a 75b
N
O N R
R
X =Cl X = Br
88 a: R = i-Pr b: R = t-Bu loading 0.5-0.56 mmol/g poly styrene N
O N i-Pr
O
styrene (6eq), DVB (7.3 eq) AIBN, 80°C, toluene (toluene/monomers: 40/60 w/w)
N
O N
N i-Pr
O N
i-Pr 89 loading 0.52 mmol/g
i-Pr
Scheme 42.
4.1.4. Immobilization of PyBox on natural polymers Some natural polymers have also been used as support for Schiff base ligands. A starch support has also been used as support for a PyBox. A telomerized starch 96 [91] presenting a final double bound reacted with a thiol-PyBox in the presence of AIBN (Scheme 45) to form the corresponding immobilized PyBox. [92] Although the Ru-97 complex exhibited lower activity (up to 44% after the third reuse) and selectivity (trans/cis: 76/24 with 50% ee for the
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
79
trans and 16% ee for the cis) towards the homogeneous complex formed with 98 (yield 67%, trans/cis: 89/11 with 77% ee for the trans and 63% ee for the cis) this supported catalyst showed better performance than that immobilized on silica [93]. These results pointed out a great influence of the support.
7
x
93-x
Ph O
O +
N N
AIBN toluene
+
O
N
O +
N N
i-Pr
i-Pr
N
i-Pr
i-Pr
90
O N O
O O NH2
N
CHO
O
Br
O
N N
92 0.45-0.5 mmol ligand/g
N O
i-Pr
i-Pr
O
N N
N i-Pr
i-Pr SH
S NaH, DMF, 100°C
O i-Pr
Scheme 43.
O
N N
i-Pr
NH
O
O
N 91
i-Pr
EtOH, reflux
O
N
O
N
Br i-Pr
N i-Pr
O
N N
i-Pr 93 0.45-0.5 mmol ligand/g
80
Christine Saluzzo and Stéphane Guillarme
O
O
N N
O
N
N
i-Pr
i-Pr
N
O
O
N
N i-Pr
95
i-Pr
i-Pr
O
N N
N
i-Pr
i-Pr
94
1) AIBN toluene/1-dodecanol 2) washing with THF
+
+
O
N
i-Pr
or
O
N
Scheme 44. OH O
(
OH
HO
(
(
O OH O
96
(
O O HO
1) Ac 2 O, pyridine 2) AIBN, CHCl3 , reflux
O N HS
N
SH N
O O
OH O
N
OH
HO
(
(
O N
S
( )5
O O HO
OH O
N 97 N
Scheme 45.
O
non supported ligand
O
(
(
O
N N
98
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
81
The homogeneous catalyst formed by reaction of non supported oxazoline 98 with RuCl2 was very efficient, the ratio cis/trans was about 89/11 and enantioselectivities were 63% and 71% respectively. These values are lower than those achieved using 4H PyBox-Ru catalyst (88 and 70%) respectively, owing to the presence of a slightly electron-withdrawing group on the pyridine moiety of the ligand. The Ru-complex of the polymer supported ligand 97 exhibited lower activity and selectivity toward the cyclopropanation reaction of styrene. Three recycles were performed showing a trans/cis ratio and the enantioselectivity constants.
4.1.5. Soluble supported-polymer Box as ligands Box 74a and 74b have also been immobilized onto the soluble polymer MeOPEG (a monomethylether of polyethylene glycol, Mn > 2000 Da) and employed as ligand in the Cucatalyzed cyclopropanation of styrene and 1,1-diphenylethylene with EDA with up to 93% ee. Taking into account the best parameters found with insoluble polymer, which were necessary to enhance the stereoselectivity of Box promoted reaction [94], i.e. disubstitution at the Box bridging atom and the necessity of introducing a spacer in order to separate the polymer moiety (PEG) to the ligand itself, Cozzi synthesized the supported polymers 99a and 99b (Scheme 46) [95]. With styrene, ligand 99b gave a trans/cis stereoselectivity in favour of the trans product (70%), the trans isomer presenting 87% ee. An increase of the yield (about 20%) was observed if the time of addition of EDA and of the reaction increased. In the same conditions, the results were inferior to those obtained with the gem dimethyl Box 74b but the two MeOPEG-supported catalysts presented similar behaviour. Recycling of the catalytic system was possible with marginal erosion of the catalytic activity and very limited loss in ee. OH
1) NaH or Cs2CO3 or Bu4N+OH-
2)
O
O
O N
N
O
O spacer
R
R
O
OMs
N
N R
R 99
: MeOPEG
a: R = Ph b: R = t-Bu
Scheme 46.
4.1.6. Soluble supported-polymer AzaBox as ligands In 2000, Reiser [96] reported the first immobilization of aza-bis(oxazolines). Azabis(oxazoline) 74b has been easily grafted to a soluble polymeric support ((methoxy(polyethyleneglycol) MeOPEG 5000) in order to form soluble catalysts which could be recovered by precipitation. This polymer (100 Scheme 47) was obtained successfully (55% yield) if a benzylidene spacer linked to the PEG was employed. Complexed with Cu(II), this ligand was able to promote asymmetric cyclopropanation of styrene (Scheme 36: R1 = Ph, R2 = H, R3 = Me, R4 = H) and 1,1-diphenylethene (scheme 36:
82
Christine Saluzzo and Stéphane Guillarme
R1 = R2 = Ph, R3 = Me, R4 = H) with methyl diazoacetate in dichloromethane. With styrene, a diastereoselectivity in favour of the trans cyclopropane with a predominance of C over D was observed (C/B: 71/29, C: 91% ee B: 87% ee) with a yield of 69% (Scheme 36). Compared to the non-supported catalytic system (ligand 75b), the results in terms of diastereoselection and ee were 10% lower. The possibility of the reuse of this catalytic system was examined. 13 cycles were conducted without loss of ee, but to keep a good yield after the 10th use, it was necessary to reactivate the catalytic system with phenyl hydrazine. O
O N t-Bu
O
H N
O
n
Br
N
n
N
O N
n-BuLi, -78°C-0°C
t-Bu 75b
O
O
t-Bu
O N
100
t-Bu
Scheme 47.
4.2. Porphyrins More recently, chiral Ru-porphyrin polymers were used for catalytic asymmetric carbene transfer. A C2-symmetric group containing two norbornane moieties fused to the central vinyl substituted benzene ring of a porphyrin was chosen in order to induce the chirality. Then the chiral ruthenium vinylporphyrin 101 was involved into radical copolymerization with styrene and DVB or EGDMA to lead respectively to monolithic resins chiral 102 and 103 which were crushed (Scheme 48). In order to form cyclopropane esters and cyanocyclopropanes, EDA and diazoacetonitrile were respectively used in the cyclopropanation of various substituted styrenes. Although less enantioselective than their homogeneous counterpart, these catalysts in presence of EDA led to high stereoselectivities. For example with 102a, up to 92/8 for styrene and pmethoxystyrene and 97/3 for p-bromostyrene, in favour of the trans isomer were obtained. Yields were respectively 77%, 88% and 75%. The best ee (90%) was obtained with the pbromo derivative. Moreover, diastereoselectivity and enantioselectivity were influenced by the nature of the porogenic solvent employed during the formation of the catalyst. Compared to 102b, better ee and diastereoselectivities were obtained with catalyst 102a (trans/cis ratio: 82/18 and 92/8; 82% ee and 71% ee respectively), these two catalysts having the same DVB/styrene ratio. This behaviour was attributed to the reduced accessibility of the catalytic sites, also evidenced with polymers prepared from higher DVB/styrene ratio. With 102 slightly lower results than those of 102a were observed. Concerning the cyclopropanation with diazonitrile, moderate activities from 35 to 53% yield and correct enantioselectivities around 70% were obtained, the best results being found with catalyst 102a. These catalysts were stable upon recovery and recyclability (no leach of metalloporphyrins) but a sligh decrease in activity was observed [97].
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
83
R
N CON Ru N N
N CON Ru N N
styrene DVB or EGDMA R AIBN, porogen
R
R
101 catalyst 102 crosslinker: DVB R = porogene: 102a: toluene 102b: CH2Cl2/dodecane
( catalyst 103, crosslinker: EGDMA R = porogene: toluene
)
O
O
O O
Scheme 48.
Supported metalloporphyrins have also been synthesized by electropolymerization leading to films of polymers. They were formed on the platinium working electrode during the oxidative electrosynthesis (Scheme 49) [98]. Contrary to the preceding catalysts 102 and 103, these electropolymerized catalysts led to low enantioselectivities for the bench-mark reaction between styrene and EDA. Up to 53% ee at - 40°C was reached in the presence of 105a. Moreover, at room temperature, the reactions proceeded efficiently (yields 80-90%). Seven recycling of polymer 105a were carried out without a significant decrease in enantioselectivity and activity.
4.3. Miscellaneous Instead of Box ligand, carboxamidate coordinate with rhodium have been tested successfully for the cyclopropanation of some functionalized alkenes [99] (Scheme 50).The carboxamidate anchored to the NovaSyn Tentagel hydroxyl resin (TG) or Merrifield resin (Scheme 50) was treated with chiral dirhodium tetrakis[methyl 2-oxypyrrolidine-5(S)carboxylate] (Rh2((S,S)MEPY)4) leading to the corresponding Rh-106 and Rh-107 precatalyst. With these catalysts, intramolecular cyclopropanation of allyldiazoacetate was investigated (Scheme 51). Compared with homogeneous catalyst, yield of bicyclolactone was similar to those obtained with the two catalysts i.e. 75% and 95% ee.
84
Christine Saluzzo and Stéphane Guillarme
Catalyst with the Merrifield support (Rh-107) presented a yield of about 75% and a slight decrease in ee after 10 runs contrary to the TG one (Rh-106) which showed 10% of the yield and of about 20% ee decrease. Ligand loading had an influence only on the yield but not on the selectivity of the reaction. A similar study was performed with the cyclopropanation of styrene and EDA. Once again, the best catalytic system in terms of reproducibility of the results on the reuse corresponds to the Merrified one. For the first use they were quite equivalents.
R1
N CON Ru N N
R2
Anodic oxidation spirobifluorene
N CON Ru N N
(R1)n
104
105 x
R=
R3
R2 =
(
)
R3 = x
(
104a: R1 = R, R2, 105a: n = 1, R1 = R, R2 104b: R1 = R2, 105b: (R1)n = R3
Scheme 49.
In 2003, Xia [100] has reported the cyclopropanation of styrene with alkyl diazoacetates by copper complexes of Shiff bases, derived from chitosan and substituted salicylaldehydes as the catalyst (Scheme 52). This natural polymer is an environmental friendly natural material: biocompatible, biodegradable, non toxic… The influence of several parameters: nature of the solvent, temperature, the molar ratio styrene/EDA and the copper content [100] has been studied. Better yields were obtained with high temperature but better ee were found at 60°C, temperature for which the nature of the solvent had a great influence. From all the solvent tested, 1,2-dichloroethane, ethylacetate, acetonitrile and toluene, the best yield (91.5%) was obtained with 1,2-dichloroethane; toluene led only to 41.5% yield. Better ee were also found in 1,2-dichloroethane. When the molar ratio styrene/EDA was about 17/1 the results of this reaction were fairly good; as this ratio diminished, the yield and ee decreased and for a ratio of 2/1, the yield reached only 45.3%. Taking into account of all these parameters, it has been shown that to obtain the best compromise between the yield (81.5%), the best cis/trans ratio (35.3/64.7) and the best ee (cis: 20.7% ee, trans: 10%ee) for the cyclopropanation of styrene, a copper content
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
85
(Cu(OAc)2) of 4.4 mol% was necessary with ligand 108a. More or less amount of the copper content led to a decrease of all these reaction results. It is noteworthy that neither the electronwithdrawing nor the bulky substituents on the salicylaldehyde of the catalysts had much influence on the yield, the ee of the cis and trans isomers but the nature of the diazoacetate had a great influence. Using t-Bu diazoacetate instead of the Et one resulted in a important increase of the ee for both the cis and trans isomers but with a dramatic drop of the yield. This behaviour could be a result of the steric hindrance of the t-Bu groups which provoked a difficulty to form metal carbene species by the reaction of t-Bu diazoacetate with the Schiff base copper complexes. These catalytic systems have also been use to transform other alkenes into cyclopropyl derivatives. In some cases, compared to the styrene reaction the cyclopropanation of 1-hepten or 1-octene gave better ee.
O(
) OH + O n
N H
TG 0.27 mmol OH/g
Cl
+ O
N H
1 mmol Cl/g
O
DCC HOBt DMF
COOH
O(
)O n
106 HN O
DMF
COO- Cs+
O
O 107 HN
O
O O(
)O
N
n
Rh2
O O(
)O
MeOOC
n
or O
O
N H
O
N H
O 3
+ Rh2((S,S)MEPY)4 PhCl
or
reflux
O
N
Rh-106 O
O O
N
O
Rh2 MeOOC
N
O 3
Rh-107
Scheme 50.
N catalyst
O O Scheme 51.
CH2Cl2 reflux
O O
86
Christine Saluzzo and Stéphane Guillarme CH2OH O
CH2OH O O
OH
O
OH derivatives of salicylaldehyde
N
HO NH2
n
R1
n
108 a: R1=R2=H b: R1=H, R2=Cl c: R1=H, R2=Br d: R1=H, R2=NO2 e: R1=R2=Cl f: R1=R2=t-Bu
R2 Scheme 52.
To conclude, for the cyclopropanation reaction, the difficulties consist in the achievement of high enantioselectivity close to a good stereoselectivity. To be useful in organic synthesis, the last requirement is preponderant and depends on the nature and the number of the substituents of the cyclopropane ring. It is the reason why, most of the catalytic systems used in heterogeneous supported catalysis cyclopropanation are formed by bis oxazolines complexes which have shown a excellent efficiency in non supported conditions. In most of the cases, this reaction seems to be very sensitive to the polymer morphology controlled by crosslinking and the nature of the porogen agent which determine the performance of the immobilized catalyst.
5. CYCLOADDITIONS AND HETEROCYCLOADDITIONS Discovered in 1928, the Diels-Alder reaction is one of the powerful synthetic methods for the construction of substituted cyclohexene that fits the modern concept of atom economy [4]. For this reason, in the last two decades, the asymmetric Diels-Alder reaction, leading to carbon-carbon bond formation, has received considerable attention and enantioselectivities greater than 90% have been reported [101]. In homogeneous conditions, a wide range of metal, ligands and dienophiles have been studied. Because of their high stabilities, most of the studies are focused on the use of N-containing complexes. Concerning the Diels-Alder reaction, polymeric supports were mostly involved in solid phase synthesis where the diene and the dienophile are grafted onto the polymer. Compared to these procedures, only few examples involving supported catalysts were reported in the literature. Most of these supported catalysts contain nitrogen as ligands such as Box, aminoalcohol, N-sulfonylamino acid and salen.
5.1. Supported Box Ligands Moberg [102] developed a polymer bound bis(oxazoline) 109 (Scheme 53) as a zinc complex catalyst for the Diels-Alder reaction of cyclopentene with 3-(2-propenoyl)-2-oxazolidinone
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
87
(Scheme 54). This supported catalyst was formed by grafting a phenolic bis(oxazoline) derivative onto an ArgoGel Wang-Cl resin and presented 0.071 mmol of ligand/g polymer. OH
O
1) K2CO3
O
2) Argogel Wang-Cl
O
O N Ph
O
O
N
N Ph
Ph
109
O
O
N
N Ph
N Ph
Ph
Scheme 53.
Compared to the monomer (conversion 100%, exo/endo ratio: 94/6, ee: 85% for the exo adduit), the polymer supported catalyst gave no selectivity (racemic compounds are formed) and lower reactivity (13% at -40°C). This behaviour is probably due to the low swelling and the rigidity of the polymer at low temperature. At the same time, Cozzi described the immobilization of Box ligands on PEG polymer (polymer 99, Scheme 49), and the corresponding copper catalyst used for the cyclopropanation reaction was also involved in Diels–Alder cycloaddition between cyclopentadiene and N-acryloyloxazolidinone (Scheme 54) [95]. This reaction was performed with 15 mol% each of Box ligand and Cu(OTf)2 in DMF in order to obtain homogeneous conditions. The reaction occurred in good yield (up to 83%) and excellent diastereoselectivity in favour of the endo stereoisomer (ratio endo/exo >98/2). However, the maximum of enantioselectivity was only 45% contrary to the non-supported Box equivalent catalyst which led to complete end at 95% ee and 88% yield. H
O
O N
H
O
O N
O (S)
O
+ O
O
N
O
(R)
Scheme 54.
In 2002, Lemaire chosed to immobilize indaBox, not only for its rigidity but also for its good enantioselectivities for the Diels-Alder cycloadditions [103]. The heterogeneization was carried out by polymerization as part of the main chain of a polymer backbone (Scheme 55). Mixed with Cu(OTf)2, polymer 110 (8 mol% relative to the oxazolidinone) was allowed to react with methacrolein and cyclopentadiene at -78°C (Scheme 56). The reaction was quantitative and led to the major endo diastereoisomer with 51-56% ee with 87-90% yields for the first three reaction cycles. Itsuno synthesized supported aminoalcohols 111-113 (Scheme 57) [104] and Nsulfonylamino acid 114 [105-107] (Scheme 58) by radical suspension copolymerization of the corresponding monomer chiral styryl aminoalcohols and styryl sulfonylamino acid derivatives with styrene and eventually with a crosslinker.
88
Christine Saluzzo and Stéphane Guillarme OH O
HO O N
+
N
OCN
NCO
DMF, dibutyltin dilaurate
O
HO O O
O N
O H
N H
N H
O
N
66%
110
n
Scheme 55. H + O
Polymeric catalyst
CHO Me
Scheme 56.
5.2. Supported Aminoalcohol and N-Sulfonylamino Acid-Derived Oxazaborolidine or Oxazaborolidinone The styryl chiral sulfonylamino acid derived from L-valine 114a, L-isoleucine 114b, Lthreonine 114c or D-2-phenylglycin 114d (Scheme 58). The loading of aminoacid residue was about 0.78-0.88 mmol/g for a polymer formed with 10% of the styryl chiral sulfonylamino acid, 80% styrene and 10% of DVB with a yield range of 85-96%. In the case of valine, for a polymer formed with 50% of the styryl chiral sulfonylamino acid, 40% styrene and 10% of DVB, it could be up to 2.55 mmol/g and 73% yield [105]. The Diels-Alder reaction was performed with cyclopentadiene and methacroleine (Scheme 56) in presence of 15 mol% of the oxazaborolidine and oxazaborolidinone catalysts derived respectively from supported aminoalcohols and from N-sulfonylamino acid polymers. The oxazaborolidine and oxazaborolidinone catalysts were formed in situ by action of BH3, BH2Br, BHBr2 or BBr3. The diastereoselectivity was excellent in favour of the exo adduct and yields from 65 to 99%. It is noteworthy that higher loading of chiral catalyst site in the polymer, lower exo selectivity and enantioselectivity. The diastereoselection depended not only on the nature of the supported ligand, the crosslinker but also on the borane and the solvent. Results are summarized in Table 6. From all the oxazaborolidine catalyst, the L-valine derived catalyst 115a gave the highest enantioselectivity and an excellent ratio endo/exo. Moreover oxazaborolidinone polymeric catalysts having oxyethylene crosslinkages (116) present better performance.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
0.1
89
0.2
0.7
styrene DVB
O H
O
Ph Ph
Ph Ph
H
OH
OH
H2N 111
H2N
0.2
0.8 styrene
NH
OH NH
OH 112
0.8
0.1
N
styrene DVB
HO Ph Ph
0.1
N HO Ph Ph
113
Scheme 57.
5.3. Supported Salen as Ligand Seebach [108] prepared non-dendritic 117 and dendritic 118 and 119 styryl-substituted salen, monomers for crosslinking suspension copolymerization with styrene (Schemes 59 and 60). Complexed with Cr, these resulting copolymers were involved in Diels-Alder reaction of Danishefsky’s diene with several aldehydes (Scheme 61). In all cases, these new Cr-salen catalysts led to slightly lower enantioselectivities than those observed with their homogeneous analogs under the same conditions. Surprisingly, upon reuse, enantioselectivities generally increased around 5% from the first to the fifth catalytic cycle. With caproaldehyde and with benzaldehyde enantioselectivities are respectively higher than 85% and 75%. Recently, Schulz was carried out electropolymerization, an original methodology to prepare polymer supported salen 113 [109][110]. It consisted in the introduction of a thiophene moiety, an electropolymerizable functionality, on the salen backbone (Scheme 62) followed by electropolymerization under cyclic voltammetry conditions on a platinum grid. These polymeric catalyst 120a and 120b were then evaluated in hetero Diels-Alder reaction (HTR) between several aldehydes and 1-methoxy-3-[(methylsilyl)oxy]-1,3-butadiene
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Christine Saluzzo and Stéphane Guillarme
(Scheme 61) using 4 mol% 120a or 6 mol% 120b. It has been shown that the counter ion has quite no influence on the selectivity but the activity is lower with 120b containing BF4.
1) styrene DVB 2) Borane
O2S HN R1
OH 114
R2 O
O2S
N
R1 R2
X
B
115
O O
crosslinkage
1) styrene crosslinker 2) BH3.Me2S
O2 S
O2S HN i-Pr
OH
N B H O 116
i-Pr
O
X = Br, X = H a: R1 = H, R2 = i-Pr b: R1 = H, R2 = t-Bu c: R1 = Ph, R2 = H d: R1 = H, R2 = CH(OH)Me
O
crosslinker: a
(CH2)8
b: n = 0; c: n = 3; d: n = 7.7 O
O
n
O
Scheme 58
Table 6. Asymmetric cycloaddition reaction of cyclopentadiene and methacroleine in presence of 15 mol% polymeric boron catalyst, at -78°C Polymer 111 112 113 113 113 113 115a 115b 115c 115d 116a 116b 116c 116d
Borane BH2Br BH2Br BH2Br BH3 BH3 BBr3 BH3 BH3 BH3 BH3 BH3 BH3 BH3 BH3
Solvent CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2 CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF CH2Cl2/THF
Yield (%) 96 98 99 95 89 96 87 99 99 65 86 85 93 88
Endo/exo 8/92 4/96 < 1/99 < 1/99 < 1/99 5/95 < 1/99 3/97 4/96 8/92 8/92 5/95 1/99 4/96
Ee (%) 16 25 44 57 65 54 65 49 49 10 84 77 92 95
Ref. [104] [104] [105] [104] [104] [105] [106] [107] [106] [106] [106] [107] [107] [107] [107] [107]
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
N
91
N
OH HO t-Bu + Styrene
t-Bu
suspension copolymerization benzene, THF/H2O, PVA, AIBN 90°C, 20h
copolymer 117 Scheme 59.
Compared to the homogeneous catalytic system analog, heterogeneous one is less selective. The difference in enantioselectivity between homogeneous catalyst and these electropolymerized ones were possibly due to the steric hindrance around the active site containing the metal and the electronic effects providing by the delocalization due to the conjugaison of the polymerized catalyst. Seven or fifteen recycles of the catalytic system have been performed with heptanal and cyclohexane carbaldehyde respectively without showing any change in enantioselectivity and activity.
6. ADDITION OF ORGANOMETALLIC REAGENTS TO ALDEHYDES, KETONES AND IMINES The carbon-carbon bond forming reaction is one of the most useful chemical processes for the construction of complex natural or synthetic organic molecules. Addition of organometallic reagents to carbonyl or imine compounds is among the most fundamental reaction [111]. Its enantioselective version producing simultaneously a carbon-carbon bond and a chiral center is particularly important. The reaction of Grignard reagents with carbonyl compounds proved to be one of the best methods for forming carbon-carbon bond. Unfortunately, Grignard reagents are unsuitable for asymmetric alkylation because of their high reactivity in opposite to organozinc reagents such as diethylzinc, diphenylzinc or alkynylzinc which are excellent nucleophiles in presence of chiral ligands (Scheme 63). Numerous studies about the asymmetric addition of these reagents to carbonyl or imine derivatives have been performed and various chiral ligands have been synthesized [112]. Among the ligands used, chiral ligands bearing nitrogen chelating donor atom have been extensively studied due certainly to their easy preparation and availability. Furthermore, a lot of chiral ligands have proved to be highly efficient for the asymmetric alkylation of carbonyl or imine compounds in homogeneous phase. But, for the reasons explained in introduction, the use of homogeneous supported or heterogeneous ligand is preferable. Among the polymer-supported ligands, derived-aminoalcohol ones have been largely used in this reaction but few are derived either from N-sulfonamideaminoalcohols, or oxazoline or salen.
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Christine Saluzzo and Stéphane Guillarme
Ph
Ph
N R
N suspension copolymerization benzene, THF/H2O,PVA, AIBN 90°C, 20h
OH HO
R
t-Bu + Styrene
Ph
copolymer 118
t-Bu
Ph
N
N
OH HO
R
R
t-Bu + Styrene
t-Bu
suspension copolymerization benzene, THF/H2O, PVA, AIBN 90°C, 20h
copolymer 119 R= O
O
O Scheme 60.
O TMSO +
O R
polymeric catalyst
H
O OMe
Scheme 61.
*
R
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
N
93
N
OH HO S
S t-Bu
t-Bu
1) CrCl2, THF 2) AgBF4, TBME 3) 50 mA, Pt/Cgraphite NBu4NBF4, CH3CN
N
N Cr O X O S
S t-Bu
t-Bu
n
120 a:X = Cl, 77% b: X = BF4, 64%
Scheme 62.
X + R
R''2M or R''M
Ligand Solvent, T°C
XH * R R"
X=O, N-R' Scheme 63.
6.1. Supported Aminoalcohols as Ligands The first work about polymer-supported β-aminoalcohol used in the asymmetric nucleophilic addition to aldehydes was reported by Fréchet [113]. The best result (95% ee) was obtained by using a polymeric catalyst derived from N,N-dialkylated (-)-3-exoaminoisoborneol 124b for the asymmetric addition of diethylzinc to o-methoxybenzaldehyde in toluene at 0°C (Scheme 64). This catalyst was prepared through reaction of aminoalcohols with 1-2% crosslinked chloromethylated polystyrene. The chiral polymeric catalyst of the reaction could be used several times in further asymmetric reactions.
Me H
Ph H
N
OH
121
Scheme 64.
O
Ph
R2N OH 122a, R=H 122b, R=Me
OH N
N R OH 123
124a, R=H 124b, R=Me
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Christine Saluzzo and Stéphane Guillarme
Soai has described the enantioselective addition of dialkylzinc reagent to aromatic and aliphatic aldehydes by using the supported-ligand 121 [114]. This latter was also prepared from (1R,2S)-(-)-ephedrine and chloromethylated polystyrene (1% divinylbenzene, 0.8 mmol Cl/g, 100-200 Mesh). In the case of benzaldehyde, the enantioselectivity was better than those reported with 124a (89% vs 80%) and with the N-alkylephedrine-based homogeneous catalyst. The authors mentioned that the catalyst could be easily recovered and reused. They also studied the reaction with norephedrine homolog and reported enhanced enantioselectivity in the case of aliphatic aldehydes [115]. Polymer-supported ephedrine 121 was also employed in the first heterogeneous enantioselective alkylation of N-diphenylphosphinoylimines and the best results (80% yield and 80 % ee) were obtained when the reaction was carried out in toluene at room temperature for 2 days [116]. Itsuno has reported interesting work about the effect of the difference of the structure of the polymeric part on the enantioselectivity of the reaction and the stability of the support [117]. They first prepared the chiral aminoalcohols 125 which could react with a crosslinked chloromethylated polystyrene or 126 which could be copolymerised with styrene and some styrene-derived crosslinking compound (Scheme 65).
OH O Ph Ph H 2N
125
Ph Ph
OH H2N
OH 126
O
O
O 3
127
Scheme 65.
Five different chiral polymers containing the same aminoalcohol moiety were synthesized (Scheme 66). Reaction of aminoalcohol 125 with a chloromethylated polystyrene crosslinked with 2% of divinylbenzene led to chiral polymer 128. When the highly crosslinked polystyrene containing 20% of DVB 129 was used, no reaction between 125 and this resin because of the lack of the swellability of the resin in organic solvents occurred. To avoid this problem, the crosslinking agent containing an oxyethylene chain 127 and the corresponding crosslinked chloromethylated polystyrene 131 were prepared. This latter swelled very well in organic solvents such as toluene and THF, even in the case of a highly crosslinked resin. Phenoxide anion of 125 could react with the chloromethyl group of poly mer 131 to give the chiral supported ligand 132. Three other chiral polymers have been synthesised by copolymerization of the chiral monomer 126 with styrene in the presence or not of crosslinking agent (Scheme 66). Chiral polymer 133 was prepared by suspension copolymerization of aminoalcohol 125 with styrene and DVB in a 1/7/2 molar ratio. Polymer 133 swelled well in organic solvents such as toluene, benzene and THF and that unusual swellability could be due to the bulkyness and polarity of the chiral monomer 125. The replacement of DVB by 127 as the crosslinking agent afforded chiral polymer 134 after copolymerization. This one also swelled well in
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
95
organic solvents and presented interesting mechanical stability. The solution polymerization of 127 and styrene led to the soluble linear chiral polymer 135. The main problem of polymers containing a high degree of crosslinking is their stability with stirring over a long period of time. Indeed, their spherical beads are not enough stable and their breakdown during stirring lead to formation of a fine powder of the insoluble polymer. So the filtration at the end of the reaction is difficult and the reuse of the chiral polymer was not possible. The authors studied the effect of the magnetic stirring on the three highly cross-linked polymer beads and found that chiral 20% 127-crosslinked polymers 132 and 134 were more stable than polymer 133 prepared by using 20% DVB. For example, when stirring at 350 rpm for 4 days in toluene, 50% of polymer 133 was transformed into a fine powder contrary to 132 and 134 which kept their original bead form. The five chiral polymers 128 and 132-135 were then tested in asymmetric addition of diethylzinc to benzaldehyde. Good enantioselectivities were obtained with chiral polymers 133-135 in opposite to the two others polymers 128 and 132. In the case of pchlorobenzaldehyde, an excellent ee of 99% was obtained with polymer 134 in oposite to oalkoxybenzaldehydes (21-54%) and longer reaction times were necessary to have satisfactory conversions with aliphatic aldehydes. The recyclability of the chiral polymers was studied in a batch system using a simple loading of polymeric ligand 134 and both yield and ee were highly reproducible. Large scale of a chiral product could be obtained by using a continuous flow system. This technique have been tested with chiral polymer 133 and showed that a column containing 5 mmol of the polymer could produce about 90 mmol of (S)-1-(p-chlorophenyl)propanol with 94% ee. The main advantage of this technique is that it avoids the stirring which destroy the polymer during repeated reactions. Soai and Watanabe have studied the effect of the introduction of a spacer between the ligand and the polymeric support [118]. In fact, when the polymer is directly connected to the nitrogen atom of the aminoalcohol, the polymer part could have an influence on the enantioselectivity since it is known that nitrogen atom is strongly involved in the formation of intermediate chiral zinc complex. The two chiral polymers 137 (content of aminoalcohol moiety is up to 0.085 mmol/g) and 138 (content of aminoalcohol moiety up to 0.25 mmol/g) were prepared from polymer 136 (1% DVB; Cl: 0.8 mmol/g; 100-200 mesh) and the corresponding chiral aminoalcohols (Scheme 67). The enantioselective addition of diethyzinc to aldehyde at 0°C with chiral polymers 137140 was examined. With aromatic and aliphatic aldehydes, higher enantioselectivities were obtained with chiral polymers 137 and 138 containing a six-methylene spacer compared to the two chiral polymers 139 and 140. The same level of enantioselectivity was found with recycled chiral polymers. The authors assumed that the spacer has here two effects. The steric repulsion between the aminoalcohol part and the polystyrene is reduced and the spacer acts as a subtituent of the nitrogen atom which assists the stereochemical control of the reaction. The four-methylene chain analog of chiral polymer 137 and chiral polymer 139 were also tested in the asymmetric alkylation of N-diphenylphosphonylbenzaldimines [119]. Contrary to aldehydes, both yield and enantioselectivity were lower with chiral polymer containing a spacer between the polymeric moiety and the catalytic part. The authors then examined the reaction conditions with 139. The result of their screening was that the best yields and ee’s
96
Christine Saluzzo and Stéphane Guillarme
were obtained when the reaction was carried out in toluene at room temperature in presence of 1 equivalent of the polymeric ligand.
a
Cl
R*O
128
a
129
Cl
R*O
130
a
Cl
O O
O
O
3
O
3
131
+
OR*
O
132
b
+
R*O
OR*
133
b
+
+ O
O
O
OR*
R*O
O O
3
O
3
134 b
+
Ph Ph
R* = OR*
135
OR*
H 2N
OH
Scheme 66. (a) R*OH, NaH, DMF. (b) AIBN, THF Ephedrine or norephedrine-derived copolymers 142a-d have also been used in the same reaction [120]. These chiral polymers were prepared by copolymerization of monomer 141a-d with styrene and DVB in a molar ratio 141a-d/styrene/DVB of 1/7/2 (Scheme 68).
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer… Ph H
Me H HN
CH 2 O
OH Bu
N
OH
5
Bu
137
CH 2O(CH 2 )6I + 136
Ph H
Me H
K 2 CO 3
K 2CO 3
HN
CH 2O 5
HO
97
N
138
PhPh
HO Ph H
Me H N
OH
PhPh
N
Bu 139
HO PhPh 140
Scheme 67.
The asymmetric addition of diethylzinc to N-diphenylphosphinylbenzaldimine was carried out in toluene at room temperature in presence of the chiral copolymerized ligands 142a-d. Although the yields were low to moderate, the enantioselectivity was moderate to high (64%-88% ee) particularly with the chiral ligands 142b and 142c. The enantioselectivities with these two polymers were comparable with those observed with the corresponding monomer 141b and 141c. The recyclability of polymer 142b was also tested and this one could be reused without loss of enantioselectivity.
CH 2
H C
m
H2 C
H C
n
DVB / AIBN
+
N R
Me *
141a-d
* Ph
OH
Benzene / THF H 2O
N R
Me *
* Ph
OH
(m/n = 1/7)
(1S,2R)-142a: R=H (1S,2R)-142c: R=Et (1R,2S)-142b: R=Me(1S,2R)-142b: R=Bu
Scheme 68.
Hosoya has reported the preparation of polymer-supported ligand which has only moderate enantioselectivity because the aim of the work was to show the effect of the type of immobilization [121]. The polymeric ligands were prepared by two different techniques, the Mix Method and the Add Method. The Mix Method was a classical polymerization: the ligand-containing monomer was dissolved in the mixture of monomers and porogenic solvent was used for the second-step swelling. After completion of this step, the polymerization step was carried out at 70°C with slow stirring for 24h to lead to the resulting polymers 144 and
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Christine Saluzzo and Stéphane Guillarme
145. The Add Method consisted in adding the same amount of monomers 143 in the aqueous polymerization medium 4h after the initiation of the polymerization to give the polymers 146 and 147 (Scheme 69). The two monomers were quantitatively incorporated in the beads produced by the Mix Method and only 44% of monomers for the Add Method. R N
OH
144: 145: 146: 147:
AIBN
PS-PG-M PS-PA-M PS-PG-A PS-PA-A
PS=polystyrene PG=phenylglycinol PA=phenyalanilol M=Mix Method A=Add Method
143a: R=Ph 143b R=PhCH 2
Scheme 69.
The catalytic activity of the different polymeric ligands was evaluated in the asymmetric ethylation of aldehyde in hexane. The polymers derived from the Mix Method gave better results in terms of activity and enantioselectivity than those from the Add Method. However, both yields and ee were slightly lower than those obtained with the monomeric ligands 137. When a mixture of toluene/hexane (1/1) was used as solvent, chemical yield dropped significantly. The effect of the level of functionalization and crosslinkage of different Merrifield resins was also tested [122]. Chiral polymers 150 were prepared in two steps from enantiopure epoxyalcohol 148 (Scheme 70). X X O Ph
NaH, DMF
OH 148
Cl Merrifield resin
R
O O
Ph 149
N H LiClO 4 CH 3CN
R
R Ph
N
R O
OH 150a: X=CH 2 , R=H 150b: X=NMe, R=H 150c: X=CH 2 , R=Me
Scheme 70.
These chiral polymers 150 with different levels of crosslinking and ligand funtionalization were evaluated in the asymmetric addition reaction of diethylzinc to benzaldehyde and the results are summarized in Table 7. Although the conversions were higher than 95%, the enantioselectivities were low to moderate and significantly lower than those observed with the corresponding homogeneous ligand [123]. In all cases, the best results were obtained with both lower level of cross-linking (1%) and level of chlorine per gram of resin (1.2 mmol) of the chloromethylated polystyrene. Higher the level of aminoalcohol residue is, more important the formation of dimeric zinc alkoxide is. These
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
99
dimeric species are suspected to be inactive in catalysis, requiring a preliminary dissociation before the catalysis step. Table 7. Enantioselectivity for the ethylation of benzaldehyde with chiral polymers 150 Ligand (%DVB ; fa) Amount (%)b ee (%) 150a (1; 0.8) 5 36 150a (2; 1.6) 4 22 150a (2; 1.8) 3 20 150b (1; 0.9) 5 39 150b (2; 1.6) 4 20 150b (2; 1.7) 3 19 150c (1; 1.0) 3 69 150c (2; 1.6) 4 57 150c (2; 1.7) 3 57 a f=mmol of ligand/g resin (calculated by elemental analysis of nitrogen). b amount of catalyst (% molar with respect tobenzaldehyde)
. It was assumed that the low enantioselectivity was probably due to a lack of bulky susbstituents on the primary hydroxy group of the chiral aminodiol. The cis 2,6dimethylpiperidine-derived aminoalcohol 151 was anchored to 2-chlorotrityl chloride resin (Barlos resin) leading to polymeric ligand 152 (Scheme 71, Table 8). The use of an amount of 5 mol% of funtionalized Barlos resin 152 in the asymmetric addition of Et2Zn to benzaldehyde in toluene was highly efficient and (S)-1-phenylpropanol could be isolated with an excellent ee (entry 2, Table 8. When the reaction was carried out at 0°C, the enantioselectivity was slightly improved (entry 3, Table 8). The asymmetric addition of Et2Zn to different aromatic and aliphatic aldehydes using the functionnalized Barlos resin 146 was also studied and both conversions (> 82%) and ee’s (>86 %) were high. X
X R
N
R
Ph
+ OH
Ph (o-Cl)-Ph
R
Cl Barlos resin
N
R
Ph
O
Ph (o-Cl)-Ph
OH 152
OH 151
Scheme 71.
Table 8. Asymmetric addition of diethylzinc to benzaldehyde using 152 Entry 1 2 3 4
f (mmol ligand/ g of resin) 0.9 1.1 1.1 1.2
% molar ligand 4 5 5 5
T (°C) rt rt 0 0
Ee (%) 79 92 93 94
100
Christine Saluzzo and Stéphane Guillarme
Hodge has also studied the reaction by using chiral polymers from ephedrine-derived monomer 141b [124]. Homopolymerization of monomer 141b in presense of AIBN in toluene and copolymerisation with styrene or 4-methylstyrene in various proportions led respectively to chiral polymers 153, 154 and 155 (Scheme 72). styrene or methylstyrene
Me H
Ph H
N
OH
1-x
AIBN Toluene
x
Me H
Ph H
N
OH
R
(1S,2R)-141b
153: x=1 154: R=H, x=0.01-0.66 155: R=CH3, x=0.08-0.52 156: R=H, x=0.06-0.51; prepared from crosslinked polymers beads
Scheme 72.
Chiral polymers 153-155 were used for the asymmetric addition reaction of diethylzinc to benzaldehyde. The reactions were carried out in toluene at room temperature for 24 hours. The results are summarized in Table 9. Although the major product was the 1phenylpropanol, benzyl alcohol was detected as a minor product with each polymer 153 (9%), 154 (0-6%) and 155 (6-7%). As we can noticed in Table 9, the higher the loading of catalyst is, the lower the yield and the ee are. It can be explained for highly loading polymers by the presence of dimeric zinc alkoxide species which drop the catalytic activity of the catalyst. Higher yield and ee were obtained with Zn-complex derived from 154f and 154g which were soluble in toluene (entries 7-8, Table 9). In the case of polymers 155 (entries 9-13, Table 9), the reaction was heterogeneous and yield and ees increased as the loading decreased. The enantioselectivities obtained with polymers 154d-154g (loading ≤ 1.32 mmol.g-1) and 154d155e (loading ≤ 1.32 mmol.g-1) were similar or better than those obtained with the model ligand, (1R,2S)-N-benzylephedrine. Table 9. Effect of the ligand loading of polymers 153-155 on the catalytic activity Entry
Polymer
1 2 3 4 5 6 7 8 9 10 11 12 13
153 154a 154b 154c 154d 154e 154f 154g 155a 155b 155c 155d 155e
Fraction of monomer 135b (x) 1 0.66 0.52 0.28 0.18 0.05 0.03 0.01 0.52 0.46 0.36 0.17 0.08
Loading (mmol.g-1) 3.52 2.98 2.65 1.81 1.32 0.44 0.27 0.09 2.56 2.38 2.03 1.16 0.61
Yield (%)
ee (%)
47 56 66 80 95 98 100 100 65 67 74 92 92
44 56 67 79 81 82 83 83 69 74 76 81 86
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
101
The authors then turned their attention to the preparation of crosslinked polymer beads which are more insoluble and are more convenient to use than the linear polymers 153-155. Different chiral crosslinked polystyrene beads 156 were prepared by reaction of various crosslinked polystyrene beads with (1R,2S)-N-benzylephedrine in presence of potassium carbonate. The catalytic properties of these various crosslinked polymer-supported ephedrines have been tested in the asymmetric addition reaction of diethylzinc to benzaldehyde and the results are summarized in Table 10. The reactions were carried out either in toluene or hexane at 0°C or 20-23°C. As it can be noticed in Table 10, the enantioselectivities were higher in toluene. Compared to linear polymers 153-155, lightly crosslinked chiral polymers led to the best results in terms of yield and ee. The use of Amberlite XAD-4 and Polyhipe™ as support proved to be not efficient. It was assumed that probably for both supports the catalytic sites are more in the inner surfaces of the pores and that result in a high local concentration of aminoalcohol residue which lead to a significant site-site interaction. Chiral polymer 156a was also used in the asymmetric ethylation of 2-methoxybenzaldehyde and 4chlorobenzaldehyde under similar conditions and ees reached 90-93% when the reaction was performed at 0°C. Three other polymers 158 were prepared by homopolymerisation and copolymerisation of aminoalcohols 157 and by grafting of the corresponding aminoalcohol onto 1% crosslinked polystyrene beads containing 0.70 mmol.g-1 of chloromethyl group leading to a polymer containing 0.64 mmol.g-1 of ligand residue (Scheme 73) [124]. Table 10. Asymmetric diethylzinc addition to benzaldehyde using crosslinked polymer beads 156. ee (%) Catalyst Degree of Yield (%) (ee)a loading substitution (yield)a b 1 156a 1% crosslinked gel 0.93 0.12 81 (81) 81 (64) 2c 156a 1% crosslinked gel 0.93 0.12 85 (-) 78 (-) b 3 156b 1% crosslinked gel 1.13 0.15 77 (83) 77 (62) 4c 156b 1% crosslinked gel 1.13 0.15 85 (81) 74 (65) b 5 156c 1% crosslinked gel 2.30 0.40 80 (82) 74 (62) 6c 156d 1% crosslinked gel 2.30 0.40 86 (97) 73 (65) b 7 156d 2% crosslinked gel 1.06 0.14 70 (-) 72 (-) 8b 156e 2% crosslinked gel 2.62 0.51 71 (76) 69 (62) 9b 156f Amberlite XAD-4 0.90 0.11 59 (62) 28 (39) c 10 156g Amberlite XAD-4 0.50 0.06 75 (70 28 (39) 11b 156h Polyhipe™ 1.51 0.21 59 (57) 42 (23) Under brackets, reaction was carried out in hexane. b Reaction was carried out at 0°C for 70h. Reaction was carried out at 20-23°C for 20h. Entry
a
Polymer
Starting polymer
c
Contrary to ephedrine-derived polymers 153-156, the use of a high loading of the catalytic residue (≥ 2.48 mmol.g-1) for chiral polymers 158a and 158b gave excellent enantioselectivies for the ethylation of benzaldehyde and similar to those obtained with the corresponding non-polymeric chiral aminoalcohol. This surprising excellent result with a high loading of aminoalcohol unit polymer is probably due to their high solubility in toluene. The
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Christine Saluzzo and Stéphane Guillarme
enantioselectivity obtained with the crosslinked polymer beads 158c with a ligand loading of 0.64 mmol.g-1 was also high (97% ee).
x
1-x
N N OH 157
OH 158a: x=1 158b: x=0.66 158c: x=0.11; prepared from crosslinked polymers beads
Scheme 73.
The authors were then interested to use the two enantiomers of ephedrine- and camphorderived polymeric ligands 153-156 and 158 in a flow system [125]. When polymeric ligand 156a and its enantiomer analog (1% crosslinking, 1.11 mmol/g of aminoalcohol residue) were employed with a flow rate of 2 mmol.h-1 of benzaldehyde and a diethylzinc/benzaldehyde ratio of 2.5/1, the ee were respectively 81% and 72%. Lightly crosslinked beads 156 (0.2% crosslinking, 1.78 mmol/g loading) produced the (R)-alcohol in a yield of 98% and in 98% ee. With a flow rate of 2.5 mmol.h-1 of benzaldehyde, the ee dropped slightly (91% vs 98%). Supported-ligand 158c (with a ligand loading of 0.64 mmol/g) was also tested in a flow system because this polymer-supported catalyst gave better results than ephedrine one 156. A loss of catalyst performance was noticed here; indeed, the chemical yields and enantioselectivity dropped significantly after aproximatively twelve runs (run 1: 95% yield and 97% ee; run 12: 59% yield and 87 % ee). It was suggested that the deterioration in performance could be due to the oxidation of the hydroxyl group of the aminoalcohol unit. (S)-aziridinyldiphenylmethanol 159 was immobilized to polymer-bound triphenylchloromethane (1.1 mequiv/g) to lead to the new chiral polymer 160 (Scheme 74) [126]. This latter was employed in the enantioselective addition of diethylzinc to aromatic and aliphatic aldehydes. For example, (S)-1-phenylpropanol was isolated in a yield of 92% and an ee of 96% when the reaction was performed in a 1/1 mixture of toluene and dichloromethane. The presence of dichloromethane proved to be essential for swelling of the polymer and making the catalytic sites accessibles to substrates. With other aromatic aldehydes and cyclohexane carboxaldehyde, the same range of chemical yield and ee was obtained. But with acyclic aliphatic aldehydes the chemical yield and ee dropped significantly. The recyclability of the chiral polymer 160 has been investigated and it was noticed a slightly decrease of the enantioselectivity of 1% per run. Styrenic L-prolinol-derived ligand N-Boc-60 was immobilized on chemical inert, mechanically stable polyethylene fibers by electron beam induced preirradiation grafting using styrene as a co-monomer to lead to polymer fiber 161 with a loading of 0.2 mmol/g [127]. Carbamate function was then removed under classical conditions to produce polymer 162 (Scheme 75). Chiral polymer 162 was used as ligand in the diethylzinc addition to
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
103
benzaldehyde and yield and ee were very low (36% and 24% respectively). A higher yield of 55% could be obtained in a titanium-mediated addition of diethylzinc to benzaldehyde by using the protected chiral polymer 161 with the same enantioselectivity.
-Tr-Cl
HN HO
Ph Ph
N
Et3N, CH 2Cl2
159
160 HO
Ph Ph
Scheme 74.
In order to improve the catalytic properties of this supported-α,α-diphenylprolinol polymer fibers, Degni has tested the immobilized ligand 67 with a loading of 0.4 mmol/g (Scheme 30) [128] in the diethylzinc addition to benzaldehyde. The chemical yields were largely superior to those obtained with polymer 162 (95-98% vs 30-36%) and the enantioselectivities were also higher and reached 40% which was comparable to the corresponding non-polymeric ligand.
OH N
O
O N-Boc 60
OH N
O
O 161
OH NH 162
Scheme 75.
Chiral polymer 67 was also tested in another enantioselective reaction, the Carreira reaction. The phenylacetylene addition to benzaldehyde in presence of zinc triflate using 67 as polymeric ligand was carried out. The corresponding propargyl alcohol was isolated in a moderate yield but with an excellent enantioselectivity of 91%. This supported ligand was also recycled for this reaction whithout loss of catalytic activity.
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Christine Saluzzo and Stéphane Guillarme
Br
OH
OH
OH N
N
Ph
N
Ph
Ph
OR
OR
OR
OR
OR
OR
OR
OR
Br 163 +
Pd(PPh 3)4 K 2CO 3 THF
OR (HO)2B
165 B(OH)2 OR
164: R= (CH 2)5CH 3
Ph
Ph
N OH
N OH
B(OH)2
RO 163 + OR
B(OH)2
OH N
OH N
166
Ph
Pd(PPh 3 )4 K 2 CO 3 THF
Ph
OR
OR
OR
OR
OR
OR
167 R=(CH 2 )5 CH3 Ph
N OH
Ph
N OH
Scheme 76.
A new strategy for optically active chiral polymers was envisaged and based on a Suzuki coupling polymerisation of (1R,2S)-ephedrine unit-bearing monomer with rigid linkers [129]. The Suzuki coupling of ephedrine-derived dibromide 163 (which was prepared in four steps from (1R,2S)-ephedrine) with the diboronic acid 164 in presence of Pd(PPh3)4 afforded chiral
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
105
polymers 165 (Scheme 76). Synthesis of chiral polymer 167 containing a longer linker between two chiral units has been performed by using diboronic acid 166. Both polymers were soluble in common solvents such as CH2Cl2, THF, CHCl3… The chiral polymeric ligands 165 and 167 were then employed in the diethylzinc addition to benzaldehyde and anisaldehyde. The enantioselectivities obtained with both polymers were between 74 and 76% for the two aldehydes. The polymers could be recovered by precipitation from methanol and the recycled polymeric ligand showed the same catalytic properties. Chiral bicyclic aminoalcohol-containing polymers 170 and 173 were produced from the industrial waste material 168a generated in the industrial process of synthesizing the ACE inhibitor Ramipril at Aventis. Supported ligand 170 was obtained by grafting of compound 168b (obtained in two steps from 168a) onto a Merrifield resin (1.1mmol Cl/g) and after reaction of the resulting polymer with phenyl Grignard (Scheme 77) [130]. Excellent yields and ee (83 and 89 % respectively) were observed for the diethylzinc addition to benzaldehyde for the heterogeneous ligand 170 and were similar to those obtained for the homogeneous reaction. In a second time, the authors turned their attention on the preparation of flow systems. They considered that in some cases the chiral supported ligands isolated by polymerisation led to higher enantioselectivity. The monolithic column was selected as polymerisation technique because the resulting material is more suitable for the preparation of flow systems because of its better porosity properties. Monoliths 173 with the desired morphology and properties were obtained by mixing 40 wt% of monomers (10 mol% of 172/90 mol% of DVB; no styrene) and 60 wt% of toluene/1-dodecanol as the porogenic mixture (10 wt% of toluene). A flow system was performed and after 24 h, (R)-1-phenylethanol was isolated with an excellent ee of 99%. This enantioselectivity is better than those obtained with the corresponding homogeneous ligand and with the grafted ligand 170 and is one of the best ee observed with a polymeric ligand. The authors assumed that this efficiency could be due to the formation of more appropiated chiral cavities because of the polymerization process or to the high level of crosslinking agent used which isolates the aminoalcohol residue. Chiral polymer 173 could be reused for four successive runs without loss of enantioselectivity. Reduction of the ester function of polymer 169 or treament with a dimagnesium bromide reagent led to the two immobilized aminoalcohols 174 and 175 (Scheme 77). Chiral polymer 174 proved to be not efficient for the diethylzinc addition to benzaldehyde (11% yield and no ee) and with chiral polymeric ligand 175, for the same reaction, the yield was excellent but the enantioselectivity was low (40%) [131]. The same group has developped a small library of supported chiral aminoalcohols from different amino acid [132]. The hydrochloride salt of different amino acid methyl esters 176 reacted with a chloromethylated polystyrene-divinylbenzene (1 mmol Cl/g, 1% DVB) under basic conditions to afford the polymeric amino acid ester 177. Reduction of the ester function or addition of Grignard reagents led to supported-amino alcohols 178-181 (Scheme 78). The nitrogen atom of these polymers was then methylated to afford the N-methylaled polymeric aminoalcohols 182-185. Chiral polymers 178-181 were first tested for the diethylzinc addition to benzaldehyde using 10 mol% of the supported catalyst. In all cases, the chemical yield and the selectivity (based on the 1-phenylethanol/benzyl alcohol ratio) were moderate and the ee were around 10 % except for the (S)-proline-derived polymer 180e (45% ee). The low catalytic activity could be due to the presence of the NH group in polymers 178a-d – 181a-d.
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Christine Saluzzo and Stéphane Guillarme H
H
H
H CO 2Me
N
b
H
N
a
170: Grafted 174: Polymerised e H
H
H
N H
169
H CO 2 R
168a: R=Ph 168b: R=Me
H
c
H
N
H CO 2Me
d
N
H
LiAlH 4, THF or BrMg(CH 2 )4 MgBr THF
H
H Ph Ph OH
172
171
169
H Ph Ph OH
H
N
H R 174: R=R'=H R' 175: R=R'=-(CH ) 2 4 OH
Scheme 77. Reagents and conditions: (a) Merrifield resin (1.1mequiv/g, 1%cross-linking), NaHCO3, THF. (b) PhMgX, THF. (c) 4-vinylbenzyl chloride, NaHCO3. (d) PhMgCl, THF. (e) A49/DVB/Toluene/1-dodecanol, AIBN.
In order to improve the catalytic activity of these suppported ligands, the nitrogen atom of these polymers was then methylated to afford the N-methylaled polymeric aminoalcohols 182-185. The catalytic activity of these heterogeneous ligands was evaluated and the results are summarized in Table 11. The analysis of the results showed that an increase in selectivity is accompanied by an increase in the enantioselectivity. Polymers 182 were poor catalysts and that is probably due to the non-presence of substituent in α position. The most surprising results are those obtained with polymers 183 bearing two phenyl groups in α position since this type of aminoalcohols give generally high enantioselectivity. The best results were obtained with immobilized aminoalcohols 184a and 185b which are derived from respectively valine and leucine. This last one gave a higher selectivity (91 vs 86 % for 184a) but a lower enantioselectivity (74 vs 80 % for 184a) (entries 9 and 14, Table 11). Supported chiral bispidine-derived aminoalcohols were screened in the addition of diethylzinc to benzaldehyde [133]. These immobilized ligands were obtained in three steps from compound 186 isolated from N-Boc-piperidin-4-one (Scheme 79). The hydroxyl function of 186 was first linked onto chloromethylated polystyrene to afford polymer 187. The carbamate was then removed under classical conditions and reaction of the resulting polymer with chiral epoxides led to chiral polymeric bispidine-derived aminoalcohols 188.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer… R
O
a, R=(CH3)2CH, R'=H b, R= (CH3)2CHCH2, R'=H H2N Cl R' c, R=Ph, R'=H d, R=PhCH2, R'=H e, R=R'=-(CH2)4-
OCH3 170
+ Cl NaHCO3 DMF
R
LiAlH4 THF
PhMgX THF
R
R OH
N
OCH3
N 171
Ph
OH
N
R'
Ph
R'
O
R' XMg(CH2)4MgX THF
R
R OH
N
BuMgX THF
R'
173a-d, R'=H 174a-d, R'=H 172a-d, R'=H 174e, R=R'=-(CH2)4172e, R=R'=-(CH2)4- 173e, R=R'=-(CH2)4172a-d, R'=H 176a-d, R'=Me CH3I 173a-d, R'=H 177a-d, R'=Me 174a-d, R'=H K2CO3 178a-d, R'=Me 175a-d, R'=H THF 179a-d, R'=Me
OH
N R'
175a-d, R'=H 175e, R=R'=-(CH2)4-
Scheme 78.
Table 11. Enantioselective diethylzinc addition to benzaldehyde using 182-185. Entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Polymer 182a 182b 182c 182d 183a 183b 183c 183d 184a 184b 184c 184d 185a 185b 185c 185d
Yield (%) 89 94 93 82 89 90 83 97 99 81 90 92 92 98 94 95
Selectivity (%) 55 65 56 48 63 70 58 70 86 53 68 70 73 91 86 85
ee (%) 10 15 7 2 15 24 11 24 80 22 23 20 34 74 17 45
107
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Christine Saluzzo and Stéphane Guillarme
Catalytic activity of both chiral polymers 188 was then evaluated in addition of diethylzinc to benzaldehyde. Although yield reached 95%, the enantioselectivity was poor (25% ee for 188a and 5% ee for 188b) contrary to the corresponding non-polymeric ligand homolog of 188a (96% ee).
1) TFA, CH 2Cl2
NaH,DMF
N Merrifield Boc resin
N
HO
4
N
O
4
N 2) n-BuLi, THF Boc
O
chiral epoxide
N R
187
186
N
4
R1 R2 188a: R=Ph, R 1 =OH, R 2 =H 188b: R=Et, R 1 =H, R 2 =OH
Scheme 79.
Pericas has reported the immobilization of (2R)-2-piperidino-1,1,2-triphenylethanol analog 190 onto several resins with different levels of crosslinking and functionalization [134]. These different chiral supported aminoalcohols 191 and 192 were prepared by attaching the aminoalcohol 190 via the benzylic hydroxyl group to different Merrifield resin (0.63-1.49 mmol Cl/g; 1 or 2 % DVB) and to two Barlos resin (1.24-1.6 mmol Cl /g) (Scheme 80). These functionalized resins were then tested in the enantioselective diethylzinc addition to benzaldehyde and the results are summarized in Table 12. The five polymer-supported aminoalcohols exhibited high enantioselectivities (>92 %) and except 192b, they presented a similar profile of catalytic activity. After six consecutive runs, the recovered 191c showed no loss of performance. This supported-ligand 191c was also used with other aldehydes including aromatic and aliphatic aldehydes and both conversion and enantioselectivities were excellent (>95%). O R
OH
O Ph
H
3 steps
Ph
Ph
189
CN
DIEA,C H 2Cl2
HO Ph
H N
190
Merrifield or Barlos resin
HO Ph Ph
H N
Merrifield resin 191a-c: R=
Barlos resin 192a-b: R=
Scheme 80.
Ph Cl
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
109
Table 12. Effect of functionnalized resins 191 and 192 on the enantioselectivity Entry
Functionalized resin (fb)
Starting resin
1 Merrifield (1% DVB; f0a = 0.63) 191a (0.375) 2 Merrifield (1% DVB; f0 = 1.49) 191b (0.850) 3 Merrifield (1% DVB; f0 = 0.63) 191c (0.389) 4 Barlos (f0 = 1.6) 192a (0.925) 5 Barlos (f0 = 1.24) 192b (0.689) a f0=mmol Cl/g. f=mmol/g (calculated by elemental analysis of nitrogen with the following f=0.714% N).
Ee 95 93 95 93 92 formula:
The asymmetric phenylation of different aldehydes was performed with chiral supported ligand 191 and the corresponding diarylmethanols were isolated in good to excellent yields (74-99%) and high enantioselectivities (85-91%) were observed but were slighly lower to those recorded with the homogeneous ligand [135]. Although the enantioselectivity obtained previously were among the best for supported ligands, the preparation of the aminoalcohol unit just before anchoring required up to five steps from commercial precursors. The supported-ligand 195 was prepared in only two steps from (S)-triphenylethylene oxide 193 (Scheme 81) [136]. The aminoalcohol 194 was anchored to a Merrifield resin (2% DVB, fo = 0.84) with an anchoring yield of 97 %. O Ph Ph
193
H Ph
HN
NH
LiClO 4
HO Ph Ph
H Ph N
N 194 H
Cs 2CO 3
HO Ph
Merrifield resin
Ph
H Ph N
195
N
Scheme 81.
The catalytic activity of the polymer-supported aminoalcohol 195 was then evaluated for the diethylzinc addition of aldehydes. After optimization of the reaction (temperature, amount of catalyst), two types of reaction conditions (2 mol% of 195, 0°C, 8h and 4 mol% of 195, 0°C, 6h) were tested with 15 different aldehydes. With the first reaction condition type, the conversion and the enantioselectivity were already high (≥ 95%) for most aromatic adehydes, the second one permit a complete conversion for all aromatic and aliphatic aldehydes and a better one for the α-substituted aldehydes. In order to evaluate the effect of the polymer part on the catalytic activity, two parallel kinetic measurements on the diethylzinc addition to benzaldehyde using either 195 or the Nbenzyl non-polymeric ligand were performed. The profiles for both ligands are pratically similar and at short reaction times, the polymer-supported ligand 195 showed a slightly better activity than its homogeneous analog (83% conversion vs 80% after 30 min). This supported ligand proved to be highly recyclable in five consecutive runs without any loss of catalytic activity (> 99% conversion) and enantioselectivity (average of 95.1 %).
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Christine Saluzzo and Stéphane Guillarme
The enantioselective phenyl transfer to different aldehydes was also carried out using the chiral polymer 195 with 5 mol % of catalyst amount and the enantioselectivities were also high (≥ 82 %) for aromatic aldehydes and moderate for aliphatic ones (68-79 %) [135]. Polymeric regioisomer of 191 and 195 was also synthesized and evaluated in the ethylation of aldehydes since the homogeneous analog was a highly active ligand [135]. Aminoalcohol 197 which was synthesised in six steps from epoxide 196 was anchored to Barlos resin with fo = 1.6 to lead to polymeric ligand 198 (f = 0.90) (Scheme 82). O Ph
H
Ph Ph
Six steps
Ph
DIEA,C H 2Cl2
OH H
Barlos resin
N
CN
196
Ph Ph N
OH
197
OH H
O
198
Ph Cl
Scheme 82.
The ethylation of aromatic and aliphatic aldehydes in toluene at 0°C in presence of an amount of 8 mol% of the supported-ligand 198 was performed. Except for α-susbituted aldehydes (around 50% conversion), both conversion and ee were high. The phenylation of aromatic aldehydes was also tested with polymeric ligand 198. Although conversions were excellent, the enantioselectivities were moderate (38-48% ee) and lower than those obtained with functionalized resin 191 and 195. In order to avoid the limitation of the use of the anchoring step as a source of diversity to prepare libraries of supported ligands containing the same type of aminoalcohol as 195, click chemistry was employed. This more convenient and efficient technique is a copper(I)catalyzed cycloaddition between an alkyne and an azide [137]. Aminoalcohol-derived alkynes 199 were prepared by reaction of 194 with different bromine reagent. The reaction between these alkynes and a variety of azido resins with different degree of functionalization (f = 0.74 – 2.25 mmol N3/g) was performed to afford the supported-ligands 200 (Scheme 83). Merrifield resin
Br 188
n
Cs2CO3
HO Ph Ph
HO Ph
H Ph N3
N
CH 3CN
N
CuI, DIPEA DMF/THF (1/1) 35°C
N 193a, n=1 193b, n=4 193c, n=8
Ph
H Ph
n
N 194a, n=1 194b, n=4 194c, n=8
n
N N N
Scheme 83.
The new functionalized resin 200a was first tested in the phenylation of p-tolualdehyde and p-anisaldehyde in toluene at 10°C using 5 mol% of the catalyst and the dependance of enantioselectivity on the level of functionalisation was studied (entries 1-6, Table 13). The
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
111
first analysis of the results was that the enantioselectivity depended strongly on the level of functionalisation of the resin. For the phenylation of p-tolualdehyde (entries 1-4), the higher the degree of functionalization, the lower the enantioselectivity. When a high ligand-loaded polymer was employed, the diarylcarbinols were isolated in their racemic form (entry 4). In this case, the triazole ring could interfere with the catalytic site involving a drop of the enantioselectivity. In order to avoid that, catalytic activity of resins 200b and 200c bearing a spacer were evaluated in the phenylation of p-tolualdehyde and p-anisaldehyde (entries 7-14, Table 13). It can be noticed that the two polymeric ligands 200a and 200b gave the best results in terms of conversion and ee. Resin 200b with a level of functionalization of 0.72 mmol/g exhibited the best catalytic activity (entries 9 and 13). This last one was chosen for phenylation of other aromatic and aliphatic aldehydes and the same range of conversion and enantioselectivity was obtained. The recyclability of this supported-ligand was studied on panisaldehyde and no decrease of the conversion was observed after three consecutive runs while a slightly drop in the enantioselectivity was recorded. The enantioselectivities were around 20% lower than those obtained with similar supported-ligand 195 with no triazole ring and for that reason the idea that the triazole ring could interact with zinc reagent is reinforced. Until now, supported diphenylprolinol were build either by copolymerisation with a vinylbenzyl group on nitrogen atom or a vinyl group on phenyl or by anchoring the ligand onto a Merrified resin via its nitrogen atom. Chen has reported a strategy to support this type of ligands using 4-hydroxydiphenylprolinol 201 [138]. Basic treatment of 201 with polymers 202 prepared in two steps from Merrifield resin (1% DVB, 100-200 mesh, 1 mmol Cl /g) led to supported-aminoalcohols 203a and 203b with a loading of aminoalcohol residue of 0.66 and 0.42 mmol/g respectively (Scheme 84). Table 13. Effect of the level of functionalisation of polymer 200 on activity and selectivity. Entry Polymeric ligand F (mmol L/g) Conversion (%) 1 200a 0.59 95 2 200a 0.74 >97 3a 200a 0.71 89 4 200a 1.16 >99 5 200a 0.59 64 6b 200a 0.71 92 7 200b 0.66 100 8c 200b 0.66 68 9 200b 0.72 95 10b 200b 0.72 99 11 200c 0.67 79 12 200b 0.66 68 13 200b 0.72 97 14 200c 0.67 54 a 3.6 % of catalyst. b10% of catalyst. cHigher crosslinked polymer (2% DVB).
ee (%) 74 68 63 0 77 71 66 66 76 78 75 77 82 76
These two immobilized ligands were tested in the diethylzinc addition to benzaldehyde and the best results were obtained when the reaction was performed in hexane at rt using an
112
Christine Saluzzo and Stéphane Guillarme
amount of 10 mol% of catalyst. Polymeric ligand 203a showed a slightly better activity than 203b (91 % yield and 63% ee vs 85% yield and 57% ee). The optimized reaction conditions in the presence of 203a as ligand were used with different aldehydes. Except for salicylic aldehyde, p-nitrobenzaldehyde and cyclohexanecarboxaldehyde, the same range of yield and ee were observed. Five consecutive diethylzinc addition to benzaldehyde reactions using the same batch of polymer 203a were carried out without loss of the catalytic activity for the three first ones while a slight decrease of the catalytic was detected for the fourth and fifth run. The replacement of the ether bond between the 4-hydroxyprolinol and the spacer by an ester link has been performed [139]. Wang has prepared the polystyrene-supported diphenyl prolinol 205 and the soluble polymer-supported analog 206 in two steps from aminoalcohol 201 (Scheme 84). The loading of aminoalcohol was evaluated to be at 0.76 mmol/g for supported ligand 205 and 0.42 mmol/g for the soluble one. With polystyrene-supported ligand 205 for the enantioselective diethylzinc addition to aldehyde, the best result (92 % yield, 68% ee) was obtained when the reaction was carried out in toluene at 25°C using 15 mol% of the catalyst. Both yield and ee were similar to those obtained with the ether-linked polymeric ligand 203 but the reaction time was shorter. The optimized conditions were also used with other aromatic aldehydes and the enantioselectivities were moderate to good (34-71%). Soluble polymer-supported ligand 206 was also tested and for all the aldehydes used both yield and enantioselectivity were higher than those using the polystyrene-supported 205. This latter could be reused at least 3 times without significant loss of performance contrary to its soluble analog. The first immobilization of a pyridine-based tridentate ligand has also been investigated since the tridentate can stabilize the zinc complex and reduce the effect of the polymer blackbone [140]. The aminoalcohol 207 was prepared in six steps from 2,5-dibromopyridine and was anchored onto a 2% DVB crosslinked Merrifield resin to afford the supportedtridentate ligand 208 (Scheme 85). This immobilized ligand was then employed in the diethylzinc addition to benzaldehyde and the excellent catalytic activity was recorded when the reaction was carried out in toluene at rt using 5 mol% of catalyst and a low benzaldehyde/Et2Zn ratio of 1/1.1 (>99% conversion and 93% ee). The best reaction conditions were used with other aromatic aldehydes and yields and ees were similar to those observed with benzaldehyde except for pyridine carboxaldehyde. In this case, nitrogen atom of pyridine ring was suspected to be too chelating. Excellent enantioselectivity (93%) was also obtained with an enolizable aldehyde, n-octanal. Furthermore the catalyst could be recovered by simple filtration and could be reused five times without loss of catalytic activity. The results obtained are interesting since the supported-ligands containing a NH group gave generally poor to moderate enantioselectivities. El-Shehawy studied the effect of the loading of the ephedrine moiety and the degree of crosslinking of polymer-supported ephedrine for the diethylzinc addition to benzaldimine [141]. Different chiral polymers were prepared by copolymerisation of monomers 141 with styrene and DVB. The compositions of these polymers are summarized in Table 14. These chiral polymers 209-213 were then tested in the asymmetric addition of diethyzinc to benzaldimines. First, the catalytic activity of the copolymers 209, 210a, 211 and 213 having a molar ratio of copolymerization of 1/7/2 were evaluated and the best result was obtained with polymer 210a (92% yield and 82% ee) when the reaction was carried out in
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
113
toluene at – 10 °C. The enantioselectivity of the reaction with polymeric ligand 210a was highly temperature-dependant and surprisingly a significant decrease of the chemical yield when the reaction was performed at 0°C or rt was observed.
O
O n
N OH
203a: n=4 203b: n=6 CH2O(CH2)nCH2Cl
NaH DMF
202a: n=4 202b: n=6 HO N OH
201 Succinic anhydride DMAP
O
HO2C O
N OH
204 NH2 MeOPEG, DMAP, DCC
DIC HOBt
O N H
O O O
O
O 2
n
O
O
N
N OH
205
Scheme 84.
OH
206
114
Christine Saluzzo and Stéphane Guillarme O
HO KI, DMF
N
207
HN
R
HO
Ph Ph
N
Merrifield resin
HN
R
HO
Ph Ph
208
Scheme 85.
Table 14. Compostions of polymers 209-213. Chiral Polymer 209 210a 210b 210c 210d 210e 210f 210g 210h 211 212 213 a
Chiral Monomer
Molar Ratioa
(1S,2R)-141a (1S,2R)-141b (1S,2R)-141b (1S,2R)-141b (1S,2R)-141b (1S,2R)-141b (1S,2R)- 141b (1S,2R)-141b (1S,2R)-141b (1S,2R)-141c (1S,2R)-141d (1R,2S)-141b
1:7:2 1:7:2 1:8:1 1:6:3 1:5:4 2:6:2 3:5:2 4:4:2 5:3:2 1:7:2 1:7:2 1:7:2
Loading of ligand (mmol/g) (DF)b 0.764 (0.10) 0.771 (0.10) 0.750 (0.10) 0.821 (0.10) 0.735 (0.10) 1.292 (0.20) 1.863 (0.30) 2.127 (0.40) 2.449 (0.50) 0.792 (0.10) 0.721 (0.10) 0.807 (0.10)
Molar ratio of copolymerization: chiral monomer:styrene:DVB. b Degree of functionalization
The increase of the crosslinking reagent (DVB) involved a significant decrease of both yield and ee. The same effect was observed when the loading degree of the aminoalcohol unit increased. The optimized procedure with chiral polymer 210a was employed with other aromatic aldimines and the same range of enantioselectivity was recorded. El-Shehawy has reported the synthesis of original polystyrene-supported dendritic chiral ephedrine [142]. Chain end-functionalized polystyrenes bearing a number of 2, 4, 8 or 16 BnBr residues at their chain-ends, PS(BnBr)2-PS(BnBr)16 were first isolated (Scheme 86). Reaction of polymers of PS(BnBr)2-PS(BnBr)16 having two-sixteen BnBr moieties with ephedrine led to the corresponding polymers PS(Ephed)2-PS(Ephed)16 bearing the same numbers of ephedrine at their chain-ends (Scheme 87). The catalytic activity of these four chiral polymer-supported dendritic aminoalcohols was evaluated in the enantioselective diethylzinc addition to N-diphenylphosphinoylbenzaldimine. The reaction was first carried out in toluene at rt for 48 h using an equimolar amount of supported-ligand (based on the total numbers of ligand unit). The best chiral polymer, PS(Ephed)8a, was also tested with other N-diphenylphosphinoylarylimine under the same reaction conditions and similar level of yield and enantioselectivity were observed.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
115
Br Br Br n
n
Br PS(BnBr)2
Br Br
PS(BnBr)4
Scheme 86.
The same trend as with imine was observed for the enantioselective diethylzinc addition to benzaldehyde and the best results (94% yield and 90% ee) were also obtained with PS(Ephed)8 [143].
Br
Me
Ph
OH
N Me
* * HN OH Me
PS
Me
*
*
Ph
PS
K 2 CO 3/DMF
Br
N Me *
Me
*
Ph
OH
* * Me
N
OH
Ph
Me
PS
(1R,2S)-PS(Ephed)2
PS
PS
PS
(1R,2S)-PS(Ephed)4 (1R,2S)-PS(Ephed)8a (1S,2R)-PS(Ephed)8b
Scheme 87.
(1R,2S)-PS(Ephed)16
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Christine Saluzzo and Stéphane Guillarme
Table 15. Enantioselective diethylzinc addition to N-diphenylphosphinoylbenzaldimine using polymer-supported dendritic aminoalcohols. Entry
Supported-ligand
Reaction Yield (%) ee (%) time (h) 1 PS(Ephed)2 48 53 81 (R) 2 PS(Ephed)2 90 85 83 (R) 3 PS(Ephed)4 48 71 84 (R) 4 PS(Ephed)4 72 90 85 (R) a,b 5 PS(Ephed)8a 48 92 90a, b (R) 6 PS(Ephed)16 48 78 79 (R) c 7 PS(Ephed)16 48 93 80 (R) 8 PS(Ephed)8b 48 91 90 (S) a 91% yield and 92% ee when using 1.0 molar of (1R,2S)-N-benzylephedrine as chiral ligand. b74% yield and 89% ee when using 1.0 molar of (1R,2S)-N-vinylbenzylephedrine copolymerized with styrene and DVB as chiral ligand. c1.5 molar equivalent of chiral polymer.
Although high catalytic activity was observed with polymer-supported aziridine-derived ligand 160, the attachment on this type of chiral ligand through the phenyl group was studied [144]. The protected monomers 214 were prepared and copolymerized with styrene and DVB to afford the chiral aminoalcohol-containing copolymers 215 with a loading of 0.1 mmol/g (Scheme 88). The three polymeric ligands were then examined in the diethylzinc addition to benzaldehyde. Protected polymer-supported ligand 215a and 215b were slighty less active than the Zwannenburg’s polymeric ligand 160. The trityl-protected supported-ligand 215a was also recycled and showed a complete loss of catalytic activity (3%) and enantioselectivity (0%). It is important to pointed out the high enantioselectivity (84% ee) obtained with the unprotected polymer-supported ligand 215c while the conversion was moderate (52 %) after 24h of reaction. Polymer-supported chiral Schiff-base amino alcohols have also been used as chiral ligands. The four polymeric Schiff-base ligands 216a, 216b, 217a and 217b have been anchored onto Merrifield resin (1 mmol Cl/g, 1% DVB, 200 mesh) using the corresponding chiral ligands (Scheme 89) [145]. These chiral polymers have been first tested as chiral ligand in the phenylacetylene addition to acetophenone and chiral polymer 217a induced the best enantioselectivity (63%) when the reaction was performed in toluene at rt using 10 mol% of the catalyst. When the reaction temperature decreased to – 18 °C, a significant increase of the enantioselectivity (75%) was observed. The optimized reaction conditions with chiral supported-ligand 217a were then applied to different functionalized acetophenone and enantioselectivities were comprised between 62 and 89%. The polymeric ligand 217a could be reused in five successive reactions with an average loss of enatioselectivity of 1% per run.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
117
Br
Me
Ph
OH
N Me
* * HN OH Me
PS
Me
*
*
Ph
PS
K 2CO 3/DMF
Br
N Me *
Me
*
Ph
OH * *
Me
N
OH
Ph
Me
PS
(1R,2S)-PS(Ephed)2
PS
PS
PS
(1R,2S)-PS(Ephed)4 (1R,2S)-PS(Ephed)8a (1S,2R)-PS(Ephed)8b
(1R,2S)-PS(Ephed)16
Scheme 87.
R N HO
214a: R=CPh 3 214b: R=CHPh 2 Scheme 88.
styrene, DVB Benzoyl peroxide Polyvinylalcohol H 2O, PhCl
R N HO
215a: R=CPh3 215b: R=CHPh 2 215c: R=H
118
Christine Saluzzo and Stéphane Guillarme Ph H N
Ph Ph
Ph H
Ph Ph
N
OH
OH
216a NaH, DMF
HO
O Ph H N
Ph Ph
Cl THF
OH
Ph H
Ph
N
OH
Ph
216b HO
O
HO O
Ph Ph
H N
Ph Ph
H
OH
N
OH 217a
NaH, DMF
Cl THF
HO H N
Ph Ph OH
O H
Ph Ph
N OH 217b
Scheme 89.
6.2. Supported Sulfonamide and N-Sulfonylated Amino Alcohols Itsuno has described the first enantioselective allylation of aldehydes using crosslinked polymer-supported N-sulfonylated aminoalcohols as chiral ligands [146]. Polymer-supported ligands 220 and 221 were prepared by copolymerisation of the norephedrine-derived monomer 218 or D-camphor-derived 219 with styrene and divinylbenzene (DVB) in a [chiral monomer]/[styrene]/[DVB] molar ratio of 1/8/1 (Scheme 90).
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer… Ph
Me * * O HO HN S O 218a: (1R,2S) 218b: (1S,2R) O * NH S O * OH 219a: (1R,2S,3R,4S) 219b: (1S,2R,3S,4S)
119
Ph
Styrene/DVB
Me * * O HO HN S O 220a: (1R,2S) 220b: (1S,2R) O * NH S O * OH 221a: (1R,2S,3R,4S) 221b: (1S,2R,3S,4S)
Scheme 90.
Reaction of these chiral polymer-supported ligands with triallyborane led to the corresponding allyboron reagents which were allowed to react with aldehydes. When the norephedrine-derived ligands 220 were employed in the asymmetric allylation of benzaldehyde, homoallylic alcohol (S) was isolated in a 93% yield and in 75% ee. The enantioselectivity was slightly higher with the camphor-derived analog 221a (85% ee). The polymer-supported ligands induced better enantioselectivities than the non-polymeric analogs. High enantioselectivities were also observed in the allylation of aliphatic aldehydes (92% for acetaldehyde and 84% for pivalaldehyde). Liskamp has reported the synthesis and the screening in asymmetric catalysis of a library of polymer-supported peptidosulfonamide [147]. (R,R) or (S,S)-pyrrolydines 222 prepared in six steps from D or L-tartaric acid were anchored onto Argonaut resin (0.41 mmol/g) to afford the corresponding supported pyrrolidines 223 (Scheme 91). The polymeric peptidosulfonamides 224 and 225 were then synthesized in four steps from the immobilized pyrrolidines 223. The different chiral polymers were tested in the titanium-mediated diethylzinc addition to benzaldehyde, p-chlorobenzaldehyde, cyclohexanecarboxaldehyde and phenylacetaldehyde. Both yield and ee were very low with all these supported-ligands for the enantioselective reaction with the two aliphatic aldehydes. With aromatic aldehydes, the best results were observed with both leucine-derived supported peptidosulfonamides 224d and 225d. Isoborneolsulfonamide-containing trans 1,2-diaminocyclohexane have been immobilized on solid support [148]. The four polymer-supported ligands 227 were synthesized by suspension copolymerization in polyvinyl alcohol (average Mw=85,000-146,000) of chiral monomer 226 with styrene and DVB (Scheme 92). These four chiral polymers 227 were then employed as chiral ligands in the enantioselective titanium-mediated diethylzinc addition to acetophenone. Although enantioselectivities were excellent (99%) with these four polymersupported ligands, the chemical yields were low (16 (227a), 23 (227b) and 36% (227d)) to moderate (56% (227c) even after a very long reaction time (17 days). A more reactive zinc reagent, the ethyl phenyl zinc reagent prepared by transmetallation of diethylzinc with triphenylboron was the employed. This reagent was used with pbromoacetophenone in the presence of 5 mol% of polymeric ligand 227 and an excess of titanium tetraisopropoxide and the corresponding tertiary alcohol was isolated with good enantioselectivities (71-86%). The amount of the crosslinking agent had a strong influence on
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Christine Saluzzo and Stéphane Guillarme
the catalytic activity and the enantioselectivity and the best results were obtained with the soluble polymer-supported ligand 227d. O HO
O 2
NHBoc
O
NH2
N
NHBoc (R,R) or (S,S)-216
O
NHBoc
N N 2 H NHBoc (R,R) or (S,S)-217
BOP DIPEA
1) TFA, Et3N
R 2) ClO2S
a, R=H b, R=Me i c, R= Pr d, R=iBu
NMM, CH2Cl2 3) Piperidine, DMF 4) Boc2O, NMM, CH2Cl2
e, R=Bn O N H
R O O S NHBoc NH
O 2
NHFmoc
N NHBoc
HN
S O O
R
218a-e (R,R) 219a-e (S,S) Scheme 91.
This ligand was employed in the enantioselective phenyl addition to different ketones, and the same level of chemical yield and enantioselectivity was observed for aromatic ketones while the enantioselectivity dropped significantly with hexan-2-one (38% ee). The reuse of the polymer-supported ligand 227d was studied and the catalytic activity decreased rapidly after only two runs. Gau has prepared three different types of polymer-supported N-sulfonylated aminoalcohols. The first one 230 was produced by anchoring the aminoalcohol 229 through the nitrogen atom to a chlorosulfonated resin (1 mmol Cl/g; 2% DVB) with a ligand loading of 2.30 mmol/g (Scheme 93) [149]. In order to decrease the ligand unit loading, the authors have also synthesized chiral monomer 231 which was allowed to copolymerize with DVB and styrene to lead to polymeric ligands 232a and 232b with a ligand loading of 1.28 mmol 1.76 mmol, respectively. It was not surprising to observe moderate yield (62%) and enantioselectivity (44%) for the titanium-mediated diethylzinc addition to benzaldehyde with the polymeric ligand 230 since a high ligand-loaded polymer is generally less active because of the proximity of the catalytic sites. The best result (100% yield and 92% ee) was obtained when 10 mol% of the low ligand-loaded 232a in the presence of 10 equivalents of titanium tetraisopropoxide
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
121
(relative to the ligand). This polymer-supported ligand was also employed with different aldehydes and both yield an ee were high in all cases. This latter could be also reused five times and the enantioselectivity decrease from 92 to 86%.
Styrene + DVB + O 2 S NH HN S O2 226 AIBN Polyvinylalcohol H 2O, THF, PhH
n
99
1
Ph
n
O2S 227a: n=32 227b: n=8 227c: n=1 227d: n=0
N H
HN
S O2
Scheme 92.
In the aim to discover other polymer-supported catalysts having a better activity and reusability, chiral polymers 234a and 234b were prepared by copolymerization with styrene and DVB with respectively a ligand loading of 0.93 mmol/g and 0.95 mmol/g [150]. The heterogeneous 234a/Ti(OiPr)4 system proved to be better than the 232/Ti(OiPr)4 systems for the enantioselective diethylzinc to aldehydes and a highly recoverable catalyst. Indeed, this polymeric ligand/Ti(OiPr)4 catalyst could be reused 9 times with enantioselectivities ≥ 87% ee. The polymer-supported tridentate ligand 234b was employed in the enantioselective triethylaluminium addition to aldehydes. To obtain the same level of enantioselectivity as precedent system, 28 equivalents of Ti(OiPr)4 relative to the chiral ligand was necessary. With most aldehydes used, the enantioselectivities were slightly lower than those obtained with the heterogeneous 234a/Ti(OiPr)4 system.
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Christine Saluzzo and Stéphane Guillarme Ph
Ph 0.98
H2N
0.02
229
OH
0.98
0.02
Et3N, CH2Cl2
SO2Cl
O2S HO
228
NH 230
Ph
Ph x
SO2 Ph HN
+ styrene + DVB
0.02
AIBN
H2O/Benzene
O2 S HO
HO
y
Ph
Ph
231
NH Ph 232a: x=0.20, y=0.78 232b: x=0.40, y=0.58
O 0.2
0.78
0.02
AIBN
+ styrene + DVB R O2S
H2O/Benzene
H N
O HO
Ph
233a: R=
R O2S
OH
H N
234a: R= OH
Cl
233b: R=
HO Cl
Ph
Cl
234b: R= Cl
Scheme 93.
6.3. Supported Oxazoline or Oxazolidine as Ligands Two polymer-supported chiral aminooxazoline 236 have been prepared because of better chelating properties of this type of ligand due to the higher nucleophilicity of the amino group [151]. Polymers 236 were produced after reaction of polymer-supported isatoic anhydride 235 (isolated from chloromethylated styrene-DVB polymer (4.8 eq. Cl/g)) and the corresponding amino alcohol under acidic conditions (Scheme 94).
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
N
O HO O
+
NH 2 R
O
123
NH Acidic clay
N
PhCl
R
O 236a, R=Bn 236b, R=i-Bu
Scheme 94.
The catalytic activity of these polymeric oxazolines was examined in the enantioselective diethylzinc addition to benzaldehyde. Chiral supported-oxazoline 236b exhibited slightly better enantioselectivity (89%) and yield (90%) than its analog 236a when the reaction was carried out in a 20% CH2Cl2/toluene mixture at 0°C. This chiral supported-ligand has been recycled three times without a significant loss of performance. O
Ph Cl Ph
Fe
Bu N CPh2 OH O 238
O
Fe
Bu N CPh 2OH O
6
O HO
Ph
O
OH
237
Ph
O 6
Fe
O 2
Bu N CPh2 OH O O
O-MPEG
6
O
O 2
O-MPEG
239
Scheme 95.
Chiral ferrocenyl oxazoline-based ligand which is often used in asymmetric catalysis have been immobilized onto two different supports, an insoluble trityl chloride polystyrene and a soluble polymer, a polyethylene glycol monomethyl ether [152]. The linker-containing compound 237 has been anchored to both polymers to produce respectively chiral insoluble polymer 238 and chiral soluble polymer 239 (Scheme 95). These polymer-supported ligands were then tested in the enantioselective phenyl and ethyl addition to p-chlorobenzaldehyde and benzaldehyde respectively. Polystyrene-supported oxazoline 238 was totally inefficient for the asymmetric phenyl transfer to p-chlorobenzaldehyde while a high enantioselectivity (87%) was observed for the ethyl addition to benzaldehyde. Contrary to the insoluble one, the soluble polymer 239 exhibited high enantioselectivity for both reactions (97 and 86% respectively). In general, the enantioselectivities were slightly lower than those obtained with the non-polymeric ligand. Futhermore, no significant loss of enantioselectivity was detected after five consecutive phenyl additions to p-chlorobenzaldehyde with the recovered polymer.
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Christine Saluzzo and Stéphane Guillarme
Portnoy focused his attention on the immobilization of PyBox ligands [153]. The polymer-supported PyBox 240 were prepared in five steps from the Wang trichloroacetamidate resin (Scheme 96). The supported-ligands 240 were allowed to react with copper (I) triflate for 24h to afford the corresponding catalysts 241. These catalysts were then used in the first heterogeneous catalyzed addition of phenylacetylene to imine. The best enantioselectivity (83%) was obtained when the reaction was performed in dichloromethane at 40°C for 24h using 10 mol% of the catalyst 241f which possesses a steric group on the oxazoline ring. When a recovered catalyst was employed for a second run, a drop of the catalytic was observed which is probably due to the oxidation of the metal. The use of other solvents such as THF in order to improve the recyclability was attempted but a decrease of the catalytic activity (80 to 56%) was detected during three consecutive reactions while the enantioselectivity increased (54 to 60%). To reduce the oxidation, ascorbic acid was added to the reaction mixture and proved to preserve the catalytic activity but the enantioselectivity was totally lost. Another strategy to immobilize a PyBox ligand has been described by Moberg and Levacher [154]. Supported PyBox 243 has been prepared using click-chemistry reaction between alkyne 241 and polystyrene-supported azide (1.2 mmol N3/g) (Scheme 97). The PyBox loading was estimated at 0.8 mmol/g. This polymer-supported PyBox/Cu(I) catalyst was then tested in the phenylacetylene to N-benzylideneaniline. The enantioselectivity was lower than that observed with the non-polymeric but slightly better than those with Portnoy’s immobilized PyBox 240. Compared to catalyst 241 similar trend was observed concerning the recyclability. When the immobilized PyBox-Cu(I) catalyst was only filtered, a drop of both catalytic activity and enantioselectivity were detected after five consecutive runs. When the supported-ligand was washed several times and reloaded with copper triflate, catalytic activity and enantioselectivity were recovered. The same level of conversion and ee were observed with other aromatic imines. The use of polymer-supported oxazolidine derived from (1R,2S)-cis-1-amino-2-indanol for the asymmetric alkynylation of aldehyde has been reported [155]. Three chiral supportedoxazolidines 244 were prepared in two steps from Merrifield resin (2.5 mmol Cl/g) (Scheme 98). The optimization of the enantioselective addition of phenylacetylene to benzaldehyde has been done with the polymeric ligand 244a. The best result (82% yield and 79% ee) was obtained when the reaction was performed in THF at room temperature for 24 h in the presence of Et2Zn, Ti(OiPr)4 and 10 mol% of the ligand. The two other polymeric ligands 244b and 244c induced higher enantioselectivity (83% and 90% respectively) under the optimized reaction conditions. The same level of catalytic activity and enantioselectivity was observed with a variety of aromatic and heteroaromatic aldehydes when the polymer-supported oxazolidine 244c was employed. This supported ligand could be reused three times without significant loss of performance.
6.4. Supported Salen as Chiral Ligands The catalytic activity of polymer-supported chiral salen has been also studied in the enantioselective diethylzinc addition to aldehydes since the importance of this type of ligand in asymmetric catalysis. Soluble polymer-supported salens have been synthesized by Venkataraman
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
125
[156]. Salen 245 was prepared and was then anchored on MeO-PEG changing only the spacer between the PEG matrix and the salen to lead to the polymer-supported salens 246-248 (Scheme 99). R
O N O
CCl3
Five steps
O
NH Wang resin
O
N
240a: R=Me 240b: R=Et O 240c: R=i-Bu 240d: R=Bn 240e: R=i-Pr 240f: R=t-Bu 240g: R=Ph
N R
CuOTf
R
O N O
N Cu OTf
O
N O
241
R
Scheme 96.
N N N N3 O N Ph
O
O
N
N
N 242
Ph
Ph
O
N
CuI/DIEA/THF
N 243
Ph
Scheme 97.
The enantioselective diethylzinc addition to benzaldehyde was then carried out using 10 mol% of polymeric salens 246-248 and the best result (90% yield and 82% ee) was obtained when the chiral polymer was used. With other functionalized aromatic aldehydes, both yield and ee were lower. Futhermore the soluble polymers could be recovered and reused whithout significant loss in selectivity or reactivity. Chiral salen-type linear polymers have been prepared and tested in the diethylzinc addition to aldehydes [157]. The symetrical dialdehyde-containing compound 249 was first synthesized in five steps from 2-tertbutylphenol and allowed to react with 1,2-diaminocyclohexane and DPEN to afford the linear polymers 250 (Scheme 100). The catalytic activity of these chiral
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Christine Saluzzo and Stéphane Guillarme
linear polymers was evaluated in the diethylzinc addition to m-nitrobenzaldehyde using 1 mol% of 250 (with respect to the monomeric unit). Although yields were satisfactory the ee were moderate and chiral linear polymer 250b proved to be more efficient for the reaction. The recovered polymer has been reused without loss of performance.
O 244a
H N O
H N 2 steps
O
244b O
CH2Cl
O H N
244c O
Scheme 98.
7. ASYMMETRIC π-ALLYLIC SUBSTITUTION Another largely studied asymmetric reaction to form new carbon-carbon bond is the palladium-catalyzed allylic substitution using particularly homogenous ligand (Scheme 101) [158]. Among the ligands employed, numerous phosphine-containing ones have been reported and we report here only the phosphorus-free nitrogen-containing ligands. The use of heterogeneous ligands has not been studied extensively and the use of some immobilized nitrogen-containing ligands has been published. To the best of our knowledge, the first polymeric ligand for this reaction was those of Lemaire’s group [20]. Their chiral poly(amide) 23 and poly(urea) 24 (Scheme 13) was employed for the catalytic allylic alkylation. Using 23 the conversion was low (38%) and the ee reached 80% while with 24, the conversion was better (72%) and enantioselectivity was lower (38%). In both cases, conversion and ee were lower than those obtained with the corresponding monomeric diamine. Moberg focused her attention on the immobilzation of oxazoline-derived ligands for the enantioselective allylic alkylation [159]. Pyridine-containing oxazolines 251 were first prepared and were supported on TentaGel resin to afford the corresponding polymeric oxazolines 252 (Scheme 102). These polymer-supported ligands were then tested in the
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
127
palladium-catalyzed allylic substitution and the sterically bulky ones 252b and 252c proved not to be active (< 5% yield).
N O
1) glutaric anhydride DMAP, CH2Cl2 2) MeO-PEG, DCC, DMAP, CH2Cl2
N
O O
O
R
R
R
O
N
OH HO
R
Cs2CO3
R
DMF
245 R R=t-Bu O
R
246
N
N
OH HO R
OH HO
3
PEG-OMs
HO
N
247
R
Cs2CO3 DMF
OMs
N
O
N
3
O
OH HO
R
3
R
248
R
Scheme 99.
When the chiral polymer 252a was used as chiral ligand, the product was isolated in a yield comprised between 60 and 100% and in 80% ee; this result was closed to that obtained with the monomeric ligand. Polymer-supported bis(oxazoline) 109 having a degree of functionalization of 0.071 mmol/g have been tested in the same reaction [102]. This polymeric ligand exhibited better enantioselectivity (94-95%) than the previous chiral polymers 252. The asymmetric π-allylic alkylation using a polymer-supported pyridyldiamide as chiral ligand was applied to the synthesis of the (R)-baclofen which is an agonist of the GABAB receptor [160]. The unsymmetrical cyclohexanediamine 253 was anchored onto a TentaGel HL-COOH resin using a classical coupling reaction to give the polymer-supported ligand 254 (Scheme 103). This one was then tested in the molybdenum-catalyzed allylation reaction of dimethylmalonate with 3-phenylprop-2-enyl methyl carbonate. A high branched-to-linear ratio (35/1) and enantioselectivity were obtained with the polymeric ligand and this result was quite similar to those observed with the non-supported ligands. The ligand could be recovered by filtration and could be reused for seven consecutive cycles without loss of catalytic activity and enantioselectivity. Bandini has described the grafting of diamino-oligothiophene compounds onto the monomethyl ether of PEG5000 to give the soluble polymer 256 (Scheme 104) [161]. High yield (98%) and ee (99%) were observed with soluble polymer 256a when the allylic alkylation reaction between 1,3-diphenylallyl and dimethyl malonate was performed in THF
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Christine Saluzzo and Stéphane Guillarme
at room temperature using cesium carbonate as the base. This result is comparable to that obtained with the non-polymeric ligand. Polymer-supported ligand 256b proved to be less efficient than 256a. The recyclability of the supported-ligand/Pd catalyst was also evaluated and loss of performance was detected only after three cycles.
OC 8H 17
N
N
OC 8H 17
N
OH HO
N
OH HO n
C 8H 17O
t-Bu
t-Bu
C 8H 17O
t-Bu
t-Bu
250a
CHCl3
H2N OHC
NH2
OC 8H 17
CHO
HO
OH
t-Bu
C 8H 17 O
t-Bu 249
CHCl3
Ph
Ph
N
N
OC8H 17
Ph
Ph
H2N
NH 2 Ph
Ph
N
N
OC 8H 17
OH HO
OH HO n
C 8H 17 O
t-Bu
t-Bu
C 8H 17 O
t-Bu
t-Bu
250b
Scheme 100.
LG R
R''
R' acyclic or cyclic LG: Leaving group Scheme 101.
Pd/L * NuH, Base Solvent
Nu R R'
*
R''
R and/or R'
R'' * Nu
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
129
O O
R' R
N
+ HO2C
O
TG
OH
N
Ph
251a: R=R'=H 251b: R=t-Bu, R'=H 251c: R=H, R'=t-Bu Scheme 102.
CO2H
O NH HN
N
N
O HN
NH HN
O DCC, DMAP
NH
TG
252a: R=R'=H O 252b: R=t-Bu, R'=H 252c: R=H, R'= t-Bu
O
NH2 N
O
O
N
Ph
O
R' R
N
N
NH
254
253 Scheme 103. R R
R S
S
R MsO
S
3
NH
O
NH
Cs2CO3 / DMF
S
S
PEG-OMe
NH NH S
OH 255a: R=H 255b: R-=O-CH2-CH2-O-
O 256a: R=H 256b: R-=O-CH2-CH2-O-
3
PEG O
Scheme 104.
8. DIHYDROXYLATION AND AMINOHYDROXYLATION The osmium-catalyzed asymmetric dihydroxylation (AD) is an excellent method to prepare chiral vicinal diols [162]. Most of the ligands employed are cinchona alkaloids and are derived from quinine or quinidine like ligand 257 for example. In 1992, Sharpless has described the improvement of the osmium-catalyzed asymmetric dihydroxylation by using bis cinchona alkaloids such as 1,4-bis(9-0-dihydroquinyl)phthalazine (258) as chiral ligand (Scheme 105) [163]. This type of ligand has then received much attention since high enantioselectivities were observed. Although the amount of OsO4 has been reduced with the possibility to use cooxidants, the high cost of osmium and chiral ligands, the toxicity, the possible contamination of the chiral products by osmium avoid the use of this process in industry. The possibility to support and to recycle the osmium/ligand catalyst and particularly
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Christine Saluzzo and Stéphane Guillarme
the use of soluble or insoluble supported ligands has been explored. In order to recycle the catalyst, number of research groups has investigated the immobilization on soluble and insoluble polymers of this type of alkaloids.
O H
N MeO
N O H
N N O
H
N O
MeO N 257
OMe N
N 258
Scheme 105.
8.1. Insoluble Supported Alkaloid as Ligand The first work about AD using heterogeneous ligand has been reported by Sharpless [164]. The polymeric alkaloids 259-262 (Scheme 106) were prepared by copolymerisation of the corresponding monomer with acrylonitrile in presence of the radical initiator AIBN. The performance of these polymer-supported cinchona were evaluated in the asymmetric dihydroxylation of trans stilbene using OsO4 and NMO or K3Fe(CN)6 as secondary oxidant. The reaction rate was slow and no ee was detected using the polymeric ligand 259 probably because of the proximity of the active site to the polymer matrix. The best results (81-87% yield and 85-93% ee) were observed when the supported alkaloid 260 was used as chiral ligand in presence of only 1 mol% of OsO4 and NMO in acetone/water at 10°C. It is interesting to note that when the OsO4/supported ligand complex was reused a second time without further addition of OsO4, the yield and enantioselectivity dropped slightly. Both yield and ee were improved when K3Fe(CN)6 was used as cooxidant with polymers 261 and 262. When the same reaction was tested with different olefins using the acetate analog of polymer 259, the polymer-supported alkaloid 263, low enantioselectivities were observed (Scheme 107) [165]. High chemical yields could be obtained when low alkaloid-loaded copolymers were used (< 15 mol%). Moreover high degree of alkaloid incorporation seemed to inhibit the reaction. Use of chiral polymers 264 and 265 did not improve the enantioselectivity of the dihydroxylation of trans stilbenes and ee were very low with polymer 264 compared to those obained with polymers 259-260. Dihydroquinidine 266 containing a p-vinylbenzoate was prepared and was copolymerized either with styrene or with 4-phenylstyrene (Scheme 108) [166]. The effect of the loading of the alkaloid monomer on the activity of the corresponding polymer for the AD of trans stilbenes was examined and the results are summarized in Table 16. As already observed with different supported ligands, higher the loading of the alkaloid monomer, more important drop of chemical yield, reaction rate and enantioselectivity were. Polymer 267a proved to be more efficient than its homolog 267b and the complex 267a/OsO4 could also be reused without
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
131
significant loss of performance. This system was employed for the asymmetric dihydroxylation of other olefins including non-aromatic and α,β-unsturated alkenes and moderate enantioselectivities were obtained while chemical yields were high. In order to improve the enantioselectivity obtained with their supported alkaloid for the AD, Salvadori has prepared polymer 269 which contained a spacer between the alkaloid and the polymer backbone [167]. This one was isolated after copolymerization of quinine-based monomer 268 with styrene and DVB in a 1/7/2 molar ratio (Scheme 109). This polymersupported quinine was then tested in the AD of trans stilbene, styrene and (E)-βmethylstyrene. The enantioselectivities were higher than those obtained with polymers 263264 with a shorter reaction time at rt (4-6h) or 0°C (7h). At 0°C, the enantioselectivity reached 87% for the dihydroxylation of trans stilbene. This polymer was also reused after recovering without significant loss of the activity. CN
CN O
S
O N
H
O
O
Cl O H
O
MeO N N CN
259
O
H
Cl O H
MeO
O S O
N
260
O O
O N
H
N
NMe2 O H
H
O H
MeO
MeO 261
N N
Scheme 106.
CN N
H
OCOR H
MeO N 263: R=CH3 264: R=p-Cl-C6H4 265: R=m-(CH3O)-C6H3
Scheme 107.
262
CN 6
132
Christine Saluzzo and Stéphane Guillarme Table 16. AD of trans stilbene using polymer 267. Entry
Polymer 267a (267b) Reaction Yield (%) ee (%) n m Time 1 9.0 1.0 24 h 86 (68)a 82 (71)a b 2 9.0 1.0 24 h 79 78b 3 8.0 2.0 24 h 72 (65) 78 (68) 4 5.0 5.0 2d 50 (50) 45 (40) 5 0.0 1.0 2-3 d 20 a Under brackets, yields and ee obtained with 267b. b Polymer containing dihydroquinine was used.
m
n
O
N O H
Styrene or
R
O
4-phenylstyrene
MeO
N O H
MeO N 266
N 267a, R=H 267b, R=Ph
Scheme 108.
Lohray has reported the first example of immobilized bisdihydroquinylpyridazine [168]. The monomer 270 has been prepared and was allowed to react with ethylene glycol methacrylate in a 1/9 ratio to afford the chiral polymer 271 (Scheme 110). When trans stilbene and styrenic olefins were dihydroxylated using supported ligand 271, OsO4 as oxidant and K3Fe(CN)6-K2CO3 as cooxidant, both yields and ee were high, ees being slightly lower than those observed with the homogeneous ligand. In opposite, the enantioselectivities dropped significantly with aliphatic alkenes. The excellent results could be due to larger pore size and hydrophily of this type of polymer which facilitate the approach of the reactants to the catalytic site. Since excellent results were obtained in the asymmetric aminohydroxylation in homogeneous phase by Sharpless [169], heterogeneous systems appeared to be of great interest. Nandanan has reported the first heterogeneous osmium tetroxide-catalyzed asymmetric aminohydroxylation of various olefins using polymer-supported bisdehydroquinine ligand 273 (Scheme 111) [170]. When chloramine T was used as nitogen source, yields and ee were moderate with all olefins. Monomer 268 was also copolymerized with hydroxyethyl methacrylate and ethylene glycol dimethacrylate as crosslinking agent in a 1/7/2 ratio to lead to the new polymersupported alkaloid 274 (Scheme 112) [171]. This one was insoluble in all solvents and swelled very well in protic polar solvents. The catalytic activity of chiral polymer 274 was tested in the AD of various aliphatic or aromatic olefins using two different reaction conditions (NMO or K3Fe(CN)6 as cooxidant in respectively acetone/H2O or t-BuOH/H2O).
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
133
With all alkenes used, the enantioselectivities were higher when K3Fe(CN)6 was employed as secondary oxidant and comparable to those obtained with the non-polymeric alkaloid. Concerning the aliphatic olefins, the enantioselectities remained low to moderate.
7
1
2
Styrene DVB
O
O
2S O O
AIBN
2R
O
O
268 R=
H
N
MeO
2S O O
2R
269
OCOC6H4p-Cl H
N Scheme 109.
N
H
N
N
O
O
H
N
MeO
OMe N
270
N O 1
N
H
N
O 9
O
N
O
O
O
H
MeO
N OMe
N
271
N
Scheme 110.
In order to improve the enantioselectivity in the AD of aliphatic alkenes, the two dihydroquinidine-derived polymers 275 and 276 were prepared by radical copolymerization of the corresponding monomers with hydroxyethyl methacrylate and ethylene glycol dimethacrylate as crosslinking agent (Scheme 112) [172]. These two polymers 275 and 276 exhibited enhanced enantioselectivities (88 and 86% vs 75% with polymer 274) for the dihydroxylation of two aliphatic alkenes, 1-decene and 5-decene compared to those observed with 274. Furthermore, when polymer 276 was used as chiral ligand in the dihydroxylation of
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Christine Saluzzo and Stéphane Guillarme
aromatic alkenes, ee were higher than 91% in all cases and close to those observed with the non-polymeric analog [173]. O S H
N
N N O
O H
O
N
MeO
OMe N
272
N EGDMA 9
O
O O
O
1
O
S
O
N
H
N N O
O H
MeO
N OMe
N
273
N
Scheme 111.
Salvadori has employed the polymer-supported bis(quinidyl)phthalazine 277 in the asymmetric aminohydroxylation of isopropyl cinnamate 278 in presence of Nchloromethanesulfonamide sodium salt and K2OsO4 in n-propanol/H2O (Scheme 113) [174]. The chemoselectivity, regioselectivity and ee are summarized in Table 17. Polymer 277 proved to be as efficient as homogeneous ligand in terms of activity, chemoselectivity and regioselectivity. Furthermore, the enantioselectivity was higher (87%) than those obtained with 273. Table 17. Asymmetric aminohydroxylation with polymer 277. Conversiona Chemoselectivityb (%) Regioselectivityc Ee of 279 (%) (%) (%) 1 98 94 91 87 2d 73 91 89 83 3d 58 84 89 81 e 4 59 79 88 78 a 279+280+281. b(279+280)/( 279+280+281). c279/(279+280). dRecycling run without addition of K2OsO4. eRecycling run with addition of 10 mol% of K2OsO4. Entry
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
7
1
O
O
2
O
O
O
OH
O
O
O 2S
135
O
O
274: R'=OC(O)-C6H4-p-Cl, R''=H 275: R'=H, R'' = O
N
H
R' R''
MeO N
S O2 N N
276: R'=H, R'' = O
H O
H
2
O
O
N OMe
N
N N 277: R''=H, R'= O
H
N
O
OMe N
Scheme 112.
When the osmium/ligand complex was recycled, both activity and chemoselectivy dropped significantly while chemoselectivity was remained. The enantioselectivity decreased slightly even when an initial amount of K2OsO4 was added to the mixture.
COOiPr
317
316 (10%) 3 MeSO 2 N(Cl)Na K 2 OsO 2 (4%) n-PrOH/H 2 O (1/1)
NHSO 2 Me COOiPr
OH COOiPr
OH 318
NHSO 2 Me 319
OH
COOiPr OH 320 Scheme 113.
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Christine Saluzzo and Stéphane Guillarme
The polymer backbone polarity could influence the outcome of the reaction since the catalytic activity is strongly dependent on the solvent system. For this reason, the catalytic activity of homopolymers 284 and 285 and the copolymers 286 and 287 having a polar group on polymer backbone were compared [175]. Homopolymers 284, 285a and 285b were prepared by polymerization of dihydroquinidine acrylate 282a, dihydroquinidine 4vinylbenzoate 283a and dihydroquinine 4-vinylbenzoate 283b respectively using AIBN as radical initiator (Scheme 114). These supported cinchona were tested in AD of trans metyl cinnamate and trans stilbenes using either NMO in acetone/H2O or K3Fe(CN)6 in tBuOH/H2O. Analysis of the results showed that better enantioselectivities were observed with alkaloid 4-vinylbenzoate-derived homopolymers 285. When the K3Fe(CN)6/t-BuOH/H2O system was used with homopolymers, enantioselectivities were excellent with both alkenes. Song assumed that in the acetone/H2O solvent system, the homopolymers 285 formed a viscous lump which did not allow a high concentration of the substrate near the active site while in the t-BuOH/H2O system these polymers swelled well. The accessibility of the active site is highly dependent on the compatibility between the polymer support and the liquid reaction medium. For this reason, copolymers 286 and 287 having a polar polymer structure were prepared by radical copolymerization of monomer 283 with methylmethacrylate or 2-hydroxyethyl methacrylate in presence of AIBN (Scheme 114). Indeed, these polymers seemed to be more compatible with the liquid reaction medium because both yield and enantioselectivity were higher with the two NMO/acetone/H2O and K3Fe(CN)6/t-BuOH/H2O systems in the AD of trans methyl cinnamate. The polymer 286 and 287/OsO4 complexes could be easily filtered from the reaction mixture and could be reused without significant loss of performance. Since 1,4-bis(9-O-dihydroquininyl)phtalazine ((DHQ)2-PHAL) 258 gave better results than the benzoate analog 257 (Scheme 105) in homogeneous catalysis, the heterogeneous ones were designed in order to check if the same trend was observed [176]. The copolymers 289a and 289b were prepared by copolymerisation of the monomer 288 with methylmethacrylate or 2-hydroxyethyl methacrylate respectively (Scheme 115). These two polymer-supported alkaloids were employed in the AD of trans metyl cinnamate and trans stilbene using OsO4 as oxidant and K3Fe(CN)6-K2CO3 in t-BuOH/H2O (1/1) at 10°C. Excellent enantioselectivities (>98%) were observed in all cases. The OsO4/289a complex was easily filtered off from the reaction mixture and reused a second time in the dihydroxylation of trans stilbene. Although the enantiosectivity remained excellent (99%), the reaction rate was slower (65% yield after 32h vs 93% yield after 15h). In opposite, the polymer 289b was difficult to filter because of its high swellability. An analog of monomer 288 has been prepared replacing the phthalazine by a 2,5diphenylpyrimidine and the corresponding monomer 290 was copolymerized with methyl methacrylate or ethylene glycol dimethacrylate to lead to the copolymers 291a and 291b respectively (Scheme 116) [177]. Unfortunately, the enantioselectivities were around 15-45% lower than those observed with the phthalazine-derived supported dihydroquinidine 289. Another type of insoluble polymer-bound cinchona alkaloid was tested in the AD of olefins. Salvadori has prepared copolymers 294 by solution or suspension copolymerization of chiral monomers 292 with the two styrenic compound 293 and 294 with different monomer ratio (Scheme 117) [178]. Both yield and ee were very high and copolymer 295a proved to be more efficient than 295b and 295c. The better swellability due to the lower crosslinking degree has certainly more influence on the catalytic properties and the
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
137
enantioselectivity than the solvent or the technique polymerisation. The supported alkaloid could be recovered and reused several times without significant loss of the enantioselectivity.
O
O 9 H C
O
8
N
MeO
O 9 H C
N
MeO N
N 283a: DHQD: (8R,9S) 283b: DHQN: (8S,9R) (CH2-CH)n (CH2-C)m
282a: DHQD: (8R,9S) 282b: DHQN: (8S,9R) (CH2-CH)n
(CH2-C)m
O C O 9 H C
COOR
COOCH3 8
O C
N
MeO
8
O 9 H C
N
8
N
MeO
284: n=1, m=0, (8R,9S)
N 285a: n=1, m=0, (8R,9S) 285b: n=1, m=0, (8S,9R) 286a: R=CH3, n=0.2, m=0.8, (8R,9S) 286b: R=CH3, n=0.2, m=0.8, (8S,9R) 287a: R=CH2CH2OH, n=0.06, m=0.94, (8R,9S)
Scheme 114.
N
N N O
H
N O
MeO
OMe 288
N
N
MMA or HEMA AIBN/Benzene
CO2R
RO2C
N H
N N O
N O
MeO
OMe N 289a: R=CH N 3 289b: R=CH2CH2OH
Scheme 115.
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Christine Saluzzo and Stéphane Guillarme
MeO2C
N
N
CO2Me
N
N
O
H
O
MeO
OMe N
N
291a
MMA, AIBN
N
N
N
N
O
H
O
MeO
OMe N
N
290
EGDMA, AIBN
O O
O
O
O N
N
O
N
O
H
O O
N O
MeO
OMe N
291b
N
Scheme 116.
8.2. Soluble Supported Alkaloid as Ligand The immobilization of a ligand on a MeO-PEG leads to the corresponding soluble polymer and it is known that the solubility could have an influence on the outcome of the reaction. For this reason, soluble polymer 296 was prepared and employed in the AD of olefins (Scheme 118) [179]. Similar yield and ee were observed for the reaction with trans stilbene than those obtained with the insoluble polymer-supported alkaloid 262 but with this soluble polymer the reaction time was very short (5h vs 48h). With other olefins used, the yield and ee were less satisfactory. The addition of diethylether allowed the precipitation of the supported alkaloid which could be reused without decrease of the catalytic activity and enantioselectivity. The soluble polymer containing the highly active ((DHQD)2-PHAL) 297 was also prepared and employed in the dihydroxylation of olefins (Scheme 118) [180]. The same level of
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
139
enantioselectivitity (> 97% for all alkenes) than that obtained with the insoluble analog 289a was observed. S
S
RO
O
6
N N
N
N
O
O
OMe
MeO N
292a: R=H 292b: R=
N
2
O 293
O
O
O
O
O O HO 6
O S O
S O O
N N
N
O
N
O
OMe
MeO N
N
295a: R=H, 292a/293/294 :5/ 93/2 (solvent: chlorobenzene) 295b: R= 292b/293/294 : 5/85/10 (solvent: toluene) 295c: R=
Scheme 117.
O 294
AIBN 2) cat. OsO4, NMO
RO
O
OH
1)
292b/293/294 : 5/85/10 (solvent:chlorobenzene/H2O)
2
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Christine Saluzzo and Stéphane Guillarme
O H
N
O
O H
O
O
n
OMe
MeO N 296 N
O
N N O
H
S O O
N
NH-PEG-OMe
O
MeO
OMe N
297
N
Scheme 118.
Bolm has reported the first anthraquinone-derived supported dihydroquinidine for the dihydroxylation reaction [181]. Two different ways about attaching the anthraquinone ligand were studied: either via the anthraquinone part or via the double bond of the natural quinidine (Scheme 119). The two families of soluble supported bisdihydroquinidine 298 and 299 were prepared and tested in the AD of allyl iodide and indene using 0.4 mol% of K2[OsO2(OH)4] as oxidant and K3Fe(CN)6-K2CO3 as cooxidant in t-BuOH/H2O (1/1). In the case of allyl iodide, the soluble supported ligands gave the same enantioselectivities than the nonpolymeric ligand. Although the yields were better for the AD of indene, the ee were slightly lower for all the supported ligands employed. The anchoring or the binding site had absolutely no influence on the enantioselectivity. RO
O
O-DHQD
N DHQD: MeO
O
O-DHQD O O
298a: R= MeO
N
O
m
O
O
298b: R= MeO RO
m
OR
S
S N
N O
H
O
MeO
OMe N
N 0
299a: R= MeO 299b: R= MeO
Scheme 119.
O
H
O m
O O
O m
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
141
After addition of MTBE, the supported alkaloid 298a and 299a could be recovered and reused in another dihydroxylation reaction and no loss of performance was detected.This type of supported anthraquinone-derived alkaloid has been also applied in a continuous flow Sharpless dihydroxylation using the continuously run chemzyme membrane reactor (CMR) in which the catalyst attached to the soluble polymeric ligand 300 is retained by a membrane (Scheme 120) [182]. The two cooxidant systems have been tested and although the NMO one gave lower ee, this system was more compatible with the CMR. The different solutions were delivered in the reactor and after a residence time of 85 minutes, fractions from the reaction mixture were collected. At the beginning, both conversion and ee were around 80% and after only six residence times the conversion dropped to 18% suggesting that a leaching of osmate occurred. Excellent results were obtained for the asymmetric aminohydroxylation using the soluble polymer-bound bisdehydroquinine 301, prepared in two steps from dehydroquinine (Scheme 121) [183]. When analogs of trans cinnamate 278 were used using K2[OsO2(OH)4] as oxidant, AcNHBr as nitrogen source in presence of LiOH in t-BuOH/H2O (1/1), regioselectivity (>20/1) and ee (> 95%) were excellent particularly with 301b and 301c. Results were less satisfactory when the acetone/water was used as solvent. After precipitation and filtration, the PEG-bound ligand 301c could be reused in five consecutive runs with an average decrease of 0.8% per run for the enantioselectivity. DHQ O
O
DHQ O
O
O
O O ca. 450 300
O
O-DHQ
O
O-DHQ
N DHQ:
O
H MeO N
Scheme 120. N N O
H
O PEG O
MeO
H N
N
Scheme 121.
OMe
O
301a: HO-PEG-OH, MW ca. 4000 301b: HO-PEG-OH, MW ca. 6000 301c: HO-PEG-OH, MW ca. 8000
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Christine Saluzzo and Stéphane Guillarme
Anthraquinone-derived quinine has also been anchored onto PEG-OMe to produce soluble polymeric alkaloid 302 (Scheme 122) [184]. Good to excellent yields (80-94%) and ee (80-99%) were observed with different olefins using K2[OsO2(OH)4] as oxidant, K3Fe(CN)6-K2CO3 as cooxidant in t-BuOH/H2O (1/1).
N
O
MeO
O-PEG-OMe
O
O
N 302 Scheme 122.
9. EPOXIDATION Epoxides constitute a class of versatile intermediates, as they can be easily transformed into a wide variety of functional groups involving regioselective ring opening reactions [185]. Thus, asymmetric epoxidation (AE) of olefins is a key reaction for the synthesis of enantiomerically enriched compounds. Complexed with Mn, Cr or Co, N,N-ethylenebis(salicylidene aminato) derivatives known as salen compounds, first described in 1990 by Jacobsen, are widely used as catalysts. From them, manganese complexes have been reported as highly active and enantioselective catalysts in the epoxidation of unfunctionalized alkenes in homogeneous phase using a wide range of oxidants [186]. Number of structure presenting the same backbone have been synthesized and involved in these oxidation reactions (Scheme 123). Easy to handle and to obtain, rather cheap, uses of Jacobsen type catalysts represent one of the most relevant methods of building chiral epoxides. Immobilization of such a compound was extensively studied because it restrains the formation of μ-oxoMn(IV) dimers by isolation of the catalytic sites. R
R
N
N
M: Mn
M O X O
R1 R2
R3
R1 R3
R2
Scheme 123.
The immobilization of salen onto an organic polymer was achieved according to two strategies: a copolymerization of a salen monomer or by grafting onto a polymer followed by
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
143
subsequent solid phase synthesis of the salen. In the latter case, only unsymmetrical salen are formed.
9.1. Supported Salen as Ligand The first example of a polymer-supported Jacobsen catalyst was reported by Dhal [187]. It involved the radical copolymerization of the corresponding distyryl monomer with EGDMA in a ratio of 10/90 to give the macroporous polymer 303 (Scheme 124). The epoxidation of unfunctionalized olefins led to moderate yields (55-72%) and low ee (up to 30%). Moreover, this catalytic system could be used at least five times without any significant loss of its catalytic activity. Ph
Ph
N
O
N Mn O Cl O t-Bu
O
t-Bu EGDMA, toluene, AIBN
Ph N O
Ph N
Mn O Cl O
O 303
t-Bu
t-Bu
Scheme 124.
A similar approach permitted to prepare a macroporous polystyrene-based polymer 306 via a radical copolymerization of a divinylsalen 304 or of a “distyryl spaced” salen 305 with styrene and DVB (ratio 10/75/15) (Scheme 125) [188]. The oxidation was performed with m-CPBA/NMO instead of PhIO as oxidant because this latter was transformed into PhIO2 its disproportionation product which was insoluble in the medium. The catalytic reaction was performed with 10 mol% of the catalyst at 0°C. After 30 minutes, modest to excellent conversions (67-99%) with low to moderate ee (10-62%) were observed. Moreover, the catalytic system could be recycled 5 times, the activity and the selectivity remained identical. Compared to homogeneous systems, enantioselectivity was still low. The epoxide of styrene, cis-β-methylstyrene and indene were formed in 16%, 62% and 60% ee respectively with conversions up to 99% in each case with polymer 305a as ligand. First it was considered that as the salen ligand was localized at the crosslink it may induced a steric hindrance and a conformational rigidity responsible of the low ee. Therefore, Laibinis [189-190] and Sherrington [191] synthesized non symmetrical immobilized salen attached specifically to the polymer in a pendent fashion. The polymer was a styrene base resin.
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Christine Saluzzo and Stéphane Guillarme
N
N Mn O Cl O t-Bu R N
304
N
Mn O Cl O
S
t-Bu O CO
Mn Cl +
t-Bu R
S
t-Bu
OO C
305 a: R = R'= -(CH2)4b: R = R' = Ph
+
AIBN toluene Δ
Mn Cl 75 10
15
306
Scheme 125.
Laibinis used a three steps synthesis for the polystyrene-supported salen 307 (Scheme 126) [189-190]. Asymmetric epoxidation was carried out in biphasic conditions with NaOCl as the oxidant in presence of 307Mn as catalyst. The enantiomeric excesses and the yields of AE with styrene (9% ee, 7% yield), cis-β-methylstyrene (79% ee, 2% yield) or dihydronaphtalene (42% ee, 46% yield) were low to modest. Sherrington [191] synthesized gel type and macroporous resins from 4-(4vinylbenzyloxy)salicylaldehyde (Scheme 126). The gel type catalyst was rather inactive due to the formation of the oxo-bridged dimer of the catalytic center contrary to the macroporous one with an effective site isolation of the catalytic center. With the macroporous catalyst 310 (Scheme 127) although yields were good, ee were always low (up to 20% ee). With an EGDMA polymer matrix (Scheme 128), polymer 311 afforded the epoxidation of 1-phenylcyclohex-1-ene with a high ee (91%) at 49% conversion. With the soluble Jacobsen catalyst, 92% ee and 72% conversion were observed [192-193]. The key factors for this result could be the low loading of Mn sites (0.08 mmol/g) and the high surface area. From all these results a highly active and selective immobilized salen ligands should result in a molecular structure close to those of Jacobsen’s catalyst. Sherrington [193] thought also that to provide good catalytic performance of the polymer-supported catalyst the following design criteria could be met. The complex could be attached to a single flexible linkage to the polymer support in order to minimize the steric hindrance. Moreover, a low
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
145
loading of the catalyst linked to the polymer was required to maximize site isolation of catalytic centres and hence to minimize the formation of the inactive oxo bridge dimer. The polarity of the resin should be such as to provide the optimum microenvironment for the catalyst and finally, the morphology of the resin must permit a high level of mass transfer to the active sites. OH CHO
HO
CH2Cl
R
H2N
NH2
t-Bu CHO
Merrifield resin
t-Bu OH
HO 307Mn M = Mn 307Cr M = Cr O
N
N M O Cl O
t-Bu
t-Bu
Scheme 126.
In 2000, taking into account the criteria edicted by Sherrington, Janda [194] described a convergent strategy to attach a salen ligand containing an appropriate linker to a low loading preformed polymer. A glutarate spacer (5 carbon atoms) was then used and the polymer 312 was prepared from styrene and polyTHF-derived crosslinker to form beads (Scheme 129). The AE in presence of m-CPBA as oxidant was effective. Goods yields were obtain in the AE of styrene and cis-β-methylstyrene with 51% ee and 88% ee respectively. These results were similar to those achieved with the homogeneous Jacobsen catalyst. The supported catalyst could be reused 3 times without loss of activity and selectivity. Another approach, keeping the C2 symmetry, consisted in the immobilization of the catalyst on a TentaGel amine resin via the pyrrolidine part of a pyrrolidine-salen instead of the aromatic rings (Scheme 130) [195]. In presence of NaOCl or m-CPBA, with 4 mol% of catalyst 313 all underwent AE of 2,2-dimethylchromene, 6-cyano-2,2-dimethylchromene and 1-phenylcyclohex-1-ene in high yields, more than 70% and with enantioselectivities of 82%, 86% and 68% respectively. In these conditions, decomposition of this catalyst was observed. To conserve the C2 symmetry, Zhen [196-197] formed poly-salen complexes by condensation of a slight excess of chiral diamino cyclohexane with subsequent insertion of Mn ion (Scheme 131). These catalysts were soluble in CH2Cl2 but almost insoluble in ether. The oxidation reaction was performed in dichloromethane. To avoid any degradation of the polymer NaOCl was used in the presence of 4-phenylpyridine-N-oxide (4-PPNO) or m-CPBA in the presence of NMO as cooxidants. With m-CPBA/NMO, enantioselectivities and activities are higher than those obtained with NaOCl/4-PPNO. With salen 314a or 315 (Scheme 131), the catalytic system was similar to Jacobsen’s one; at 0°C, up to 45% ee were observed for the AE of styrene. The lower temperature, the higher catalytic efficiency. For
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Christine Saluzzo and Stéphane Guillarme
styrene epoxide formation, at room temperature and at -78°C the yields were 61% and 91% respectively with no significant improvement of the enantioselectivity of about 40%. Recovery and reuse of the catalytic system by precipitation was possible with only slight loss in enantioselectivity. The behaviour of catalyst 314b (Scheme 131) was a little different [197]. At -78°C, it presented much lower activity but at -22°C, 83% yield and 51% ee were observed which was represented the best ee for the epoxidation of styrene. This catalytic system unstable in the reaction conditions was not reused.
+
O
suspension polymerization
+
CHO
O CHO 308
OH
OH
H2N
N
N Mn O Cl O O
t-Bu
1) salicylaldehyde derivative dioxane 18C6 2) Mn(II) 3) air 4) NaCl
O
310 t-Bu
NH2
N OH 309
Scheme 127.
N
N Mn O Cl O
O
t-Bu
t-Bu
t-Bu 311
O
O O
O
OAc O O
EGDMA 68%
Scheme 128.
19%
13%
NH2
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
O O
N
N Mn O Cl O
O O
t-Bu
t-Bu
t-Bu 312 Scheme 129.
O O
O NH
N N
t-Bu
N Mn O Cl O
t-Bu
t-Bu
t-Bu 313 Scheme 130.
314 a: R = H b: R = Me
N
N Mn O Cl O
R C R
t-Bu
t-Bu
n
N
H2C O H2C
N Mn O Cl O t-Bu
t-Bu 315 Scheme 131.
CH2 O CH2
n
147
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Christine Saluzzo and Stéphane Guillarme
Kureshy [198] had also carried AE with a poly-salen. The poly-salen 316 derived from chiral diphenylethylenediamine (2 mol%) (Scheme 132) and NaOCl in the presence of cooxidant (NMO, 4-PPNO, PyNO, DMSO) forming the oxidative reagent were used for the epoxidation of styrene, indene and 6-cyano-2,2-dimethylchromene. In all cases, the best results in term of activity (yields up to 99%) and enantioselectivity were obtained with PyNo as cooxidant. The epoxide of styrene, indene and 6-cyano-2,2-dimethylchromene were formed in 32%, 78% and 100% ee respectively. Comparatively, Poly-salen 314a (n = 12, Scheme 131) led to 55% ee for epoxystyrene but for epoxyindene and epoxy 6-cyano-2,2-dimethylchromene 69% and 100% ee were observed. The turn over frequency (TOF) was higher with poly-salen 314a (Scheme 131). The activity of the recycled catalyst gradually decreased upon successive use possibly due to minor degradation under epoxidation conditions and/or weight loss during recovery process of the chiral catalyst, but the ee remained constant. Ph
Ph
N
N Mn O Cl O
CH2
t-Bu
t-Bu
n
316 n = 10 Scheme 132.
N
N Mn +
O O Ph Ph
X
-
317: R = CH2CH2CH2OH 318: R = H 319: R = CH2CH2CH2OCOC6H4 a: X = PF6 b: X- = AcO-, c: X- = CF3SO3Scheme 133.
R
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
149
Smith [199] involved immobilized Katsuki-type (salen)Mn complex 318 (R = H, Scheme 133) for the AE of 1,2-dihydronaphtalene with NaOCl/4-PPNO. The Katsuki-type (salen) 317 presenting an hydroxyalkyl group (R = CH2CH2CH2OH Scheme 133) on the 6-position was grafted onto a polystyrene resin via an ester link by means of polystyrene carboxyl chloride (1% crosslinked, 200-400 mesh, 1.17 mg/mol of chloride) (salen 319, Scheme 133). The counter ion seemed to have no influence on the catalytic efficiency and selectivity. For all of them, yields were about 40% with excellent ee (upper than 90%). Each catalytic system was reused after filtration, ee and yields remained quite unchanged. To increase the yield upon the recycle (up to 70%), concentration of the reaction mixture was required. The molecular imprinting polymer (MIP) was tested by Gohdes [200]. The manganese salen precatalyst 320 (Scheme 134) having two polymerizable acryloyl groups could be covalently embedded in the crosslinked polymer network. In order to mimic the bound styrene substrate, phenylacetate ligand was fixed on the Mn atom which after its removal will leave behind the substrate-binding cavity. The MIP synthesis was performed with the precatalyst 320 in presence of EGDMA as the crosslinking agent, styrene as a non linking polymer and dichloroethane as a porogen. Exploration of porosity and crosslinking effects on activity on epoxidation of the styrene in presence of PhIO or m-CPBA was carried out. In the optimum conditions 40-50% porogen and 70% crosslinking and m-CPBA as oxidant, the ee was up to 14%. This imprinted polymer could be reuse at least 3 times without loss of ee. Rigid macroporous polymer substrate analog
shape selective binding pocket
O O L M L
L M L
Ligands covalently attached to the polymer matrix N
N Mn + O O O O O O
O 320 molecular precatalyst
O
Scheme 134.
Another way to immobilize the salen catalyst was to use glucose as a linker and to graft it on CHO modified Wang resin (Scheme 135) [201]. The activity of the catalyst 322 in the AE of styrenes has been investigated with four oxidants: H2O2, NaOCl, (n-Bu4N)HSO5 and mCPBA. The best results in terms of activity (99%) have been achieved with m-CPBA/NMO;
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Christine Saluzzo and Stéphane Guillarme
the respective ee were 30% and 80% for styrene and cis β-methylstyrene. As the reaction was accompanied by extensive lack of metal from the solid, no recycle has been considered. HO
O O
O
HO
O
O N
+
O N
N
O t-Bu
+
N
Mn
Mn O
O PF6-
t-Bu
t-Bu
homogene catalyst 321
O PF6t-Bu
supported catalyst 322
Scheme 135.
OH O t-Bu
R
R
NH2 H2N Mn(OAc)2 t-Bu O HO
OH t-Bu
O
OAc t-Bu Mn O O
t-Bu N R
N R
323 a R = Ph b R = -(CH2)4-
Scheme 136.
A new approach consisting in the formation of rigid and amorphe salen-polymer (323, Scheme 136) was reported by Gothelf [202]. It involved a condensation of a rigid trisalicylaldehyde with substituted ethylene diamines (Scheme 136). The AE of cis βmethylstyrene at 0°C, with m-CPBA in presence of NMO and polymer 323a led to 78% conversion with a cis/trans epoxide ratio of 17.0, with a catalyst loading of 15 mol%. Up to
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
151
67% ee for the cis stereoisomer and 23% ee for the trans diastereoisomer are obtained. After isolation from the reaction mixture, the catalyst was reused at least 6 times without significant drop in reactivity and selectivity. Weck [203] developed a monomer salen complex linked to a norbornene via a stable phenylene-acetylene linker and its subsequent polymerization by means of the controlled ROMP method using 3rd generation Grubb’s catalyst (Scheme 137). This polymerization methodology led to fully functionalized immobilized metal-salen catalyst. By this way, the supported catalyst showed catalytic activities and stereoselectivities similar to the nonsupported Jacobsen catalyst. Moreover, activities and selectivities seemed to depend on the density of the catalytic moieties: homopolymer 324 were less selective than their copolymer analogs 325. For example, AE of 1,2-dihydronaphtalene led in both cases to total conversion and 76% ee for the homopolymer 324 vs 81% ee for copolymer 325a. Recycle was possible and after 3 recyles a drastic decrease in ee was observed. AE of dihydronaphtalene led to 81% ee for the first cycle vs 6% ee for the third one.
N
O O
O t-Bu
n
N
M
t-Bu
O
[Ru]
t-Bu
Mes N N Mes Cl Cl Ru N N Ph
324 M = Mn-Cl, n = 50 [Ru] CDCl3 50°C
N
O O
O t-Bu
M
Br
Br
O
N
O n-octyl
t-Bu
O t-Bu
[Ru] CDCl3 rt, 2h
O O n-octyl x
N
O O
y
O t-Bu
M
N O t-Bu
t-Bu 325Mn a M = Mn-Cl, x/y = 1.1, x+y = 50 b M = Mn-Cl, x/y = 3.1, x+y = 100 c M = Mn-Cl, x/y = 9.1, x+y = 100 325Co a M = Co, x/y = 1.1, x+y = 50 b M = Co, x/y = 3.1, x+y = 100 c M = Co, x/y = 9.1, x+y = 100
Scheme 137.
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Christine Saluzzo and Stéphane Guillarme
9.2. Porphyrins Polymer RuCO-porphyrins 102 and 103 (Scheme 48) were used for the cyclopropanation reaction of styrenes but they were also tested in the epoxidation reaction. Contrary to cyclopropanation for which moderate enantioselectivities and low activities were observed, these Ru porphyrin complexes gave good ee (up to 76%) and activity (up to 89%) for AE of unfunctionalized olefines [204]. Recently, Simmoneaux showed that iron catalyst 326 derived from electropolymerized tetraspirofluorenyl porphyrin (Scheme 138) led to moderate yields without chiral induction [205]. CH2OAc
N Cl N Fe N N
AcOH2C
CH2OAc
CH2OAc )
n
)
anodic oxidation n
H2COAc Fe(Cl)
)n )
n(
AcOH2C
CH2OAc
n(
n
)n )
n
CH2OAc
326
CH2OAc CH2OAc
Scheme 138.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
153
9.3. Soluble Polymers In 2006, Yu [206] prepared a polymeric chiral salen Mn(III) complex containing chiral diamine spacer, acting as a solvent regulated phase transfer catalyst (scheme 139). This catalyst presented a Mn ion content of 1.49 mmol/g and a molecular weight of 9100. Evaluation of its catalytic behaviour in AE of styrene was performed in the presence of the oxidative system m-CPBA/PyNO, m-CPBA/4-PPNO, m-CPBA/4-PPPyNO or mCPBA/NMO. Enantiomeric excesses up to 35% were found similar to those observed in the same reaction conditions with Jacobsen catalyst. This was probably due to the solubility of the polymeric catalytic system in the reaction mixture acting thus as an homogeneous catalyst by minimizing the diffusion of the reactants to the active sites of the catalysts. The nature and the amount of the N-oxide cocatalyst showed some impact on both yield and ee. Without this additive, the yield was rather low and no ee was detected; and its presence led to an increase of both yield and ee. The optimum yield and ee were obtained when 2 equivalents of each additive were used. But NMO was the best one leading to 98% yield and 43% ee. Recycle was possible but after the third one, poor yields and ee were observed.
O ClH2C
1) K2CO3 H2N NH2
OH t-Bu
2) Mn(OAc).4H2O 3) LiCl
N
N Mn O Cl O NH t-Bu
NH
t-Bu
327 n ~14
n
Scheme 139.
Recently, Liese [207-208] used a hyperbranched polyglycerol support as high loading Mn-salen complex (chemzyme) as a catalyst fo AE 6-cyano-2,2-dimethylchromene for a continuous application in a membrane reactor. This polymer supported catalyst was performed using a polyglycerol which molecular weight is 8000 g/mol and OH group loading 13.5 mmol/g. The polymer purification was carried out by ultrafiltration leading to 65% of the desired polymer 328 (Scheme 140). The following metal insertion led to polymersupported catalyst 329. The oxidative reagent was formed by m-CPBA and NMO. Upon recycling it was observed an enhancement of the stability of the catalyst, the total turnover number increasing from 23.5 for a single batch to 80 in four repetitive batches. Moreover, some metal leaching occurred because the maximum conversion decreased from 98 to 75%, but enantioselectivities were less affected; they decreased from 95 to 88% ee from the first to the fourth batch. Nevertheless, in a continuously operated chemical membrane reactor, the TTN reached up to 240 after 20 residence times with conversions up to 70% and ee up to 92%.
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Christine Saluzzo and Stéphane Guillarme
-
O
O
+
OH
+
ClH3N
N t-Bu
HO
t-Bu Et3N
N
t-Bu
N
OH
HO
O t-Bu
t-Bu
t-Bu loading 1.6 mmol/g Yield 65%
328 metal insertion
N
+
N
M O
O X- O
t-Bu
t-Bu
t-Bu 329Co M = Co, X = OAc, conversion = 54%, Co loading: 0.76 mmol/g 329Mn M = Mn, X = Cl, conversion = 75%, Mn loading 1 mmol/g Scheme 140.
10. KINETIC RESOLUTION OF TERMINAL EPOXIDES Close to Mn-salen complexed which were effective in the epoxidation of unfunctionalized alkenes Co or Cr-salen complexes have shown their ability in asymmetric ring opening such as hydrolytic kinetic resolution (HKR) of racemic epoxides. This reaction, performed in homogeneous conditions was able to furnish chiral epoxides and diols with high enantioselectivities. Numbers of inorganic supports have been used however some organic supports have also been studied. Other kinetic resolution could be performed such as ring opening epoxide with other nucleophiles and dynamic resolution. Contrary to the catalytic systems employed for the epoxidation, mechanistic studies of asymmetric ring opening have shown that cooperative interactions between catalyst units are needed [209]. Thus, high local concentration of catalyst was necessary and high-loading support should increase the catalytic reactivity. In 1999, Jacobsen [210] prepared a polystyrene-supported Co-salen complex 332 by grafting a monophenol derivative 330 of an highly efficient chiral salen onto hydroxymethyl polystyrene beads (90 μm) derivatized as their p-nitrophenyl carbonate 331 (Scheme 140).
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
155
The same polystyrene complex 332 could be synthesized by resin capture of the salen 330. The preparation of salen 330 was carried out using an excess of di-tert-butyl salicylaldehyde relative to 2,5-dihydroxy-3-tert-butylbenzyldehyde and enantiopure cyclohexane diamine (ratio: 3/1/2) yielding a 9/6/1 ratio of ditertiobutyl, monophenolic (330) and diphenolic ligands. Then, addition of the carbonate derived polystyrene resin to the mixture allowed the selective capture of the two phenolic ligands. After washing, the polymeric mixture is formed by the incorporation of the diphenolic ligand in the resin bound ligand close to ligand 332. Complexed with Co, this small amount of the incorporated diphenolic ligand in the resin catalyst did not affect the rate and the enantioselectivity; compared to material prepared with pure ligand 332, similar results were observed.
H N
H +
OH HO
HO
t-Bu
330
H
O 331
H N
N
O
O
1) DMF, DIPEA, 1.5h 2) Co(OAc)2, MeOH/PhMe 3) PhMe/HOAc (9/1)/ air
t-Bu
t-Bu
NO2
O
N
Co O
O
O
O OAc t-Bu t-Bu 332
OH = 90 μm hydroxymethylpolystyrene 160 μmol of Co-salen per gram of resin
Scheme 140.
The HKR of epichlorhydrine (X = Cl, Scheme 141) and 4-hydroxy-1-butene oxide (X = OH, Scheme 141), the dynamic kinetic resolution of epibromhydrin and the enantioselective ring opening of epoxides by phenol were examined. In the first experiment, combination of the crude organic soluble products of the five recycle reactions and concentration led to the (S)-epichlorhydrine in 41% overall yield and > 99% ee and the (R)-chlorodiol in 93% ee. In the second one, the sum of five experiments provided (S)-triol in 36% overall yield and 94.4% ee while the enantioselectivity of the epoxide was only 59% ee. The dynamic resolution of epibromohydrine provided only the bromodiol in 94% overall yield and 96% ee (sum of the five experiments). Excellent results were also obtained for the ring opening epoxides with phenol, ee could reach more than 99% and yield 98%. The kinetic resolution of other epoxides with phenol was also studied in enantioselective parallel synthesis with the same catalyst, providing efficient access to important precursors of pharmacologically active compounds [211]. For all the studied substrates, the catalytic system was proved to be very efficient. β−aryloxyalcohols were formed in high yield (90-100%), purity ((93-100) and ee (81->99%).
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Christine Saluzzo and Stéphane Guillarme
H H N N Co O O OAc t-Bu t-Bu
O O O
O
X
OH
OH
OH
X +
O
X X = Cl, OH HKR conditions
X = Br Dynamic resolution conditions
Co-salen (0.25 mol%) H2O (0.7 eq.), CH2Cl2, rt, 3h
Co-salen (0.5 mol%) H2O (1.5 eq.), rt, 24h
HO
O
R
R
PhO
R = Br or n-Pr Co-salen (1 mol%) (CF3)3OH (0.2 eq.), rt, 2h
Scheme 141.
t-Bu O OHC
t-Bu
CHO
O O
t-Bu O O
CHO t-Bu
t-Bu t-Bu CHO t-Bu t-Bu + O
333 O
O
O
O
O
O
CHO t-Bu
t-Bu
O
O O
334
1) (R,R)-diaminocyclohexane, THF 2) Co(OAc)2.4H2O, toluene/MeOH 2,6-lutidinium p-toluenesulfonate
polymeric salenCo(III)complexes O
O
salen
salen
en
335
O
O 336
n=6
salen:
sal
n le sa Scheme 142.
O
O O
O
O
O
O
O
N O
O
N M
O
t-Bu t-Bu M = Co(III)OTs
O O
n
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
157
Zheng [212] prepared crosslinked polymeric Co(III)-salen complexes in order to induce cooperative effect. Trialdehyde 333 and dialdehyde 334 are formed via condensation of 2,5dihydroxy-3-tert-butylbenzyldehyde with diacid and triacid derived from hydroquinone and chloroglycinol. Different proportions of tri and di aldehydes (tri/di) were subsequently condensed with enantiopure cyclohexane diamine to afford polymeric ligands 335 (Scheme 142). An average molecular weight between 4000 and 10000 were observed for these polymers. For the HKR of epichlorhydrine, styrene oxide, and phenylglycidyl ester, in a wide range of tri/di proportion, similar behaviour of the catalytic system was observed in the same reaction conditions. For example, with epichlorhydrine conversion based on racemic mixture varied from 50-52%, epoxide ee from 98-99% and diol ee from 91-97% using 0.02 mol% of the catalyst, at 10°C. Most of the crosslinked polymeric catalyst presented better activities than those of the oligomer 336, showing a positive effect on the cooperation on catalytic centers with the crosslinked polymer. But with the complete crosslinked polymeric catalyst 335 activities and enantioselectivities are slightly lower, behaviour probably due to the poor solubility of the catalyst. Moreover, attempts to recycle the catalytic system were unsuccessful. This fact was ascribed to the sensitive ester linkage to the reaction medium.
N
N
OH HO HO
OH
O
O
O
t-Bu t-Bu
n
337 N
N
O N
OH HO
Cl
t-Bu
Cl
t-Bu
OH HO O
O
OH
t-Bu t-Bu
338 HO
N O
n
OH
OH
N
O
OH HO O
HO OH
N
t-Bu t-Bu
O 339
O
n
Scheme 143.
Kim [213] studied the effect of the counter ion on the HKR of epichlorhydrine, 1,2epoxybutane, 1,2-epoxyhexane and epoxystyrene. The salen polymer catalyst were synthesized by copolymerization of salen bearing chloromethyl groups with sodium phenoxide derivatives of hydroquinone, 1,3,5-trihydroxybenzene or 1,1,1-tris(phydroxyphenyl)ethane in presence of N-methylpyrrolidine and NaH in THF (Schemes 143 and 144). Co(II) type polymeric chiral salen ligands were formed by reaction of the corresponding salen ligand with hydrous Co(II) acetate. To obtained Co(III) polymeric chiral
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Christine Saluzzo and Stéphane Guillarme
salen catalysts, the Co(II) polymer salen was treated with ferrocenium hexafluorophosphate or ferrocenium tetrafluoroborate noted respectively Co(III).(PF6), Co(III).(BF4). The HKR of epichlorhydrine, 1,2-epoxybutane, 1,2-epoxyhexane and epoxystyrene was performed. With all these substrates, excellent enantioselectivities (up to 99.8%) were observed with 337-, 338-, 339-, 340-Co(III).(PF6), -Co(III).(BF4) catalysts. Both type of catalysts exhibited almost the same ee as monomeric salen catalyst. These catalytic systems were stables; they could be recovered and reused at least seven times without further treatment without loss of activity and selectivity and without regeneration of the catalyst. However, it is noteworthy that 340Co(III)OAc catalyst, the regeneration with AcOH/air is necessary to avoid an ee decreased to 20% in the second hydrolysis. Moreover with 340-Co(III)OAc catalyst a racemization during the reaction and the product isolation by distillation could occurred The ee decreased slowly via racemization which is not the case with 340-Co(III).(PF6).
O HO t-Bu
O CH2
OH t-Bu
N H2N
NH2
N
OH HO t-Bu
t-Bu
CH2 n
340
Scheme 144.
Other 340-Co-salen complexes containing Lewis acid were studied [214](Scheme144). Monomeric salen Co(II) or Co(III) unit are supposed to be attached to the polymer salen catalyst by means of the Lewis acid. Thus, salen units were linked together as a dimeric form (polysalen 346 and 348, Scheme 145). It was observed a great increase of the catalytic activity for the HKR reaction of various epoxides. For example, in presence of 0.4 mol% polysalen catalysts 346 and 348 the HKR reaction of epichlorhydrine led to about 98% ee, after 6h whereas with complex 345 ee was up to 55%. As dissociation of monomeric salens from the polymer backbone partially occurred after their first use, the polymeric salen 346 and 348 lost their catalytic activity. It is noteworthy that the activity of the salen catalyst could be recovered upon treatment with monomeric salen. Contrary to Mn salen 256Mn (Scheme 126), which is used for the AE, Laibinis used the heterogeneous Cr salen 256Cr catalyzed ring epoxide opening in the presence of TMSN3 [190]. In the conditions of ring opening epoxide, ee reach 36% and yield 47% for the propylene oxide. But the catalytic system is stable and could be reused 3 times without loss of activity and enantioselectivity. Weck [203] has performed the HKR of several epoxides by means of polymer-supported cobalt salen catalysts containing different counterions (325/Co-OAc, 325/Co-I, 325/Co-OTs, 325/Co Scheme 137). 325/Co-I and 325/Co-OTs catalysts have shown higher activities than 325/Co-OAc and ee could be up to > 99% with a conversion of 55% after 1h with 325/Co-I for the HKR of epichlorhydrine. These catalysts can only be recycled once as a result of their decreased solubility after the reoxidation step. Weck [215] has also described polystyrene-supported chiral salen ligands 349 and 350 synthesized respectively by radical homopolymerization or copolymerization of an
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
159
unsymmetrical styryl-substituted salen monomer (Scheme 146). They were easily converted into their Co(III) acetate complexes and then tested in the HKR of epichlorhydrine at room temperature. The reaction was carried out with 0.5 mol% of the catalyst and after 1h, conversion is about 55% and ee >98% even after four recycling (catalyst 350b). OH N N Co O O t-Bu t-Bu 341
t-Bu N F
BF3.2H2O t-Bu
O
t-Bu F B F
Co
t-Bu
O
BF3.Et2O
t-Bu t-Bu N
t-Bu
Co
t-Bu
N
O Cl t-Bu t-Bu O
t-Bu
N N Co O O t-Bu
N
F t-Bu F B F 342
t-Bu
343
N Co O
N
N O
t-Bu BF3 t-Bu
344
Co O
N O
O
t-Bu
t-Bu
N
Co
BF3.Et2O MC/N2
345
n
N
N
n
N
N
O
O
O
O
t-Bu BF3 t-Bu n
345
t-Bu t-Bu F B F F t-Bu t-Bu H t-Bu O O Co N t-Bu N
N
N
O
O
BF3 t-Bu
t-Bu
N Co
Co
Co
t-Bu
346
O
t-Bu t-Bu F B F F t-Bu H t-Bu O O N Co N t-Bu n
N
N
N
Co OCl O
O
t-Bu BF3 t-Bu 347
N
N
n
Cl
O
O
t-Bu t-Bu F B F F t-Bu t-Bu H t-Bu O O N Co N t-Bu
N
N
t-Bu
O
O Cl BF3 t-Bu t-Bu
348
N Co
Co
Co
Cl
O
t-Bu t-Bu F B F F t-Bu H t-Bu O O Co N t-Bu N n
Scheme 145.
Polyethylene glycol–salen-Co-OAc (Scheme 140) were applied in the HKR of several terminal epoxides with a catalyst loading of 0.5 mol% using 0.55 equiv. of water in THF. A positive dendritic effect was observed; the dendritic backbone was supposed to increase the intramolecular cooperation between Co active site by bringing the reactive site of the reactant
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Christine Saluzzo and Stéphane Guillarme
adjacent to each other (cooperative bimetallic catalysis) [208]. This reason was evoked to explain that better results were obtained with the polymeric catalyst contrary to the non polymeric one. With epichlorhydrine, the conversion reached 52.5% and the enantioselectivity 90.5%.
N
N (
t-Bu
OH
HO )
t-Bu
349 a: n = 12, Mn = 7200 b: n = 24, Mn = 14600
AIBN
N t-Bu
OH t-Bu
n
t-Bu
N HO t-Bu styrene AIBN
N OH
HO t-Bu
)(
t-Bu
N (
t-Bu
350 a: n = 15, m = 14, Mn = 10200 b: n = 6, m = 48, Mn = 8600
n
)
m
Scheme 146.
11. MISCELLANEOUS Few other asymmetric reactions have been performed using insoluble or soluble polymersupported ligands. The first example is a Mukaiyama-aldol condensation between silyl ketene acetal and different aldehydes using polymeric Box analog of 99 as chiral ligands and Cu(OTf)2 as metal source in water (Scheme 147) [216]. When using benzaldehyde as substrate, yields were very low (12-34%) and ee were moderate (40-62%) whatever the polymer-supported Box. The same level of enantioselectivity was observed with other aldehydes while the yield was better with all the ligand/Cu complexes used. Insoluble polymer-supported Box 82 (n=1) (Scheme 40) was also used in an enantioselective glyoxylate-ene reaction (Scheme 148) [217]. In most cases, although the yields and the ee were satisfactory, they were slightly slower than those observed with the corresponding non-polymeric Box.
Nitrogen-Containing Ligands Anchored onto Polymers as Catalyst Stabilizer…
O
O
O
161
O
N
N
R
R
351a: R=i-Pr 351b: R=Bn
= MeO-PEG5000
352: R=Bn
=
(OCH 2 CH 2)40 -
t-Bu
OTMS + RCHO
OMe
OH
Cu(OTf)2 L* (20 mol%)
CO 2Me
R
H 2O
Scheme 147.
R2 R1
R3 +
H
R2
cat. Box/Cu(OTf)2
O CO2Et
CH2Cl2
R1
OH CO2Et
R3 Scheme 148.
The possibility to recycle the catalyst was examined in the ene reaction of αmethylstyrene and ethyl glyoxylate. The conversion dropped from 90 to 60% after the fourth recycle (conversion was evaluated after six hours for each run) while the enantioselectivity remained constant. Furthermore, a continuous flow-system has been developed using the polymer 82 and overall 23 mmol of α-methylstyrene have been transformed into the corresponding product after 5 consecutive batches. The enantioselectivity did not change between the successive runs (close to 90% ee). The TTN was estimated at 44 or 51 versus the supported Box and copper salt respectively. The complexes of polymer 243 (Scheme 97) with YbCl3 and LuCl3 have been used for the ring opening of cyclohexene oxide by trimethylsilylcyanide [154]. Although the conversions were excellent, the ee were low to moderate. Furthermore a significant drop of the enantioselectivity was observed when the recovered supported Yb-complex was reused. The same complexes were then evaluated for the enantioselective TMSCN addition to benzaldehyde. For this reaction, both yield and ee were better with the Lu-complex. The asymmetric TMSCN addition to benzaldehyde was also performed in a microreactor using the polymeric catalyst 353 (Scheme 149) [218]. A normal batch procedure was first carried out and the protected cyanohydrin was obtained with 91% conversion and 73% ee.
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Christine Saluzzo and Stéphane Guillarme
When a microreactor using this catalyst 353 was employed, same result was observed with a flow rate of 0.8µL. Enantioselective sulfoxidation has been also studied using polymeric aminoalcoholderived Schiff bases. Polymers 354-357 have been prepared by copolymeristation of the corresponding monomer with either MMA and EGDMA or styrene and DVB (Scheme 150) [219]. These four polymers were then stirred with VO(acac)2 for 4h at rt to lead to the vanadium complexes. For the enantioselective oxidation of thioanisole, although the yields were similar to those obtained with the homogeneous analogs, the enantioselectivity were lower. The best selectivity was obtained with the catalyst derived from 355. N
N Ti O O O
O
N O O O O
O N
N
Ti O O O O O O Ti N
353 N N
N
N OH HO
OH
O
OH 3
HO
O
O
Scheme 149. NO2 HO
HO OH
HO HO
OH
N
OH
N
N
H
H
OH
OH
N
H
H O
O
O O
O
O
MMA EGDMA AIBN Toluene
styrene DVB AIBN Toluene
MMA EGDMA AIBN Toluene
styrene DVB AIBN Toluene
HO
HO OH
NO2
HO HO
OH
N
OH
N
N
H
H
OH
OH
H O PS
PS
O PA2
PA1 355
356
PA=polyacrylate, PS=polystyrene
Scheme 150.
N H
O
O 354
O
357
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163
12. CONCLUSION The performance of the soluble catalysts supported on a polymer or of the heterogeneous catalysts is highly dependant on the polymer morphology which is, in the last case, mainly controlled by the degree of crosslinking, the nature of the crosslinker and nature of the porogen. For soluble polymer catalysts, the catalytic performances are generally close to that observed with homogeneous non-supported catalysts. This behaviour is mainly inherent to high mobility of the bound catalyst and a good mass transport. However, the stability of the catalyst depends on the reaction conditions, some catalytic systems being not stable in some solvents. Their recovery needs filtrations or precipitation, sometimes under inert atmosphere. But, their use could be interesting for an industrial point of view, particularly in continuous flow systems involving membranes where important conversions of the substrate with good selectivity could be involved. As for insoluble catalysts they could be separated into two series.The ligand could be anchored onto swellable or unswellable polymers. In the first case, the support is formed by a crosslinked polymer, mostly a polystyrene crosslinked with 0.5 to 3% DVB. In the second case, pratically unswellable polymers formed the support. This latter is a highly crosslinked polymer. A large variety of solvent could be used without changing the texture of the catalyst. In order to obtain a good mass transport, it is generally necessary to obtain immobilized catalysts with a pore size enough larger to include the size of the metal complex catalyst and the substrate. This parameter depends on the nature of the porogen. These immobilized catalysts must present a large surface area For the specific molecular imprinting polymer, the catalyst is efficient if a good mass transport is possible and particularly if the chiral cavity fits perfectly with the size and the structure of the substrate. It is the reason why in most of the cases the shape of the polymer is specific to a single substrate. Reaction performed with monolithic polymer-supported catalysts, lead generally to good to excellent behaviour in terms of catalytic activity and enantioselectivity and as the reaction is performed without stirring, the catalytic system is generally very stable. The catalyst ability for recycling and reuse is in correlation to the stability of the catalytic system itself. A simple change of the nature of the polymer support could increase the stabilization and thus the recyclability of the catalytic system. Concerning the nature of the nitrogen-containing ligand itself, immobilized derivatives of DPEN revealed to be the most efficient in the reduction of prochiral ketones by catalytic hydrogenation in the presence of BINAP or by HTR and CBS-supported ligands for the enantioselective hydride reduction. As for C-C bond formation via an addition of organometallic reagent to aldehyde, ketone or imine or allylation, supported-complexes of prolinol, ephedrine and oxazoline derivated constitute the best catalytic systems. Most of the polymeric-catalytic systems used for the dihydroxylation or aminohydroxylation of C=C bond is issued from alkaloid, mainly cinchona which has been proved to be the best ligand for this type of reaction.
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For the epoxidation or kinetic resolution of terminal epoxide, polymer-supported salens have shown to be either as efficient as or more efficient than their non-supported homologs. It is noteworthy that concerning the loading, low loading in ligand and metal are required to achieve asymmetric hydrogenation, C-C bond formation or dihydroxylation or aminohydroxylation. But, high loading is necessary for epoxidation or resolution of terminal epoxides if salen derivatives ligands are employed. To conclude, the nitrogen-containing ligands are easy to handle, to synthesize and to support them on polymers, thus this constitutes a crucial advantage for using them in various separation systems (precipitation, ultrafiltration, membrane reactor, biphasic solid/liquid or liquid/liquid).
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 4
SMALL MOLECULE STABILIZATION: A NOVEL CONCEPT FOR THE STABILIZATION OF SMALL INORGANIC NANOPARTICLES Georg Garnweitner* TU Braunschweig, Institute for Particle Technology, Braunschweig, Germany
ABSTRACT In the last 20 years, the synthesis of nanoparticles with defined size and shape has been studied with strongly growing interest, leading to a multitude of synthetic approaches and strategies. Whereas the synthesis of the nanocrystals has been studied in great detail, far less effort has been directed towards the stabilization of the obtained materials against agglomeration. This is surprising as the stabilization determines their dispersibility in various solvents, which is a crucial parameter for most applications. For conventional colloids, the classical theories of electrostatic, steric and electrosteric stabilization are well established, but application of these theories to the stabilization of small nanomaterials leads to some peculiarities and at the same time has some limitations, which is known from experimental experience but has not been studied in a systematic fashion yet. One important conclusion from the theories is that short organic molecules sufficiently serve to provide steric stabilization of nanoparticles less than about 50 nm in size, without a need for long-chain polymeric stabilizers. This concept has been successfully applied using commercial metal oxide nanoparticles in the 50 nm size range, and it is even possible to tailor nanoparticle dispersions with respect to their rheological properties by adjustment of the stabilizer size. Through proper choice of the stabilizer, nanoparticle slurries with high solids content but at the same time low viscosity can be realized, which is highly advantageous for applications especially in the field of ceramic processing. For ultrasmall nanoparticles in the sub-10-nm regime, the picture is somewhat different. On the one hand, the dispersions of such particles in a stabilized state show *
Corresponding author, Prof. Dr. G. Garnweitner, TU Braunschweig, Institute for Particle Technology, Volkmaroder Str. 5, 38104 Braunschweig, Germany
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Georg Garnweitner very special properties on the verge to molecular solutions, rendering them highly relevant for applications and thus their preparation highly important. On the other hand, due to the lack of suitable model materials, the fundamentals of interaction and stabilization of such small nanoparticles remains largely in the dark. Only a small number of reports were specifically directed to adress these problems and systematically investigate the effects of stabilizer chemistry and structure as well as solvent influence. A brief overview of these studies is provided to show that first concepts have been presented, but the general applicability of these concepts still remains to be seen, and to demonstrate the substantial need for further research in this field in order to develop concepts for the rational stabilization and preparation of dispersions with tailored nanoparticle interactions and thus tailored properties.
INTRODUCTION The strong tendency of nanoparticles and nanomaterials to agglomerate and aggregate constitutes probably the most important challenge for their synthesis and application [1, 2]. Already since the work of Graham and Ostwald [3] it is known that dispersions of particles in aqueous (but also gaseous) media are thermodynamically unstable. The agglomeration of particles, which is termed coagulation in the field of colloids chemistry, results in a reduction of the surface free energy, and therefore occurs instantly [4]. In the case of nanoparticles, which by common definition are colloidal particles less than 100 nm in size, the formation of agglomerates usually is observed already during the particle synthesis [5, 6]. The agglomerates, also termed secondary particles, in many cases exceed the size of the individual nanocrystals, or primary particles, by orders of magnitude [1]. For many applications, such agglomerates are undesired as they corrupt the materials’ performance and complicate processing and storage, especially if the nanoparticles are to be processed or applied as dispersions. In these cases, agglomerates usually lead to an increase in viscosity of the system, are prone to sedimentation (as the sedimentation velocity increases with the square of the particle diameter), and strongly increase the turbidity. Another important application of nanoparticles where the agglomeration state has a crucial importance is the field of nanocomposites. Commonly, nanocomposites are fabricated by incorporation of inorganic nanoparticles into a polymer matrix, resulting in a material that can be processed and shaped like a polymer but possesses strongly enhanced mechanical or optical properties. The mechanical properties of nanocomposites on the one hand were reported to be strongly dependent on the secondary particle size of the nanomaterial filler [2], and on the other hand of course also the optical properties, especially transparency, are greatly influenced by aggregation of the nanoparticles [7]. Since the beginning of colloids science, however it is also known that the agglomeration of colloids and dispersed particles can be prevented or controlled by stabilization [8]. The attractive interactions between the colloidal particles, caused by van-der-Waals forces, need to be compensated by repulsive interactions. The latter can be based either on electrostatic repulsion due to same-sign surface charges (electrostatic stabilization), or on repulsion via a polymer shell formed through adsorption of polymers to the particle surface (steric stabilization, in presence of polyelectrolytes termed electrosteric stabilization due to additional charged-induced repulsion) [9, 10]. The stabilization by control of the interaction forces between colloidal particles has been in the focus of extensive research efforts. Already
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in the early days of colloid science, the forces between particles were studied theoretically [11], and for electrostatic stabilization the so-called DLVO theory was proposed [12, 13] and its validity for a broad range of systems and conditions is accepted until today [14]. A special focus of research has been the steric stabilization of colloidal particles by adsorption of organic polymers to the particle surface [15-17]. Typically used polymer stabilizers possess a molecular weight ranging between 5,000 Da and 15,000 Da. These polymers are targeted to adsorb to the particle surface and form an organic layer around the particles (often termed adlayer), of about 3-10 nm thickness. The general unterstanding of steric stabilization is that when two particles approach each other, the polymer layers interpenetrate, creating an osmotic pressure that counteracts the attractive interparticle force, thereby efficiently preventing close contact of the particles that would result in agglomeration [17]. The stabilization of colloidal particles can be achieved by addition of the stabilizer in a distinct stabilization step following the synthesis (post-synthesis method) [18], or by addition to the reaction system already prior to the particle formation (in-situ method) [19-22]. The latter strategy has the potential benefit of a perfect stabilization as the forming particles are instantly capped with the stabilizer, but in many cases possesses the disadvantage of a modification of the particle formation process, as strongly binding stabilizers may lead to altered particle sizes, different morphologies or even completely prevent the particle formation [23-25]. In the post-synthesis method, particle fabrication and stabilization are carried out independently, thus rendering this strategy suitable also for solventless hightemperature particle synthesis methods that would prevent the use of a stabilizer during the formation process. During their synthesis in the absence of stabilizers, however, the nanoparticles may strongly agglomerate or aggregate, which in many cases necessitates the use of mechanical dispersion methods such as ultrasonic treatment additionally to a chemical process to achieve a successful stabilization. For many applications, steric stabilization is the strategy of choice due to the higher stability, its suitability also for hydrophobic media and matrices, and the lower sensitivity towards pH and additives. Besides the stabilization effect, the adsorbed organic layer around inorganic particles can additionally be used for functionalization, which is a more encompassing concept that is based on achieving chemical functionality of the particles, resulting in functions such as selective binding to biological molecules, affinity to certain media or surfaces, environmentally responsive behavior, etc. [26-28].
STERIC STABILIZATION OF NANOPARTICLES Whilst a great deal of research has been carried out to study the interactions of particles featuring adsorbed polymer chains, especially with respect to the chemistry and conformation of the polymer chains, the dependence of these interactions on the size of the involved particles is an aspect that has not so much been in the focus of research activities. Whereas the particle interaction is relatively independent of the particle size for larger colloids, for particles smaller than 100 nm in size, this is no longer true [29]. The thickness of the adlayer formed by common polymer stabilizers for such particles starts to become considerable
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compared to the particle size, and for small nanoparticles can even exceed the size of the core particle, which has severe, and mainly undesired, consequences [20]. On the other hand, it is generally known that the stabilization of particles with sizes below the critical range of 100 nm also follows some special principles. Already from Hamaker’s theory of the attraction between particles, it can be inferred that for very small particles, the range of attractive potential decreases, and already for 50 nm sized particles only amounts to a few nm. This has severe consequence on electrostatic interaction, as is described by the DLVO theory, as well as for steric stabilization [13, 30]. It needs to be stated, however, that the attractive potential strongly depends on the nature of the material, as is illustrated in Figure 1, presenting calculations by Sigmund et al. of the attractive potential between two 100 nm sized particles of SiO2 and Al2O3 [31]. It is clearly visible that the attraction beween Al2O3 particles is much more long-ranged than beween silica spheres, which would imply that alumina particles need a stabilizing layer with substantially larger thickness to prevent them from reaching an interparticle distance small enough to result in a high attractive potential. The horizontal line in Figure 1 indicates the stabilization range, as the thermal energy is sufficient to easily overcome an attractive interaction of 2 k·T. Hence, the critical separation distance for the SiO2 spheres amounts to about 3.5 nm, whereas for the Al2O3 spheres it is increased to 12 nm [31].
Figure 1. Comparison of van der Waals interaction energies for SiO2 (– – –) and Al2O3 (- - -) spheres of 100 nm radius in water, and of SiO2 spheres in n-dodecane (––––). Reproduced from Ref. [31] with kind permission of Wiley-VCH.
These results imply that for the steric stabilization of SiO2 particles, polymers with substantially lower molecular weight, corresponding to a thinner stabilizing layer, are required than for Al2O3. The molecular weight MW of an adsorbed homopolymer can be approximately related to the adlayer thickness by [17] L [nm] = 0.06·(MW)0.5
(Eq. 1),
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indicating that the polymer used for the formation of an adlayer of sufficient thickness would require a minimum molecular weight of 40,000 Da for the alumina particles, but only of about 3,000 Da for the silica. For smaller particles, the minimum required stabilizing layer thickness decreases substantially. Lu performed theoretical calculations of the particle-particle interaction energies of aqueous dispersions of Al2O3 nanoparticles with a particle size of 20-45 nm stabilized by a protective adlayer of an adsorbed polymer [32]. The distance-dependent steric interaction energy, which was calculated based on the work of Vincent and Edwards [33] assuming a uniform polymer segment density at small interparticle distances, as well as the total interaction energy are presented for various protective layer thicknesses in Figure 2. For this system, a protective layer of 5 nm in thickness was found to be more than sufficient to achieve stabilization of the particles, with repulsion steadily increasing as the particles approach each other, whilst for a layer thickness of 2.5 nm and less, the interparticle forces become dominantly attractive at low separation distance. The system with a stabilizing layer thickness of 2.5 nm however still shows an energy barrier of several k·T in total interaction energy at intermediate separation (Fig. 2, b). Consequently, a layer thickness of 2.5 nm should be sufficient to prevent coagulation of the particles, and these findings nicely correspond to experimental results of successful stabilization when using a polymer with a molecular weight of 1,800 g mol-1 [32].
Figure 2. Theoretical calculation of the dependence of the steric interaction energy Ester (a) and the total interaction energy ET (b) on interparticle distance for different values of polymer layer thickness. Reproduced from Ref. [32] with kind permission of Elsevier.
As the particle size is further reduced, successful stabilization is achieved with even smaller stabilizers. Here, an adlayer of sufficient thickness may even be formed by adsorption of olilgomers or even of common organic molecules [34]. Experimentally, the stabilization of such small nanoparticles by adsorption of simple organic molecules such as surfactants or complexating agents is indeed broadly applied. Many well-known and successful strategies to nanoparticle dispersions involve the use of surfactants as stabilizers, such as fatty acids or alkylammonium salts, for the preparation of dispersions of small nanoparticles, especially for metal nanoparticles [35-37]. In many cases, e. g. for the famous citrate stabilization technique for gold nanoparticles [38], it has been shown that in fact the binding of the utilized surface modifier results in the immobilization of charges to the particle surface, leading to stabilization via an essentially electrostatic rather than steric mechanism [39-41]. The preparation of magnetic fluids and other nanoparticle dispersions in hydrophobic media on the other hand prove the feasibility of steric stabilization via molecular species, as in these cases an electrostatic mechanism can be excluded due to the absence of charges. In order to
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distinguish the mechanisms of steric stabilization by adsorption of low-molecular weight species from polymer-based stabilization concepts, the term “small molecule stabilization” has been coined and shall also be used in this Chapter.
UTILIZATION OF SMALL MOLECULE STABILIZATION CONCEPTS FOR TAILORING THE PROPERTIES OF NANODISPERSIONS Even though the concepts of small molecule stabilization are hence in broad use for many applications, knowledge about the underlying fundamentals and mechanisms is by no means complete. Often the stabilization is applied in a trial-and-error fashion, without any general rational concept for a directed stabilization that would be optimized for a specific system. Additionally, the usual goal of a perfect stabilization in fact does not always represent the optimum case with respect to handling, processing and application issues. For example, in ceramic processing, but also in many other fields, weakly flocculated dispersions are highly advantageous [42, 43]. Such dispersions are easy to purify, due to the presence of larger structures that can be centrifuged or filtered, and afterwards can be restabilized via simple processes [44]. Therefore, concepts need to be developed that reach beyond the usual blackand-white image of stabilization or agglomeration, rather targeting improved control and even tunability of the particle interactions [45]. This offers the possibility of tailoring dispersions with respect to their rheological properties [46, 47], and it has been shown that also processes such as the fractionation and size selection of particles are greatly facilitated or even only made possible through customization of the particle interactions [48]. One of the most straightforward strategies to tailor the particle interactions for a given material and particle size is an adjustment of the thickness of the stabilizing steric layer. It is generally known that a thinner stabilizing layer, equivalent to smaller chain length or lower molecular weight of the stabilizer, results in increased attractive interaction between the particles, whereas a thicker layer vice versa leads to better shielding of the core particles and thus to lower attractive particle interaction, although it needs to be stated that this rule-of-thumb implies analogous chemistry of the stabilizers which does not apply in all cases. However, it becomes clear that tailoring of the stabilizer and its size is an essential means to control and optimize the properties of nanoparticle dispersions [31, 44]. One important case illustrating the practical relevance of controlled stabilization is found in dispersions with high solid content, as used in ceramic processing. Here, solid bodies are formed from particle slurries via various shaping methods, followed by drying and calcination to result in a dense and dry rigid body. A high solid content of the slurry is generally desired to facilitate the drying process, leading to a lower risk of cracks and lower shrinkage. In the recent years, exact control of the size of the utilized particles and of their surface interactions has become more and more important for the fabrication of advanced ceramics with enhanced properties [49, 50]. One particular focus has been the emerging field of nanoceramic processing, where nanostructured ceramic bodies are obtained through special processing strategies [51, 52]. For the fabrication of such ceramics, two important requirements are a high solid content to allow fast and facile drying, and on the other hand a relativley low viscosity in order to enable processing of the slurry. Both of these requirements strongly depend on the stabilizing layer thickness. The maximum solid content that can be achieved in
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stable slurry is realized when the particles are packed in an optimized fashion. As illustrated in Figure 3, the packing of small particles is however strongly influenced by the adlayer thickness Δ. For a large Δ in the range of the particle radius, within a given volume less particles can be packed (solid lines) as compared to small Δ (dashed lines). This can also be expressed as the effective volume fraction Φeff which is based on the entire particle including the adlayer and is related to the true volume fraction Φ of the core particles by
Φ eff
Δ⎞ ⎛ = Φ ⎜1 + ⎟ r⎠ ⎝
3
(Eq. 2),
where r refers to the particle radius [53]. Hence, the maximum achievable solids volume fraction based on the core particles increases as Δ is decreased. Vice versa, the maximum allowable layer thickness for reaching a random close packing state (Φrcp=0.64) at a given volume fraction Φ can be calculated from [54]
⎡ Φ rcp ⎤ Δ max = r ⎢3 − 1⎥ ⎢⎣ Φ ⎥⎦
(Eq. 3).
Figure 3. Illustration of the packing of spherical particles dependent on the thickness Δ of a stabilizing layer [54]. Image reprinted with kind permission of Elsevier.
On the other hand, the adlayer thickness has also an influence on the viscosity of the dispersion. It is generally known that the viscosity η of a particle dispersion depends on its solids volume fraction Φ. The particles start to form a network as Φ is increased, which leads to an increase in relative viscosity ηr as described in the modified Krieger-Dougherty equation
η ⎡ Φ ⎤ η r = = ⎢1 − η l ⎣ Φ max ⎥⎦
−n
(Eq. 4),
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where ηl is the viscosity of the liquid medium, Φmax is the maximum filling level at which any shearing is blocked, and n is a fitting parameter [55, 56]. The combined effect of a decrease in dispersion viscosity together with higher maximum loading levels creates a strong impetus for investigations on the influence of stabilizer size on the dispersion properties [43]. Due to the high practical relevance of the rheological properties of suspensions and slurries, it is thus no surprise that the tailoring of interparticle properties by variation of adlayer thickness has been increasingly studied. Most studies were directed towards ceramic processing and utilized metal oxide nanoparticles in the 50 nm size range that by now are also commercially easily available. As an example, the validity of the modified KriegerDougherty equation (Eq. 4) for highly concentrated suspensions of nanoscale ZrO2 particles about 40-50 nm in size was investigated by Renger et al. [54]. The authors used different stabilizers, including triammonium citrate (TAC) and 2-(2-(2-methoxyethoxy)ethoxy)acetic acid (TODA). The relative viscosity ηr of suspensions stabilized with each stabilizer was thereby determined for various high solid contents, as shown in Figure 4. It is clearly visible that TODA is capable of stabilizing dispersions with much higher volume fraction of solids than TAC. The authors applied fittings to the obtained data points utilizing the modified Krieger-Dogherty equation, and it is clearly visible that the TODA-stabilized system follows this equation (Φmax = 0.43, n = 8.24). For the TAC stabilizer, the fit is rather bad, indicating that the particles in this case probably were not well-dispersed, which agrees with the observed higher viscosity of dispersions of TAC-stabilized zirconia nanoparticles as compared to TODA-stabilized dispersions at the same solids content. Moreover, due to the smaller thickness of the repulsive barrier layer, the maximum achievable filling level for TODA is much higher as compared to TAC, both experimentally and in theory [54]. This example illustrates that the suitability of a stabilizer cannot only be judged on the size of the repulsive layer that is formed upon its adsorption, but also other factors such as the adsorption strength and affinity play a key role.
Figure 4. Relative viscosity of suspensions of ZrO2 nanoparticles stabilized with TODA (♦) and TAC (▲) vs. solid content. The experimental data were fitted using Eq. 4 (---) [54]. Image reprinted with kind permission of Elsevier.
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Studart and Gauckler performed extensive studies on the stabilization of 65 nm-sized Al2O3 nanoparticles, and first investigated the minimum adlayer thickness sufficient for stabilization to then be able to prepare dispersions with highest solids concentrations [57, 58]. They stabilized the alumina particles with alkyl chains as well as polyether chains that were linked to the particle surface in a very stable and controlled fashion via a gallol linker. The minimum thickness of the stabilizing shell was determined to 3.6 nm, corresponding to a molar weight of about 500 g mol-1; smaller stabilizers resulted in agglomeration of the nanoparticles and a significantly higher viscosiy of the medium [57]. Optimization of the stabilizing shell allowed the authors to prepare suspensions with volume concentrations above 40 % [58]. However, it was also demonstrated that the nature of binding to the nanoparticles is of crucial importance to stability: besides the gallol linker, also poly(methacrylic acid) was used as linker, and additionally purely electrostatic stabilization was employed (Figure 5). Whereas under certain conditions, the poly(methacrylic acid)-coupled polymer provided good stabilization, and also the electrostatic stabilization mechanism was efficient at low salt concentrations, only the strongly binding gallol-coupled polymer stabilizers provided high stability at all tested conditions. These results by Gauckler et al. show that only a strong, in the best case covalent linkage of the stabilizer can ensure stability in a broad range of salt concentrations, pH levels, temperatures, etc.
Figure 5. Stabilization of alumina nanoparticles with PEG chains via gallol linkers (a), with block copolymer chains (b), or electrostatic stabilization (c); left: schematic, right: obtained particle size distributions at various salt concentrations. Reprinted from Ref. [58] copyright 2007 American Chemical Society.
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Aside from aqueous and hydrophilic systems, also the rheology of suspensions of alumina nanoparticles in hydrophobic organic solvents has been of high interest [53, 59, 60]. Again, slurries with high solid contents but low viscosity are desired, which also sparked investigations as to the optimization of the utilized stabilizers. Bell et al. performed extensive tests of the rheology of suspensions of alumina nanoparticles in decalin (cis/transdecahydronaphthalene). Various fatty acids were employed as stabilizers, and the authors clearly observed a dependence of the stability on the chain length of the used stabilizer (Figure 6). Propionic up to heptanoic acid were found not to create sufficiently thick layers, leading to strongly flocculated systems with elastic properties, whereas the use of oleic acid resulted in only a very week overall attraction between the particles, thus creating stable fluid-like dispersions [53].
Figure 6. Interparticle total energy potential for alumina nanoparticles stabilized with various fatty acids in decaline (a…particle radius). Reprinted from ref. [53] with kind permission of Elsevier.
An interesting schematic by Studart et al. illustrating the general correlation beween adlayer thickness and the maximum achievable volume fraction is depicted in Figure 7. At small stabilizing layer thickness, the particles can be regarded as attractive spheres, with strong attraction beween the particles. As the adlayer thickness increases, the attraction is diminished, leading to higher volume fractions that can be realized for a given viscosity. As the adlayer thickness reaches and then exceeds a critical value, full stabilization is realized, and the particles can be considered as hard spheres. However, the increasing exclusion volume results in a decrease in maximum volume fraction. This nicely illustrates that indeed, an optimum adlayer thickness needs to be found if one wants to realize concentrated ceramic suspensions with low viscosity.
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Figure 7. Volume fraction of Al2O3 nanoparticles resulting in a relative viscosity ηr of 1000 in dependence of adlayer thickness. Image reproduced from Ref. [58] copyright 2007 American Chemical Society.
SMALL MOLECULE STABILIZATION OF ULTRASMALL NANOPARTICLES: PROBING THE LIMITS Within the world of nanoscale objects, smallest nanoparticles in the size regime below 10 nm play a special role [61]. In this size range, a significant amount of atoms is located at the particle surface, resulting in extreme surface energies, and additionally quantum effects start to emerge [62, 63]. Also dispersions of such ultrasmall nanoparticles are unique, in that they have the appearance of molecular solutions even at high solid concentrations due to the small dimensions of the particles, bringing about very low light scattering and low viscosity. This renders the dispersions of ultrasmall nanoparticles difficult to characterize with regard to the nanoparticle properties, but on the other hand leads to exceptional and fascinating properties that enable numerous new applications. Therefore, the preparation of such dispersions by stabilization of ultrasmall nanoparticles is of special importance. Again, the elaboration of rational concepts for the stabilization, ideally being able to specifically tailor the particle interactions to realize desired properties, is an ambitious but highly important goal. For many applications, especially in the optical field, agglomeration of the nanoparticles is practically not tolerable, whereas for others, weakly flocculated systems are advantageous also in this case. The stabilization of nanoparticles in the size range below 10 nm on the other hand is still also of fundamental interest. As stated before, all calculations based on the colloidal models of particle interaction predict that the thickness of any stabilizing adlayers would now lie in the Ångström regime and could in principle be reached already by the adsorption of organic solvent molecules. The colloidal
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models are, however, based on assumptions that reach their limitations when the particle size approaches the lower nanometer regime, such as treatment of the solvent as continuum [64] or approximating the surfaces of polymer-coated particles as planes [34]. Recent theoretical work by Roan and Kawakatsu on the interaction of nanoparticles with end-grafted homopolymers challenges the results of earlier models of steric stabilization such as the Edwards model [16], suggesting attractive forces between particles at large interparticle distance, repulsive forces at intermediate distance, and again attractive forces at small separation [65-69]. Even though the stabilization of ultrasmall metal nanoparticles with small organic species has already been applied since the early days of colloids chemistry, the stabilization of metal oxide nanoparticles at the frontier to the molecular scale is still only partially explored. Although numerous reports deal with the preparation of dispersions from nanoparticles, the specific study of the interactions between stabilizer and the nanoparticle surface, as well as of their precise effect on the particle agglomeration and disagglomeration and the general influence of stabilizer size and geometry, has not been a strong focus of research, much in contrast to the particle synthesis itself. Moreover, due to the general lack of suitable materials with required homogeneity, the systematic and reliable study of size effects on the particle stabilization and the elucidation of fundamentals have proven difficult. To the knowledge of the author, no studies exist so far as to the effects of a systematic variation of stabilizer size and chemistry on the properties of small nanoparticle dispersions. Only a small number of studies have been explicitly devoted toward the investigation of specific aspects of the stabilization of ultrasmall nanoparticles, and some examples are presented below to highlight recent findings in this area. Most of these investigations were performed on metal oxide nanostructures due to their high application relevance. Of these systems, titania nanoparticles certainly represent one of the most explored systems, due to their high application potential especially in photocatalysis. Numerous synthesis approaches have been reported, ranging from gas-phase pyrolysis to solvent-based approaches, especially aqueous and nonaqueous sol-gel chemistry, and mainly lead to ultrasmall nanoparticles only a few nm in diameter. The further processing of these nanoparticles requires the preparation of particle dispersions, and therefore it is no surprise that also the stabilization of these particles against agglomeration has been a focus of research. TiO2 nanoparticles used for the preparation of aqueous dispersions are usually charge-stabilized, whilst diverse strategies are pursued for their stabilization in organic media. In the latter case, most often organic polymers or surfactants are added to adsorb to the nanoparticles, for example fatty acids [70, 71]. In basically all cases, this results in substantial deagglomeration of the nanoparticles, however in the most cases no full stabilization, equivalent to the exclusive presence of individual nanoparticles, is observed. The reason for this might in fact be an only partial suitability of the stabilizer, which forms an insufficiently thick shell to be able to prevent attraction between the particles, or even induces these interactions, or shows insufficient binding, thereby not forming a compact and uniform shell. Not many studies are dedicated to a detailed investigation on these mechanisms, whilst generally a still very empirical approach is pursued here, often solely being based on trialand-error principles [72].
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Figure 8. TEM images of TiO2 nanoparticles without stabilizer (a) and stabilized with both propionic acid and n-hexylamine (c); the inset images (b) and (d) present the corresponding high-resolution images of one particle [73]. Reproduced with kind permission of Elsevier.
Further insight into the stabilization of small nanoparticles can be gained by looking at a very interesting study by Nakayama and Hayashi, who investigated the stabilization of TiO2 nanoparticles prepared via a chemical method. Their aim was to realize good stability of the nanoparticles using organic surfactants of the smallest possible quantity in weight by reducing the chain length of alkyl surfactants [73]. Thereby, the binding of the surfactants to the nanoparticle surface turned out to be a crucial aspect: the authors identified the surface of titania to show different acid sites, both Lewis acid sites (Ti+) and Brönsted acid sites (Ti– OH). Therefore, a combination of two stabilizers, both a carboxylic acid and an alkyl amine, was used in order to have binding and saturation of both sites. Indeed, when applying a twostep approach with both classes of stabilizers, much better stabilization was achieved as compared to the individual agents. Figure 8 shows images of transmission electron microscopy (TEM) indicating that the individual nanoparticles, about 5 nm in size and highly crystalline, are strongly agglomerated after the synthesis, whereas after the treatment they are much more evenly distributed on the TEM grid after drying, indicating their lower agglomeration state and lower attractive interaction. This is also evident in photographs of dispersions of the stabilized nanoparticles in various solvents (Figure 9). Due to the formation of hydrochloric acid as side product of the synthesis, a good dispersion of the nanoparticles in water is achieved even in the absence of stabilizers (a). In this case, the mechanism of stabilization is clearly of electrostatic nature. Such “bare” TiO2 nanoparticles however cannot be stabilized at all in organic solvents, necessitating the use of a stabilizer. Whereas the sole use of hexanoic acid (b, c) and n-hexylamine (d) however resulted in significant turbidity, the two-step strategy employing a mixed stabilization led to a completely transparent dispersion (e), proving that the utilized combination of stabilizers strongly reduces or even fully prevents the agglomeration. An important conclusion from this work therefore must be a careful study of the various surface sites of the particles, as the use of a stabilizer only binding to one particular type of site may result in an insufficiently dense stabilizing layer.
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Figure 9. Photograph (top) and UV-vis spectra (bottom) of bare TiO2 nanoparticles in water (a) and of TiO2 nanoparticles stabilized with alkyl carboxylic acid in methanol and chloroform (b, c) and with an n-alkylamine (d) as well as the mixed stabilizers in chloroform (e). Reproduced from Ref. [73] with kind permission of Elsevier.
As mentioned in the Introduction section, the adsorption of stabilizers to achieve stabilization of particle dispersions can not only be carried out in a separate step, but also already during the particle synthesis (in-situ modification or in-situ functionalization), which
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can be especially beneficial for small nanoparticles with their extremely high specific surface area. For titania nanoparticles, thorough studies on the in-situ modification with organic adsorbands were pursued, not only with the goal of optimizing the particle stabilization, but also as this is a means to alter and enhance the photocatalytic properties [74-76]. It has been shown that special ligands can form charge-transfer complexes with titania surface species, which results in a shift of the absorption edge towards the visible range and thus, a change in color of the material [77]. In these studies, ortho-catechols were identified as an especially suitable class of ligands, because they bind to the titania surface in a very stable fashion due to the confined geometry of the two hydroxyl groups [77]. Usually, they cannot be replaced by other ligands and thus can be considered as covalently bound [78]. This brings about a great advantage, as the stabilization of titania nanoparticles modified with such ligands can be studied independently of the effect of ligand affinity or exchange. Niederberger and the author utilized this concept to perform studies of the dependence of stabilization on the chemical structure of the stabilizer [78]. It was shown that via modification of the side chain of catechol ligands, the stabilization of the nanoparticles could be optimized for a specific solvent. By using an aminoethyl sidechain, the nanoparticles were tailored to form highly stable aqueous dispersions, being redispersible even after full drying; when employing more hydrophobic side groups, transparent dispersions were formed in various organic solvents [78]. Figure 10 shows a photograph depicting dispersions of nanoparticles capped with two kinds of catechol species in the respectively suitable solvent. It however also needs to be stated that due to the employed synthesis conditions, also a high quantity of charges (protons) was located on the nanoparticle surface, allowing the preparation of aqueous dispersions also in the absence of ligands under certain pH conditions [79, 80].
Figure 10. Titania nanoparticles modified with various catechol ligands as stabilizers in the dry state (a) and dispersed in water (b) and tetrahydrofuran (c). Reproduced with alterations from Ref. [78] copyright 2004 American Chemical Society.
Stabilization studies have also been carried out with other metal oxide nanoparticles. Due to the demanding requirements as to transparency and homogeneity of nanocomposites for optical applications, which are greatly determined by the agglomeration state of the
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nanoparticles, the stabilization of nanoparticles of high-refractive index materials such as zirconia is of high practical relevance. The author has recently performed an extensive study of the stabilization of ultra-small zirconia nanoparticles about 3 nm in size that were utilized for the fabrication of holographically structured optical devices [81]. The particles were prefabricated via the benzyl alcohol-based nonaqueous synthesis strategy, which represents one convenient route to highly crystalline, well defined and homogeneous metal oxide nanoparticles and nanostructures [82, 83]. The synthesis was performed in the absence of stabilizers; however the used solvent was detected to bind to the particle surface to a considerable extent. As the solvent has insufficient stabilizing properties, the particles were initially obtained as weak agglomerates, but could easily be stabilized via the addition of a suitable stabilizer. Stabilization experiments in polar organic solvents such as THF revealed that silanes are highly suitable for particle stabilization due to their stable linkage to the particle surface, and the results suggested that the stability of the formed dispersion can be correlated to the amount of stabilizer adsorbed to the particle surface [84]. Investigations in hydrophobic organic solvents on the other hand mainly were focused on long-chain fatty acids, the addition of which resulted in instant stabilization and even disintegration of previously formed agglomerates without any need for additional mechanical treatment, as is illustrated in the photograph and the TEM micrographs presented in Figure 11. The nanoparticles could be redispersed in very small volumes of solvent, which resulted in concentrated dispersions reaching up to 200 mg mL-1 solids content whilst being completely transparent. The TEM image of a dried dispersion (Figure 11, b) proves that the particles are completely free-standing in the dispersions, and the repulsive forces even prevent their reagglomeration on the sample grid upon drying [81].
Figure 11. Photograph (left) and TEM images (right) of ZrO2 nanoparticles before (a) and after (b) stabilization with oleic acid. Images taken from Ref. [81] with kind permission of Wiley-VCH.
In this system, the adsorption of the stabilizer was characterized throroughly employing various spectroscopic techniques. Especially, 1H and 13C liquid state NMR spectroscopy proved as a useful probe for the surface chemistry of nanoparticles in concentrated dispersions, as species adsorbed to the surface can be identified, however the functional groups directly adjacent to the surface are motionally hindered, which results in spectral broadening [85]. It is hence possible to assess the amount of surface-bound species, determine the functional groups binding to the particle surface, and qualitatively investigate the chemistry of both particle surface and bulk solution. In the zirconia case, it was detected that indeed only partially the initially bound benzyl alcohol solvent is replaced by the stabilizer
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arachidic acid (Figure 12, a). The stabilizer is bound via a deprotonated carboxylic acid functional group, and no free arachidic acid was detected. A quantitative evaluation of the adsorbed species was possible via complementary TGA measurements (Figure 12, b), and revealed further interesting details of the stabilization process. For example, it turned out that the addition of higher amounts of stabilizer did not increase the total amount of organics on the particle surface but merely led to an increased exchange of the arachidic acid stabilizer with previously adsorbed benzyl alcohol, indicating that after the initial synthesis, the particle surface is saturated with benzyl alcohol [81].
a)
b)
Figure 12. Characterization of the binding mode of fatty acid stabilizers to the surface of ZrO2 nanoparticles can be carried out via 13C NMR spectroscopy (a; BA=benzyl alcohol) and thermogravimetric analysis (b; AA=arachidic acid, HD=hexadecanoic acid, OD=octadecanoic acid, OA=oleic acid). Image (a) reproduced from Ref. [81] with kind permission of Wiley-VCH; image (b) is courtesy of the author.
CONCLUSION In this Chapter, the concept of small molecule stabilization of inorganic nanoparticles has been introduced. It has been shown that whilst the steric stabilization of colloids by adsorption of polymers has been intensively studied both experimentally and in theory, the stabilization of nanoparticles is a much less understood process. The established theories of colloidal stabilization, based on electrostatic or steric stabilization mechanisms, can also be applied for nanoparticles, revealing that already small adsorption layers created by small molecules rather than polymers can suffice to achieve stabilization, which has also been confirmed by experimental results. Indeed, practical experience even in the early days of colloids chemistry has already shown that the small molecule stabilization of nanoparticles is a feasible and highly advantageous strategy. Despite this long-time utilization, only selective aspects of the precise influence of the stabilizer on the microscopic (particle size and agglomeration) and macroscopic (rheology) properties of nanoparticle dispersions have however been studied in more detail. For larger nanoparticles in the 50 nm size range, investigations have been performed by several groups in the last years, showing the applicability of concepts that utilize a specific tailoring of stabilizer length and chemistry to achieve particle dispersions with tunable rheological properties.
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For small nanoparticles in the 10 nm size range that are of key interest for novel applications, only some aspects of small molecule stabilization have been explored so far, such as the advantage of using mixed stabilizers that can provide a true, dense particle adlayer via the specific saturation of several different surface sites, or the applicability of a “twoface” concept where double-functional stabilizers are used, featuring a head group that selectively and in a stable manner can bind to the particle surface, and a tail that is specifically adapted to be compatible with the solvent. Many other fundamental aspects still remain unclear, as it is known that the colloidal theories reach their limitations precisely in the low nanometer size regime, with novel studies predicting severe deviations and a higher interaction complexity when applying more elaborate and realistic models. Moreover, smallest nanoparticles are on the limit to the molecular scale, and the general question arises as to the validity of the conventional concepts of stabilization for such structures. It remains to be seen whether it will be possible to bridge the gap between theoretical explanation and practical experience in this field, whilst in view of the high relevance of particle stabilization for almost all areas of applications, the development of rational concepts for tailoring the particle interactions would be highly desirable and will certainly create a strong research impetus in the future as small nanoparticles in high quality and homogeneity will become increasingly available.
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[49] Kendall, K. Influence of powder structure on processing and properties of advanced ceramics. Powder Technol., 1989, 58, 151-161. [50] Adair, J. H.; Crampo, J.; Mandanas, M. M.; Suvaci, E. The role of materials chemistry in processing BaTiO3 in aqueous suspensions. J. Am. Ceram. Soc., 2006, 89, 18531860. [51] Ahn, E. S.; Gleason, N. J.; Nakahira, A.; Ying, J. Y. Nanostructure processing of hydroxyapatite-based bioceramics. Nano Lett., 2001, 1, 149-153. [52] Garnweitner, G. Zirconia nanomaterials: Synthesis and biomedical application. In: Nanomaterials for the Life Sciences Vol. 2: Nanostructured Oxides (Ed.: C. Kumar), 2009, Wiley-VCH, Weinheim (Germany). [53] Bell, N. S.; Schendel, M. E.; Piech, M. Rheological properties of nanopowder alumina coated with adsorbed fatty acids. J. Colloid Interf. Sci., 2005, 287, 94-106. [54] Renger, C.; Kuschel, P.; Kristoffersson, A.; Clauss, B.; Oppermann, W.; Sigmund, W. Rheology studies on highly filled nano-zirconia suspensions. J. Eur. Ceram. Soc., 2007, 27, 2361-2367. [55] Krieger, I. M.; Dougherty, T. J. A mechanism for non-newtonian flow in suspensions of rigid spheres. Trans. Soc. Rheol., 1959, 3, 137-152. [56] Bergström, L. Shear thinning and shear thickening of concentrated ceramic suspensions. Coll. Surf. A, 1998, 133, 151-155. [57] Studart, A. R.; Amstad, E.; Antoni, M.; Gauckler, L. J. Rheology of concentrated suspensions containing weakly attractive nanoparticles. J. Am. Ceram. Soc., 2006, 89, 2418-2425. [58] Studart, A. R.; Amstad, E.; Gauckler, L. J. Colloidal stabilization of nanoparticles in concentrated suspensions. Langmuir, 2007, 23, 1081-1090. [59] Bergström, L.; Schilling, C. H.; Aksay, I. A. Consolidation behavior of flocculated alumina suspensions. J. Am. Ceram. Soc., 1992, 75, 3305-3314. [60] Bergström, L. Sedimentation of flocculated alumina suspensions: γ-ray measurements and comparison with model predictions. J. Chem. Soc., Faraday Trans., 1992, 88, 32013211. [61] Schmid, G. E. Nanoparticles: From Theory to Application, 2005, Wiley-VCH, Weinheim (Germany). [62] El-Sayed, M. A. Small is different: Shape-, size-, and composition-dependent properties of some colloidal semiconductor nanocrystals. Acc. Chem. Res., 2004, 37, 326-333. [63] Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chemistry and properties of nanocrystals of different shapes. Chem. Rev., 2005, 105, 1025-1102. [64] in 't Veld, P. J.; Petersen, M. K.; Grest, G. S. Shear thinning of nanoparticle suspensions. Phys. Rev. E, 2009, 79, 021401. [65] Roan, J.-R. Attraction between nanoparticles induced by end-grafted homopolymers in good solvent. Phys. Rev. Lett., 2001, 86, 1027-1030. [66] Roan, J.-R. Erratum: Attraction between nanoparticles induced by end-grafted homopolymers in good solvent. Phys. Rev. Lett., 2001, 87, 059902. [67] Roan, J.-R.; Kawakatsu, T. Self-consistent-field theory for interacting polymeric assemblies. I. Formulation, implementation, and benchmark tests. J. Chem. Phys., 2002, 116, 7283-7294.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 5
MOLECULAR IMPLICATIONS IN THE SOLUBILIZATION OF THE ANTIBACTERIAL AGENT TRICLOCARBAN BY MEANS OF BRANCHED POLY (ETHYLENE OXIDE)-POLY (PROPYLENE OXIDE) POLYMERIC MICELLES Diego A. Chiappetta1,2, José Degrossi3, Ruth A. Lizarazo4, Deisy L. Salinas4, Fleming Martínez4 and Alejandro Sosnik1,2* 1
The Group of Biomaterials and Nanotechnology for Improved Medicines (BIONIMED), Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina 2 National Science Research Council (CONICET), Buenos Aires, Argentina 3 Department of Toxicology, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina 4 Department of Pharmacy, Faculty of Sciences, National University of Colombia, Bogotá, Colombia
ABSTRACT Aiming to gain further insight into the complexity of drug/polymeric micelle interaction phenomena, the present chapter investigated the incorporation of the poorly water-soluble topical antibacterial agent triclocarban (TCC) into polymeric micelles of the branched pH/temperature-responsive poly(ethylene oxide)-poly(propylene oxide) block copolymers Tetronic® 1107 (MW = 15 kDa, 70 wt% PEO) and 1307 (MW = 15 kDa, 70 wt% PEO). Solubility extents showed a sharp increase of up to 4 orders of magnitude. Due to the pH*
Corresponding author: Department of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Buenos Aires, 956 Junín St, 6th Floor, Buenos Aires CP1113, Argentina, Phone #: 54-11-49648273, Fax #: 54-11-4964-8273, Email:
[email protected].
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dependent character of both the carrier and the drug, studies were performed under different pH conditions. Due to a more efficient poloxamine aggregation at higher pH-values, a clear increase in the solubilization capacity was apparent under these conditions. However, ionization of TCC at pH 12.7 constrained the formation of hydrogen bonds between the urea moieties and the polyether chain, leading to a decrease in solubility above this pH. The size and size distribution of drug-loaded micelles was evaluated by Dynamic Light Scattering (DLS). Findings indicated the increase in the size of the aggregates with the incorporation of the drug. The morphology of the nanostructures was visualized by transmission electron microscopy (TEM). The stability of the systems over time was also evaluated. Finally, the antibacterial activity of different TCC/poloxamine complexes was assayed on different bacteria collections. For example, while a poloxamine-free TCC aqueous solution (pH 7.4) was not effective on Staphylococcus aureus, a 10% drug-containing T1307 system inhibited the bacterial growth to some extent. These results supported the release of the drug from the polymeric reservoir. However, as opposed to previous reports, overall findings indicated the limited intrinsic activity of TCC against the investigated pathogens.
1. INTRODUCTION Adhesion of bacteria to the surface of biomedical devices, tissues and working areas and the later progression of the colonization process involves a first stage (phase one) of physical and reversible surface/pathogen interactions [1]. Then, a second stage (phase two), entails a permanent time-dependent adhesion and the secretion of a slimy extracellular matrix known as biofilm [2,3]. Microorganisms embedded in a biofilm frequently display higher minimal inhibitory concentrations (MIC) and resist common antibiotic chemotherapy [4,5]. This phenomenon would stem from a modified metabolism. Biomaterial-centered infections (BCIs) originate in the colonization of biomaterial-made devices by bacteria and the generation of a biofilm [3,6]. Among the microorganisms, Staphylococcus epidermidis and other staphylococci were indicated as highly prevalent actors in the development of BCIs [4,7,8]. Generation of biofilm by antibiotic-resistant clinical strains (e.g. methicillin-resistant staphylococci or MRS and vancomycin-resistant enterococci or VRE) plays also a central role in the spreading of hospital-acquired infections [9,10]. The low aqueous solubility displayed by more than 50% of the FDA-approved drugs constitutes a main obstacle towards the development of formulations for oral, parenteral and topical administration [11,12]. Triclocarban (TCC, 3,4,4´-trichlorocarbanilide, see structure in Table 1) is a synthetic antibacterial agent used as antiseptic in cosmetic and health-consumer products [13,14]. It presents low acute and chronic toxicity. Due to its molecular nature, TCC remains unionized in a broad range of pH-values (pKa = 12.7). Contrary to triclosan (TS), another well-known topical and broadly used biocide, reports indicated that TCC displays a more limited activity (essentially against Gram positive microorganisms) [15,16]. The extremely low solubility of TCC in water (~50 ng/mL) constrains the development of water-based antibacterial TCCcontaining formulations [17]. In order to enhance the soubility of TCC, different approaches have been investigated [18]. For example, complexation of the drug with different cyclodextrins (CDs) led to good solubility improvements [19,20]. In this context, whether the
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low antibacterial activity stems from the intrinsic resistance to the drug or, by the contrary, from the extremely low concentrations attainable in aqueous media remains unclear. Polymeric micelles are nanostructures generated by the self-assembly of polymer amphiphiles in water [21]. Micelles are conformed by two zones: (A) an inner and hydrophobic core and (B) an outer and hydrophilic shell. The hydrophobicity of the core enables the inclusion and solubilization of poorly water-soluble molecules [22]. Solubilization is governed by two main parameters, namely the micellization ability of the polymer and the drug/micelle interaction. Poly(ethylene oxide)-poly(propylene oxide) block copolymers (PEO-PPO) are one of the most extensively investigated groups of micelleforming biomaterials [23,24]. A number of advantageous features support their application: (A) a broad range of commercially available molecular weights and EO/PO ratios, (B) a proven cell and tissue-compatibility and (C) the approval of several derivatives by different regulatory agencies (e.g., FDA, EMEA) as additives in pharmaceutical and cosmetic products [25-27]. The most thorough work was performed on micelles of the linear PEO-PPO-PEO poloxamers (Pluronic®) [24]. On the other hand, the 4-arm poloxamines (Tetronic®) present a central ethylenediamine segment that renders the molecule two tertiary amine moieties. Thus, molecules are both temperature and pH sensitive [28]. Also, amine groups enable further chemical modification to produce more cell-adhesive substrates [29]. Our research group dedicates efforts to investigate the application of polymeric micelles as nanocarries for poorly-water soluble biocides in the context of hospital biofilm prevention and treatment. In a recently published work, we investigated the inclusion of TS into polymeric micelles of the poloxamine Tetronic® 1107 at different pH-values [30]. Solubility levels increased up to 4 orders of magnitude. Microbiological assays demonstrated that TSloaded micelles were active against a broad spectrum of pathogens, including MRS and VRE. Moreover, TS/poloxamine systems were effective against Staphylococcus epidermidis biofilm. Aiming to gain further insight on the molecular aspects controlling the solubilization process of TCC the present chapter reports on the inclusion of the drug into polymeric micelles of two branched PEO-PPO block copolymers, Tetronic® 1107 and 1307. The size and size distribution of drug-loaded micelles were studied by Dynamic Light Scattering (DLS) and the morphology of the aggregates visualized by Transmission Electron Microscopy (TEM). Finally, the antibacterial activity of TCC-containing systems was investigated on different collections and clinical pathogens.
2. MATERIALS AND METHODS Materials. Tetronic® T1107 (MW = 15 000 Da, 70 wt% PEO, HLB 18-23) and 1307 (MW = 18 000 Da, 70 wt% PEO, HLB > 24) were a kind gift of BASF (NJ, USA). Triclocarban (TCC, Sigma, St. Louis, MO), KH2PO4 (Carlo Erba, Italy), KCl (Merck, Germany), NaOH (Merck, Germany) and concentrated HCl (Anedra, Argentina) were used as received. Buffers of pH 2.0, 5.8 and 7.4 were prepared in distilled water according to the USP 24 edition. Buffer pH 12.7 was prepared using Na2HPO4 (Merck, Germany) and the pH adjusted with NaOH 0.2M.
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Thermal analysis. In order to establish the enthalpy of fusion of TCC (ΔHf) a sample (~ 5 mg) of the drug was sealed in a 40 μL Al-cruicible pan and analyzed by Differential Scanning Calorimetry (DSC, Mettler TA-400 differential scanning calorimeter) in a single heating ramp (25 to 300oC, 10oC/min.). The value of enthalpy was normalized to the weight of the sample and expressed in kJ/mol. Preparation of poloxamine micelles. Poloxamine micellar systems (1 to 10%) were prepared by dissolving the required amount of polymer in the different media at 4oC. Systems were equilibrated at 23oC, 24 hours prior to use in order to allow the formation of the micelles. Concentrations are expressed in % w/v. Preparation of TCC-loaded micelles. TCC (in excess, ~ 20 mg/mL poloxamine solution) was added to T1107 or T1307 micellar systems (3 mL, 1 to 10% w/v) in dark glass vials (10 mL) and sealed appropriately with Parafilm®. Specimens were vigorously shaken (48 h) in a temperature-controlled horizontal shaker at 23oC (Minitherm-Shaker; Adolf Kuhner AG, Switzerland). Suspensions were filtered through clarifying filters (0.45 μm, cellulose nitrate membrane, Microclar, Argentina) and dried in a vacuum oven at room temperature. Dry samples were re-dissolved in ethanol and the concentration adjusted as required. Drug concentrations were determined by measuring the absorbance in a UV spectrophotometer (265 nm, CARY [1E] UV–Visible Spectrophotometer Varian, USA) at 23oC using a calibration curve of TCC solutions in ethanol covering the range between 2.7 to 13.7 μg/mL (correlation factor was 0.9985). Mean concentrations are expressed in μg/mL ± S.D. (n = 3). Ethanol was used as blank. Molar solubilization ratios (MSR) were calculated by ratioing the molar concentration of the drug by the molar concentration of the polymer, at every condition. Measurement of the micellar size by Dynamic Light Scattering (DLS). The average hydrodynamic diameter of drug-loaded poloxamine micelles was measured in a Zetasizer Nano Series (Zetasizer Nano-Zs, Malvern Instruments, UK) provided with a He-Ne (633 nm) laser and a digital correlator ZEN3600. Measurements were performed in PMMA disposable cuvettes at 23°C. Samples were filtered by clarifying filters (0.45 μm) prior to the assay. Results are expressed as the average of five measurements. Visualization of the drug-containing micelles. In order to investigate the morphology of drug-containing micelles, samples were visualized by Transmission Electron Microscopy (TEM, EM 109T Zeiss Transmission Electron Microscope, Karl Zeiss, Berlin, Germany). Briefly, TCC-containing poloxamine systems were placed on grids covered with Fomvar film and stained with 2% w/v phosphotungstic acid solution in water. Then, samples were dried in a closed container with silicagel and observed under the microscope. Stability of the TCC-loaded micelles upon storage. In order to study the stability of the drug-containing micelles along the time, specimens previously prepared were stored at room temperature for 1 month and the concentration of TCC in the different samples determined by UV (see above). Samples were worked up as previously described. Results of % of remaining TCC (% TCC) are expressed as Mean ± S.D. (n = 3).
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Antibacterial activity of TCC/poloxamine systems. Antibacterial evaluation of different TCC-containing T1107 and T1307 micelles (pH 7.4) was evaluated in plate experiments. Bacteria were cultured on Tryptone Soy Agar (TSA, Britannia, Argentina) plates and were incubated at 35oC for 24 h. Then, microorganisms were removed from the isolation medium, suspended in 0.9% NaCl to a final concentration equivalent to an optical density of 0.6 (at 600 nm) and diluted 1/10 (0.9% NaCl). Suspensions (0.1 mL) were diluted in cold molten TSA (10 mL) containing 2,3,5-triphenyltetrazolium hydrochloride (TTC, 0.007% final concentration, Sigma, USA), plated in sterile Petri dishes and allowed to solidify. Paper discs (6 mm diameter) were embedded in the corresponding samples, namely, poloxamine-free saturated TCC solution in buffer (pH 7.4, control), drug-free poloxamine T1107 and T1307 micelles (blank) and TCC/poloxamine systems, incubated (35oC, 24 h) and the inhibition zone in disc diffusion tests was measured. Results are expressed in diameter. Inhibition zones of =O –NH–
(a)
Quant
Fedors V/ cm3 mol–1
Van Krevelen Fd / J1/2 cm3/2 mol–1
Fp2 / J cm3 mol–2
Uh / J mol–1
1
1 × 52.4
1 × 1270
1 × (110)2
-
1
1 × 33.4
1 × 1110
1 × (110)2
-
3 1 2
3 × 24.0 1 × 10.8 2 × 4.5 177.6
3 × 450 3 × (550)2 1 × 290 1 × (770)2 2 × (210)2 2 × 160 4340 1612800 δd = δp = ((1612800)1/2/177.6) (4340/177.6) = 7.15 MPa1/2 = 24.44 MPa1/2 δ = (24.442 + 7.152 + 7.282)1/2 = 26.48 MPa1/2
Calculated according procedures described by Barton [31].
3 × 400 1 × 2000 2 × 3100 9400 δh = (9400/177.6)1/2 = 7.28 MPa1/2
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TCC solubility (μg/mL)
700 600 500
2
400
5.8
300
7.4
200
12.7
100 0 0
2
4
6
8
10
12
T1107 concentration (% w/v) A
TCC solubility (μg/mL)
600 500 400 2 5.8 7.4 12.7
300 200 100 0 0
2
4
6
8
10
12
T1307 concentration (% w/v) B Figure 1. Sa of TCC versus poloxamine concentration at four different pHs. A) T1107 and B) T1307.
3. RESULTS AND DISCUSSION 3.1. Water-Solubilization of Triclocarban The goal of the present work was to investigate the molecular implications governing the solubilization process of TCC into the core of poloxamine polymeric micelles and the further understanding of the phenomena associated with the limited antibacterial performance of the drug.
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Table 3. Triclocarban/poloxamine molar ratios (MSR) in poloxamine systems between 1 and 10% at four different pH-values.
Poloxamine
T1107
T1307
Polymer concentration (%) 1 3 5 7 10 1 3 5 7 10
2.0
5.8
pH 7.4
12.7
13.88 5.55 3.55 2.74 3.09 21.16 9.32 6.89 8.62 20.90
7.76 5.69 4.63 4.84 6.02 10.04 6.03 7.63 24.44 67.39
30.18 11.49 20.42 42.60 196.08 20.22 99.54 166.46 264.07 289.39
11.74 8.75 12.27 24.03 60.66 17.17 36.90 119.50 103.24 241.57
The ideal solubility of a solute is controlled by the melting point and the enthalpy of fusion [32]. The molecular form of TCC could interact with solvents and among itself by hydrogen bonding, namely, as hydrogen-donor through the N-H groups present in the urea moiety, whereas it could act as hydrogen-acceptor through the carbonyl-oxygen in the same functional group. In addition it could interact through weak interactions, such as London dispersion forces due to the presence of highly hydrophobic aromatic rings. The ideal solubility of a crystalline solute in a liquid solvent can be calculated by Eq. 1:
ln X 2id = −
ΔH fus (Tfus − T ) ⎛ ΔC p + ⎜⎜ RTfusT ⎝ R
⎛ T ⎞⎤ ⎞ ⎡ (Tfus − T ) ⎟⎟⎥ ⎟⎟ ⎢ + ln⎜⎜ T T ⎠⎣ ⎝ fus ⎠⎦
(1)
Where, X 2 is the ideal solubility of the solute as mole fraction, ΔHfus is the molar enthalpy id
of fusion of the pure solute (at the melting point), Tfus is the absolute melting point, T is the absolute solution temperature, R is the universal gas constant (8.314 J⋅mol–1⋅K–1), and ΔCp is the difference between the molar heat capacity of the crystalline form and the molar heat capacity of the hypothetical super-cooled liquid form, both at the solution temperature. Since ΔCp cannot be easily determined, in this investigation it is assumed that ΔCp may be approximated to the entropy of fusion (ΔSfus = 79.4 J⋅mol-1⋅K-1). Values of ideal and aqueous solubility are presented in Table 1. Since the thermal behavior is a function of the solutesolute interactions in the crystalline lattice, the stronger these forces are, the more insoluble the solute is. As opposed to triclosan that melts around 56oC (and shows an enthalpy of fusion of 17.75 kJ/mol) [33], TCC displays an extremely high melting point around 255oC, supporting the presence of strong solute-solute forces. In order to characterize the ideal solubility of crystalline TCC, the enthalpy of fusion was determined by DSC. The high value observed (41.94 kJ/mol) supported the very poor solubility of the drug observed in water (~50 ng/mL) (Table 1). The low solubility of TCC was also supported by calculations according to the group contribution methods of Fedors and van Krevelen (Table 2). Based on the analogy
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that ‘like dissolves like’, the gap between the value of the parameter of solubility (δ ) of TCC (δ = 26.5 MPa1/2) and the one of water (δ = 48.0 MPa1/2) supported the limited drug solubility in aqueous medium. Even though some minimal solubilization improvement could be theoretically attained below the critical micellar concentration (by means of the interaction between isolated amphiphile molecules known as unimers and drug molecules), maximization of the inclusion phenomenon relies on the presence of the micelles. Due to their structure, poloxamines are both temperature and pH-responsive. In general, pKa values are between 4 and 6 for the first amine group and around 6-8 for the second [34]. Thus, depending on the pH of the medium, from diprotonated to unprotonated forms (going through a monoprotonated one) are available. When positively-charged (pH < pKa), central ethylenediamine groups repel each other and hinder micellization, leading to an increase in the CMC; repulsion is stronger for diprotonated than for monoprotonated forms. Once the molecule displays an uncharged form (at pH > 8), the CMC usually decreases sharply. Consequently, the efficiency of the solubilization usually increases at higher pH-values. On the other hand, the affinity of TCC/poloxamine affinity also plays a fundamental role. Thus, the present study focused on polymer concentrations above the CMC for both T1107 and T1307, in the whole range of pHvalues [30,34]. Two main factors influencing the aggregation phenomenon and the solubilization were evaluated: (A) the concentration of the polymer and (B) the pH. Due the high enthalpy of fusion shown by TCC and the tremendously low solubility in water, a more limited solubilization of the drug in the hydrophobic polymeric core in comparison to triclosan (a previously investigated biocide) was predicted. The apparent solubility (Sa) of TCC in T1107 and T1307 micellar systems (1 to 10%) saturated with the drug was determined at pH-values between 2.0 and 12.7 (Figure 1). Findings indicated a sharp increase as the concentration of both amphiphiles rose. For example, for T1107 at pH 7.4, Sa increased from ~ 50 ng/mL in poloxamine-free medium to 6.4, 21.48 and 412.6 μg/mL for 1, 5 and 10% systems, respectively. It is worth stressing that these results meant an increase in solubility of up to more than 3 orders of magnitude. A decrease in the pH (see pH-values between 2.0 and 7.4) hindered the micellization due to the repulsion between the positivelycharged ethylenediamine blocks and rendered lower Sa levels. These findings were in agreement with previous reports that indicated the concomitant decrease in solubility as the pH decreased [35]. Due to the unprotonated character of poloxamine molecules at pH 12.7, an even higher solubility was a priori envisioned. However, TCC contains a ureide moiety in the structure that displays the ability to form hydrogen bonds with the polyether chain. Ionization of this functional group at pH-values above the pKa (~ 12) is supported by the increase in the intrinsic solubility of the drug above this pH from 50 to 300 ng/mL. Thus, even if micellization was favored under these conditions, a more limited interaction between the drug and the polymer resulted in lower solubilization extents between 2.5 and 127.6 μg/mL for 1 and 10% systems, respectively. T1307 displays a higher molecular weight and a consequent enlargement of the micellar core (from about 4500 to 5400 Da) that could favor a more efficient solubilization. On the other hand, HLB data indicate that T1307 presents, overall, a more hydrophilic structure. Results with T1307 followed a similar trend for similar polymer concentrations and pH conditions; solubility gradually increased from pH 2.0 to 7.4 and then decreased at pH 12.7. It is worth remarking that in this case values increased utmost 4 orders of magnitude. The sharp increase in the apparent solubility of TCC as the pH rose can be
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further evaluated in Table 3, where calculations of MSR are presented. In general, values steadily increased in the pH range between 2.0 and 7.4 due to the higher aggregation tendency of the amphiphiles. Then, under more basic conditions (pH 12.7), where the drug was ionized, a sharp decrease was apparent. A similar trend was observed as a function of the polymer concentration in the range 3-10% and pH-values between 5.8 and 12.7. The analysis of (A) 1% systems and (B) specimens at pH 2.0 (at every concentration) was less straightforward. In both conditions, the apparent solubility (expressed in μg/mL) was relatively low. However, MSR values were, often, higher than those calculated for 3 and 5%, indicating a higher number of solubilized TCC molecules per poloxamine molecule. An interesting phenomenon is also the one observed for T1107 at pH 2.0 where MSR values gradually decreased between 1 and 7% and then showed a slight recovery for a 10% sample. T1307 (pH 2.0) showed a similar tendency though the recovery was apparent at a lower concentration: 7%. These results were particularly observed under conditions where micellization was strongly hindered due to (A) very low polymer concentrations slightly above the CMC (~0.5-1%) and (B) repulsion of poloxamine molecules derived from diprotonation. Previous investigations showed that the lower the pH the smaller the size of poloxamine aggregates is [35,36]. In the proximity of the CMC (1% systems) at pH 2.0, the presence of very small aggregates (including a high incidence of unimers in the composition) probably led to higher MSR values. This, even with very low Sa levels. Then, as a consequence of the increase in the polymer concentration, more micelles were generated and a sudden decrease in the molar ratio was found (even if higher TCC amounts were solubilized). Finally, additional increase in the poloxamine concentration led to a substantial recovery in MSR. These findings also suggested the presence of a phenomenon where larger micelles are formed, enabling the inclusion of a higher number of TCC molecules per polymer molecule. On the other hand, the solubilization ability of poloxamines for TCC was lower than the one found for TS [30]. These findings expectedly emerged from the stronger solute-solute forces showed by the former and a more limited tendency to interact with the liquid-like hydrophobic micellar core.
3.2. Effect of TCC on the Aggregation of Poloxamine Inclusion of TCC into the polymeric micelles was expected to affect the size and size distribution of the aggregates [37]. TCC-loaded micelles of 3, 5 and 7% poloxamine systems (at pH 7.4) were evaluated by means of DLS (Figure 2). An increase in the micellar size due to the enlargement of the core associated with the incorporation of the solute molecules could be anticipated [37,38]. Findings of T1107 and T1307 indicated the presence of two main populations (Table 2). Small size aggregates (~6-8 nm) were mainly consistent with the presence of regular drug-loaded micelles. However, this population decreased in intensity as the polymer concentration rose (and consequently the drug loading); the area of the small-size peaks was 82.9 to 56.5% for 3 and 7% samples, respectively, for T1107 micelles. By the contrary, for the same amphiphile, the presence of larger aggregates (~65-110 nm) suggested the enlargement of the micelles due to the incorporation of TCC into the core. In addition, the higher the polymer concentration was, the higher the incidence of this large-size fraction. Results for T1307 followed a similar trend as the recurrence of the large-size micelles increased for high polymer concentrations from 46.3 to 63.0% for 3 and 7% T1307 systems, respectively. On the other hand, these aggregates displayed a smaller size than those
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generated by T1107 and a slight decrease in the size of the aggregates was found as the polymer concentration rose. This behavior was probably associated with both the higher hydrophilicity and the larger shell of T1307 micelles that rendered more stable aggregates. It is worth mentioning that TCC/poloxamine systems were completely transparent and supported the fact that micellar fusion was not involved in the aggregates enlargement process [30]. Visualization of TCC-loaded systems by means of TEM is exemplified for 10% T1107 micelles in Figure 3. Findings showed the presence of spherical aggregates of different sizes and supported the DLS data. Also here, no evidence of secondary aggregation was found [30]. It is noteworthy that size determination by TEM was unreliable due to the nonrepresentative character of the sample.
12
Intensity (%)
10 8 3% 5% 7%
6 4 2 0 1
10
100
1000
Size (nm)
(A) 10
Intensity (%)
8 6
3% 5% 7%
4 2 0 1
10
100
1000
Size (nm)
(B) Figure 2. Micellar size and size distribution (% intensity) of TCC-loaded 3, 5 and 7% T1107 (A) and T1307 (B) systems.
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Figure 3. TEM micrographs of TCC-saturated 10% T1107 micelles negatively stained with 2% phosphotungstic acid at pH 7.4. Different sizes are apparent. Scale bar = 100 nm (microscope mag. = 100,000x).
Table 4. Micellar size and size distribution (% intensity) of TCC-loaded 3, 5 and 7% T1107 and T1307 systems Poloxamine
T1107
T1307
Poloxamine concentration (%) 3 5 7 3 5 7
Peak 1 Size (nm) %
Peak 2 Size (nm) %
8.03 6.89 6.51 8.05 6.97 6.05
89.81 64.11 112.7 76.4 54.3 50.3
82.9 68.8 56.5 52.2 53.3 37.0
17.1 31.2 43.5 47.8 46.7 63.0
3.3. Stability of the TCC-Loaded Micelles upon Storage Solubilization encompasses the inclusion of TCC molecules into the hydrophobic core of the aggregates. Usually, under regular storing conditions, temperature can fluctuate, affecting the micellization of the thermo-responsive poloxamines. In this context, a decrease in temperature would lead to the disassembly of the micelles and a decrease in the micellar concentration, often to concentration levels close or below the CMC. Moreover, according to
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the thermodynamic calculations (strong solute-solute interactions), TCC displays a high tendency to aggregate. Consequently, in the case of TCC/poloxamine complexes, a precipitation phenomenon and a titer loss over time was envisioned. Different drugcontaining specimens (1-10% of pH 7.4) were maintained at room temperature and the concentration of the drug determined after 1 month. A T1107 1% system displayed a sharp drug-load loss of about 64% and the appearance of insoluble drug crystals. Then, higher polymer concentrations improved the stability upon storage. For example 10% systems of T1107 and T1307 lost only 16.6 and 15.6% of the initial TCC loading, respectively. Also, a slight increase in stability was observed for T1307, consistent with its more stable structure in water due to a higher hydrophilicity [24]. It is remarkable that TCC/poloxamine complexes presented a more limited stability than the highly stable TS/poloxamine ones that remained almost unchanged for at least 3 months [30]. Solubilization relies on the generation of strong solute-solvent interactions in order to overcome solute-solute and solvent-solvent ones. The ΔHf displayed by TCC was substantially higher than the reported for TS and indicated much stronger solute-solute forces. Thus, as expected, weaker drug/core and stronger drug/drug interactions resulted in a higher sensitivity of the complex to small changes in temperature and a higher tendency of the drug to precipitate.
3.4. Antibacterial Activity of TCC/Poloxamine Systems According to previous reports, TCC displays a relatively limited antibacterial spectrum and effectiveness. Studies of the antibacterial activity of the complex aimed to explore if the low activity of the drug was an intrinsic limitation or, otherwise, it stemmed from the poor solubility attainable under regular conditions (e.g., in water). In contrast to a previously published study on TS where 5% polymer were used, herein, due to the more limited solubility of TCC and its lower antibacterial activity, more concentrated poloxamine solutions (10%) were tested. Plate assays indicated a slightly broadening of the inhibition area when Gram positive Staphylococcus aureus and methicilin-resistant Staphylococcus aureus (clinical strain) were exposed to the substantially higher biocide concentrations attained in micellar systems. On the other hand, findings stressed the substantially lower activity of this agent compared to the previously investigated TS and suggested that the poor antibacterial activity relied on the intrinsic resistance of the different pathogens to the antibacterial agent. These results contrasted with previous reports indicating minimal inhibitory concentrations of TCC in the 0.5-5 μg/mL range. In addition, inhibition for drug-loaded T1107 specimens was more limited probably due to the lower concentration attained.
4. CONCLUSION Inclusion of the poorly water-soluble drug triclocarban into poloxamine polymeric micelles enhanced the aqueous solubility in up to 4 orders of magnitude. In agreement with previous reports, higher solubilization extents were found at higher pH values in the range between 2.0 and 7.4. In contrast, a pronounced decrease in solubility was found at pH 12.7, regardless the more efficient micellization of the polymers under these conditions. This
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phenomenon could be explained by the pH-sensitive ionization of the -NH-CO-NH- moiety of TCC under this pH. At a lower pH (e.g., 7.4) where aggregation was less favored, unionized ureide groups formed H bonds with the poly(ether) chain and solubilization was enhanced. By the contrary, once unprotonated, TCC molecules displayed a weaker interaction with the polymer and a lower solubility was found. Finally, the activity of the complexes was assayed on a number of bacteria. Even though complexation slightly increased the effectiveness of the antibacterial agent under study, activity was dramatically lower than that demonstrated by TS. Overall findings supported the relatively limited activity of TCC even at concentrations significantly higher than those attainable in poloxamine-free media.
ACKNOWLEDGMENTS This work was partially supported by the University of Buenos Aires and the CONICET (Grant UBACyT-B424).
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[10] Witte, W. (2004). International dissemination of antibiotic resistant strains of bacterial pathogens, Infect Gen Evol, 4, 187-191. [11] Kasim, N. A., Whitehouse, M., Ramachandran, C. h., Bermejo, M., Lennernäs, H., Hussain, A. S., Junginger, H. E., Stavchansky, S. A., Midha, K. K., Shah, V. P. & Amidon G. L. (2004). Molecular properties of WHO essential drugs and provisional biopharmaceutical classification. Mol Pharmaceutics, 1, 85 -96. [12] Lindenberg, M., Kopp, S. & Dressman, J. B. (2004). Classification of orally administered drugs on the World Health Organization Model list of essential medicines according to the biopharmaceutics classification system. Eur J Pharm Biopharm, 58, 265-278. [13] Breneman, D. L., Hanifin, J. M., Berge, C. A., Kewick, B. H. & Neumann, P. B. (2000). The effect of antibacterial soap with 1.5% triclocarban on Staphylococcus aureus in patients with atopic dermatitis. Cutis, 66, 296-300. [14] Luby, S., Agboatwalla, M., Feikin, D., Painter, J., Billhimer, W., Altaf, A. & Hoekstra R. (2005). Effect of handwashing on child health: a randomised controlled trial. Lancet, 366, 225-233. [15] Beaver, D. J., Roman, D. P. & Stooffel, P. J. (1957). The preparation and bacteriostatic activity of substituted ureas. J Am Chem Soc, 79, 1236-1245. [16] Black, J. G., Howes, D. & Rutherford, T. (1975). Skin deposition and penetration of trichlorocarbanilide. Toxicology, 3, 253-264. [17] Loftsson, T., Leeves, N., Bjornsdottir, B., Duffy, L. & Masson, M. (1999). Effect of cyclodextrins and polymers on triclosan availability and substantivity in toothpastes in vivo, J Pharm Sci, 88, 1254-1258. [18] Sosnik, A., Carcaboso, A. M. & Chiappetta, D. A. (2008). Polymeric Nanocarriers: New endeavors for the optimization of the technological aspects of drugs. Rec Pat Biomed Eng, 1, 43-59. [19] Loftsson, T., Össurardótti, Í. B., Thorsteinsson, T., Duan, M. & Másson, M. (2005). Cyclodextrin solubilization of the antibacterial agents triclosan and triclocarban: Effect of ionization and polymers. J Incl Phenom Macrocycl Chem, 52, 109-117. [20] Duan, M. S., Zhao, N., Össurardóttir, Í. B., Thorsteinsson, T. & Loftsson, T. (2005). Cyclodextrin solubilization of the antibacterial agents triclosan and triclocarban: Formation of aggregates and higher-order complexes. Int J Pharm, 297, 213-222. [21] Kataoka, K., Harada, A. & Nagasaki, Y. (2001). Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv Drug Deliv Rev, 47, 113-131. [22] Croy, S. R. & Kwon, G. S. (2006). Polymeric Micelles for drug delivery. Curr Pharm Design, 12, 4669-4684. [23] Moghimi, S. M. & Hunter, A. C. (2000). Poloxamers and poloxamines in nanoparticle engineering and experimental medicine. TIBTECH, 18, 412-420. [24] Chiappetta, D. A. & Sosnik, A. (2007). Poly(ethylene oxide)–poly(propylene oxide) block copolymer micelles as drug delivery agents: Improved hydrosolubility, stability and bioavailability of drugs. Eur J Pharm Biopharm, 66, 303-317. [25] Bromberg, L. E. & Ron, E. S. (1998). Temperature-responsive gels and thermogelling polymer matrices for protein and peptide delivery. Adv Drug Del Rev, 31, 197-221.
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[26] Kabanov, A. V. & Alkhov, V. Yu. (2002). Pluronic® block copolymers in drug delivery: From micellar nanocontainers to biological response modifiers. Critical Rev Therap Drug Carrier Syst, 19, 1-72. [27] Kibbe, A. H. (2000). Handbook of Pharmaceutical Excipients, American Pharmaceutical Association: Washington D. C.; 386-388. [28] Dong, J., Chowdhry, B. Z. & Leharne, S. A. (2003). Surface activity of poloxamines at the interfaces between air–water and hexane–water. Colloid Surface A: Physicochem Eng Aspects, 212, 9-17. [29] Sosnik, A. & Sefton, M. V. (2006). Methylation of poloxamine for enhanced cell adhesion. Biomacromolecules, 7, 331-338. [30] Chiappetta, D. A., Degrossi, J., Teves, S., D’Aquino, M., Bregni, C. & Sosnik, A., (2008). Triclosan-loaded poloxamine micelles for enhanced antibacterial activity against biofilm. Eur J Pharm Biopharm, 69, 535-545. [31] Barton, A. (1991). Handbook of Solubility Parameters and Other Cohesion Parameters. 2nd Ed. New York: CRC Press; 157-193. [32] Martínez, F., Ávila, C. M. & Gómez, A. (2003). Thermodynamic study of the solubility of some sulfonamides in cyclohexane. J Braz Chem Soc, 14, 803-808. [33] Veiga, M. D., Merino, M., Cirri, M., Maestrelli, F. & Mura, P. (2005). Comparative study on triclosan interactions in solution and in the solid state with natural and chemically modified cyclodextrins. J Incl Phenom Macrocycl Chem, 53, 77-83. [34] Gonzalez-Lopez, J., Alvarez-Lorenzo, C., Taboada, P., Sosnik, A., Sandez-Macho, I. & Concheiro A. (2008). Self-associative behavior and drug solubilizing ability of poloxamine (Tetronic®) block copolymers. Langmuir, 24, 10688-10697. [35] Alvarez-Lorenzo, C., Gonzalez-Lopez, J., Fernandez-Tarrio, M., Sandez-Macho, I. & Concheiro, A. (2007). Tetronic micellization, gelation and drug solubilization: Influence of pH and ionic strength. Eur J Pharm Biopharm, 66, 244-252. [36] Kabanov, A. V., Nazarova, I. R., Astafieva, I. V., Batrakova, E. V., Alakhov, V. Y., Yaroslavov, A. A. & Kabanov, V. A. (1995). Micelle formation and solubilization of fluorescent probes in poly(oxyethylene-β-oxypropylene-β-oxyethylene) solutions. Macromolecules, 28, 2303-2314. [37] Riess, G. (2003). Micellization of block copolymers. Prog Polym Sci, 28, 1107-1170. [38] Allen, C., Maysinger, D. & Eisenberg, A. (1999). Nano-engineering block copolymer aggregates for drug delivery. Colloid Surface B: Biointerfaces, 16, 3-27.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 6
SILOXANE-CONTAINING COMPOUNDS AS POLYMER STABILIZERS Carmen Raclesa* , Thierry Hamaideb and Etienne Fleuryb a
“Petru Poni” Institute of Macromolecular Chemistry, Iasi, Alea Gr. Ghica Voda 41A, RO700487, Romania. b Université de Lyon F-69003 Lyon, France. Ingénierie des Matériaux Polymères. CNRS, UMR 5223. 69622 Villeurbanne, France.
ABSTRACT Generally, surfactants are used as stabilizers of interfaces or particles and their applications are very wide, from foams or adhesion modifiers to the orientation of chemical reactions. Siloxane surfactants are known for their ability to decrease the surface tension of liquids in such extent that is comparable only with some fluorinated compounds, which are thought to exhibit potential toxicological problems. On the other hand, polysiloxanes are unique by their set of properties, like for example low glass transition temperature, hydrophobic behavior, transparency to visible and UV light, high permeability to various gases (especially oxygen), physiological inertness, excellent blood compatibility (low interaction with plasma proteins). In addition, their chemistry is very versatile, and as a result, a very broad range of siloxane-organic compounds can be synthesized, including amphiphilic macromers or polymers. The most commonly known siloxane surfactants are the so called „silicone polyethers”, but other nonionic, as well as ionic surface active agents have been prepared and used over the years in cosmetics, textile conditioning, foam stabilization, coatings or agriculture. Recent developments in this research field and especially our experimental results on the synthesis, properties and applications of siloxane-containing surfactants will be reviewed. Our main interest is to propose new surfactants or alternative synthetic procedures, and new stabilization systems for polymeric nanoparticles. Carbohydrate modified (poly)siloxanes with different architectures have particularily been studied and tested, due to their biocompatibility and bioavailability. *
Corresponding author: e-mail:
[email protected] 214
Carmen Racles, Thierry Hamaide and Etienne Fleury
INTRODUCTION Surfactants are crucial for certain applications in the modern technique, from laundry and heavy washing to personal care or medicine. The most recent applications of surfactants are related to nanotechnologies; they are used as templates for nano-patterning, as well as for the stabilization of nanoparticles -the „new wave” in science- [1]. Various surface active agents have been synthesized and investigated, as well as their interaction with polymers [2]. New surfactants continue to appear, and even slight modifications of the molecular structure are very useful for understanding of their properties [3]. In the domain of polymer stabilization (polymerization in dispersed media as well as using physico chemical procedures), it becomes of first importance to anchor the surfactant onto the surface of the particles not only to avoid flocculation, but also to limit water pollution. In the case of emulsion polymerization, a good way to overcome these drawbacks is to use polymerizable surfactants (also called "surfmers") [4-6]. Polymeric surfactants, and particularly block and grafted copolymers, offer another quite versatile way to stabilize nanoparticles [7], with enhanced properties with respect to the molecular emulsifiers. The consequence is excellent surface properties at low concentrations, which allows using lower amounts of emulsifier for the stabilization of emulsions [8]. This is an obvious economical benefit, and the fact that the residual concentration of emulsifier in the aqueous phase is lower, brings another advantage limiting secondary effects such as air entrapment and foaming, or adsorption at various interfaces. Among polymeric surfactants, polyethylene oxide - polypropylene oxide (PEO-PPO) diblock and triblock copolymers (Pluronics® or Poloxamers®) are well-known and their properties and uses are broadly documented [9, 10]. The hydrophobic PPO block can advantageously be replaced by a biodegradable block polyester such as polycaprolactone or polylactide [11-16]. In the field of surface active compounds, one cannot disregard the unique properties of siloxanes as hydrophobic components in connection with such applications. Siloxane surfactants are used since the 1960s in textile conditioning, polyurethane foams, as adjuvants in agriculture, paint additives, cosmetic formulations and so on. Their uniqueness is their solubility both in water and in organic solvents, reducing the surface tension of all these media and exhibiting hydrophobic as well as oleophobic properties. The reasons for this unique behavior are the high flexibility and low cohesive energy of the siloxane bond. The siloxane backbone can adapt to the interface geometry very easily, without creating steric hindrance due to this flexibility [17, 18]. On the other hand, there are some additional properties that make siloxane compounds very attractive. For example, polydimethylsiloxanes (PDMS) have a very low glass transition temperature, transparency to visible and UV light, high permeability to various gases (especially oxygen), physiological inertness, excellent blood compatibility (low interaction with plasma proteins) [19]. The very low surface tension of the polysiloxanes, along with their versatile chemistry and biocompatibility increased the interest for siloxane-containing surfactants. Thus, amphiphilic compounds of all kinds of molecular architectures and variable chemical structures have been synthesized and studied as surface active materials. A wide range of organic moieties, namely nonionic, anionic, cationic and zwitterionic organic groups, have been linked to polysiloxane backbones in order to get soluble products [20]. Siloxane surfactants are usually required in small amounts and often provide better performances then their purely organic counterparts. For example, it was calculated that one
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Si(CH3)2 unit can be compared to 4 CH2 units, while a C(CH3)2 unit is equivalent to 2 CH2 groups in terms of surface activity [21]. Many of these surfactants are largely used and commercially available. The first and thus the most studied were those having polyethyleneoxide as hydrophilic part, either as block copolymers or as trisiloxanes (Schemes 1-3). Only some of the most recent developments in this class of surfactants will be reviewed here, since the subject is quite vast and other comprehensive references are available. In various domains, the choice of surfactants is not dictated only by their surface properties, but is limited by many other constraints. This is particularly true for applications that concern living organisms, environment and humans. That is why a more and more important topic is that of biocompatibility and / or biodegradability. Carbohydrate-containing siloxane surfactants have attracted much attention, since they can be obtained from a natural resource and thus be more eco-friendly. The reports on this particular class of siloxane surfactants will be summarized, including our own contribution.
1. SILICONE POLYETHERS (SPE) The most commonly known siloxane surfactants contain polyoxyalkylenes (polyethers) as hydrophilic parts. They have been investigated since the 1960s and silicone polyethers have practically become the definition itself of siloxane surfactants. Their effectiveness in organic systems as well as in water and their use in cosmetics, textile conditioning, foam stabilization, coatings and agriculture has been reported [22-28]. The polyether groups are polyethylene oxide (PEO) and/or polypropylene oxide (PPO), and are attached as side chains to the siloxane backbone through a hydrosilylation or condensation process. They can have a linear or grafted (comb) architecture and a great diversity of structures are available (Schemes 1-4), since molecular weight and composition of the polyether chain (PEO/PPO) as well as the functionality of the end group and the ratio of siloxane to polyether can also be varied. Siloxanes with a very high molecular weight are more suitable for forming water/oil emulsions [29]. Depending on their solubility parameters, silicone polyethers can stabilize foams or act as antifoaming agents [30].
Me3SiO
Me3SiO
Me
O
Si
(CH2)3 O CH2 CH2 O
SiMe3 y
m
R
Scheme 1. Polyether trisiloxane surfactants
Me Si
O
(CH2)3 O CH2 CH2 O
SiMe3 y
m
CH2CH O H CH3 n Bluestar Silicone H3303.
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Carmen Racles, Thierry Hamaide and Etienne Fleury CH3
CH3
(CH3)3-Si-O
Si O
Si O
x
y
CH3
CH2
Si-(CH3)3
3
(OCH2CH2) OR n
CH3
CH3
RO(CH2CH2O) (CH2)3 Si O n CH3
CH3
Si O x
CH3
Si (CH2)3 (OCH2CH2)n OR CH3
Scheme 2. General structures of the main types of SPE copolymers
Me3SiO
Me Si Me
O
Me Si
O
x (CH ) 2 2
Me Si
Me
O
Si
y (CH ) 2 3
O Si(CH3)2 CH2 O n CH 2 SiMe3 OH
O
(CH)p
Me3SiO
SiMe3 z
Si
O
Me
Me
CH3
Me
O
Si x (CH ) 2 2 Si(CH3)2 O SiMe3
m
O
Si y
(CH2)3
SiMe3 z
O CH2 n CHOH CH2
3
OH
Shin-Etsu - KF 6038 Me3SiO
Me
Shin-Etsu - KF 6104 Me Si
Me
O
Me
O
Si x
(CH2)3 O CH2 CH2 O
SiMe3 y
m
CH2CH O R CH3 n
Wacker-Belsil® DMC 6031 PEG/PPG - 25/25 Dimethicone Goldschmidt. ABIL B 8851. n = 20, m =5, x/y = 75/25 Goldschmidt. ABIL B 8873. n = 20, m =5, x/y = 35/65 Bluestar silicone SP3300. n = m = 22. x/y = 90/10 Scheme 3. Examples of commercial surfactants
Extensively studied nonionic surfactants are PDMS / polyether block copolymers [31] and poly(ethylene oxide) substituted trisiloxanes [32-35]. They are able to achieve a maximum surface tension depression to about 21 mN/m and a low critical micelle concentration (CMC) [36]. These siloxane surfactants can self-assemble into a variety of
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ordered morphologies in the melt as well as in solution, depending on the volume fractions of the hydrophilic units [37].
1. A. RECENT DEVELOPMENTS IN THE SYNTHESIS AND INVESTIGATION OF SPES The preparation, uses, and physical chemistry of silicone surfactants have been described in a well-known reference [38], focusing on silicone polyoxyalkylene copolymers. This book explores many aspects of SPEs as surfactants, like interfacial processes, surface viscoelasticity, and aggregation, explains the unusual wetting behavior of the trisiloxane surfactants and the ternary phase behavior of mixtures of silicone surfactants with water and silicone oils. That is why we only try to emphasize some newer insights into the subject, reported mainly after year 2000. From the chemical point of view, some improvements in the structure and synthesis of SPEs have been made. In order to avoid Si-O-C linkages, which are susceptible to hydrolytic instability, hydrosilylation reactions are used to prepare these compounds, starting from unsaturated precursors [21, 39-41]. Triblock PEO-PDMS-PEO copolymers have been obtained by anionic polymerization of ethylene oxide using a PDMS diol as transfer agent. These copolymers have been fully characterized by NMR and Maldi-Tof mass spectrometry. As expected, the solubility depends on the PEO/PDMS ratio [42]. It was found that, when the hydrophilic functional groups are linear or when the endcapped units of the hydrophilic groups are of hydroxy type, the hydrolytic stability decreases and chemical attack of silicone surfactants backbone occurs easily in the aqueous solution. A way to avoid this is to synthesize grafted copolymers or to use PEO blocks having methoxy end groups [43]. Although such siloxane surfactants were extensively exploited, their surface active properties are still investigated [21, 27, 44]. All types of aggregates known for hydrocarbon surfactants comprising micelles, lyotropic liquid crystalline phases from lamellar to the inverse hexagonal phase, vesicles, and "sponge" (L3) phases have been found in aqueous solution [37, 45]. It was found that the minimum ratio which insures water solubility in siloxane surfactants is 9 PEG units to 29 siloxane groups. The critical aggregation concentration decreases systematically with the decrease in the hydrophilic PEG substitutions. The surface and selfassembling properties have been investigated with surface tension, viscosity, fluorescence, and contact angle measurements [21]. Recent investigations still approach different aspects of this complex subject, like selfassembling behavior, adsorption on different substrates, rheology. Many studies have been undertaken for siloxane surfactants in dilute aqueous solutions, but their potential applications in organic solvents or as nanoreactors are not yet fully investigated. The CMC, micelle hydrodynamic diameter, microviscosity in the micelle core in water and in mixtures of water with polar solvents have been investigated for a polyoxyalkylene siloxane surfactant, by means of fluorescence [46].
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The self-aggregation in long PEO-PDMS surfactants showed their ability to form inverse micelles and liquid crystals in nonpolar oils, including octamethylcyclotetrasiloxane (D4). These structures, which are not found in conventional hydrocarbon surfactant systems, were explained by the segregation tendency of the PEO chains and the flexibility of the siloxane chains [27]. A polyethyleneoxide-b-polydimethylsiloxane-polyethyleneoxide surfactant, (EO)15(DMS)15-(EO)15, was studied with freeze-fracture transmission electron microscopy and pulsed-field gradient nuclear magnetic resonance spectroscopy, in order to establish the effect of glycerol on the permeability of vesicle membranes. Small vesicles with diameters of less than 25 nm and multilamellar vesicles with diameters larger than 250 nm were observed in pure water, which were modified when water was gradually replaced with glycerol [47]. Polydisperse (PPO-b-PEO) allyl ether siloxane surfactants were synthesized by the hydrosilylation reaction of 1,1,1,3,5,7,7,7 octamethyltetrasiloxane (MD2'M) with oligo(PO-bEO) allyl ethers. In this series, the surface tension increased with increasing the EO chain length and it decreased with increasing the PO ratio, while the sedimentation time of the aqueous solution showed opposite trend. The cloud point temperatures tended to increase with the increase in the EO chain length and decrease of the PO ratio [48]. Recently, a series of well defined amphiphilic poly[methyl(3,3,3-trifluoropropyl)siloxane]-b-poly(ethylene oxide) (PMTFPS-b-PEO) diblock copolymers with different block lengths have been prepared by hydrosilylation [49]. Trisiloxane surfactants containing polyethyleneoxide chains of different lengths, known as superwetters, have been studied by soft-contact AFM imaging and direct force measurements at the solid-liquid interface, using different substrates [50]. The surface aggregate structures for these siloxane surfactants correlate with those of their hydrocarbonbased equivalents and resemble bulk structures. Surface force measurements of ABA type SPEs (Scheme 4), compared to hydrocarbonbased Pluronic surfactants, at the solid/liquid interface in water/ethanol mixtures showed that SPEs are capable of providing steric stabilization at a hydrophobic surface up to 80% and even up to 95% ethanol level (depending on structure), while in the case of hydrocarbonbased surfactants this barrier was 40% [51]. CH3
CH3 Cap
(CH2CH2O) n
(CH2-CH-O) m (CH2)3 CH3
Si O CH3
x
Si CH3
(CH2)3 (OCH2CH2) n
(OCH-CH2)
m
Cap
CH3
Cap: OH or OAc Scheme 4. General formula of triblock SPE surfactants [51]
Although the nature of superwetting remains enigmatic, the surface aggregate structures are studied in order to better understand colloidal phenomena and to approach nanotechnologies by self-assembly of hierarchical structures [50].
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219
1.B. SPEs as Polymer Stabilizers: Applications of SPEs Oil-in-water emulsions obtained with certain SPEs are stable in the presence of alcohol, which is an important advantage for many emulsion and dispersion formulations, where alcohols are common co-solvents or additives. At the same content of ethanol, hydrocarbonbased Pluronic surfactants do not emulsify oil-in-water [51]. Silicones give a unique dry-lubricity feel to surfaces such as textiles, hair, and skin [38]. That is why graft polysiloxane-poly(oxyalkylene) copolymers are used for making water-insilicone oil (w/o) emulsions for personal care products [52]. The question of how to solubilize polymeric silicone oils, preferentially in microemulsions, is of high technological importance [53] and has received special attention. Silicone surfactants alone or in combination with short-chain alkanols are efficient emulsifiers for short chain siloxanes. It was found that organic surfactants are not as suitable as silicone surfactants for stabilizing silicone/water emulsions [54]. The chemical affinity of the hydrophobic part of the surfactant to the silicone oil is a benefic factor, helping the solubilization process [53]. Silicone oil in water (o/w) emulsions are more difficult to stabilize. To prepare such dispersions, nonionic PDMS-polyoxyalkylene copolymers with high degree of polyoxyalkylene substituents are generally used, in order to render the surfactant more hydrophilic. Thus, the resulting emulsions are sterically stabilized by the polyether chains [54]. Viscoelastic and viscosimetic measurements have been performed on model systems and on commercial products [29]. Water-in-silicone emulsions have been prepared, in which the oil phase consisted of decamethylcyclopentasiloxane (cyclomethicone, D5) and the surfactant used was a branched type silicone copolymer. The interfacial rheology study of such surfactants revealed that the ability of these copolymers to effectively stabilize water-in-D5 emulsions is a result of a process involving the nucleation, growth, and accumulation of surfactant-rich particulates at the D5/water interface [55]. The w/o/w or o/w/o emulsions are important, in particular when the protection of sensitive ingredients or controlled release of active substances is required [54]. Silicone-based emulsifiers are suited for these applications. They are strongly adsorbed at the oil interface, and do not migrate from one interface to the other, thus preventing the destabilization. Two emulsifiers have to be used to stabilize these multiphase emulsions. The hydrophilic– lipophilic balance (HLB) values of the emulsifiers should be above 10 for the hydrophilic emulsifier and below 6 for the hydrophobic emulsifier [54]. The ability of SPEs to form vesicles, which are structurally comparable to liposomes, has opened a wide range of possibilities for the incorporation of active ingredients. Silicone vesicles can have a diameter from about 0.05 to 1 micron and an internal volume of 10-6 μm3. The membrane thickness of these vesicles is about 3 to 4 nm [56]. The main application of siloxane-based surfactants vesicles is in cosmetics. For example, using silicone vesicles, hydrophilic and hydrophobic active substances can be separated and protected from each other, thus reducing for example skin irritancy. Active delivery systems include non-aqueous emulsions of polyols in silicone fluids, multiple-phase emulsions, and polar solvent-in-oil emulsions. Silicone-based surfactants offer many benefits in waterborne coatings. The coating surface tension can be modified to improve substrate wetting, in particular when trisiloxane polyethers are used, since these structures provide excellent wetting to low-energy substrates,
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Carmen Racles, Thierry Hamaide and Etienne Fleury
like plastics. Copolymers with linear or grafted structures give moderate wetting, but can provide mar resistance, slip and de-aeration [30]. For waterborne coatings and ink formulations, polymeric surfactants are used in conjunction with polar organic solvents. The numerous patents covering this area of research are discussed in a review [57]. In view of such applications, the adsorption of a grafted (rake-type) polymeric siloxane surfactant containing 48% PDMS, 39% PEO, and 13% PPO on carbon black particles dispersed in mixtures of water with polar organic solvents has been investigated [58]. The adsorption was found to obey the Langmuir isotherm below the critical micelle concentration and a sharp increase in the adsorbed amount was observed at higher surfactant concentrations. DLS and SANS data indicate that the structure of the adsorbed layer is similar to that of micelles. An aqueous laundry stain pretreater composition which would provide outstanding cleaning performance on both oil and water based stains on a variety of fabrics has been developed using polyalkylene oxide modified trisiloxanes [59]. Trisiloxane surfactants have been used as adjuvants in agriculture. They increase the foliar uptake of pesticides, due to reduction of surface tension and superspreading. A method for improving the performance of agricultural compositions under conditions of low humidity has been developed, by using silicone surfactants that reduce the surface tension to less than 30 mN/m in 0.10% (w/w) aqueous solution, without concomitant spreading of the spray solution [60]. SPEs can act as stabilizers in chemical reactions. For example, silver nanoparticles with an average diameter of about 3 nm have been obtained inside the aggregates of a PDMS-gPEO copolymer, without adding a reducing agent [61]. Polymerization of halogen-containing monomers in aqueous medium using siloxane surfactants instead of potentially toxic fluoroalkyl surfactants has been described [62].
2. SILOXANE SURFACTANTS WITH CARBOHYDRATE MOITIES Siloxane-polyethers usually have a poor emulsification activity at the siloxane-water interface, which has been explained by the weak intermolecular interactions between the surfactant molecules [63]. This is one reason for seaking surfactants with stronger intermolecular interactions. Carbohydrates are bioavailable, biocompatible, biodegradable, biologically active and suitable for molecular recognition. They are hydrophilic and capable of self-association. So, there are a lot of reasons for using them as partners for siloxane compounds, in order to produce more environmentally friendly surfactants with superior properties. Glycopolymers are often defined as synthetic polymers bearing sugar moieties [64-68]. This definition can also be enlarged to any macromolecular structure resulting from chemical modification of natural polymers (cellulose, dextran ...). They are used in a lot of biomedical application fields including drugs and drug delivery devices because of the ability of sugar moieties to interact with protein receptors (the so-called cluster glycoside effect). In most cases, glycopolymers are amphiphilic polymers because of the presence of hydrophilic and hydrophobic units and they can act as polymer surfactants. These glycopolymers can therefore offer a unique combination of macromolecular recognition and colloidal
Siloxane-Containing Compounds as Polymer Stabilizers
221
stabilisation of nanoparticles in dispersed media to get opportunities for the production of novel targeted drug delivery systems. For instance, maleic copolymers with different contents of galactose moieties and dodecyl chains were synthesized and used as both a stabilizer and a surface coating for the preparation of poly(ε-caprolactone) nanoparticles by the emulsification-diffusion technique. The surface modification of nanoparticles was confirmed by ζ-potential measurements. Nanoparticles were also shown to be recognized by a galactose-specific lectin, demonstrating the presence of galactose units on the particle surface [69]. More recently, 8-amino-3,6-dioxaoctyl α-D-mannopyranoside and 8-amino-3,6dioxaoctyl β-D-galacto-pyranoside were prepared for coupling with a copolymer made from ε-caprolactone macromonomers and maleic anhydride. The glycopolymers thus obtained were used in conjunction with Pluronic® F-68 for the stabilization of PCL nanoparticles prepared by the emulsification-diffusion technique [70]. The combination of carbohydrates and silicones has received a great attention in both scientific and economic media [71]. Hybrid materials, from small molecules, to polymers, oligomers and nanostructured composites have been prepared and their applicability especially in biomedical fields has been proved.
2.A. Synthetic Methods for Carbohydrate – Modified Siloxane Surfactants A broad variety of carbohydrate-modified siloxane surfactants have been synthesized and tested as surfactants [72-75] or in cosmetic formulations [76-79]. Such compounds are currently produced on an industrial scale (see for example Scheme 5) and their use has gradually increased due to their excellent biodegradability, good dermatological compatibility, and the absence of toxic effects. The chemistry of carbohydrate modified polysiloxanes is not easy, starting from the totally different solubility of the reagents and continuing with the instability of the siloxane and glycosidic bonds in certain conditions. Nevertheless, various structures have been obtained, sometimes applying very ingenious solutions to overcome such problems. Different carbohydrate moieties have been used, as well as various HO-protecting procedures (mainly using acetyl or trimethylsilyl derivatives) and subsequent deprotection methods. The most common method for linking carbohydrate derivatives with siloxanes is the hydrosilylation. An alternative is the ring-opening reaction between an amino-functional siloxane and a carbohydrate lactone [79, 81-83], but other methods have also been described and are still developed. Allyl glycosides, allyl ethers and allyl amides of glucose, gluconic acid and glucuronic acid-γ-lactone with protected hydroxyl groups have been used to obtain silane precursors for modified polysiloxanes [84]. A great variety of carbohydrate-modified siloxane surfactants have been prepared by Wagner and coll. [72-75, 78, 79]. They described a synthetic path yielding to straight-chained glycosides-functionalized siloxanes with amide linkages, via allyl-glycidyl ether –modified intermediates [78]. Similar compounds have been prepared from silanes, carbosilanes, polysilanes and non-permethylated siloxanes [73] and the role of the key intermediates for the surface properties of the surfactants has been thoroughly investigated.
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Carmen Racles, Thierry Hamaide and Etienne Fleury Me
Me3SiO
Si
Me
O
Me
Si
Me
O
x (CH ) 2 3
Si y
O
SiMe3 z
(CH2)w
O
CH3 CH2 OH H2C H O O O OH HO ~ 1,8
Scheme 5. A commercially available carbohydrate-modified polysiloxane (Wacker Belsil SPG128VP): Caprylyl Dimethicone Ethoxy Glucoside, 20% in cyclopentasiloxane (D5)) [80]
Different approaches have been used for the hydrosilylation of allylglycosides with various H-functional polysiloxanes [71, 78, 85-87]. Glucopyranosyl- and cellobiosylterminated oligodimethylsiloxanes with thioether or ether linkages have been prepared and tested as transdermal penetration enhancers [87]. The monosaccharide-modified compounds exhibited a pronounced permeation acceleration for antipyrine, while the disaccharide ether siloxane had no enhancing activity. High molecular weight polysiloxanes modified with mono-, di- or oligosaccharides showed different aggregation behavior in solution, depending on the detailed chemical structure of the carbohydrate moiety and its stereochemistry [88]. Gemini surfactants have been obtained from a trisiloxane amine, D-gluconic acid δ−lactone and oligoethylene glycol diglycidyl ethers [89] (Scheme 6). These surfactants reduced the surface tension of water to approximately 21 mN/m at a concentration of around 10-5 mol/L and showed two critical aggregation concentration values. H3C H3C
H3C
CH3
CH3 Si
Si O
CH3
CH3
CH3
H3C H3C
Si CH3 O
O
N
O(CH2CH2O) OH
HO
OH H
H
OH
H
OH CH2OH
Scheme 6. Gemini surfactants [89]
O
N
n OH
NH O
O H
Si
Si
Si
HN
CH3
H3C
H HO
OH H
H
OH
H
OH CH2OH
CH3
Siloxane-Containing Compounds as Polymer Stabilizers
223
Amphiphilic polysiloxanes have been recently obtained by reacting a water-soluble poly(3-aminopropyl)siloxane with fatty acid chlorides to attach hydrophobic moieties and subsequently with gluconolactone which provided the hydrophilic groups. The regularity of the nanoaggregates in water formed from the amphiphilic polysiloxanes was relatively controlled by the chain lengths and functionalities of the hydrophobic parts [90]. Glucose functionalized polysiloxanes have been obtained by transacetalation [63]. Different acidic catalysts have been tested and activated clay was found to be the best, due to the great advantage of avoiding the contamination of the final product. We have described the synthesis of mannose, glucose, galactose or cellobiose modified siloxanes: telechelic difunctional oligosiloxanes, siloxane copolymers with pendant monosaccharide groups and cyclic oligosiloxanes (Scheme 7) by a method involving hydrosilylation and cation exchangers as deprotecting catalysts [91-93]. OH
OH O
O
HO HO
OH
O R
OH
OH O
HO HO
Si O Si n
O
OH
OH
Si
N
Me O
CH 3
OH OH
I
HO
Me
HO O
R O
Me O
Si
N
Me
n
H 3C
Ce llo bio se
II
O
Cellobiose
Si O
Si O R
OH O Si
O
O
III
x
O
Si y
O
OH OH
HO
Si
O
OH
HO OH OH
IV
Scheme 7. Carbohydrate modified siloxanes [91-93]
In this approach, the protection-deprotection method was based on the fragile solubility equilibrium. In a first version [91, 92], the trimethylsiloxy groups were removed in the presence of a cation exchange resin which was used in wet state (gel form), in order to prevent the cleavage of the Si-O bonds. Indeed, cation exchangers with similar exchange capacity are largely used for the polymerization of cyclosiloxanes, in laboratory and in industry [94-97]. The only difference is that these polymerization catalysts are thoroughly dried before use, by azeotropic distillation of toluene, and only traces of water remain in their structure. Thus, the highly hydrophobic siloxanes have access to the active sites. For the deprotection of the saccharide OH groups, the catalyst that we used had a high content of water (50%) and in this situation the active sites are not available for the hydrophobic part of
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Carmen Racles, Thierry Hamaide and Etienne Fleury
the molecules: in other words, the water has a screening effect between the siloxane backbone and the catalytic sites and therefore prevents the breakage. On the other hand, the protected compounds were dissolved in a mixture of THF and methanol. The deprotection occurs gradually and the solubility of the deprotected compounds shifts towards methanol. The higher the content in hydrophilic groups the better the methanol and water solubility. In this case, cleavage of the siloxane bonds may occur, but the phenomenon is limited, especially if the reaction time is strictly controlled. The polymer conformation and self-assembling in solution have probably a great importance in preserving the integrity of the siloxane chains. On the contrary, when the same cation exchanger was used, but after drying to constant weight, the siloxane chain break became more important [93]. The reaction time was significantly reduced, but the chain length control was not possible anymore. However, the glycosidic bonds in cellobiose moieties were not affected by the deprotection catalyst. This deprotection method proved to be as „tricky” as effective, but the overall advantages of ion exchangers have to be considered, and especially the easy removal from the reaction mixture, without significant contamination of the final product. OH O
HO HO
Si
OH HO O
O N Ac
O
OH
N3
O
Si O
OH
OH
Si
OH O
HO HO
OH HO O
N Ac
O
OH
OH
H
Si O
N
N
O
N
Si O
OH
Si OH OH
HO
O O
HO HO
HO m
OH O HO HO
HO HO
O OH OH HO
O
OH HO O O
OH O OH
O
O HO
O O
OH
OH HO O
O OH
N Ac
H
OH
Si O
N
N N
O n
O
O
O OH
Si O Si
OH
Scheme 8. Cellobiose and xyloglucan modified heptamethyltrisiloxane (MD’M) obtained by click chemistry [98]
Although the procedure based on Fischer glycosidation and deprotection of HO- groups using a cation exchanger may be adjusted to produce as less damage to the siloxane chain as possible, it remains dangerous for the integrity of oligosaccharide segments, since those are also cleaved and reformed according to an equilibrium process. Oligosaccharide modified
Siloxane-Containing Compounds as Polymer Stabilizers
225
polysiloxanes can be obtained, but the precise control of their structure remains uncertain with this procedure. An alternative may be the „click chemistry” applied to silicon-based materials. This reaction is quite versatile and appropriate to be used with compounds with different solubilities. Thus, the application of click chemistry to generate surfactants appears to be promising and particularly valuable. Well-defined glyco-polyorganosiloxanes with cellobiose and xylogluco oligosaccharide have been synthesized by „click” chemistry (Scheme 8) [98]. The method is based on Huisgen 1,3-dipolar cycloaddition from azido-containing PDMS with plant polysaccharide xyloglucan and cellobiose bearing a terminal alkyne functionality. It involves non-protected sugar derivatives, easy purification steps and it allows regioselective introduction of the alkyne function onto the carbohydrate moiety.
2.B. Applications of Carbohydrate Modified Siloxanes in Polymer Stabilization Attempts have been made to prepare microemulsions of hexamethyldisiloxane or longer PDMS, using carbohydrate modified siloxane surfactants [78, 79], but the approach was not succesful due to the siloxane insolubility of the amphiphile [53]. The addition of ethanol was helpful, but only in certain cases. The surface active properties of a xyloglucane modified trisiloxane (Scheme 8) at the dodecane/water interface, as well as at the silicone/water interface, were studied and compared with those obtained with other surfactants usually involved in cosmetic formulations. The tests indicated that this glyco-organosiloxane was a poor emulsifier, but its foaming behavior is promising for detergent and cosmetic applications [98]. Glycopolysiloxanes with cellobiose moieties have been used for the steric stabilization of vinyl acetate miniemulsion polymerization [93]. The first attempts to get stable PVAc nanoparticles by using only the glycosiloxane derivatives as polymer surfactants failed, but stable nanoparticles were obtained using a mixture of glycosiloxane and a non-ionic triblock PEO-b-PPO-b-PEO copolymer. The observation that glycosiloxanes employed alone are unable to stabilize miniemulsion polymerizations is in good agreement with the fact that mannose derivatives grafted onto vinylic polymer backbones are ineffective in assuring the colloidal stability of polycaprolactone nanoparticles [70]. This may tentatively be interpreted in terms of the layer thickness of the polymer surfactant adsorbed onto the particles. The siloxane chain is highly hydrophobic and forms a flattened alignment with the interface. The sugar moieties grafted either onto the backbone or at the chain ends are solvated by the aqueous phase but their small molecular size does not allow a sufficient extension into the solution to give a thick enough polymer layer to ensure a good steric stabilisation, contrary to silicone polyethers. Adding a triblock copolymer such as Pluronics is therefore needed to provide a good colloidal stability since the PEO segments extend further into water. Polymer nanoparticles are more and more investigated and one of their most spectacular applications is in the medical field, where they are largely used for encapsulation of drugs [10]. For such a purpose, the polymers have to be biodegradable, biocompatible or bioresorbable [10, 99, 100]. Siloxane surfactants containing monosaccharides, as well as
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potassium salts of siloxane-aliphatic carboxylic acids, have been used for the stabilization of polymer nanoparticles by nanoprecipitation [91, 92]. In the emulsification process, it was assumed that the chemical affinity of the hydrophobic part of the surfactant to the silicone oil promotes solubilization [53]. Nevertheless, this compatibility seems to be insufficient and other factors also intervene. For the same surfactant and experimental parameters, we observed differences in the size and size distribution from one polymer to another, and especially the stability after drying was greatly influenced by the thermo-mechanical properties of the polymer core. Well-defined spherical nanoparticles were obtained with polysulfone (PSF), a rigid polymer with high Tg. Polycaprolactone, PCL (negative Tg, but positive Tm) formed stable monodisperse particles in water, which exhibit a tendency of aggregation after drying. The nanoparticles obtained by nanoprecipitation of high molecular weight PDMS (both negative Tg and Tm collapsed) [91, 92]. In our attempt to obtain stable silicone nanoparticles, we have tested the possibility of crosslinking polysiloxane chains in the presence of siloxane surfactants [101]. The crosslinking reactions occur in the formed particles and improve their colloidal and dry-state stability. However, the average diameter and size distribution of the particles measured by DLS were not very good, which may be due to the insufficient hydrophilic content, or insufficient sterical repulsion. Other chemical reactions can be performed in the presence of carbohydrate modified siloxane surfactants. For example, a glucose substituted cyclosiloxane has been successfully used to stabilize silver nanoparticles (Figure 2) [102].
3. OTHER SYSTEMS Alkali metal salts of siloxane-aliphatic acids can play the role of anionic surfactants. Such compounds have been described in a patern long time ago and their possible use as anticorrosion agents and emulsifiers has been claimed [103]. We studied potassium salts of mono- and di-substituted sebacomethyl disiloxane (Scheme 9) as surface active compounds and we used them as stabilizers for polymeric nanoparticles [92, 101]. H3 C
H3C
O Si
CH3 Si
O
O K
(CH2)8
+
CH3 H3C
O
O
Scheme 9. Example of anionic siloxane surfactant
For example, polymer nanoparticles have been obtained combining nanoprecipitation with polycondensation. We have prepared poly(siloxane-azomethine) nanoparticles in a one step procedure, using a siloxane surfactant [101]. These particles were small and monodisperse (Figure 1d), as in the case of polysulfone. The core polymer has a positive Tg, as well, and this seems to be an important criterion for obtaining small and stable polymeric nanoparticles.
Siloxane-Containing Compounds as Polymer Stabilizers
227
a
bb
c d
5 μm
Figure 1. SEM and AFM images of nanoparticles stabilized with nonionic or anionic siloxane surfactants: (a) polycaprolactone, (b) polysulfone, (c) crosslinked PDMS, and (d) poly(siloxaneazomethine)
Figure 2. UV-Vis spectrum and AFM (phase) image of Ag nanoparticles obtained with a glucosemodified cyclosiloxane
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Carmen Racles, Thierry Hamaide and Etienne Fleury
Well-defined particles have been obtained from PDMS containing different additives, using the surfactant depicted in Scheme 9. For example, metal nanoparticles, or crystalline compounds may be encapsulated in PDMS and the resulting particles are better defined and stable after drying (Figure 3), probably due to a reinforcing effect. The same result was observed in the case of PCL nanoparticles with encapsulated indomethacine. In that case, the colloidal stability was visibly increased compared to the neat polymer [92]. Another class of siloxane surface active agents is that of cationic surfactants, which usually contain amine groups (Scheme 10) and are widely used in cosmetic products, especially as hair softeners and conditioners [54]. A quaternary ammonium chloride has been used to modify montmorillonite, in order to obtain nanocomposites with polymathylsilsesquioxane [104].
Figure 3. TEM images of crosslinked polysiloxane nanoparticles reinforced with metal nanoparticles (left) and with an organic crystalline compound (20%) (right)
Si HO
O
Si
Si m
O
nO
Si O H Si
O
HO
Si O
Si a
O
NH
NH
NH2
NH2
CH3
CH3
(CH3)3-Si-O
Si
m
CH3
O
Si n
CH2
O Si-(CH3)3
3 CH2CH3
O CH2
+
CH CH2 N OH
Scheme 10. Cationic siloxane surfactants.
b
CH3 Cl
CH2CH3
Si O
c
Si OH
O O d
H
Siloxane-Containing Compounds as Polymer Stabilizers
229
The ability of siloxane surfactants to stabilize different chemical reactions allows the preparation of different nano-materials. For example, gemini surfactants containing a siloxane moiety have been used as templates for the preparation of mesoporous metal oxides such as zirconium, titanium, and vanadium oxides. The siloxane segment seems to play an important nano-propping role during the surfactant removal by direct calcination [105]. Amphiphilic ABA-type triblock copolymers containing PDMS, two poly(2-methyloxazoline) –PMOXA- side blocks and methacrylate terminal groups have a complex selfassembling behavior, which allows for the synthesis of covalently cross-linked hydrogels, with applications as contact lenses [106]. Cross-linked vesicles of such polymers are nanoand microcapsules which act as stable nanoreactors with controlled permeability [107-109]. Siloxane copolymers with polar groups may form aggregates in nonaqueous media, too. Their behavior is similar to that of surfactants in water. The relationship between nanostructure and rheological properties of such systems, and their potential use as nanoreactors are still under investigation [61, 110, 111]. For example, the micellization of a siloxane- ketimine in organic media may lead to metal complex nanoparticles [112].
CONCLUSION Although siloxane surfactants and especially silicone polyethers are known and produced for some decades, they continue to incite the interest of the scientific world. New structures are emerging, new methods are used for their investigation and new applications are discovered. In particular, biocompatible surfactants are of great importance nowadays, as well as those obtained from renewable sources. Nano-materials may be prepared with siloxanecontaining surfactants, which are active both in water and in organic media.
ACKNOWLEDGMENT The financial support from the Romanian Ministry of Education and Research under grants: Idei_233 no. 5/2007 and Bilateral Franco-Romanian Program Brancusi (14760TM) is gratefully acknowledged. We acknowledge the support provided by Région Rhone-Alpes for a postdoctoral fellowship (C.R.), which allowed the begining of our collaboration a few years ago. The authors are fully indebted to Prof. Sylvie Boileau for fruitfull discussions.
REFERENCES [1] Lee, YS. Self-assembly and Nanotechnology – A Force Balance Approach, 2008, Wiley, Hoboken. [2] Holmberg, K; Jönsson, B; Kronberg, B; Lindman, B. Surfactants and Polymers in Aqueous Solution, 2002, Wiley. [3] Chevalier, Y. Current Opinion in Colloid & Interface Science, 2002, 7, 3. [4] Abele, S; Sjöberg, M; Hamaide, T; Zicmanis, A; Guyot, A. Langmuir, 1997, 113, 176.
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Reviewed by Prof. Sylvie Boileau, Institut de Chimie et des Materiaux, Paris-Est (ICMPE), LRP-CNRS, 2-8 Rue Henri Dunant, 94320 Thiais, France
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 7
AMPHIPHILIC BLOCK COPOLYMERS: POTENT EFFLUX PUMP INHIBITORS FOR DRUG DELIVERY AND CANCER THERAPY Martin Werle and Hirofumi Takeuchi Gifu Pharmaceutical University, Laboratory of Pharmaceutical Engineering, 5-6-1 Mitahora, Gifu, Japan.
ABSTRACT The ability of amphiphilic block copolymers to modulate multi drug resistance related processes has been demonstrated the first time more than 10 years ago. Nowadays, the efflux pump inhibitory activity of amphiphilic block copolymers is used in two main areas. First, to improve the transport of efflux pump substrates across the blood brain barrier (BBB) and second, in cancer therapy. It has been shown that in the presence of amphiphilic block copolymers higher concentrations of certain anticancer drugs, which are known as efflux pump substrates, can be found in the brain. Within the current chapter, recent developments in the field of amphiphilic block copolymer mediated efflux pump inhibition are discussed. Besides presenting data from in vitro and vivo studies, also the mechanisms involved in efflux pump inhibition are addressed. In addition, the influence of hydrophilicity/lipophilicity of various amphiphilic block copolymers as well as factors such as micelle formation on the efflux pump inhibitory activity are explained.
1. INTRODUCTION Efflux pumps are transmembrane located transporter proteins which are expressed in various tissues including liver, placenta, the proximal tubule in the kidney, capillary endothelial cells of brain and testis, and epithelial cells of the intestine [1,2]. In addition, they are over-expressed in cancer cells and are involved in the multi drug resistance (MDR) mechanisms of tumors. Besides other mechanisms such as CYP3A, efflux pumps constitute an integral part of the body´s natural detoxification system. Due to their localisation in various tissue they affect absorption, distribution, metabolism and elimination [3]. They are
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known to actively transport a wide range of structurally diverse compounds out of the cells; including anticancer agents [4], immunosuppressants [5], steroid hormones [6], calcium channel blockers [7], beta-adrenoreceptor blockers [8] and cardiac glycosides [9]. The most prominent representative of the group of efflux transporters is P-glycoprotein (P-gp)[10], but also other efflux pumps such as the multidrug resistant proteins (MRP) 1 and 2 as well as the breast cancer resistant protein (BCRP) [11] gain increasing attention. Efflux pumps are ATPdependent proteins which are encoded by the ATP Binding Cassette (ABC) gene family. It has been demonstrated that a broad variety of substances are capable of inhibiting efflux pumps. As MDR can be regarded as a main problem in cancer therapy, an inhibition of efflux pumps can contribute to the development of improved cancer therapeutics by increasing the transport of efflux-pump substrates into MDR cells. Another promising approach of efflux pump inhibition is to improve the transport of efflux pump substrates through physiological barriers, such as the blood-brain barrier (BBB) or the small intestine. Various therapeutic and already marketed drugs were identified to exhibit efflux pump inhibitory properties, in addition to their main pharmacological activity. Representatives of these so called “first generation” inhibitors are for example Verapamil or Tamoxifen [7,12]. In the following years, effort has been put in the development of inhibitors that lack therapeutic effects but still retain their efflux pump inhibitory properties. Therefore, “second generation inhibitors” where synthesised by chemically modifying first generation efflux pump inhibitors. Examples of second generation efflux pump inhibitors, which are based on the lead structure of first generation inhibitors are KR30031, a verapamil analogue, and PSC833 (Valspodar®), which is based on cyclosporine D [13,14]. Finally, “third generation inhibitors” which often lack CYP3A4 interactions and which are capable of inhibiting P-gp in a non-competitive manner were developed. Representatives which entered clinical trials are Elacridar (GF120918; GlaxoSmithKline), XR9576 (Tariquidar; Xenova), LY335979 (Zosuquidar; Eli Lilly), R101933 (Laniquidar) and ONT-093 (Ontogen). Recently, it has been discovered that some widely used polymeric auxiliary agents can interact with efflux pumps. Many of these polymeric efflux pump inhibitors have been used for decades because of their proposed lack of pharmacological activity. The use of polymeric efflux pump inhibitors in pharmaceutical applications has been comprehensively reviewed previously [15,16]. Representatives of polymeric or polymer based efflux pump inhibitors are anionic gums and sodium alginate [17], polyethylene glycol (PEG)[18,19], PEG based detergents [2023], thiolated polymers [24-26] and poloxamers such as pluronics [27]. Among the amphiphilic copolymers which are capable of inhibiting efflux pumps, pluronics have been evaluated in most detail so far. By modifying the length of the ethylene oxide (EO) and propylene oxide (PO) segments which are arranged in the basic A – B - A structure, polymers with various hydrophilicity/lipophilicity ratios can be synthesized. These “functional excipients” are known to affect immune response [28] and wound healing [29]. In addition, it was discovered already 10 years ago, that pluronics can modulate MDR related processes.
2. EFFLUX PUMP INHIBITION MECHANISMS OF PLURONICS Various mechanisms which are responsible for the efflux-pump inhibitory properties of pluronics have been identified and discussed in detail previously [30]. Among them are inhibition mediated by ATP depletion, membrane fluidization and ATPase inhibition. As
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already mentioned, efflux pumps are ATP dependent, which means that ATP is required in order to sustain the function of efflux pumps. It was demonstrated that the intracellular ATP level of cancer cells was depleted following a 2 hour incubation period with pluronic P85 [31]. Additional studies by Batrakova et al. using various MDR and non-MDR cell lines showed that the pluronic mediated ATP depletion is reversible and that MDR cells are much more responsive to pluronic than non-MDR cells [32,33]. Another mechanism which appears to be responsible for the efflux pump inhibitory activity of pluronics is mediated by the ability of pluronics to be absorbed on cell membranes. This effect leads to changes in the microviscosity of cell membranes, due to alterations in the structure of the lipid bilayers [34]. In particular, the presence of pluronic P85 leads to fluidization of membranes of healthy and cancer cells [35]. It has been suggested that non-specific changes in lipid and protein conformation and mobility can lead to ATPase inhibition, which would consequently lead to decreased efflux pump activity [36]. Studies showed that exposure of cells to pluronic P85 led to dramatically decreased ATPase activity in comparison to the control which contained no polymer [35].
3. FACTORS INFLUENCING THE EFFLUX-PUMP INHIBITORY ACTIVITY OF PLURONICS The hydrophilicity/lipophilicity ratio was identified to have a direct impact on the effluxpump inhibitory activity of pluronics [37]. In a study focussing on this topic, the efficacy of 20 pluronics with different hydrophilic (EO) / lipophilic (PO) ratios on the accumulation of rhodamine 123 into MDR cell lines (KBv) was investigated. Based on their efficacy, pluronics were divided into three groups. Group 1 comprised of hydrophilic pluronics with a hydrophilic/lipophilic balance (HLB) between 20 and 29. Representatives of this group are F68, F88, F108 or F127. Group 1 pluronics showed no or only minor effects on P-gp. The most effective P-gp inhibitors belonged to group 2 and were identified to be hydrophobic pluronics with a HLB below 19 and with 30 – 60 repeating PO units in the PO block. In this group, P85, L81 and L61 can be found. Group 3 pluronics, which are less effective than group 2 pluronics, are also hydrophobic copolymers with a HLB below 19, but the number of PO units is either lower than 30 or higher than 60. Representatives of group 3 pluronics are L35, L44 or L121 [37]. It was shown that only the group 2 pluronics where transported to the inside of the cells, and that group 2 pluronics showed the strongest effect on ATP depletion as well as on ATPase inhibition. The properties of the 3 groups are summarized in Table 1. Another important factor which is not directly related to the hydrophilicity/lipophilicity ratio of various pluronics but which strongly influences the efflux pump inhibitory activity of pluronics is micelle formation. Above a certain concentration, the so called “critical micelle concentration” (CMC), amphiphilic block copolymers self assemble into micelles. It has been demonstrated, that the efflux pump inhibitory activity of pluronics increases with increasing pluronic concentrations, but only until the CMC is reached. Above the CMC, substrate accumulation in cancer cells could not be further increased or was found to even decrease [37]. Therefore, the occurrence of pluronic unimers can be regarded as the crucial prerequisite for the efflux pump inhibitory activity of pluronics [37]. As one mechanism for efflux pump inhibition has been identified to be ATP depletion, it seems necessary that the pluronic unimers are transported into the cells in order to exert this action.
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Martin Werle and Hirofumi Takeuchi Table 1. Properties of the three groups of pluronics Group 1 F68, F88, F108, F127 hydrophilic 20 - 29
Group 2 P85, L81, L61 hydrophobic < 19
Group 3 L35, L44, L121 hydrophobic < 19
29 - 65
30 - 60
30 < or > 60
Cell accumulation behaviour
No cell accumulation
Accumulation in the cytoplasma
Inhibitory activity
-
+++
Representatives Character HLB Number of PO units per PO Block
Accumulation in the intracellular endosomal compartments, not released in the cytoplasma +
4. APPLICATION IN CANCER THERAPY AND DRUG DELIVERY Based on their ability to inhibit efflux pump transporters, pluronics have been utilized in cancer therapy as well as in order to overcome the blood brain barrier (BBB). The BBB is known to prevent the transport of certain drugs to the brain. Brain microvessel endothelial cells which form tight extracellular junctions and display low pinocytic activity as well as efflux pumps that remove drugs from the epithelial cells have been identified to be responsible for the limited transport [38]. As pluronics are capable of inhibiting efflux pumps, an improved transport of efflux pump substrates through the blood brain barrier in presence of pluronics was anticipated [39]. The potential of pluronics for BBB delivery has been reviewed in detail previously [30]. Miller et al. [27] were maybe the first who demonstrated a concentration dependent inhibitory activity of pluronic P85 by monitoring the accumulation of rhodamine 123 in brain microvessel endothelial cell (BMVEC) monolayers. Extensive data of in vitro permeation studies using bovine brain microvessel endothelial cells (BBMEC) and a broad variety of efflux pump substrates including e.g.: etoposide, doxorubicin and paclitaxel that show an improved apical to basolateral drug transport are available [40]. In addition, in vivo studies that demonstrate the efficacy of pluronics to improve the transport of efflux pump substrates through the BBB have been performed. As way of example, the brain accumulation of digoxin in wild type mice, mdr1a knockout mice and wild type mice treated with pluronic P85 was investigated and it was reported that in the presence of pluronic a prolonged residence time and an increased concentration of digoxin in the brain was observed [39]. Another physiological barrier besides the BBB which displays efflux pump expression and which can limit drug transport is the intestinal mucosa. Drug transporters located in the intestine have been identified to be responsible for the low oral bioavailability of a number of efflux pump substrates. Moreover, it has already been demonstrated in various studies, that the oral bioavailability of efflux pump substrates can be improved by co-administrating efflux pump inhibitors [20,26]. A review article focussing on the applications of pluronics in cancer therapy has been published previously [41]. Apart from their efflux pump inhibitory activity, pluronics are promising agents in cancer therapy because they affect drug sequestration and the GSH/GST detoxification system, which is also discussed in the mentioned review article [41]. The in vivo efficacy of pluronic formulated doxorubicin for the treatment of various different kinds of cancer has been demonstrated [42]. It was concluded that the enhanced cytotoxicty of
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anticancer agents in the presence of pluronics can at least partly be assigned to P-gp inhibition. An enhanced doxorubicin accumulation in P-gp expressing cells was observed in the presence of pluronics, whereas such an effect was not observed when using non P-gp expressing cell lines [37,43]. These findings were supported by studies in which it was shown that the accumulation and permeation of various P-gp substrates was enhanced in the presence of pluronics when using MDR1 transfected cell lines. In contrast, no or only minor effects were observed in non-transfected cells [39,44]. In addition, no enhanced accumulation of non-P-gp substrates in neither resistant nor sensitive cell lines was observed [27,39,45]. Besides the capability of pluronics to inhibit P-gp, there is evidence that pluronics can also inhibit other efflux pumps including MRP1 and MRP2. However, a comparison of the effect of pluronics on P-gp ATPase activity and MRP1/MRP2 ATPase activity revealed that the effect on P-gp ATPase activity was more pronounced than that on MRP1/MRP2 ATPases [46].
CONCLUSION The potential of pluronics to inhibit efflux pumps and their efficacy in cancer therapy as well as drug delivery has been demonstrated in numerous studies. Especially an intended use of pluronics for cancer therapy is very promising and a plethora of data is already available. In conclusion, the ability of pluronics to inhibit efflux pumps is believed to play an important role in the development of novel drug formulations in the near future.
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Martin Werle and Hirofumi Takeuchi of verapamil outward across the plasma membrane of K562/ADM cells. Cancer Res, 49, 5002-5006. Karlsson, J., Kuo, S. M., Ziemniak, J. & Artursson, P. (1993). Transport of celiprolol across human intestinal epithelial (Caco-2) cells: mediation of secretion by multiple transporters including P-glycoprotein. Br J Pharmacol, 110, 1009-1016. de Lannoy, I. A. & Silverman, M. (1992). The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin. Biochem Biophys Res Commun, 189, 551-557. Juliano, R. L. & Ling, V. (1976). A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta, 455, 152-162. Choudhuri, S. & Klaassen, C. D. (2006). Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters. Int J Toxicol, 25, 231-259. Kirk, J., Syed, S. K., Harris, A. L., Jarman, M., Roufogalis, B. D., Stratford, I. J. & Carmichael, J. (1994). Reversal of P-glycoprotein-mediated multidrug resistance by pure anti-oestrogens and novel tamoxifen derivatives. Biochem Pharmacol, 48, 277285. Woo, J. S., Lee, C. H., Shim, C. K. & Hwang, S. J. (2003). Enhanced oral bioavailability of paclitaxel by coadministration of the P-glycoprotein inhibitor KR30031. Pharm Res, 20, 24-30. Friedenberg, W. R., Rue, M., Blood, E. A., Dalton, W. S., Shustik, C., Larson, R. A., Sonneveld, P. & Greipp, P. R. (2006). Phase III study of PSC-833 (valspodar) in combination with vincristine, doxorubicin, and dexamethasone (valspodar/VAD) versus VAD alone in patients with recurring or refractory multiple myeloma (E1A95): a trial of the Eastern Cooperative Oncology Group. Cancer, 106, 830-838. Werle, M. (2007). Natural and Synthetic Polymers as Inhibitors of Drug Efflux Pumps. Pharm Res in press, Werle, M. (2008). Polymeric and low molecular mass efflux pump inhibitors for oral drug delivery. J Pharm Sci, 97, 60-70. Carreno-Gomez, B. & Duncan, R. (2002). Compositions with enhanced oral bioavailability. ed., US. Johnson, B. M., Charman, W. N. & Porter, C. J. H. (2002). An in vitro examination of the impact of polyehtylene glycol 400, pluronic P 85 and vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate on p-glycoprotein efflux and enterocyte-based metabolism in excised rat intestine. AAPS PharmSci, 4, Shen, Q., Lin, Y., Handa, T., Doi, M., Sugie, M., Wakayama, K., Okada, N., Fujita, T. & Yamamoto, A. (2006). Modulation of intestinal P-glycoprotein function by polyethylene glycols and their derivatives by in vitro transport and in situ absorption studies. Int J Pharm, 313, 49-56. Varma, M. V. & Panchagnula, R. (2005). Enhanced oral paclitaxel absorption with vitamin E-TPGS: Effect on solubility and permeability in vitro, in situ and in vivo. Eur J Pharm Sci, 25, 445 - 453. Collnot, E. M., Baldes, C., Wempe, M. F., Hyatt, J., Navarro L., Edgar, K. J., Schaefer, U. F. & Lehr, C. M. (2006). Influence of vitamin E TPGS poly(ethlyene glycol) chain length on apical efflux transporters in Caco-2 cell monolayers. J Control Rel, 111, 3540.
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[22] Friche, E., Jensen, P. B., Sehested, M., Demant, E. J. & Nissen, N. N. (1990). The solvents cremophor EL and Tween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascites tumor. Cancer Commun, 2, 297-303. [23] Liu, C., Wu, J., Shi, B., Zhang, Y., Gao, T. & Pei, Y. (2006). Enhancing the bioavailability of cyclosporine a using solid dispersion containing polyoxyethylene (40) stearate. Drug Dev Ind Pharm, 32, 115-123. [24] Werle, M. & Hoffer, M. (2006). Glutathione and thiolated chitosan inhibit multidrug resistance P-glycoprotein activity in excised small intestine. J Control Rel, 111, 41-46. [25] Föger, F., Schmitz, T. & Bernkop-Schnürch, A. (2006). In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan. Biomaterials, 27, 42504255. [26] Föger, F., Hoyer, H., Kafedjiiski, K., Thaurer M. & Bernkop-Schnürch, A. (2006). In vivo comparison of various polymeric and low molecular mass inhibitors of intestinal P-glycoprotein. Biomaterials, 27, 5855-5860. [27] Miller, D. W., Batrakova, E. V., Waltner, D. O., Alakhov, V. & Kabanov, A. V. (1997). Interactions of pluronic block copolymers with brain microvessel endothelial cells: evidence for two potential pathways for drug absorption. Bioconjug Chem, 8, 649 - 657. [28] Hunter, R. L., McNicholl, J., Lal, A. A. (1994). Mechanisms of action of nonionic block copolymer adjuvants. AIDS Res Hum Retroviruses, 10, 95-98. [29] Rodeheaver, G., Turnbull, V., Edgerton, M. T., Kurtz, L. & Edlich, R. F. (1976). Pharmacokinetics of a new skin wound cleanser. Am J Surg, 132, 67-74. [30] Kabanov, A. V., Batrakova, E. V. & Miller, D. W. (2003). Pluronic block copolymers as modulators of drug efflux transporter activity in the blood-brain barrier. Adv Drug Deliv Rev, 55, [31] Slepnev, V. I., Kuznetsova, L. E., Gubin, A. N., Batrakova, E. V., Alakhov, V. & Kabanov, A. V. (1992). Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP. Biochem Int, 26, 587-595. [32] Batrakova, E., Li, S., Alakhov, V. & Kabanov, A. V. (2000). Selective energy depletion and sensitization of multiple drug resistant cancer cells by Pluronic block copolymers. Polym Prep, 41, 1639-1640. [33] Batrakova, E., Li, S., Elmquist, W. F., Miller, D. W., Alakhov, V. & Kabanov, A. V. (2001). Mechanism of sensitization of MDR cancer cells by Pluronic block copolymers: selective energy depletion. Br J Cancer, 85, 1987-1997. [34] Melik-Nubarov, N. S., Pomaz, O. O., Dorodnych, T. Y., Badun, G. A., Ksenofontov, A. L., Schemchukova, O. B., Arzhakov, S. A. (1999). Interaction of tumor and normal blood cells with ethylene oxide and propylene oxide block copolymers. FEBS Lett, 446, 194-198. [35] Batrakova, E. V., Li, S., Vinogradov, S. V., Alakhov, V., Miller, D. W. & Kabanov, A. V. (2001). Mechanism of pluronic effect on p-glycoprotein efflux system in blood brain barrier: contributions of energy depletion and membrane fluidization. Pharmacol Exp Ther, 299, 483 - 493. [36] Regev, R., Assaraf Y. G. & Eytan G. D. (1999). Membrane fluidization by ether, other anesthetics, and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells. Eur J Biochem, 259, 18-24.
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[37] Batrakova, E., Lee, S., Li, S., Venne, A., Alakhov, V. & Kabanov, A. V. (1999). Fundamental relationships between the composition of pluronic block copolymers and their hypersensitization effect in MDR cancer cells. Pharm Res, 16, 1373-1379. [38] Pardridge W. M., editors Introdcution to the Blood-Brain Barrier. Methodology, Biology and Pathology ed., Cambridge: University Press., 1998; 486. [39] Batrakova, E. V., Miller, D. W., Li, S., Alakhov, V., Kabanov, A. V. & Elmquist, W. F. (2001). Pluronic P85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies. J Pharmacol Exp Ther, 296, 551-557. [40] Batrakova, E. V., Li, S., Miller, D. W. & Kabanov, A. V. (1999). Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers. Pharm Res, 16, 1366-1372. [41] Kabanov, A. V., Batrakova, E. V. & Alakhov, V. Y. (2002). Pluronic block copolymers for overcoming drug resistance in cancer. Adv Drug Deliv Rev, 54, 759-779. [42] Alakhov, V., Klinksi, E., Li, S., Pietrzynski, G., Venne, A., Batrakova, E., Bronitch, T. & Kabanov, A. V. (1999). Block co-polymer based formulation of doxorubicin. From cell screen to clinical trials. Colloids Surf B: Biointerfaces, 16, 113-134. [43] Venne, A., Li, S., Mandeville, R., Kabanov, A. V. & Alakhov, V. (1996). Hypersensitizing effect of pluronic L61 on cytotoxic activity, transport, and subcellular distribution of doxorubicin in multiple drug-resistant cells. Cancer Res, 56, 3626-3629. [44] Evers, R., Kool, M., Smith, A. J., van Deemter, L., de Haas, M. & Borst, P. (2000). Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport. Br J Cancer, 83, 366-374. [45] Batrakova, E. V., Han, H. Y., Alakhov, V., Miller, D. W. & Kabanov, A. V. (1998). Effects of pluronic block copolymers on drug absorption in Caco-2 cell monolayers. Pharm Res, 15, 850-855. [46] Batrakova, E. V., Li, S., Li, Y., Alakhov, V. Y. & Kabanov, A. V. (2004). Effect of pluronic P85 on ATPase activity of drug efflux transporters. Pharm Res, 21, 22262233.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 8
THE ABSENCE OF PHYSICAL AGING EFFECTS IN THE SURFACE REGION OF GLASSY POLYMERS Z. Yang Department of Physics, The Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong
ABSTRACT The effects of physical aging near the surface region of glassy polymers are studied via the relaxations of (1) surface topographic features created by rubbing, and (2) the rubbing induced birefringence (RIB). Extensive experimental results are presented to show that physical aging processes that would have drastic effects on the relaxations of bulk polymers have little effects on the relaxations of rubbed surfaces. We also found that surface topographic features, such as ditches and ridges created by rubbing, relax at temperatures at about 20 °C below the bulk glass transition temperature of the polystyrene for the molecular weight of 442 kg/mol, even though the Laplace Pressure driving the relaxation is 1/500 of the yield limit. The relaxation of RIB in polystyrene (PS), its derivatives with modified side group, and polycarbonate (PC), involves only the length scale of the order of an individual segment. A phenomenological model based on individual birefringence elements is proposed for the RIB relaxation. The relaxation times (RT’s) of the elements are found to be independent of the thermal or stress history of the samples, either before or after the formation of the birefringence. The RT’s are also independent of the molecular weight, rubbing conditions, and film thickness, while the RT’s distribution function does depend on the molecular weight and rubbing conditions. The model provides quantitative interpretations that agree very well with all the reported experimental results, and sheds important light to the novel behaviors of the RIB relaxation. The absence of physical aging effects is probably due to the combined effects of small length scale of the RIB relaxation, and the accelerated aging speed in the near surface region. This is consistent with the mobility enhancement in the surface layer previously reported in the literature.
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PART I. INTRODUCTION Upon cooling a polymer melt or rubber, its volume (enthalpy) begins to deviate from the equilibrium liquid value at glass transition temperature (Tg) that depends upon the cooling rate. When the cooling is stopped the glass spontaneously evolves towards its equilibrium thermodynamic state (volume or enthalpy) [1]. Associated with the changes in thermodynamic structure are the changes in the mechanical, dielectric, etc., responses of the material that have come to be referred to as physical aging [2]. In general, the behavior of the physical aging response for the linear viscoelastic properties of polymers is widely accepted to follow the broad outlines put forward by Struik [2] and often referred to as following a time-aging time superposition principle analogous to the time-temperature superposition found in viscoelasticity of polymer melts [3]. For creep compliance, typical value of the characteristic time τ0 of polystyrene (PS) at 60 ºC in thermodynamic equilibrium state is well beyond the realistic experimental time scale [3], but its value of a sample freshly quenched from above Tg (~ 100 ºC) is many orders of magnitude smaller than the one in equilibrium state [1]. PS pre-strained below Tg also exhibits much shorter τ0 at 40 ºC [4]. The compliance curve then shifts to longer time along the logarithmic time axis as the aging time increases, following the time-aging time superposition principle. Compliance curves at different aging times can be shifted to form a master curve. If the evolution of the creep compliance is viewed as the relaxations of microscopic segmental units, then all the time constants of the relaxation elements are shifted by the same factor (thermorheologically simple) [1]. Thermal quench aging and stress aging behave qualitatively the same [1] [2] [4]. Quantitatively there are differences. The time-aging time exponential factor is smaller for increasing strain [4]. It is modeled in the strain dependent aging clock theory [5]. Recent constitutive theory based on Holmholtz free energy can fit a wide range of physical aging phenomenon quantitatively [6]. Aging in glassy polymer films were also investigated in the frame of solvent induced glass transition [7] [8]. Results are utterly different from those obtained with equivalent temperature cycles. Hysteresis at small activity as well as complex behavior of the melting time depending on the quench amplitude had been observed. This behavior was attributed to the possible coupled evolution of the free volume and the solvent fraction. Physical aging also exists in thin films. The gas permeability of 400 nm thick polysulfone decreases when the films continued to densify [9]. Thin films age faster in gas permeability [10]. Kawana and Jones reported [11] thin supported films of PS aged below Tg exhibit clear overshooting in expansivity temperature curves when reheated for thicknesses of 18 nm or more, but not for a thickness of 10 nm, suggesting the existence of a surface layer of the order of 10 nm in which aging is complete within the time scale of 103 s, while the rest of the film essentially has thermal properties identical to bulk samples. In this article we review the work on the effects of physical aging in the region near the surface of glassy polymers. In Part-II the relaxation of topographic features of PS surface is examined. In Part-III the relaxation of RIB in PS is examined. In Part-IV the relaxation of RIB in PS derivatives and polycarbonate (PC) is examined. In Part-V we conclude.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 245
PART II. TOPOLOGICAL RELAXATION OF POLYSTYRENE SURFACE 1. Introduction The dynamics of polymer surfaces is an intriguing subject in polymer physics, in addition of being of significant technological importance. There has been much experimental evidence that demonstrate the existence of a surface mobile layer [12 – 24]. Positron annihilation [12] and muon spin probes [13] indicated the presence of a surface mobile layer about 2 nm in thickness near the polystyrene (PS) surface. The Tg of PS thin films were found to decrease drastically with the reduction of thickness [14] [15], and later studies revealed the broadening of the glass transition in polymer thin films with depth [16] [17] [18]. These phenomena were attributed to the existence of a liquid-like surface layer that extended deeper with the rise of temperature and diverged at bulk Tg. Surface friction force microscopy also showed a significant decrease in Tg of the surface layers [19] [20] [21]. The higher mobility of the surface layer was attributed to the chain ends enrichment at the surface, which was confirmed by experiments [20]. Gold spheres of 10 nm and 20 nm in diameter placed on PS surfaces were found to sink into PS at temperatures well below bulk Tg [22] [23], and the phenomenon was interpreted as due to the presence of a liquid-like surface layer. Rough PS surfaces were found to turn mobile at temperatures well below Tg under the driving force of Laplace Pressure [24]. Despite of the rich evidence listed above, there are a few equally strong experimental evidence that seem to contradict the presence of such mobile layer [25] [26]. Using a vigorous viscoelastic mechanic contact analysis, Hutcheson and McKenna showed that the sinking of gold spheres on PS surface could actually take place without the decrease in surface Tg [25]. The creep compliance of 30 nm thick PS films was found to have a lower Tg but a much stiffened rubbery plateau than the bulk value [26]. A direct measurement of the local segmental mobility as a function of depth from surface is strongly desired. But such an experiment without disturbing the segmental motions near the surface would be a rather difficult challenge. However, if physical aging is fast in the near surface region and can come to completion within reasonably short time (~ 103 s), the complication of physical aging effects on the dynamic properties of surface will no longer be present and the analysis of the properties of surface relaxation could be simplified. Gentle rubbing with a velvet cloth on PS surfaces can create molecular segment alignment and distortion [27] [28], in addition to surface ridges and ditches with depths and heights of the order of nanometers. The relaxations of the rubbing induced optical birefringence [29] [30] [31] and the segmental alignments [27] [28] [32] were investigated. These relaxations, however, occurred at even lower temperature than the surface topography relaxations [31]. Preliminary experimental evidence showed that the birefringence relaxation is also independent of the aging effects [31]. We study the evolution of the topological features of rubbed surfaces of samples undergoing various aging processes. The approach is similar to creep experiments that are widely employed in the study of segmental relaxations in bulk polymers, but with two distinctions. One is that here the stress due to the Laplace Pressure (LP) from the surface topographic features, such as ditches and ridges created by rubbing, is present at all time until
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the surface is mostly flattened. The other more important one is that the process to generate the initial roughness (by rubbing with a velvet cloth) introduces large mechanical deformation on the surface, and deformation is known to greatly accelerate the relaxations in bulk samples [4]. Extensive experimental results will be presented to reveal the relaxations of PS surfaces deformed by rubbing with velvet cloth. We found that the dominant driving force of the relaxations is the LP due to the surface topographic features. The surface layer turns to rubbery at about 20 °C below the bulk glass transition temperature for the molecular weight of 442 kg/mol, even though the LP is only about 1/500 of the yield limit. However, this is not due to the accelerated relaxations caused by large mechanical deformation often observed in bulk samples, because the relaxation is independent of the thermal aging before the rubbing process, and post rubbing aging below 55 °C. In other words, the effects of these physical aging processes that would have great influence on the relaxations of bulk polymers are completely absent in the relaxations of surface strain.
2. Experiments Mono-dispersed PS with molecular weights 442 kg/mol (Mw/Mn < 1.1) in toluene solution was spin coated on silicon wafers with 20 nm of thermal SiO2 to form 62 nm films. The samples were then annealed in vacuum at 150°C for several days to completely drive off the solvent before slowly cooled down (< 0.1 ºC/min) to room temperature. These will be referred to as fresh samples from now on. Rubbing was done at normal pressure of 90 kg/m2 and at a constant speed of 1.0 × 10-2 m/s at 23 ºC. Each rubbing run consisted of 90 passes, with each pass covering 2.0 cm distance. All were done in ambient atmosphere so no solvent induced effects occurred [7]. The samples were thick enough that possible influence of the PS/substrate interface was avoided. This was confirmed by PS films on two types of substrates, namely the SiO2 which is known to have weak interaction, and hydrogen terminated Si which is known to have strong interaction with the PS films [33]. No difference in surface strain relaxations was observed for the films on the two types of substrates. For the sake of completeness, the samples investigated were divided into two series, namely the A-series and the B-series. Starting from fresh samples, the A-series samples underwent thermal aging processes before rubbing, while the B-series samples underwent thermal aging processes after rubbing. There are three possibilities of aging effects. The first is that the aging prior to rubbing affects the morphology of the rubbed surfaces and their subsequent relaxations. The second is that due to large mechanical deformation the previous thermal history is ‘erased’ by rubbing so the relaxation depends on post rubbing aging only. The third is that the aging has no effect on the surface relaxation. The study of the A-series samples will substantiate or rule out possibility-1. If possibility-1 is ruled out, the study of the B-series samples will then decide whether possibility-2 or -3 is true. The surface topography was measured on a scanning probe microscope (SPM) in tapping mode. The lateral resolution is about 10 nm and the height resolution is about 0.1 nm. An onstage heater provided the controlled thermal processes (aging and annealing). The measurement sequences were the same for both the A-series and the B-series samples. The samples were measured using either type-I or type-II measurement sequences. In the type-I measurement sequence, the sample was first raised quickly from 23 ºC to the annealing
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 247 temperature (say 40 ºC), kept there for 0.5 hours, then quickly cool down to 23 ºC. SPM measurements were then carried out on a selected 5 × 5 μm2 area and completed within 1 hour at 23 ºC. The process was then repeated but with a higher annealing temperature, until the surface had flattened. In the type-II measurements, the sample temperature was quickly raised to a fixed value (say 85 ºC) for a certain duration (say 0.5 hours), then quickly cooled down to 23 ºC for SPM measurements. The process was then repeated except that the duration (annealing time) the sample temperature was maintained at the given value was progressively longer. In all, the annealing times were 0.5 hours, 1.0 hours, 3.0 hours, and 10 hours. This resulted in the accumulative annealing time of the samples as 0.5 hours, 1.5 hours, 4.5 hours, and 14.5 hours. In both measurement sequences the total time taken to measure the viscoelastic response of the samples (< 20 hours) was much shorter than the aging times (up to 7 days). Several images of nearly the same area were analyzed in both sequences. Using recognizable topological features, a smaller but identical area was selected from the set of micrographs for analysis. This enabled us to track the evolution of particular topological features such as ditches and ridges, as well as statistical averages such as the root mean square roughness < h 2 > − < h > 2 , over the whole area. The main reason SPM was carried out only at 23 ºC was that the PS surfaces remained frozen at such low temperature, where no detectable change of the topographic features was found in any samples up to 7 days. The surfaces undergoing relaxation at elevated temperatures were frozen once they were quickly cooled to 23 ºC, allowing ‘snap shots’ to be taken without the surfaces going through continuous changes.
3. Results and Discussions 3.1. Typical surface relaxation behavior A reference sample (Sample-A1), which was a fresh sample rubbed and then measured within one hour, was studied first. Figure 1 shows a typical topographic image of the sample surface just after rubbing (Figure 1(a)), and after consecutive 30-minute annealing at 40 °C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C (Figure 1(b)), following the type-I measurement sequence. The fresh surface contains ditches and ridges along the rubbing direction. A ‘roughness spectrum’ obtained from the Fourier transform of a line scan across the rubbing direction is shown in Figure 1(c). The dominant features are about a fraction of micrometer in width. Features less than 20 nm are nearly absent. However, it is possible that there are smaller features that cannot be resolved by the limited lateral resolution of the AFM tip. Our analysis is therefore limited to the features that can be resolved in our instrument, which is comparable to the study reported in Ref. [24]. The surface remained unchanged up to 80°C. The deep ditches that had survived the annealing at 100°C can be matched well with the original ones before annealing. The images taken after annealing above 80°C show that the general evolution pattern of the surface was a nearly uniform reduction of the height of the ridges and the depth of the ditches, so the shallow ditches and low ridges disappeared first. No lateral (horizontal and perpendicular to ditch/ridge) movement of the ditch/ridge walls was observed. The center positions of the ridges and ditches remained unchanged. At 100°C the reduction in height or depth across the entire area ranged between 2 – 3 nm, and the shallow ditches with original depth < 2 nm were smeared out. There was no irregular local
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stress driven relaxation [34], indicating that the LP was the main driving force [24]. This is consistent with the statistical aspect ratio data in Ref. [24]. The average height differences < Δh >line between several ditch/ridge pairs, a typical one being marked by a pair of small arrows at the bottom of Figure 1(a), are plotted as a function of annealing temperature in Figure 1(d). It is seen that the reduction of < Δh >line with annealing temperature follows the same trend, regardless of the initial height differences of the pairs. In particular, the reduction from 23 °C to 100 °C is 3.4 ± 0.4 nm for all the pairs with initial height difference ranging from 5 nm to 11 nm. This is true for all other samples reported in this study. Figure 2 depicts the average roughness over the whole area < Δh > area as a function of annealing temperature together with that of the other four samples underwent various aging processes to be described later. The behavior of the average roughness resembles that of the height differences of the ridge/ditch pairs shown in Figure 1(d). Noticeable changes of morphology features (≥ 0.1 nm) occurred only at ≥ 80 ºC, and the decrease in roughness was rather rapid beyond this point. Because the average roughness < Δh > area and the individual ridge/ditch < Δh >line relaxations behaved the same, the average roughness < Δh > area will be used as the indicator of the surface relaxations in the rest part of the article. The main driving force to flatten a rough polymer surface is the Laplace Pressure P = γ 0 / ρ [24], where γ0 = 0.035 N/m is the surface energy of PS [25] [35], and ρ is the surface curvature along the direction normal to the surface. In the case of an indent of 2 radius w and depth h, the effective radius is approximately given by ρ = w , assuming that 4h the arc passes through the middle point of the bottom and the edge of the indent. The same applies to a long ditch of width w and depth h. The LP becomes P = 4γ 0 h / w2
(1)
Typical depth (height) and width of ditches (ridges) in Figure 1 (h ≈ 10 nm, w ≈ 100 nm) gives rise to LP ≈ 1.4 × 105 N/m2, and varies by a factor of < 4 across the surface. This is about 1/500 of the yield stress of PS [4], so the relaxation was under a driving force well below the yielding limit [36]. The reduction of roughness on the surface is given by, in analogy to conventional creep theory, C = PJ (T , t )
(2)
where C is the ‘relative deformation’ with the freshly rubbed surface as the reference , P is the Laplace Pressure, t is the time and J(T, t) is the surface effective compliance (SEC), which is expected to be a sensitive function of temperature T. Its counterpart, the bulk creep compliance, also depends sensitively on the thermal history of the sample, i. e., the effects of physical aging [1]. As the width w remains unchanged the average LP of the area under investigation is proportional to the roughness, and the ‘relative deformation’ is proportional to the change of roughness.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 249 23 ºC
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Figure 1. (a) SPM image of a rubbed PS surface; (b) SPM image of the same area as (a) after annealing at consecutive temperatures up to 100 ºC described in the text; (c) A roughness spectrum obtained from the Fourier transform of a typical line scan across the ridges and ditches in Figure 1(a); (d) The height difference of selected ridge/ditch pairs as a function of annealing temperature. All the images were taken from a reference Sample-A1, i. e., it was in thermal equilibrium before rubbing and the measurements were taken right after rubbing.
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area (nm)
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Temperature (°C) Figure 2. (a) The surface average roughness versus temperature curves of the reference sample and the A-series samples underwent various aging processes before rubbing. The points for A-1 have been shifted upwards by 1.0 nm, while that of the A-3 have been shifted down by 0.5 nm. (b) The surface average roughness versus temperature curves of the reference sample and the B-series samples underwent various aging processes after rubbing. The points for A-1 have been shifted upwards by 1.0 nm, while that of the B-3 have been shifted down by 0.5 nm.
Consider the red curve in Figure 1(d) as an example. At 90 °C the reduction of height difference < Δh >line as compared to the fresh surface is about 1.5 nm from the initial 6.0 nm value. For the sake of discussions we take two extreme scenarios. If the entire 62 nm film is involved in the relaxation one obtains a relative change along the film depth of about 2.5 %. Since the LP is only 1.4 × 105 Pa, it results in an average compliance over the entire film depth of about 1.8 × 10-7 Pa-1, much higher than the bulk compliance in glassy state (JG = 8.5 × 10-10 Pa-1). If only the initially deformed layer (~ 6.0 nm) is taken as reference the relative change is then 25 %, and the compliance is close to that of the bulk rubbery plateau (JR = 6.1 × 10-6 Pa-1). The actual situation is probably somewhere in between, but even in the extreme case the effective compliance is still much higher than JG. In the meantime, as no lateral movement of ditches and ridges was observed, and considering the fact that the spacing between ditches is hundreds of nanometers as compared to the film thickness of 62 nm, the randomly distributed local stress [34], if ever present, must be at least 200 times smaller than the LP.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 251 Vs
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Figure 3. (a) The schematics of the free volume evolution of A-series samples. (b) The schematics of the free volume evolution of B-series samples.
The change of surface topographic features in glassy state under the present LP is expected to be about ~ 1.2 × 10-4 in relative change and ~ 8 × 10-3 nm in actual displacement, which is well below the SPM detection limit. Therefore, only when the surface is near the rubbery state can the displacement be detected. For a ditch 10 nm deep, a height reduction of 3 nm due to the increase of temperature from 23 °C to 100 °C reduced the original LP by only 30 %, while the SEC changed by several orders of magnitude. It is therefore a good approximation to take LP as nearly constant when analyzing the drastic change of SEC with temperature, and the roughness (height difference) versus annealing temperature curves in Figure 1(d) and Figure 2 therefore represent a measure of the viscoelastic SEC as a function of temperature within a fixed time scale of 30 minutes. In the following, we examine the physical aging effects on the roughness versus temperature curves of samples undergone different thermal processes. In particular, we concentrate on the onset temperature of the glassy-to-rubbery transition, and examine the curves up to 110 ºC when there was still considerable roughness left and the driving force, while somewhat weakened, was still present.
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3.2. Aging before rubbing In Figure 3(a) the bulk specific volume of the surface layers of the PS samples undergoing various physical aging processes is shown schematically. Following Struik [1] [2] we use the term ‘specific volume’ to describe the degree of deviation from thermodynamic equilibrium of the system and the enhancement of segmental mobility in general term, rather than the actual excessive volume, since mechanical deformation may not actually decrease the actual mass density but does increase the segmental mobility, or ‘free volume’ [4]. In the following discussions the speculation about the thermodynamic states of the samples is based upon the assumptions that (i) the aging behavior of the surface region of the rubbed PS follows the general trend of bulk PS undergoing the same processes; and (ii) the rubbing process places the samples away from thermodynamic equilibrium but not enough to ‘erase’ the effect of previous thermal history. As shown in Figure 3(a), Sample-A1 was in thermodynamic equilibrium before rubbing, while after rubbing it was away from the thermodynamic equilibrium state as indicated by the dashed line below Tg. During the subsequent measurement sequence, its specific volume evolved close to the thermodynamic equilibrium line. It served as a reference sample and its surface relaxation has already been presented in sub-section 3.1. Sample-A2 was quenched from 130°C to 23°C and rubbed within 10 minutes. Its specific volume is expected to be far away from the equilibrium line. During the subsequent measurement sequence, it might age slightly towards equilibrium, but it would evolve far away from the equilibrium line, and its segmental mobility is expected to be well above that of Sample-A1. As a result, its surface is expected to turn rubbery at lower temperature than Sample-A1. Sample-A3 was quenched from 130 ºC to 70 ºC, aged there for 7 days, then quenched to 23 ºC, and rubbed. Due to the 7 days aging its specific volume is expected to be closer to the equilibrium line than Sample-A2, but may not be as close as Sample-A1, in the subsequent SPM measurements. Although the three samples had been under different thermal treatments before rubbing, their surface morphology characteristics after rubbing were the same. The typical ditches and ridges shown in Figure 1 were observed on all three surfaces, and the average roughness over some typical areas was about the same. This implies that before rubbing the surface of Sample-A2 was not much ‘softer’ than Sample-A1 in thermal equilibrium, which is in stark contrast with the bulk cases where a freshly quenched sample would have much higher creep compliance [1]. The subsequent relaxation behaviors of the three surfaces were the same. All the surfaces remained frozen below 80 ºC, and started to turn rubbery above 80 ºC. Figure 2(a) shows the < Δh > area versus temperature curves of the three A-series samples. For clarity, the red points for the Sample-A1 are shifted up by 1 nm, and the green points for Sample-A3 are down shifted by 0.5 nm. As the LP was comparable in all three samples, their surface roughness evolution is a direct measure of the SEC as a function of temperature. Adopting the approach in determining Tg from thermal expansion curves, we use the cross point of two lines, one horizontal when the surface remains frozen and the other when the surface is relaxing, as the onset temperature of the glassy-to-rubbery transition TR. The values of TR are (86 ± 2) ºC, (88 ± 2) ºC, and (86 ± 2) ºC for samples A-1, A-2, and A-3, respectively. The results imply that the SEC remained unchanged by the thermal history before rubbing. Such results rule out
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 253 possibility-1 speculated in Figure 3, leaving possibility-2 and -3 for further investigations. One is that the rubbing process had effectively erased the effects of all the previous thermal history, and placed the surface layers of all three samples in nearly the same state far from thermodynamic equilibrium, such as in the neighborhood of point-A in Figure 3(a), which is well above all the volume curves due to various thermal quenches. The other possibility is that the surface layers of all samples were at point-B (in thermodynamic equilibrium) before the measurement sequence was started, due to the quick aging of the surface layer despite of the large mechanical deformation caused by rubbing. The original viscoelastic properties of the surface were therefore restored before the start of the SPM measurements.
3.3. Aging after rubbing As discussed above, rubbing introduces large mechanical deformation to the surface, which may effectively ‘erase’ all the previous thermal history and place the surface in nearly the same non-equilibrium state. Here we study the effects of aging after rubbing. Since we found that surface features would change significantly at above 80 ºC, all post rubbing aging processes reported below were done at < 60 ºC. Similar to the discussions in sub-section 3.2, in Figure 3(b) the bulk specific volume of PS samples undergoing various physical aging processes is shown schematically. We take the first scenarios as suggested at the end of sub-section 3.2, and assume that the rubbing processes placed the samples far away from thermal equilibrium, i. e, at point-A in Figure 3(b). Therefore, the reference Sample-A1 was at point-A after rubbing. Sample-B2 was aged at 23 ºC for 4 days after rubbing, before being tested in the same way as the A-series samples. Assuming that the surface layer aged at a rate comparable to bulk, Sample-B2 should be closer to the thermodynamic equilibrium line than Sample-A1. Sample-B3 was aged at 50 ºC for 2 days after rubbing, then quenched to 23 ºC and tested. Right before testing the sample was probably closer to the equilibrium line than Sample-B2. Despite of the large differences in their thermal history after rubbing, the observed surface morphology evolution of the two samples is the same as the A-series samples. Their roughness at different annealing temperature as plotted in Figure 2(b). The transition temperature for the three samples are (86 ± 2) ºC, (84 ± 2) ºC, and (88 ± 2) ºC for samples A-1, B-2, and B-3, respectively. These experimental results rule out the possibility that the surface layer of PS follows the aging behavior of bulk polymers. Physical aging affects the temperature dependence of a number of dynamic responses of bulk polymers. It is well known that in differential scanning calorimetry there is a large enthalpy peak right below Tg for quenched samples, and the peak reduces as the samples age [37]. Physical aging of PS was investigated by dynamic viscoelasticity measurements [38]. The effect of physical aging was observed for samples aged at 60 and 80 ºC after quenched from 190 ºC. The temperature curves of the dynamic viscoelastic functions of the bulk samples right after quench and that of well aged were significantly different. The curves of the samples partially aged at 60 ºC coincided with that of the quenched sample in the low temperature range. It then started to deviate from the curve at above 65 ºC and gradually merged to the curve of the well aged sample at high temperature. The ‘merge temperature’ increased with the aging time. On the other hand, the curves of the samples aged at 80ºC differed from the curves of the well aged ones and the quenched samples in the entire temperature range. This is significantly different from the aging behavior of the surface viscoelastic response of our samples.
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We now study the post rubbing aging using the type-II measurements, which according to the discussion in subsection 3.1, are effectively the measurements of the viscoelastic SEC versus time at a fixed temperature. The annealing temperature is 85 ºC. Sample-C1 is a reference sample similar to Sample-A1 and tested within one hour after rubbing. Before the measurements Sample-C2 was aged at 50 ºC for 2 days, and then quenched to 23 ºC. SampleC3 was aged at 23 ºC for 7 days after rubbing. The speculation on their thermodynamic states follows the general arguments on the samples underwent similar processes. Figure 4 shows the isothermal temporal evolution of the average roughness at 85 ºC of the three samples. Individual surface topological features behaved the same as the average roughness. Following the same argument above, it is clear that at 85 ºC the surface is near rubbery state. Furthermore, the temporal evolutions of the three samples are identical within experimental uncertainties. In particular, since the aging times of Sample-C1 and Sample-C3 differed by about 170 times, even at a shift rate of 0.6 [2], the bulk creep compliance curve of Sample-C3 should have shifted by a factor of 22 (= 1700.6) relative to Sample-C1 along the time axis. Had the SEC followed the bulk creep compliance, it would have taken 22 hours for the roughness of Sample-C3 to decrease by the same amount as Sample-C1 in 1.0 hour. Similarly, the bulk shift factor at 50 ºC is about unity, and the aging time of Sample-C2 was 48 times of that of Sample-C1, so the time shift factor between Sample-C2 and Sample-C1 is about 48, i. e., it would have taken more than 48 hours for the roughness of Sample-C2 to decrease by the same amount as Sample-C1 in 1.0 hour. No detectable drop in its roughness should have been observed within the experimental time span of 14.5 hours. The fact that the SEC of all three samples behaved the same is consistent with a surface layer in which aging effects are absent.
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3.4. Discussions The experimental results in this paper show that the aging effects are absent in the relaxation of surface of PS. This implies that in the surface region either aging was complete within an hour at room temperature, or aging was extremely slow that even after 7 days no noticeable effects could be observed, a very unlikely scenario that is in stark contradiction to
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 255 the previous reports [9] [12 – 24]. As the surface ages quickly, the deformed surface recovers its thermodynamic properties within the 1 hour time scale. We therefore suggest that their viscoelastic properties determined from the evolution of surface topographic features at elevated temperatures are the intrinsic properties of PS surfaces. Indeed, relaxations of surfaces deformed in two different ways, namely by rubbing below Tg (like cold rolling), and by deformation above Tg (like hot drawing) [24], behaved much the same. It is consistent with the findings in Ref. [11] that physical aging effects were absent in PS films 10 nm thick, and is consistent with the general expectation of the enhanced mobility in the surface layer. The observed surface relaxation behaviors, apart from their independence on physical aging, are themselves intriguing. For example, in bulk creep experiments, the deformation of the specimen creates internal stress that counter balances the external force until reaching the balance point before the rubbery plateau is reached over a long period. In the surface relaxation studied here, the only ‘counter balance’ is the reduction of LP due to the reduction of roughness. As seen in Figure 4, the roughness decrease in the first 4 hours is about 0.2 nm, which is twice the decrease (0.1 nm) in the following 10 hours. In other words, the relaxation rate became slower with increasing time. Such behavior cannot be accounted for by a film with uniform compliance that remains constant or increases with time as in the bulk, because it cannot explain why under nearly the same LP (reduced by only 10 %), the reduction of roughness becomes much slower at longer time. Instead, it hints a depth dependent compliance which is much enhanced at the surface. As the absence of aging effects has been established in this work, the stage is set for the detailed analysis of these surface relaxation behaviors, which will be reported in our subsequent publications.
4. Conclusion Extensive experimental results have been presented to reveal the relaxations of polystyrene surface deformed by rubbing with velvet cloth. We found that the relaxations were mostly driven by the Laplace Pressure due to the surface topographic features, such as ditches and ridges created by rubbing. The relaxation at elevated temperatures was dominated by a nearly uniform reduction of the depth of the ditches and the height of the ridges across the surface, while the centers of these topological features remained fixed. There was no lateral movement (perpendicular to the ditches/ridges) of ditch/ridge walls. The randomly distributed stress, if present, was at least 200 times smaller than the Laplace Pressure. For PS with molecular weight of 442 kg/mol the surface layer turned to rubbery at about 20 °C below the bulk glass transition temperature, even though the Laplace Pressure was only about 1/500 of the yield limit. This, however, was not due to the accelerated relaxations due to mechanical deformation, a phenomenon well known in bulk polymer samples, because the relaxation was independent of the thermal history before the rubbing process, and post rubbing history below 50 °C. Physical aging processes at 23 °C for up to 7 days and at 50 °C for 2 days, which would have drastic effects on the relaxations of bulk polymers, have little effects on the relaxations of strained surfaces. This is consistent with the observed mobility enhancement in the surface layer. The observed enhanced mobility of the surface layer is most likely the intrinsic property of a polymer surface, which is consistent with many previous experimental results.
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PART III. RELAXATION OF RUBBING INDUCED BIREFRINGENCE IN POLYSTYRENE 1. Introduction The relaxations and related dynamic properties of glass forming polymers have been extensively studied in the past decades [39 - 48]. The birefringence of polystyrene (PS) uniaxially drawn at above the glass transition temperature (Tg) remains almost unchanged at temperatures below Tg. Above Tg there are three well separated relaxation steps with distinct time scales τ1, τ2, and τ3 [40]. The relaxation of the segmental alignment dichroism of cold rolled PS at 60 °C can be fitted by two single exponential functions of time with relaxation times of 76 s and 3600 s [39]. Many other relaxation processes below Tg follow the Kohlrausch-Williams-Watts (KWW) stretched exponential function [41] [42] [43]:
φ (t ) = φ (0)e − (t / τ
0
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These include creep compliance [41], density fluctuations [42], volume relaxation of quenched PS [43], etc.. Unlike the cold rolled or hot drawn PS where the polymer specimen is usually elongated by several times of its original length, rubbing with a velvet cloth on a PS surface at a modest pressure of (~ 9 g/cm2) and speed (~ 1 cm/s) creates segmental alignment and segmental distortion, mostly within the top 20 nm layer near the surface [27] [31]. The surface layer is severely deformed, with ditches up to 5 nm deep, but the average thickness of the PS film, as measured by ellipsometry, remains unchanged, implying that little materials are removed by the rubbing cloth. The majority (> 89 %) of the rubbing induced birefringence (RIB) in PS comes from the segmental distortion. Only less than 11 % comes from the segmental alignment [27]. For a 20 nm thick PS, the RIB can be as large as 20 % of the intrinsic value of undistorted PS segments. The RIB relaxations of a number of polymers were first studied by Kovacs and Hobbs [44], and more recently by a number of groups [27] [28] [30] [31] [45], but there is no consistent theoretical interpretation [30] [45] [46]. Upon cooling a polymer melt, its volume (enthalpy) begins to deviate from the equilibrium liquid value at Tg that depends upon the cooling rate. When the cooling is stopped the glass spontaneously evolves towards its equilibrium thermodynamic state (volume or enthalpy) [1]. Associated with the changes in thermodynamic structure are changes in the mechanical, dielectric, etc., responses of the material that have come to be referred to as physical aging [2]. In general, the behavior of the physical aging response for the linear viscoelastic properties of polymers is widely accepted to follow the broad outlines put forward by Struik [2] and often referred to as following the timeaging time superposition principle analogous to the time-temperature superposition in the viscoelasticity of polymer melts [3].
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 257
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The characteristic time τ0 of the creep compliance of PS at 60 ºC in thermodynamic equilibrium state is well beyond the realistic experimental time scale, but its value of a sample freshly quenched from above Tg (~ 100 ºC) is many orders of magnitude smaller [1]. PS pre-strained below Tg also exhibits much shorter τ0 at 40 ºC [4]. The compliance curve then shifts to longer time along the logarithmic time axis as the aging time increases, following the time-aging time superposition principle. Compliance curves at different aging times can be shifted to form a master curve. If the evolution of the creep compliance is viewed as the relaxations of microscopic segmental units, then all the time constants of the relaxation elements are shifted by the same factor (thermorheologically simple) [1]. Thermal quench aging and stress aging behave qualitatively the same [5]. Quantitatively there are differences. The time-aging time exponential factor is smaller for increasing strain. It is modeled in the strain dependent aging clock theory [5]. Recent constitutive theory based on Holmholtz free energy can fit a wide range of physical aging phenomenon quantitatively [6]. This is the characteristics of the cooperative relaxation that it depends sensitively on global thermodynamic states. As the RIB is due to segmental alignment and distortion, it is most relevant to examine the RIB relaxation in the context of segmental relaxations below Tg. And in such regime physical aging is expected to play an essential role. Our preliminary results indicated [31], however, that the RIB relaxes in a way that is characteristically different from any other known forms of relaxation in glass forming polymers. In this part, we explore further along this line. In particular, conventional segmental relaxation times (RT’s), such as the parameter τ0 in Eq. (1), strongly depend on the thermal and strain history, in addition to temperature [1] [4]. Extensive results will be presented to show that the RIB RT’s are independent of the thermal and strain history, while having about the same temperature dependence as the conventional segmental relaxations. This part is divided in the following way. After Introduction (Section-1) and Experiments (Section-2), experimental evidence to prove the absence of physical aging effects on the relaxation of RIB is presented in section-3. In section-4 a phenomenological model based on a generic energy barriers distribution is presented. In section-5 extensive experimental results are analyzed based on the model. The model provides quantitative interpretations that agree very well with all the reported experimental results, which in turn further confirms the absence of physical aging effects. And in section-VI we summarize the main conclusions.
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2. Experiment The details of the experiments can be found in Ref. [46]. Briefly stating, PS in toluene solutions was spin-coated on thermally grown SiO2 on silicon substrates. The samples were then annealed at 130 °C in vacuum for at least 24 hours, and slowly cooled down (< 0.1 °C/min) and stored at room temperature before testing. The thickness of the resulting PS films ranged from 7 nm to 50 nm depending on the solution concentration and spin speed. These samples will be referred to as fresh samples. As many samples were used in the study, a standard film thickness of 30 nm is taken unless otherwise specified. Fresh samples rubbed to saturation but without going through any other rubbing and thermal processes will be referred to as reference samples. Rubbing was done on a home-made apparatus. Each rubbing pass covered 2 cm distance at a speed of 1 cm/s in one direction, with a normal pressure of 9 g/cm2. The optical RIB of the PS films was measured using the reflectance difference spectroscopy at 633 nm in wavelength. Various thermal and rubbing processes were imposed on the test samples, and their effects on the RIB relaxation behaviors of the samples were studied by using the following ‘standard’ tests. The first was to measure the normalize birefringence (NB) as a function of temperature when the sample temperature was raised continuously at 2 K/min. The results are referred to as the Continuous Curves Ω(T). The second was the isothermal relaxation of NB, i. e., the NB of a sample was measured as a function of time at a fixed temperature. Note that the isothermal relaxation process also served as physical aging as both are expected to take place. The third was the combination of the two, i. e., a continuous temperature increase at 2 K/min after an isothermal process at a given temperature for a given period of time.
3. Physical Aging In this section, we examine the effects of physical aging on the relaxation of RIB.
3.1. Rubbing induced birefringence Figure 5 depicts a typical plot of RIB vs rubbing pass at 23 ºC of two fresh PS samples with molecular weights (Mw) of 99 Kg/mol. For the first sample, the RIB was recorded after each rubbing pass up to 10 passes. For the second sample the birefringence was recorded at every 5 passes. The birefringence increased quickly with the first few rubbing passes, and then gradually approached the saturated value. More than 70 % of the total birefringence was generated by the first 20 passes, and the subsequent 65 passes created the remaining birefringence. Further rubbing after saturation resulted in destroying roughly the same amount of birefringence as creating it, and the net birefringence fluctuated around the saturation value with amplitude of about 1 % of the saturation value. There was also a birefringence transient, which is not shown in the figure, of Δn ≈ 1.0 × 10-4 that relaxed within 30 s after each pass. The net birefringence value of each sample was measured within 100 s after the completion of the relaxation of the transient birefringence due to the last rubbing pass. It was then used as the normalization factor in determining the NB in the subsequent relaxation studies. After the completion of the transient, the birefringence relaxed slowly, decreasing by about Δn = 1.0 × 10-4 in the first hour after rubbing. The maximum
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 259 saturation birefringence was about 20 % of the intrinsic segmental alignment value [40]. However, since only about 10 % of the birefringence was from the segmental alignment and the rest from the segmental distortion [27], the degree of segmental alignment was around 2 % [40]. This could be highly non-uniform, however, with higher concentration close to the surface and decreasing with depth. The intrinsic maximum birefringence of segmental distortion is not known to the best of our knowledge, leaving the degree of segmental distortion still undetermined.
3.2. Typical relaxation behaviors of RIB and surface morphology Besides the RIB, rubbing also creates grooves on the surface which flatten and finally disappear at elevated temperatures, as reported in Part-II. One issue of significance is whether the relaxations of RIB and surface morphology are related. A typical Continuous Curve of RIB of a reference sample with Mw = 99 Kg/mol is shown in Figure 6(a), along with the evolution of surface morphology roughness (points) measured by atomic force microscope (AFM) in tapping mode. The RIB had completely relaxed near 114 ºC. If a freshly rubbed sample is heated to 130 ºC within seconds, all its RIB will disappear within 30 s. A portion of the AFM images are shown in Figures 6(b) and 6(c). The micrographs of nearly the same 5 × 5 μm2 area were recorded at 23 ºC after each annealing at the given temperature for 30 minutes. Using recognizable topological features, such as the crossing of two ditches as markers, a smaller but identical area was selected from the set of micrographs for analysis. The surface Laplace Pressure is estimated as 1.4 × 105 N/m2, and varies by a factor of < 4 across the surface. This is about 1/500 of the yield stress of PS [4]. Careful tracking of the individual topographic features showed that noticeable relaxation of morphology features (≥ 0.1 nm) occurred only at ≥ 85 ºC, and the decrease in roughness was rather rapid beyond this point. On the other hand, at 80 ºC about 30 % of the RIB had already relaxed, while no noticeable change ≥ 0.1 nm in any morphology features occurred. Also, no change in morphology could be detected if the sample was left at 23 ºC for up to 7 days, but during the same period there was significant RIB relaxation (See subsection 3.4 below). It is seen that significant RIB relaxation occurred at much lower temperatures than surface morphology relaxations, a first sign that the two are not directly related. The relaxations of RIB can take place without any collective segmental movement beyond 0.1 nm in length, which is an indication of their very small length scale. The possibility that the morphology birefringence (MB) is or contributes significantly to the observed RIB has been ruled out in our previous works [31] [46]. For the sake of completeness in the discussion, we revisit the main findings. First, the amplitude of MB can be estimated from the surface morphology, and the result showed that the amplitude is too small to account for the observed birefringence [46]. Second, we performed the following experiments [31]. Poly(vinyl alcohol) (PVA) dissolved in water was spin coated on top of some PS samples of different molecular weights after rubbing to saturation. The samples were then dried at 20 ˚C in dry air for two weeks, together with the reference samples rubbed under the same condition but without PVA cover. This resulted in a flat surface PVA cover of about 15 nm with a refractive index (= 1.5) almost the same as PS. The MB will be completely masked even though the PS/PVA interface still preserves the original PS surface morphology. However, the observed birefringence of the PS samples after PVA coating remained the same as before, varying by no more than 1 % of the original Δn, indicating that the observed birefringence was not due to surface morphology, and little birefringence was disturbed by the PVA cover. Third, if the RIB of PS was concentrated only within the very top layer, the PVA coating would certainly alter its relaxation behavior at elevated
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temperature. However, no noticeable difference in relaxation behavior at elevated temperatures was observed for samples covered with PVA as compared to the reference samples. For example, the Continuous Curves for both the PVA covered and the reference samples were essentially the same. This has two implications. First, the relaxations of PS surface grooves are expected to be hindered by the PVA, due to the reduction of Laplace Pressure and the presence of PVA that impedes the movements. But this has no effects on the RIB relaxations. Second, the RIB relaxation is mainly from the segments below the very top PS surface layer, away from the influence of the PVA cover. Combining all the evidence, we conclude that the RIB is not due to the MB of surface, and the relaxations of surface morphology and RIB are not directly related. 4 1.0
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The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 261
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Figure 7. (a) The isothermal relaxation curves at 90 °C of a reference sample and a sample which underwent repeated rubbing process as described in the text. (b) The Continuous Curves of two PS samples. The solid curve is for a reference sample, while the dashed curve is for a sample which underwent repeated rubbing process.
Figure 6(d) shows the NB of a reference sample under a step-like temperature rise sequence. The solid curve is the NB as a function of time as its temperature underwent the sequence shown as the dashed curve in the figure. When the sample was subjected to a fast rise in temperature from 45 °C to 60 °C, a sizable portion of its NB disappeared along with it, followed by a slower and smaller decay as the sample temperature was maintained at 60 °C. After 30 minutes at 60 °C, subsequent rise-then-hold in temperature from 60 °C to 80 °C and from 80 °C to 100 °C brought about similar responses of the NB, until all RIB disappeared. An isothermal relaxation curve of a reference Sample-A is shown in Figure 7(a). The temperature was raised at t = 0 from 23 °C to 90 °C in less than 3 seconds, and then was maintained there for 10 hours. Within the first 10 s about 52 % of the original birefringence had relaxed. The NB then decayed slowly, approaching logarithm in time. Simple isothermal relaxations of reference samples at other elevated temperatures behaved in similar way in that there was always an initial quick drop in NB followed by a slow decay [31].
3.3. Thermal quench before rubbing Several fresh samples were first heated up to 130 ºC and quenched to 23 ºC in less than 300 s, and then rubbed to saturation. Their saturation RIB and Continuous Curves show no
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noticeable difference from the typical ones presented in Figure 5 and Figure 6. This is inconsistent with the established physical aging theory, according to which the samples quenched to 23 ºC should have preserved the majority of the segmental RT’s at 130 ºC. If a reference sample is quickly heated to 130 ºC, all its RIB will disappear within 30 s, implying that at 130 ºC the RIB RT’s are all shorter than 30 s. Therefore, had the RIB RT’s followed the segmental RT’s, all of them in the quenched sample would have been short (≤ 30 s). All RIB in the sample would have relaxed quickly even at 23 ºC, and no net RIB should have even been obtained. This is in clear contradiction to the experimental results.
3.4. Repeated rubbing Conventional segmental relaxations are greatly sped up by high strain/stress [4]. In rubbed PS the RIB is due to the segmental alignments and distortion, so it is taken as a measure of the degree of distortion in the discussion here. More experimental results are presented in Figure 7. Here sample-A was one of the reference samples. Sample-B was from group-B where the samples were first rubbed to saturation (> 80 passes), heated up at 2K/min till 90 ºC, then quenched to 23 ºC and rubbed again until the RIB was back to its original saturation value at the end of the first rubbing run. The entire quenching and rubbing process was completed within 30 minutes. The molecular weight of the samples in both groups was 99 Kg/mol. The isothermal relaxation curves, the dashed curve for sample-B from group-B and the solid curve for a reference sample-A, are depicted in Figure 7(a). The Continuous Curves of a reference sample-A’ and a sample-B’ from group-B are depicted in Figure 7(b). The two curves coincide well in both cases. No significant difference is seen. All these again show that once the RIB is created, its RT’s are not affected by further deformation, in clear contradiction with the behavior of the conventional segmental relaxations. 3.5. Disrupted continuous curve The experimental results presented so far demonstrate that the RIB relaxation does not depend on the rubbing and thermal history before the final rubbing process and the start of the subsequent relaxation measurements. There could be a small possibility that somehow the final rubbing process ‘resets the clock’ and puts the PS all to a state that relax in a very similar way, i. e., similar to mechanical ‘rejuvenation’ in segmental relaxations even though the term itself is not exact [5] [47]. To clarify, we studied the effects of thermal processes conducted after rubbing. The NB curves as a function of temperature of two originally identical samples are shown in Figure 8. For a reference sample-A, the temperature was raised at 2 K/min from 23 °C to 130 °C without interruption. The result is a typical Continuous Curve. The second sample, sample-C, underwent more complicated temperature sequence. First the temperature was raised continuously from 23 °C to 90 °C at 2 K/min. Its NB decreased in almost the same way as sample-A up to 90 °C. Upon reaching 90 °C the temperature was lowered at 2 K/min till 44 °C (when ambient cooling could no longer maintain the 2 K/min rate). The NB continued to decrease by a small amount until the temperature reached about 80 °C, then maintained its value from 80 °C to 44 °C. Upon reaching 44 °C the temperature was raised again to 90 °C at 2 K/min. The NB remained unchanged during the second temperature rise to 80 °C. Upon reaching 90 °C again, the sample was dropped into liquid nitrogen, and then let to warm up in a dry air ambient environment (~ 23 °C) for 16 hours. During the period no RIB change was observed. Finally the temperature was raised continuously from 23 °C to 130 °C at 2 K/min. The NB again
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 263 traced the curve where the temperature was raised from 44 °C to 90 °C, and joined the NB of sample-A after 94 °C. Several points are noted in the above temperature sequence. First, the relaxation of NB of sample-C was identical to that of sample-A for T > 94 °C, i. e., the relaxation above 94 °C was not affected by the thermal history below 90 °C, within the time scale of ~20 hours. Second, no relaxation of NB was observed below 80 °C after the first temperature rise, indicating that by the first time the temperature reached 90 °C the relaxation of NB below 80 °C was already complete. That is also the reason why the NB curves of the second (before liquid nitrogen quench) and the last temperature rise (after quench and annealing at 20 °C for 16 hours) are identical. Third, above 80 °C the relaxation was still active, and the relaxation of NB was again activated when 80 °C was reached from below in both the second and the last temperature rises. Fourth, unlike in the case of physical aging where further nonequilibrium can be introduced by additional thermal process, quenching or other thermal process below 90 °C on rubbed PS brought no increase to the remaining RIB. According to the conventional physical aging theory, the quench in the above case will freeze the thermodynamic state and the segmental RT’s at 90 °C. If the linear compliance of the sample is tested at 20 °C, its characteristic time will be close to that at 90 °C, and many orders of magnitude shorter than the one in thermodynamic equilibrium [1]. If the RIB RT’s had followed the trend of the conventional segmental RT’s in the quench process, then the measured NB would have decreased during the 16 hours at 20 °C and when the temperature was raised again from 20 °C, because the RT’s should have been close to those at 90 °C. The portion of curve-C above 94 °C should have shifted horizontally to the left (lower temperature), and the amount of shift would have depended on the elapse time at 20 °C, as is indicated by the imaginative curve labeled ‘Following PA’ (PA stands for physical aging) in the figure. When the temperature reached 90 °C for the third time the thermodynamic state of sample-C was certainly different from that of sample-A at the same temperature. The fact that the RIB in sample-C relaxed in the same way as sample-A at above 94 °C indicates that although the thermodynamic state of sample-C was different from sample-A, the portion of RIB that would relax at above 94 °C was still the same as before, unaffected by the quenching and reheating.
1.0
NB
0.8 Sample-C
0.6
Sample-A
0.4 PA
0.2 0.0 20
40
60
80
Temperature (°C)
100
120
Figure 8. Normalized birefringence versus temperature of two PS samples. The solid curve is for a reference sample-A, while the dashed curve is for sample-C which underwent several temperature rise and fall sequences as described in the text.
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Z. Yang 1.0
TLag
NB
0.8 0.6
56 K, 70 rubs
6 K, 44 rubs 6 K, 5 rubs
0.4
99 K, 10 rubs
0.2 0.0 20
99 K, 80 rubs
56 K, 10 rubs
40
60
80
Temperature (°C)
100
120
Figure 9. Normalized birefringence of three pairs of samples versus temperature at a heating rate of 2 K/min after annealing at 23 ºC for 7 days. The sample parameters are given in the text.
3.6. The temperature lag phenomenon The Continuous Curve of any rubbed sample following a period of isothermal annealing differs from the ones without the annealing. This is the temperature lag phenomenon first reported in Ref. [31]. Three pairs of samples were studied here. The molecular weights of the pairs were 6.4 Kg/mol, 56 Kg/mol, and 99 Kg/mol, respectively. For each pair of samples, one was rubbed for only a few passes (5 for the Mw = 6.4 Kg/mol sample and 10 for the other two samples) and the other one till saturation (44 passes for the 6.4 Kg/mol sample, 70 passes for the 56 Kg/mol sample, and 80 passes for the 99 Kg/mol sample). The net RIB of the lightly rubbed samples was about 40 % of the saturation values (Δn = 0.012 for the 6.4 Kg/mol sample, Δn = 0.017 for the 56 Kg/mol sample, and Δn = 0.021 for the 99 Kg/mol sample). The rubbed samples were then left at 23 ºC for 7 days, during which they aged and the RIB relaxed but no surface morphology change could be detected by AFM. Figure 9 shows the Continuous Curves of the samples after the aging. Several features are noted. The temperature at which the NB vanished was around 90 ºC for the Mw = 6.4 Kg/mol samples, 104 ºC for the Mw = 56 Kg/mol samples, and 110 ºC for the Mw = 99 Kg/mol samples. These temperatures are close to but do not strictly follow the Tg’s of PS at these molecular weights. Also, they are almost rubbing condition independent. This effectively rules out the possibility of significant chain scissions by rubbing, as shorter chains are equivalent to smaller molecular weights which would lower the Tg and the temperature at which RBI disappears. The same were observed for the reference samples without aging. Within the 7 days, for samples of the same molecular weight, the decrease in NB for lightly rubbed sample was larger than the saturated one. For example, for the 99 Kg/mol samples, the RIB of the lightly rubbed one reduced to 81 % of its original value right after the rubbing, a drop of 19 %, while that of the saturated one reduced to 91 % of its original value, a drop of 9 %. This is in contradiction with the strain accelerated relaxation mechanism [4]. The NB’s did not immediately decrease as the temperature was raised above the aging temperature TA until it reached a higher temperature TD. Above TD the Continuous Curves resembled those reference samples rubbed in the same ways but without the 7-day aging at 23 °C. Despite the large difference in molecular weights and in the number of rubbing passes, the temperature lag, defined as TLag ≡ TD – TA, of the six samples were the same within the experimental uncertainty of ± 1ºC.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 265 More results measured under similar conditions on other samples are summarized in Table 1. The aging/annealing times tA ranged from 1 hour to 7 days. The molecular weights ranged from 6.4 Kg/mol to 550 Kg/mol. The film thicknesses ranged from 7 nm to 30 nm. The annealing temperatures were 23 ºC and 60 ºC. At a given annealing temperature, TLag increased with the logarithm of annealing time, in a similar way as reported earlier [31]. At a given annealing temperature and for a given annealing duration tA, the TLag’s of all the samples were the same within the experimental uncertainty of ± 1ºC, regardless of the molecular weight, the film thickness, and the rubbing passes. In a similar experiment as in subsection 3.4, several group-B samples, after repeated rubbing and heating, were tested for their TLag at 60 ºC, and the outcome showed no discrepancy to the normal values shown in Table-1 and in Figure 10. Therefore, the TLag is also independent of the thermal and rubbing history. Table 1. The temperature lags of the PS samples investigated. Film Thickness (nm)
TA (°C)
Duration tA (s)
3600
22
23 36,000
6.05 × 105
30 7 10
60
36,000
Molecular Weight (kg/mol) 6.4 6.4 12 12 56 56 99 99 6.4 6.4 34 34 6.4 6.4 56 56 99 99 6.4 22 99 550 99 99
TLag (°C)
15.3 15.7 15.4 15.7 15.8 16.1 15.8 15.6 24.2 25.1 25.9 24.6 29.1 28.8 28.4 29.6 27.1 29.1 20.0 20.8 18.9 19.0 20.1 20.1
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Z. Yang
14
Ln(Time (s))
13 12 11 10
60ºC, 7 nm 60ºC, 10 nm 60ºC, 30 nm 90ºC, 30 nm 90ºC, 18nm* 22ºC, 30 nm
9 8 7 5
10
15
20
TLag (°C)
25
30
35
Figure 10. Logarithm of annealing time tA versus temperature lag TLag at several annealing temperatures. The 18* nm sample was quenched from 130 °C to 23 °C several minutes before it was rubbed at 23 °C.
The logarithm of annealing time tA as a function of TLag is plotted in Figure 10 at several annealing temperatures. A nearly linear relation between the two quantities can be confirmed. More quantitative analysis will be present in later sections. Here we concentrate the discussions on its implication on the effects of physical aging. The temperature lag phenomenon is one of the most important characteristics of RIB relaxation, and plays a critical role in the quantitative analysis of its relaxation behaviors. The RIB remains unchanged when the temperature is still in the range of TLag, while above the range the RIB decreases with the increasing temperature in exactly the same way as a sample without going through the annealing and aging. For example, for a sample annealed (and aged) at 23 ºC for 7 days, there was no change in RIB in the temperature range of the subsequent Continuous Curve from 23 ºC to 50 ºC, while the portion of the Continuous Curve above 60 ºC was the same as the one in Figure 5. In other words, the annealing at 23 ºC for 7 days eliminated all the low temperature RIB relaxation up to about 50 ºC, but left the one well above 50 ºC unchanged. This is in stark contradiction with the effects of physical aging, where the segmental relaxations at all temperatures sufficiently below Tg, regardless whether they are above or below the aging temperature, are affected by the aging process [1].
4. Model We have conducted many similar experiments on many other samples. The outcome, together with the experimental results presented in Section-3, can be summarized in the following. (i) The relaxation of RIB does not depend on thermal history before rubbing. Representative experimental evidence is presented in subsection 3.3. (ii) The relaxation of RIB does not depend on the thermal and rubbing history prior to the last rubbing run. Representative experimental evidence is presented in subsection 3.4.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 267 (iii) The dependence of the RIB relaxation on the thermal history after the last rubbing run is governed by the temperature lag effect. If a rubbed sample is aged at temperature TA for a duration of tA, then a ‘lag temperature’ TLag can be found experimentally from its subsequent Continuous Curve such that below TA + TLag the Continuous Curve is flat, and the portion of Continuous Curve above TA + TLag resembles the one obtained from reference samples without aging. Furthermore, TLag is proportional to the logarithm of tA. Representative experimental evidence is presented in subsections 3.5 and 3.6. Optical probes have been used to study the relaxations of PS and other polymers, and substantial physical aging effects were observed. Thurau and Ediger reported photobleaching studies on the segmental relaxations of PS [48]. The rotational probe relaxation followed the KWW function, while the translational probe decayed in a simple exponential form. After quenching from above Tg, the RT’s at 33 ºC were much shorter than the equilibrium values. The aging effect was much less drastic than the typical ones seen in creep compliance experiments [1]. Aging from 103 s to 105 s, a change of time scale of 100 folds, brought RT increase by only three times. The fluorescence intensity of chromophore-doped/labeled PS thin films studied by Ellison et al. [49] was insensitive to aging, changing by only 6 % in total intensity over 5 hours of aging. These results indicate that the relaxations involving smaller length scales are not as sensitive to aging as the ones involving larger scales in creep experiments. Besides the small length scale, the absence of physical aging effects in the RIB relaxation could also be due to the accelerated aging speed in the near surface region. In fact, the aging speed could be depth dependent, similar to the speculated depth dependent Tg [50]. Kawana and Jones reported [11] thin supported PS films aged below Tg that exhibited clear overshooting in expansivity temperature curves when reheated for thicknesses of 18 nm or more, but not for a thickness of 10 nm, suggesting the existence of a surface layer of the order of 10 nm in which aging was complete within ~103 s, while the rest of the film essentially had thermal properties identical to bulk samples. The absence of the physical aging effects in the RIB relaxations could therefore be due to the combined effects of accelerated aging of the near surface region and the small length scales involved in the relaxation process. As was pointed out earlier [31], the RIB relaxation does not follow the KWW function. One essential element for the KWW function to be applicable is that the start and end values of the relaxation observable must be well defined, as in the case of photobleaching where the values are 1 and 0, or in the glass to rubbery transition of creep compliance where the end values are the well established JG and JR values. In isothermal RIB relaxation, the only well defined values are 0 and 1 for NB and there is no ‘level off’ in between. Taking the short time (~ a few hours) end value as ‘level off’ is mistaking [31]. Since KWW function does not apply here, we must start with the generic distribution function and proceed to determine it using experimental results. One of the characteristics of the relaxation processes in glass forming polymers is the heterogeneity. A monotonic relaxation process can in general be expressed as ∞
α (t ) = ∫ g (τ )e −t / τ dτ 0
(2)
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Z. Yang
where α is the relaxation function of an observable, such as the segmental alignment dichroism, the optical birefringence, etc., τ is the relaxation time (RT), and g(τ) is the RT density distribution. In thermodynamically simple cases g(τ) = G(κτ) where the shift factor κ is temperature and thermal history dependent but the functional form of G(x) remains fixed, following the well known time-temperature superposition law and the time-aging time superposition law. There is no general and systematic way to extract G(κτ) from experimental results, but fortunately in many cases [41] [42] [43] G(κτ) takes the simple form of KWW function in the time scale within which the change of κ can be ignored. The G(κτ) after various length of aging times can then be determined and the shift factor κ extracted. The dominant effect of physical aging in the conventional cases is on the time shift factor κ, which leads to the well known time-aging time superposition principle [1]. Extensive experimental evidence in the last section has shown clearly that the RIB relaxation depends strongly on temperature but is insensitive to physical aging. The RIB relaxation can therefore be described by an RT distribution function g(τ) that depends only on temperature but not on thermal or strain history. This is the most significant difference between the conventional segmental relaxations and the RIB relaxation. As the RIB g(τ) depends on temperature only, the existing models that predict the physical aging effects [5] [6] are no longer applicable here. Instead, a new and systematic approach [31] can be devised to determine the distribution function from carefully designed experiments. The phenomenological model we propose is based on the generic model for the relaxations in glass forming polymers that consists of individual relaxing units overcoming an energy barrier under thermal excitations. In the model, the total RIB of a PS sample is taken as the combined contribution of individual RIB elements. To remove its contribution to the total RIB each element must overcome an energy barrier E. The RT is expressed in the conventional Arrhenius form E
τ = Ae ( 273+T ) R
(3)
where R = 8.315 J/ ( K ⋅ mol ) is the gas constant, A is a pre-exponential factor, and T is the temperature in the unit of °C. Once an RIB element is created by rubbing, its RT will not be altered by further thermal process or strain, until it has relaxed and disappeared. By expressing the energy barrier in the form
E = (273 + ξ )η 0
(4)
where η0 is the same for all RIB elements, and ξ (in the unit of °C) is element specific, the RT becomes
τ (T , ξ ) = τ 0 e γ (ξ −T )
(5A)
τ 0 ≡ Aeη / R
(5B
with 0
and
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 269
γ ≡ η 0 / R(273 + T )
(5C).
The parameters γ and τ0 in Eq. (5A) are the same for all elements and depend weakly on temperature, while ξ is generally different for different RIB elements and is independent of temperature. At a given temperature T the parameter ξ alone determines the RT of the element. The prefactor A for all the RIB elements is the same at any given temperature, which means that all elements have the same ‘vibration frequency’ in their potential wells. This results in the RT’s in the ‘threshold temperature’ form as expressed in Eq. (5A), with the ‘threshold temperature’ proportional to the well depth, as is hinted by the step-like drop in NB following a step-like rise of temperature in Figure 6(d). At a given temperature T, the elements with ξ ≤ T will have short RT’s (~ seconds) and relax quickly, while those with ξ > T will have RT’s that increase exponentially with ξ. The rubbing process generates an initial density distribution N (ξ , 0) of elements, where ∞
∫ N (ξ , 0)d ξ = 1 . After spending tA at temperature T the distribution becomes
0
N (ξ , t A ) = N (ξ , 0)e
−
tA
τ (T ,ξ )
(6A)
The isothermal relaxation of the normalized RIB is then given by ∞
NB(T , t ) = ∫ N (ξ , 0)e
−
t
τ (T ,ξ )
dξ
(6B)
0
The expression is equivalent to Eq. (2).
5. Analysis We now proceed to the analysis of the experimental results presented in Section-III.
5.1. Range of ξ where N (ξ , 0) is non-zero According to Eq. (5A), any elements with ξ < 23 °C created by rubbing will have relaxed quickly before any subsequent measurements are carried out. The relaxation of these elements is the origin of the transient birefringence right after each rubbing pass reported in subsection 3.1. On the other hand, the RIB relaxation is complete when the temperature is near Tg (see Figure 6). Therefore, taking into account of the experimental Continuous Curve in Figure 6, we expect N (ξ , 0) to be a smooth and continuous function of ξ in the range of 23 °C ≤ ξ ≤ ~Tg. According to the experimental results in Figure 9, in the Mw = 6.4 Kg/mol samples there were no RIB elements with ξ > 90 °C, while in the Mw = 99 Kg/mol samples there were RIB elements with ξ > 90 °C but not the ones with ξ > 114 °C. When the temperature of a freshly rubbed sample is raised quickly from 23 °C to 90 °C there must be a quick drop of birefringence because the elements with barrier height 23 °C ≤ ξ ≤ 90 °C will relax within ~ 10 s. This quick drop of NB has indeed been observed if one keeps track of the original
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birefringence before temperature rise, as is shown in Figure 7(a). Likewise, a step-like rise of temperature will bring a step-like drop in NB, which was indeed observed (See Figure 6(d)). More analysis on the distribution function will be presented in subsection 5.7. Note that although ξ is in the unit of °C, it describes the potential well depth and itself is independent of temperature.
5.2. Interpretation of temperature lag phenomenon The RT of an RIB element increases exponentially with ξ at a given temperature T. Equation (6) then predicts the ‘temperature lag’ phenomenon reported in subsection 3.6. For a freshly rubbed sample, its initial density distribution is N (ξ , 0) . As its temperature T(t) is raised at a constant rate, the elements with ξ = T(t) will relax quickly as their RT’s become short enough. As a result, the NB decreases with temperature, leading to a typical Continuous Curve as shown in Figure 6(a). However, after a freshly rubbed sample has gone through an isothermal annealing process for certain duration tA at temperature TA, subsequent temperature rise at a constant rate will not bring immediate decrease in NB until a higher temperature TD is reached. This is because the elements with ξ0 ≥ ξ, where τ(TA, ξ0) = tA, have already relaxed. Subsequent rise in temperature will not bring any significant decrease in NB until it reaches TD ≥ ξ0. By measuring the temperature lag TLag (≡ TD – TA) at different annealing time tA, the relation between τ(TA, ξ) and ξ can be obtained, namely tA = τ(TA, ξ = TA + TLag). According to Eq. (5A), TLag is linearly dependent on the logarithm of tA. Using linear fit we obtained the parameters [31] for τ(TA, ξ) at TA = 60 °C, namely γ = 0.42 ± 0.05 K-1 and τ0 = 14 ± 8 s, which lead to η 0 / R = 140, and E ranging from 340.4 kJ/mol to 445.7 kJ/mol. Similarly, in the subsequent part of the paper the parameters at other temperatures from 23 °C to 90 °C and their possible dependence on PS properties will be extracted from the experimental results presented in Section-3. We found that Eq. (5) is followed, and the parameters γ and τ0 can be extracted in all the cases studied.
5.3. Molecular weights, film thickness, and rubbing distance dependence As presented in subsection 3.6, at a given temperature the TLag of samples with different thermal histories, thicknesses, molecular weights, and prepared by different rubbing distances were all the same. This implies that the dependence of τ(T, ξ) on ξ, the energy barriers the RIB elements must overcome, is the same regardless of the molecular weight, the film thickness, thermal history, and the rubbing conditions. This indicates that only the local environment immediately surrounding an individual element determines its energy barrier, rather than the global parameters such as the chain length, the net RIB, or the film thickness. 5.4. Temperature dependence of γ and τ0 For a fixed annealing time tA, TLag is smaller at higher temperatures. The NB curves of four samples are plotted against (T – TA) in Figure 11, where T was the temperature at a constant heating rate of 2 K/min and TA was the annealing temperature prior to the temperature rise. The annealing time was tA = 10 hours for all the samples. The (T – TA) at which NB started to drop was the largest for TA = 45 ºC and smallest for TA = 90 ºC. The TLag data extracted from the curves are plotted against TA in the insert of Figure 11. A linear relation is seen between TLag and TA.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 271 0.90 60 ºC 45 ºC
0.85
NB
90 ºC 22
Tlag (°C)
20
0.80
18
75 ºC
16 14 12 10 40
0.75 0
50
60
70
80
TA (°C)
5
90
100
10
15
20
25
T - TA (K) Figure 11. The NB curves of four PS samples vs (T – TA), where T is the temperature at a constant heating rate of 2 K/min and TA is the annealing temperature prior to the temperature rise. The insert shows the TLag obtained from the curves as a function of TA. The molecular weight of the samples is 550 Kg/mol.
Table 2. The parameters of the relaxation times obtained from the linear fitting in Figure 6. The 18* nm sample was quenched from 130 °C to 23 °C several minutes before it was rubbed at 23 °C. Film Thickness (nm) 7 10 30 30 18* 30
TA (°C) 60 60 60 90 90 23
τ0 (s) 15 ~ 44 15 ~ 33 11 ~ 24 4.5 ~ 5.5 3.3 ~ 33 5.5 ~ 45
γ (K-1)
η0 /R
0.35 ± 0.05 0.37 ± 0.04 0.41 ± 0.03 0.64 ± 0.02 0.64 ± 0.09 0.37 ± 0.04
116 ± 17 123 ± 13 136 ± 10 232 ± 7 232 ± 32 110 ± 12
According to Eq. (5), the above results indicate that either or both the parameters γ and τ0 increases with temperature. To quantitatively determine the two parameters, TLag’s at fixed annealing temperatures were measured at different annealing time tA, and the results are shown in Figure 10. Straight lines of ln(tA) vs. TLag are found. The linear fitting results of the data in Figure 10 are summarized in Table 2. The value of τ0 in all the cases are of the order of 5 ~ 50 s, although at 90 ºC the parameter τ0 does seem to be smaller than at lower temperatures. The data at 60 ºC hint some weak thickness dependence of γ, but the difference is still within the experimental error. The value of γ increases by about 50 % from 60 ºC to 90 ºC. Also to be noted is the 18* nm sample, which was quenched from 130 °C to 23 °C several minutes before it was rubbed at 23 °C. The entire quenching and rubbing process was completed in 30 minutes. Compared to conventional physical aging, such drastic thermal quench had surprisingly little, if any, effects on the RIB RT’s. Summarizing the results presented so far, we conclude that the RT’s of the RIB elements follow Eq. (5A) in the temperature range investigated. The parameter τ0 is at most weakly dependent on temperature, changing by no more than a factor of 10 over the temperature range from 23 ºC to 90 ºC. As a comparison, the actual RT’s change by about 1011 times in
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the same temperature range. The RT’s are independent of the molecular weights, the number of rubbing passes, the film thickness down to 7 nm, and the thermal history. The parameter γ increases linearly with temperature at a rate of 7 × 10-3 K-2, and its value at 90 ºC is 0.64 K-1. According to Eq. (5C) an increase of γ leads to η0 to increase with temperature, indicating that the well depth of the RIB elements not yet relaxed increases with temperature. But since the pre-factor A, according to Eq. (5B), decreases exponentially with η0, an RIB element with ξ = 100 ºC, for example, will still relax quickly only when the temperature reaches 100 ºC.
5.5. Disrupted Continuous Curve If the temperature is lowered shortly after T1 is reached, the RIB elements with ξ ≤ T1 will have relaxed and those above T1 will simply go ‘hibernation’ until the temperature is raised back to the neighborhood of T1. This explains the relaxation behavior of sample-C in subsection-3.5. By the first time the temperature reached 90 °C, all elements with ξ ≤ 90 °C in sample-C had already relaxed while most of those with ξ ≥ 90 °C were still intact. During the first and the second temperature drop and rise, the NB remained constant because there were no ξ ≤ 90 °C RIB elements left, and the RT’s of the remaining elements with ξ ≥ 90 °C were very long. Therefore there were few elements that would relax in that temperature range within the time scale of the experiment. The small decrease in NB above 80 °C was due to the slow relaxations of the elements with ξ slightly above 90 °C. Once the temperature was finally raised above 90 °C, the elements with ξ ≥ 90 °C then relaxed in earnest in the same manner as the reference sample, because their RT’s remained unaltered by the thermal processes. 5.6. Density distribution N(ξ, 0) The analysis so far shows that the dependence of the RT of an RIB element τ(T, ξ) follows Eq. (5A), and the parameters γ and τ0 are the same for all the PS samples investigated so far. As shown in Eq. (6B), τ (T , ξ ) and N (ξ , 0) together determines the relaxation of RIB in a sample. The difference in the relaxations of samples with different molecular weights or rubbing conditions lies in the difference between their density distributions. For example, the reduction of NB at a fixed temperature is faster if there are more elements with lower barrier heights and therefore shorter RT’s. As N (ξ , 0) approaches zero when ξ is near Tg, the upper bound of ξ, above which N (ξ , 0) is zero, of a lower molecular weight sample is lower than that of a higher molecular weight sample. In this part, we concentrate on the extraction of the density distribution N (ξ , 0) from the experimental data, using the relaxation times obtained in subsection 5.5. In our early work [31], a qualitative argument was given to show how to extract the density distribution from the experimental Continuous Curve Ω(T). Here we present a quantitative derivation. It is noted that Ω(T) is a special form of NB(T, t) with
T (t ) = T0 + αt
(7)
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 273 At 2 K/min, α = 1 K/s, and T0 is the starting temperature at t = 0. For Ω(T) after 30 annealing at temperature TA for a certain duration, T0 = TA. To obtain the initial density distribution N (ξ , 0) , the measurement of Ω(T) must start from the rubbing temperature within 100 s after rubbing. The general expression for the relaxation of NB with given temperature T(t) is ∞
NB(T (t ), t ) = ∫ N (ξ , 0)e − f (ξ , t ) d ξ
(8A)
0
with
dt ' 0 τ (T (t '), ξ ) t
f (ξ , t ) = ∫
(8B)
The relaxation time τ (T , ξ ) is given by Eq. 5(A). We take τ0 = 15 s, and γ = 0.64 K-1 at 90 °C and increases linearly with temperature at a rate of 7 × 10-3 K-2, and proceed to show how to extract the density distribution from the measurements of Ω(T). We define the relaxation function as
K (t , ξ ) ≡ e − f (ξ ,t )
(9)
where T(t) is given by Eq. (7). From the numerical results with α = 1 K/s shown in Figure 30 12(a), it is seen that K(t, ξ) is nearly a step function of ξ, the rise being at the point where ξ = T(t). The dashed curve is the negative derivative − ∂K = − ∂K . It is a narrow peak function α∂t ∂T with half width at half height proportional to 1/γ and a total area of unity. It then follows that ∞
dΩ = dT
d ( ∫ N (ξ , 0) K (t , ξ )d ξ ) 0
α dt
∞
= ∫ N (ξ , 0) 0
∂K d ξ ≈ − N (ξ , 0) |ξ =T ∂T
(10)
i. e., the density distribution N (ξ , 0) can be extracted from the experimentally measured Continuous Curve by taking its derivative with respect to temperature. The same conclusion was reached in the qualitative argument [31]. A typical density distribution N (ξ , 0) of a reference sample is shown in Figure 12(b), extracted from the experimental Continuous Curve in the same figure. The density distribution was then put back to Eq. (8), and by using Eq. (5A) for the relaxation times, to generate a calculated Continuous Curve at the heating rate of 2 K/min. It is seen that the calculated one coincides with the experimental one very well. It should be noted that the relaxation times used in the calculation were extracted from the TLag measurements from other samples. The fact that the two curves agree well with one another provides a strong evidence to support the theoretical model.
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1.0
0.20
0.8
0.15
0.6 0.10 0.4 0.05
0.2 0.0 40
20 1.0
(a)
50
40
60
70
ξ (°C) ξ (°C) 60
80
90
80
100
2K/min
0.00 100
120 4 3
0.2K/min
0.6
2 0.4
Density Function
1
0.2 0.0 20
N(ξ)
NB
0.8
Derivative of K(t, ξ)
K(t, ξ)
274
(b)
40
60
80
100
0 120
Tempearture (°C) Figure 12. (a) The calculated decay function at a constant heating rate of 2 K/min (solid curve) along with its derivative (dashed curve) as a function of the energy barrier parameter ξ at the time when the corresponding temperature is 60 ºC. (b) The experimental Continuous Curve and the density distribution N(ξ) together with the numerical simulation results. The solid curve is the experimental curve (left and bottom axes). The circular points are the density distribution N(ξ) obtained from the experimental Continuous Curve by taking derivative versus temperature (right and top axes). The two dashed curves are numerical simulations of the Continuous Curve at two heating rates. The molecular weight of the sample is 550 Kg/mol.
Using the same data, we also simulated the Continuous Curve for heating rate at 0.2 K/min, 10 times slower than the one used in the measurements. It is seen that because the elements now have more time to relax, the amount of remaining elements at any temperature is smaller than at a faster heating rate. Extracting the density distribution from such Continuous Curve therefore requires more complicated numerical work than simple derivative operations.
5.7. Rubbing dependence of N (ξ , 0) In the sections above we have shown that the RT’s are independent of the rubbing conditions. In this section, we explore the effects of rubbing on the density
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 275 distribution N (ξ , 0) . Shown in Figure 13 are the density distributions of the six samples obtained from the Continuous Curves in Figure 9. It is seen that below ξ ≈ 45 ºC the density is zero for all the samples, due to the relaxation for 7 days at 23 ºC. The ‘temperature lag’ phenomenon is clearly manifested. For the two Mw = 6.4 Kg/mol samples, the density distributions are about the same, and are nearly uniform between ξ ≈ 60 ºC and ξ ≈ 80 ºC. Both drop to zero at ξ ≈ 90 ºC. For the Mw = 56 Kg/mol and Mw = 99 Kg/mol samples, the density distribution of the lightly rubbed ones are nearly constant between ξ ≈ 50 ºC and ξ ≈ 100 ºC, but the distribution of the ones rubbed to saturation have a hump above ξ ≈ 90 ºC. The results indicate that in relatively high molecular weight PS, the rubbing process first creates birefringence elements with a nearly uniform distribution of energy barriers, and further rubbing creates elements with barriers more concentrated between ξ ≈ 90 and 100 ºC. For light molecular weight PS, the rubbing process creates a uniform density distribution all the way to the saturation birefringence. This could be due to the entanglements of the PS molecules, as 6.4 Kg/mol is below the entanglement limit of Mw ≈ 20 Kg/mol [51] while 56 Kg/mol is above it. Further study is being carried out to understand the microscopic mechanism of the rubbing process.
5.8. Repeated rubbing As has already been shown above, the RT’s of the RIB elements, once created, are independent of the subsequent thermal or rubbing processes until the elements have relaxed. Repeated rubbing processes were attempted in the hope to generate higher concentration of deeper barriers in the samples. As presented in subsection 3.4, a rubbed sample was first raised to 90 ºC and quenched to 23 ºC to eliminate all the RIB elements except for the ones with the barriers ξ ≥ 90 °C. It was rubbed again, in the hope that if the chance of generating barriers of all depths had been the same, then there would have been more barriers with ξ ≥ 90 °C after the second rubbing run than the first run. The experimental results presented in Figure 7, however, imply that the distribution density function N (ξ , 0) at the end of the second rubbing run was nearly the same as the first run. This is somewhat surprising. A likely scenario is that the second rubbing run destroyed and created about the same amount of elements with ξ ≥ 90 °C. As a result, the relaxations of the repeatedly rubbed samples were the same as the reference ones. 5.9. Discussions The major findings presented so far seem to contradict the cooperative motion characteristics of segmental relaxations. For example, the RT’s of creep compliance can change by many orders of magnitude during aging. Yet such change has little effects on the local environment of the remaining RIB elements, such as their energy barrier heights and/or well width, either before or after they are formed. Such finding, however, is consistent with the very small length scale involved in the RIB relaxations. The change in thermodynamic state changes the large scale cooperative motions, but has diminishing effects on the smaller length scale RIB relaxations. The small length scale of RIB relaxation could imply that it is of the nature of βrelaxation due to side group motions. However, the β-relaxation relaxation times are much shorter than 1 s at 20 ºC and above, while the RIB relaxation times can exceed 1010 s, comparable to that of the cooperative segmental relaxations (α-relaxation). This effectively
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rules out the possibility that the RIB relaxation is of the nature of β-relaxation. The microscopic picture of RIB is still largely unknown other than that most of it is due to the segmental distortions, a subject that is still poorly understood.
1000 x N(ξ, 0)
20 Mw = 6.4 K 5 rubs 44 rubs
15 10 5 (a)
0 20
40
60
80
100
120
80
100
120
100
120
ξ (°C)
1000 x N(ξ, 0)
20 15
Mw = 56 K 10 rubs 70 rubs
10 5 0 20
(b)
40
60
ξ (°C)
1000 x N(ξ, 0)
20 15
Mw = 99 K 10 rubs 80 rubs
10 5 (c)
0 20
40
60
80
ξ (°C)
Figure 13. The density distributions of the three pairs of samples extracted from the Continuous Curves in Figure 8.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 277
6. Summary A systematic study of the relaxation of rubbing induced birefringence in PS has been conducted. Extensive and clear experimental evidence have been found that show the absence of the physical aging effects in the relaxation of RIB, and the relaxation of RIB involves very small length scales. The RIB relaxation is then modeled by a relaxation times distribution function that depends only on temperature but not on thermal or strain history. An individual birefringence elements model has been proposed and a systematic way has been devised to extract the parameters in the model from specifically designed experiments, namely the Temperature Lag measurements and the Continuous Curve measurements. The results predicted by the model agree well with experiments. The absence of physical aging effects in RIB relaxation is in stark contrast to the conventional segmental relaxations, where the relaxation times can be changed by many orders of magnitude by thermal processes or stress, a phenomenon that has been extensively studies in the last four decades. This is probably due to the much accelerated aging process in the near surface region, and the very small length scale of the RIB relaxation. A model for the RIB relaxation and for the absence of the physical aging effects based on microscopic first principles is still lacking. The results presented here provide a concrete test ground for the development of future theoretical models.
PART IV. RIB IN OTHER GLASSY POLYMERS 1. Introduction The viscoelastic properties and segmental relaxations of glass forming polymers above the glass transition temperature (Tg) are generally well understood. The viscoelasticity follows the time-temperature superposition principle [52]. In the segmental alignment relaxation of polystyrene (PS) drawn at above Tg there are three well separated relaxation steps with distinct time scales τ1, τ2, and τ3 [40]. The relaxation time constants in these cases all follow the so-called Vogel–Fulcher temperature law [3] [52] [53]. Below Tg the relaxation phenomena are much more diversified and without a unified picture as in the cases above Tg. The segmental relaxation of polycarbonate (PC) still follows the time-temperature superposition, and the relaxation time follows the Arrhenius form with activation energy of 958 KJ/(mol·K). The relaxation becomes much slower at temperatures below Tg – 20 °C [52]. Additional α-relaxation besides the normal α-relaxation peak was observed in dielectric relaxations [54] [55]. The relaxation times follow the Arrhenius form with activation energies in the range of 17 – 55 KJ/(mol·K). Dielectric probe molecules were mixed with PS and their size dependence of dielectric relaxations was investigated [56] [57]. Below a particular size-1, no coupling between the probe and host PS was found. Above size-1 but below size-2, both α-relaxation and β-relaxation coupled to the probes occurred. Above size-2 no β-relaxation coupling was observed [56]. In Ref. [57], the shortest probes revealed β*-relaxation in additional to the normal α-relaxation and β-relaxation. Reorientation of detrapped molecules dissolved in PS revealed by electron parametric resonance is much faster even at low temperatures (190 GHz at 200 K) and encounters much lower barriers (~600 J/(mol·K)) [58].
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Non-linear optics probe molecules dispersed in PS [59] revealed that below Tg the relaxation time τ follows the Arrhenius form with activation energies of 45 – 50 kcal/(mol·K), or 188 – 209 kJ/(mol·K). Similar relaxation has been found in labeled PS [60]. Other non-linear optical probe molecules have much longer decay time (~ days) [61]. Relaxation of poled order in dye doped PS was probed by isothermal and non-isothermal current measurements [62]. Neutron scattering revealed that the fast process of phenyl rings starts at 200 K, and main chain starts at 250 K [63]. The relaxation of the segmental alignment dichroism of cold rolled PS at 60 °C can be fitted by two single exponential functions of time with RT’s of 76 s and 3600 s [39]. The rubbing induced birefringence (RIB) relaxations of a number of polymers were first studied by Kovacs and Hobbs [44]. Significant relaxations were observed below Tg. In PartIII, we found that the physical aging effects are absent in the relaxation of RIB in PS, and the relaxation involves very small length scale. A phenomenological model based on individual birefringence elements was proposed for the RIB relaxations. The relaxation times (RT’s) of the elements were found to be independent of the thermal or stress history of the samples, either before or after the formation of the birefringence. The RT’s were also independent of the molecular weight, rubbing conditions, and film thickness, while the initial RT’s distribution function did depend on the molecular weight and rubbing conditions. The model provided quantitative interpretations that agreed very well with all the reported experimental results, and shed important light to the novel behaviors of the RIB relaxation in polymers below Tg. In this part, we report on the RIB relaxations of other glass forming polymers, including PC and PS derivatives with various modifications to the phenyl ring side group. It is found that the RIB relaxations follow qualitatively the same way as PS, but there is quantitative difference in minor details among them. On the other hand, they are qualitatively different from any of the other reported relaxations mentioned above [39, 57 – 68]. No physical aging effects in the relaxations of RIB are found. The relaxations are then analyzed under the same theoretical framework as in Part-III. The energy barrier height decreases with decreasing temperature in all the cases, leading to a sub-Arrhenius form of RT dependence on temperature at temperatures below Tg.
2. Experiments Polymers in toluene solutions were spin-coated on thermally grown SiO2 on silicon substrates. The samples were then annealed at 20 °C above Tg in vacuum for at least 24 hours, and slowly cooled down (< 0.1 °C/min) and stored at room temperature before testing. The thickness of the resulting polymer films ranged from 38 nm to 70 nm. Rubbing was done on a home-made apparatus. Each rubbing pass covered 2 cm distance at a speed of 1 cm/s in one direction, with a normal pressure of 9 g/cm2, except for the B5 samples which required 25 g/cm2. The optical RIB of the PS films was measured using the reflectance difference spectroscopy at 633 nm in wavelength. The RIB vs rubbing pass at 23 ºC of the polymers behaved in a similar way as PS. The birefringence increased quickly with the first few rubbing passes, and then gradually approached the saturated value. For PC about 10 % of the total birefringence was created in the first 5 rubbing passes, and the subsequent 75 passes created the remaining birefringence.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 279 Further rubbing after saturation resulted in eliminating roughly the same amount of birefringence as creating it, and the net birefringence fluctuated around the saturation value with amplitude of about 1 % of the saturation value. Other polymers behaved almost the same. The RIB approached saturation at about 80 rubbing passes, except for B2 samples, the saturation pass of which being 60 due to its low molecular weight. The net birefringence value of each sample was measured within 100 s after the last rubbing pass. It was then used as the normalization factor in determining the normalized birefringence (NB) in the subsequent relaxation experiments. Details about the samples parameters and the maximum birefringence are listed in Table 3. The maximum birefringence ranges from 8.0 × 10-3 for B2 samples (poly(4-methylstyrene), Mw = 2.7 kg/mol) to nearly 6.0 × 10-2 for B3 samples (poly(4-methoxystyrene), Mw = 10.5 kg/mol), as compared to the maximum birefringence of 2.5 × 10-2 for PS. This indicates that the side groups contribute significantly to the RIB. The RIB relaxation behaviors of the samples were studied by using the following ‘standard’ methods. The first was to measure the NB as a function of temperature when the sample temperature was raised continuously at 2 K/min. The results are referred to as the Continuous Curves Ω(T). The second was the isothermal relaxation of NB, i. e., the NB of a sample was measured as a function of time at a fixed temperature. The third was the combination of the two, i. e., continuous temperature rise at 2 K/min immediately following an isothermal process at a given temperature for a given period of time.
3. Results and Discussions 3.1. Disrupted continuous curve Rubbing deforms the surface region of the sample and puts the sample in nonequilibrium. To verify the absence of physical aging effects, we studied the effects of thermal processes conducted after rubbing, namely the disrupted Continuous Curve experiment, in the same approach as in Part-III. The NB curves as a function of temperature of two originally identical PC samples are shown in Figure 14. For a reference sample-A, the temperature was raised at 2 K/min from 23 °C to 180 °C without interruption. The result is a typical Continuous Curve. The second sample, sample-B, underwent more complicated temperature sequence. First the temperature was raised continuously from 23 °C to 100 °C at 2 K/min. Its NB decreased in almost the same way as sample-A up to 100 °C. Upon reaching 100 °C the temperature was lowered at 2 K/min till 30 °C. The NB increased slightly and linearly with the lowering of temperature due to thermal expansion. Upon reaching 30 °C the temperature was raised again to 100 °C at 2 K/min. The NB traces back the nearly flat line formed during the lowering of temperature. Upon reaching 100 °C again, the sample was dropped into liquid nitrogen, and then let to warm up in a dry air ambient environment (~ 23 °C) for 16 hours. During the period no RIB change was observed. Finally the temperature was raised continuously from 30 °C to 180 °C at 2 K/min. The NB again traced the straight line where the temperature was raised from 30 °C to 100 °C for the second time, and joint the NB of sample-A after 104 °C.
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1.0 PC Sample-A Sample-B
NB
0.8 0.6 0.4 PA
0.2 0.0 20 40
60 80 100 120 140 160 180
Temperature (ºC)
Figure 14. Normalized birefringence versus temperature (Continuous Curve) of two PC samples. The solid curve is for a reference sample-A, while the dashed curve is for sample-B which underwent several temperature rise and fall sequences as described in the text.
Table 3. Physical parameters of the samples used in the study. Mw (kg/mol) 2.7 K
Thickne ss (nm) 58
RIB (10-3)
10.5 K
40
B4
poly(4-methoxy styrene) poly(4-methyl styrene)
90 K
49
B5
poly(α-methyl styrene)
112 K
38
B6 B7
poly(4-methyl styrene) poly(4-tertbutylstyrene) Poly(4-hydroxyl styrene)
50 K 3.2 M
45 40
47.4 ~ 59.3 23.6 ~ 27.4 16.4 ~ 19.6 11.6 42.0
69.5 K
50
Sample Name B2 B3
T3
PC
Polymer poly(4-methyl styrene)
Polycarbonate
Polydisperse d
70
8.0
13.0 ~ 16.8
11.4 ~ 15.2
TA (ºC) 45
τ0 (s)
η(C-1)
30 ~ 90
85
16 ~ 25
95
22 ~ 33
140
37 ~ 180
N/A 130
N/A 1.7 ~ 3.7
60 60
0.33 ~ 2.5 74 ~ 134
80
90 ~ 245
100
15 ~ 40
120
5.5 ~ 15
0.36 ± 0.04 0.54 ± 0.02 0.45 ± 0.02 0.32 ± 0.04 N/A 0.59 ± 0.02 0.38 ± 0.04 0.24 ± 0.01 0.25 ± 0.02 0.41 ± 0.03 0.49 ± 0.03
Several points are noted in the above temperature sequence. First, the relaxation of NB of sample-B was identical to that of sample-A for T > 104 °C, i. e., the relaxation above 104 °C was not affected by the thermal history below 100 °C, within the time scale of ~ 20 hours. Second, no relaxation of NB was observed below 95 °C after the first temperature rise, indicating that by the first time the temperature reached 100 °C the relaxation of NB below 95 °C had already completed. That is also the reason why the NB curves of the second (before
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 281 liquid nitrogen quench) and the last temperature rise (after quench and annealing at 23 °C for 16 hours) are identical. Third, above 95 °C the relaxation was still active, and the relaxation of NB was again activated when 95 °C was reached from below in both the second and the last temperature rises. Fourth, unlike in the case of physical aging where further nonequilibrium can be introduced by additional thermal process, quenching or other thermal process below 100 °C brought no increase to the remaining RIB. According to the conventional physical aging theory, the quench in the above case will freeze the thermodynamic state and the segmental RT’s at 100 °C. If the linear compliance of the sample is tested at 30 °C, its characteristic time will be close to that at 100 °C, and many orders of magnitude shorter than the one in thermodynamic equilibrium [1]. If the RIB RT’s had followed the trend of the conventional segmental RT’s in the quench process, then the measured NB would have decreased during the 16 hours at 23 °C and when the temperature was raised again from 30 °C, because the RT’s should have been close to those at 100 °C. The portion of curve-B above 104 °C should have shifted horizontally to the left (lower temperature), and the amount of shift would have depended on the elapse time at 23 °C, as is indicated by the imaginative curve labeled ‘PA’ (PA stands for physical aging) in the figure. When the temperature reached 100 °C for the third time the thermodynamic state of sample-B was certainly different from that of sample-A at the same temperature. The fact that sample-B relaxed in the same way as sample-A at above 104 °C indicates that although the thermodynamic state of sample-B was different from sample-A, the portion of RIB that would relax at above 104 °C was still the same as before, unaffected by the quenching and reheating.
3.2. Continuous Curves The two solid curves in Figure 15 are the Continuous Curves of two PC samples, one rubbed for 5 passes and the other rubbed till saturation. The curves are smooth and monotonic, without step-like features which could be caused by possible discrete RIB domain relaxations. The temperature T0 at which RIB reaches zero is 156 ºC, which is close to the glass transition temperature Tg of PC. The curve for the lightly rubbed sample is lower than that of the one rubbed to saturation. The density distribution functions N (ξ , 0) of the two samples (dashed curves in Figure 15) obtained by using Eq. (10) in Part-III are depicted as well. Unlike low molecular weight PS or lightly rubbed high molecular weight PS, where the density function is nearly flat between ξ ~ 23 ºC and ξ ~ Tg , the density function of even the lightly rubbed PC sample has a peak right below T0, and the rest of the density is nearly flat. For the rubbed to saturation sample, the peak is even higher. This indicates that unlike in PS, initial rubbing on PC already creates more favorably the high energy barrier birefringence elements. Like high molecular weight PS, further rubbing creates even more high-barrier elements and relatively less low-barrier elements. The width of the peak of PC, however, is narrower than that of PS. More Continuous Curves for other polymers rubbed till saturation are shown in Figure 16(a). All curves behave qualitatively the same. The RIB approaches zero near the Tg of the corresponding polymers, except for poly(4-tert-butylstyrene) (B7) which has a tail extending well above its Tg. Again, all curves are without step-like features, which rules out domain-like relaxation behavior. The density functions of the polymers obtained from the curves in Figure 16(a) are depicted in Figure 16(b). It is seen that except for poly(α-methyl styrene) (B5), all the others show a peak in their density functions. The shape of the density function of the
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poly(4-methyl styrene) (B4) sample is close to that of high molecular weight PS, with a peak near ξ = 90 ºC. For the same type of polymer (B2) with lower molecular weight (Mw = 2.7 kg/mol) the peak of the density is near the barrier value of ξ = 50 ºC. The density functions of poly(4-methoxy styrene) (B3) and poly(4-tert-butylstyrene) (B7) have high peaks below their Tg’s but with somewhat different shapes. The detailed differences between these polymers indicate different microscopic details of the birefringence elements in different polymers, which remain to be further explored.
ξ (°C) 20 1.0
40
60
60 Passes 5 Passes
0.8
30 25 20
0.6
15 0.4 0.2 0.0 20
3
10
N(ξ) × 10
NB
80 100 120 140 160
5 40
60
80 100 120 140 160
0
Temperature (°C)
Figure 15. Continuous Curve (left and lower axes) and distribution of barriers (right and upper axes) of two PC samples, one lightly rubbed and the other rubbed till saturation.
1.0 B2 B3 B4 B5 B7
NB
0.8 0.6 0.4 0.2 (a)
0.0 20 40 60 80 100 120 140 160 180
Temperature (°C) Figure 16 (Continued)
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 283
N(ξ) × 10
3
40 B2 B3 B4 B5 B7
30 20 10 (b)
0 20 40 60 80 100 120 140 160 180
ξ (°C)
Figure 16. (a) Continuous Curves and (b) Density distribution of barriers of several polymers.
The surface layer of PS-like polymers is expected to have a gradient of mobility enhancement, with the top layer at the surface being much more mobile than the one deep in the bulk. The density function of the polymers presented here might be related to such depth dependent mobility, with the birefringence elements with higher barriers being in the deeper region from the surface, and the elements with lower barriers closer to the surface. Further investigations are required to verify the hypothesis.
3.3. Isothermal relaxation curves Figure 17 shows the RIB isothermal relaxations of several samples as a function of time, together with a PS sample for comparison. The NB(t) curve for B2 has been shifted upward by 0.3 for clarity. All curves behave qualitatively the same. For example, when the temperature is raised quickly from 23 °C to the isothermal relaxation temperature TA (60 °C for PC, PS and T3, 45 °C for B2), there is a quick drop of NB within ~ 10 s, because the elements with barrier height 23 °C ≤ ξ ≤ TA will readily relax. The NB then decayed slowly, approaching logarithm in time. The RT’s of the polymers span a wide range. Some are shorter than 10 s, while others are longer than 104 s. For example, for PC, 17 % of the elements have RT’s shorter than 10 s, while over 70 % of the elements have RT’s exceeding 104 s. At such rate, it would probably take a time in the geological time scale for the remaining RIB elements of PC to relax completely. Similar conclusions can be drawn for the other polymers. 3.4. Temperature lag The temperature lag phenomenon has been observed in the RIB relaxations of all the polymers we have investigated in this study. Figure 18 shows a typical set of experimental temperature lag Continuous Curves. Notice that after the samples had stayed at a certain temperature TA for certain duration tA, subsequent temperature rise above TA did not bring the RIB to decrease immediately. Instead, the RIB remained unchanged until a higher temperature TA + TLag was reached. Beyond TA + TLag the RIB started to decrease again, in much the same way as the sample without having spent time tA at TA. The temperature lag TLag was larger for longer annealing time tA. According to the discussion in Part-III, the dependence of RT of the birefringence elements on the barrier height parameter ξ, in the form
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of Eq. (5A), can readily be obtained from the measured TLag and tA, namely t A = τ 0 e
γ TLag
. The
experimental tA vs TLag data are presented in Figure 18 together with the least square fittings. The obtained parameters τ 0 and γ are listed in Table 3. It is seen that the value of τ 0 differs by up to a factor of 10 for all the polymers except for T3, its smaller than the rest of the polymers. The exponential parameter
τ 0 being about 100 times
γ varies by a factor less than
3, ranging from 0.23 for PC at 60 °C to 0.59 for B7 at 130 °C. The variation is within that of the temperature dependent γ of PS. There is no correlation between γ and T0 of the corresponding polymer, even though their T0 varies from about 80 °C (B2) to nearly 180 °C (B7), indicating that the relaxation of RIB is influenced only by a very small scale local environment. Taking into account the fact that in PS nearly 90 % of the RIB is due to segmental distortion and the fact that the RIB relaxations of all the polymers studied here behave in much the same way, we speculate that the majority of RIB in these polymers is due to the segmental distortion in the polymers.
1.0
PC, 60°C T3, 60°C PS, 60°C B2, 45°C
0.9
NB
0.8 0.7 0.6 0.5 0.4 1 10
10
2
3
10
10
4
5
10
Time (S) Figure 17. Isothermal relaxation curves of RIB of several polymers.
The samples listed in Table 3 are all thick enough that the finite thickness effects are avoided. This can be verified by the fact that none of their T0 is substantially lower than their Tg. In Part-III, we have shown that the amount of temperature lag of PS is independent of the film thickness. We expect the same for the polymers presented here. We want to emphasize again that the temperature lag phenomenon is in contradiction with the effects of physical aging, proving once again that aging effects are absent in the relaxation of RIB. In the temperature lag phenomenon, the RIB remains unchanged when the temperature is still in the range of TA + TLag, while above TA + TLag the RIB decreases with the increasing temperature in the same way as a sample without going through the annealing (aging) at TA. The annealing at TA eliminated all the RIB elements up to about ξ ≤ TA + TLag, but left the ones with ξ’s well above TA + TLag unchanged. This is in stark contrast with the effects of physical aging, where the segmental relaxations at all temperatures sufficiently
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 285 below Tg, regardless whether they are above or below the aging temperature, are affected by the aging process [1].
3.5. Temperature dependence of energy barriers At fixed annealing times the temperature lag TLag is usually temperature dependent. A typical one is shown in Figure 19(a). The general trend is that TLag decreases with increase temperature. Figure 19(b) summarizes TLag at fixed annealing time of 10 hours at different temperatures for several polymers. It is consistent with the increase of parameter γ with temperature shown in Tab. 3. For PC, TLag is nearly temperature independent above 100 ºC, and increase with decreasing temperature below 100 ºC at about the same rate as PS. For B6, the TLag increases with decreasing temperature in the entire temperature range of investigation and at a rate faster than PS. For T3, TLag is nearly constant below 80 ºC and then decreases with increasing temperature at a rate nearly the same as B6. These detailed differences among the polymers suggest the influence of detailed microscopic environment on the RT’s of the RIB elements.
0.9 PC
NB
0.8
30 Min. 3 hrs. 10 hrs. 3 days
0.7 TLag
0.6 0.5
(a)
0.4 60
70
80
90
100
Temperature (ºC)
110
120
13
Ln[tA (s)]
12 11
PC
10
60°C 80°C 100°C 120°C
9 8 7 5
Figure 18 (Continued)
(b)
10
15
20
25 TLag(°C)
30
35
40
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13
Ln[tA (s)]
12 11 10
T3 B2 B3 B4 B5 B7
9 8
(c)
7 5
10
15
20
25
30
35
40
TLag (°C) Figure 18. (a) The Tlag curves of PC at fixed temperature and different annealing times; least square fittings of logarithmic annealing time tA vs Tlag for (b) PC at different temperatures, and (c) other polymers.
1.0
TLag
T3 45 °C 60 °C 75 °C 90 °C 110 °C 130 °C 150 °C
NB
0.9 0.8 0.7
(a)
0.6 0
20
40
60
80
100
120
Tlag (°C)
TLag (°C)
40 B6 T3 PS PC
30 20 10
(b)
40
60
80
100
120
140
160
Termperature (°C) Figure 19. (a) Typical Continuous Curves of PC showing Tlag at fixed annealing time of 10 hours and at different temperatures; (b) Tlag vs temperature at fixed annealing time of 10 hours of several polymers.
The Absence of Physical Aging Effects in the Surface Region of Glassy Polymers 287 The highest energy barrier for PC at T = 120 ºC for the RIB elements with ξ = 150 ºC is E = 677 kJ/mol, which is lower than the value for segmental relaxations of 958 KJ/(mol·K) [52]. The energy barriers of the same elements at 60 ºC are E = 273 kJ/mol, which is less than half of the value at 120 ºC, mainly due to the temperature dependence of γ . Similar results are obtained for other polymers. Such results have an interesting implication. It is well known that above Tg the segmental RT’s follow the Vogel – Fulcher form [3] [52] [53], which increases faster than the Arrhenius form with decreasing temperature. It is equivalent to an Arrhenius form with an energy barrier that increases with decreasing temperature. Below Tg the relaxation times of other forms of relaxation reported so far are in Arrhenius form with a smaller but temperature independent barrier ranging from 50 kJ/mol to 209 kJ/mol [54] [55] [59] [60]. What we have observed here is an energy barrier that decreases with decreasing temperature. The RT still increases with decreasing temperature but at a rate that is slower than the Arrhenius form, because the energy barrier is also decreasing. We call it subArrhenius form of relaxation times. Figure 20 shows the simulation RT in an Arrhenius form and that of PS RIB elements with ξ = 100 ºC, based on the temperature dependent RT’s obtained in Part-III. The Arrhenius energy barrier E and the pre-factor are so chosen such that the two curves have the same slope and value at T = 100 ºC. It is clear that the increase of RT of RIB elements with ξ = 100 ºC is indeed slower than the Arrhenius form at temperatures much lower than 100 ºC. Above T = 100 ºC, however, the RT of RIB increases faster than Arrhenius with temperature.
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Temperature (°C) Figure 20. Simulation of RIB relaxation times of PS (solid curve) and an imaginative one following Arrhenius form as a function of temperature.
4. Summary In summary, we have shown that RIB can be generated in many glass forming polymers. Significant relaxations of RIB can take place well below the glass transition temperature Tg, similar to what Kovacs and Hobbs have reported [44]. The relaxation times span a wide range from ~ 10 s to probably geological time scale. Physical aging effects are absent in the RIB relaxations. The model proposed for the interpretation of RIB in PS works well for all the
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polymers investigated here. The energy barriers are of the order of a few hundred kJ/mol and decrease with decreasing temperature. It is in opposite of the trend of Vogel – Fulcher form for polymer segmental relaxations above Tg, and is different from the barriers found in other relaxation processes below Tg, which are temperature independent [39, 57, 60 – 68]. The relaxation behaviors of different polymers are qualitatively similar but somewhat different in quantitative details, such as in the values of the saturated birefringence, the shape of the initial barrier density distribution functions, the rates of barrier decrease with decreasing temperature, and the dependence of relaxation times on temperature and parameter ξ, etc. They are different from any of the relaxations below Tg that have been reported in the literature [39, 57, 60 – 68]. In view of the substantial amount of experimental results, a microscopic model for the relaxations of RIB is much desired.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 9
CURRENT DEVELOPMENTS IN DOUBLE HYDROPHILIC BLOCK COPOLYMERS G. Mountrichas and S. Pispas Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., 11635, Athens, Greece
ABSTRACT Double hydrophilic block copolymers (DHBCs) constitute a novel class of watersoluble macromolecules with potential utilization in a wide range of applications. The exceptional combination of features, coming from their block copolymer structure and their ability to be stimuli responsive, establishes this class of copolymers as a core of intense research interest, aiming at elucidating aspects regarding their targeted synthesis, solution behavior and application possibilities. In this chapter, the current developments in the field of double hydrophilic block copolymers are discussed. In particular, synthetic strategies leading to the preparation of DHBCs are described. Moreover, their aqueous solution behavior is examined in respect to their ability to self assemble, due to changes in the solution temperature, and/or pH, as well as due to complexation. Additionally, the potential applications of DHBCs in mineralization processes, nanomedicine, nanotechnology and so on are mentioned. Finally, future perspectives in the field of DHBCs regarding general polymer science and nanotechnology issues, as well as open scientific questions, on synthesis and solution behavior of this class of materials, are also discussed.
1. INTRODUCTION Double hydrophilic block copolymers (DHBCs) are a class of polymers that combine the self assembly ability of block copolymers with the water solubility of hydrophilic macromolecular chains. Numerous sophisticated works have been already described in the literature, indicating the potential of this class of copolymers in emerging technologies. The synthesis of novel DHBCs, using either new monomers or post polymerization functionalization schemes, is the subject of intense investigation during the current years.
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Moreover their self-assembly in aqueous media has been also studied in considerable detail. Emphasis has been given to the stimuli responsive character of the DHBCs upon environmental changes, like solution pH, ionic strength or temperature. Moreover, the potential utilization of DHBCs in a wide range of applications has been demonstrated in a number of publications. The synthesis of DHBCs has been realized by several synthetic strategies. Typically, controlled polymerization schemes are used in order to ensure the formation of block copolymers. A plethora of monomers have been polymerized following a number of polymerization mechanisms, involving, but not limited to, anionic polymerization, atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer polymerization (RAFT). The formation of supramolecular structures, like core-shell micelles or vesicles, has been indentified for a numerous block copolymers upon changes in pH, salinity or temperature. The combination of blocks, able to respond at different stimuli, within the chemical structure of the copolymer chain has immerged as an appealing way for controlling and enriching the self assembly behavior of DHBCs. Finally, DHBCs have been established as a key component in a wide gamut of applications, related to drug and gene delivery, surface functionalization or creation of smart surfaces and mineralization of inorganic matter. In this chapter recent developments in the field of DHBCs are described. Excellent review articles have already outlined the major features of DHBCs [1,2]. A number of excellent reviews summarize specific aspects of DHBCs in detail [3,4,5]. This chapter is focused on recent advances in the particular research area. The synthetic strategies followed for the preparation of DHBCs, important investigations on their aqueous solution behavior and a number of potential applications are presented. The field is growing rapidly. Therefore, the creation of a complete list of works, concerning DHBCs, is practically impossible and beyond the goal of this chapter. The works presented here have been selected in order to representatively describe the current developments in DHBC research.
2. SYNTHESIS OF LINEAR DIBLOCK DHBCS The synthesis of a variety of linear diblock DHBCs structures has been realized by the vast majority of the so called controlled polymerization techniques. Herein, we describe recent achievements in the synthesis of DHBCs categorized according to the followed polymerization mechanism.
2.1. Synthesis via Anionic Polymerization Methodologies Anionic polymerization is known to give model block copolymers with controlled molecular weights, narrow molecular weight distributions and versatile architecture. Anionic polymerization has been used for the synthesis of DHBCs in several cases although this type of polymerization technique is relatively intolerant to the presence of polar functionalities on the monomers utilized. A recent example has been described by Hadjichristidis and coworkers [6]. They have presented the synthesis of a series of poly (2-vinylpyridine-b-
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ethylene oxide) (P2VP-PEO) block copolymers with different molecular weights and composition. The synthesis was performed by sequential polymerization of the vinyl pyridine monomer followed by the polymerization of the ethylene oxide, in a polar solvent, Figure 1. The high enough molecular weights, as well as the low polydispersity index of the obtained copolymers, indicate the successful utilization of anionic polymerization techniques for the synthesis of P2VP-PEO DHBCs. The synthesis of P2VP-PEO block copolymers has been reported several years ago [7,8] with rather poor results concerning the purity of the final block copolymers. Combination of anionic polymerization and post polymerization reactions has been used for the synthesis of poly(acrylic acid-b-Ν,Ν-diethylacrylamide) (PAA-PDEA) copolymers [9]. Initially the synthesis of a precursor poly(tert-butylacrylate-b- Ν,Ν-diethylacrylamide) (PtBMA-PDEAAm) block copolymer was realized via sequential anionic polymerization of the tert-butyl acrylate and diethylacrylamide monomers. However, an amount of PtBMA homopolymer was detected in the crude reaction product. In order to remove the vast majority of the homopolymer, the authors proposed the precipitation of the crude product in hexane, where the homopolymer is highly soluble, in contrast to the block copolymer. The purified block copolymer was subjected to deprotection of the tert-butyl group in acidic media, leading to the desirable DHBC. The final block copolymer showed pH and thermosensitive solution aggregation. DHBCs of the type poly(p-hydroxystyrene-b-methacrylic acid) (PHOS-PMAA) were also synthesized via anionic polymerization followed by acidic hydrolysis [10]. Both blocks of the precursor poly(p-tert-butoxystyrene-b-tert-butylmethacrylate) (PtBOS-PtBMA) copolymers, formed by sequential addition of the protected monomers, could be deprotected in a single step giving the desired pH-responsive block copolymers, Figure 2. Hydrolysis was found to be nearly quantitative and resulted in a series of copolymers with well defined molecular characteristics and of variable composition.
Figure 1. Synthesis of P2VP-PEO DHBCs by anionic polymerization. Reproduced from [6] by permission of Elsevier.
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Figure 2. Synthesis of PHOS-PMAA by combination of anionic polymerization and post polymerization deprotection reaction. Reproduced from [10] by permission of the American Chemical Society.
A new family of neutral-ionic block copolymers were synthesized by post polymerization functionalization of anionically prepared poly(isoprene-b-ethylene oxide) amphiphilic diblock copolymers. Reaction of the double bonds of the polyisoprene block with excess chlorosulfonylisocyanate, followed by alkaline hydrolysis of the intermediate lactam functionalized copolymers, led to the synthesis of poly[sodium (sulfamate/carboxylate) isoprene-b-ethylene oxide] (PSCI-PEO) DHBCs [11]. Degrees of functionalization of the polyisoprene block were found in the range 0.70-0.78 molar. The PSCI block of the copolymers carries surfamate and carboxylate groups that can change the state of their ionization depending on the solution pH, giving to the copolymer variable degrees and type of charges. Different series of DHBCs were prepared from anionically synthesized poly(p-tertbutoxystyrene-b-ethylene oxide) (PtBOS-PEO) precursors [12]. Post polymerization acidic hydrolysis of the PtBOS block resulted in poly(p-hydroxystyrene-b-ethylene oxide) (PHOSPEO) copolymers. Further functionalization of the PHOS block via a Mannich type aminomethylation reaction gave the poly[3,5-bis(dimethylaminomethylene)hydroxystyreneb-ethylene oxide] (PNHOS-PEO) copolymers, as testified by FT-IR and NMR experiments. In these copolymers the PNHOS block carries two dimethylamino groups per monomeric unit that can be protonated in acidic media and weakly acidic phenolic groups that have their own pH sensitivity. The PNHOS-PEO block copolymers were further quaternized with
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methyliodide to give the corresponding cationic-neutral block copolymers that retain their cationic nature at all pH values.
2.2. Synthesis via Atom Transfer Radical Polymerization Methodologies The synthesis of an interesting DHBC, namely poly (4-vinylbenzoic acid-block-2(diethylamino)ethyl methacrylate) (PVBA-PDEAEMA), has been presented by Armes and Liu [13]. The synthesis was performed by ATRP using protecting group chemistry in three steps. Initially the polymerization of a tert-butyl protected PVBA macro-initiator was performed followed by the polymerization of the second monomer, DEAEMA. Finally the hydrolysis of the tert-butyl protected block was realized giving rather monodisperse block copolymers. A similar procedure has been presented by the same group for the synthesis of another block copolymer, namely poly[4-vinylbenzoic acid-block-2-N-(morpholino)ethyl methacrylate] (PVBA-PMEMA). The protection of the VBA monomer with a tert-butyl group is essential for the synthesis of the desired copolymer, otherwise a mixture of PVBA-PMEMA diblock and PVBA homopolymer was obtained. The particular copolymer is expected to have interesting solution properties since the PVBA block is a week polyacid, while PMEMA is a conjugated acid, soluble over a wide pH range at room temperature and in the absence of salt [14]. The synthesis of an ampholytic block copolymer, namely PMAA-PDEAEMA, carrying carboxylic and tertiary amino side groups, has been also realized by ATRP, as has been reported by Tam and coworkers [15]. Initially, the synthesis of the tert-butyl protected PMAA block was performed using p-toluenesulfonyl chloride as an initiator and CuCl complexed with N,N,N’,N’,N”,N”-hexamethyltriethylenetetraamine as a catalyst in 50 vol % anisole at 90 °C. The obtained polymer was used as the macroinitiator for the subsequent polymerization of the second monomer, DEAEMA, under similar reaction conditions, Figure 3. The resulted copolymer was subjected to selective hydrolysis, under acidic conditions, for removal of the tert-butyl protecting group. In the works mentioned, protecting group chemistry has been utilized for the synthesis of ampholytic/zwitterionic block copolymers. However, Armes and coworkers have also presented the synthesis of zwitterionic block copolymers without using protecting groups. In particular, various tertiary amine methacrylate monomers were copolymerized with 2hydroxyethyl methacrylate to produce diblock copolymers with a hydroxyl functionalized block. The synthesis of the amine containing block was realized in bulk, followed by the polymerization of the hydroxyl group containing monomer in methanol [16]. The obtained diblock precursor was then derivatized using succinic anhydride in order to introduce the desired carboxylic groups on the hydroxyl functional block. The procedure followed led to high monomer conversion and to copolymers having fairly low polydispersities. However, as has been revealed by the solution behavior of the synthesized polymers, the existence of some amine containing segments in the carboxylated block could not be excluded. In order to improve the synthetic procedure, the polymerization of the first block was performed in protic media under mild conditions [17]. Moreover, the order of monomer addition has been also inversed, because of the low blocking efficiency of the amine block, when it was polymerized in methanol. Finally, pure block copolymers, with polydispersities ca. 1.2, were obtained following the above synthetic procedure.
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Figure 3. Synthesis of the ampholytic PMAA-PDEAEMA by ATRP. Reproduced from [15] by permission of the American Chemical Society.
The ATRP mediated synthesis of non-zwitterionic copolymers, without the use of protecting groups, has been also presented in the literature [18]. An interesting example is the case of the poly(N-isopropylacrylamide)-b-poly(4-vinylpyridine) (PNIPAM-P4VP) copolymers. The synthesis of the particular type of copolymer was achieved by using conventional ATRP techniques. The molecular characteristics and the data obtained by size exclusion chromatography (SEC) analysis indicate the successful synthesis of copolymers with polydispersity index values ca.1.2.
2.3. Synthesis via RAFT Polymerization Laschewsky and coworkers have presented the synthesis of a series of DHBCs using RAFT polymerization methodologies, which is a “controlled” free radical polymerization. The system, that they have reported, is a temperature responsive block copolymer, namely poly(N-isopropylacrylamide-b-3-[N-(3-methacrylamidopropyl)-N,Ndimethyl]ammoniopropane sulfonate) (PNIPAM-PSPP) [19]. The synthesis was performed by sequential polymerization of NIPA and SPP monomers. The molecular weight was determined to be close enough to the theoretically calculated one. Interestingly, the followed polymerization procedure led to macromolecular chains with high enough molecular weight, up to about 60,000 g/mol. The same polymerization technique has been also utilized for the synthesis of other polyacrylamide containing DHBCs, like PNIPAM-PDEAEMA [20] or
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poly(N,N-dimethylacrylamide-b-N-isopropylacrylamide) (PDMA-PNIPAM) [21]. In the last case, Liu and coworkers have presented the synthesis of a PDMA-PNIPAM, where some azide-containing monomers (AzPAM) have been copolymerized within the PNIPAM block [21]. The above azide-containing monomer seems to be polymerized quite easily in dioxane at elevated temperatures, while the azide groups are relatively inert and compatible with RAFT processes. The authors assumed that the structural similarity of AzPAM and PIPAM monomers has as a result the statistical copolymerization of the two monomers. The RAFT process has been also used in the synthesis of novel ionic liquid containing DHBCs [22]. The polymerization of ionic liquid monomers was performed in methanolic solutions at elevated temperatures. The synthesis of copolymers with predictable molecular characteristics was described, indicating the living nature of the polymerization procedure. The synthesis of block copolymers of the type poly(ionic liquid-b-acrylamide) (PIL-PAm) and PMAA-PIL was successfully realized by using the as synthesized first block as a macrochain transfer agent for the polymerization of the second block. The final copolymer could be molecularly dissolved in water. However, chemical modification, by either esterification of the carboxylic groups of the PMAA block or ion exchange in the PIL block, could be used for the introduction of hydrophobic character in one of the blocks, leading to the formation of supramolecular self-assembled structures in aqueous media. The synthesis of a well defined block copolymer, containing a poly(N-vinylimidazole) block (PVim), has been demonstrated by Ge et al. [23]. The polymerization was realized via a RAFT procedure employing xanthates as chain transfer agents. Initially, the polymerization of NIPAM monomer, affording PNIPAM macro-CTA, was realized followed by the polymerization of Vim, Figure 4. The obtained block copolymers had controllable molecular characteristics with a Mw/Mn value ca. 1.2. The living nature of the Vim polymerization has been confirmed by kinetic experiments, for the first 150 min of the polymerization reaction. It has to be noted that the above procedure was the first example of the synthesis of PVim containing block copolymers.
Figure 4. Synthesis of a PVim containing DHBC by RAFT. Reproduced from [23] by permission of the American Chemical Society.
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2.4. Synthesis via Other Types of Polymerization Methodologies Group transfer polymerization (GTP) has been recognized as a powerful alternative methodology, toward the synthesis of methacrylate containing copolymers, with well defined structure and narrow molecular weight distribution. Armes and coworkers have presented the synthesis of a diblock copolymer, namely poly[2-(dimethylamino)ethylmethacrylate-b-2(diethylamino)ethylmethacrylate] (PDMAEMA–PDEAEMA), utilizing the GTP technique [24]. The particular synthesis was the first effort to synthesize a block copolymer in which both component blocks are polybases. The molecular weight of the synthesized polymers was up to about 20,000 g/mol with narrow molecular weight distributions (typically less than 1.15). The chemical modification of the copolymer by quaternization of the PDMAEMA block has been also presented by the same group [25]. Moreover, the synthesis of block copolymers consisting of DMAEMA and other tertiary methacrylate blocks has been also described [26,27]. Finally, Tsitsilianis and coworkers have presented the synthesis of an A-b(B-co-C) DHBC where A is PDEAEMA and B-co-C is poly[(oligo(ethylene glycol) methacrylate)-co-methyl methacrylate] (P(EGMAco-MMA)) [28]. A series of copolymers with different content of MAA were synthesized in order to obtain macromolecules with different hydrophilicity. An interesting class of DHBCs is the case of the polypeptide diblock copolymers. Lecommandoux and coworkers have presented the synthesis of such a type of copolymer, i.e. poly(γ-benzyl-L-glutamate-b-N-trifluoroacetyl-L-lysine) (PBLG-PTFALys). The synthesis was performed by sequential ring-opening polymerization of the corresponding R-amino acid N-carboxyanhydrides. The PTFALys block was synthesized first, followed by the polymerization of the second monomer. The polymerization took place in organic media and at room temperature. The final DHBC copolymer was obtained upon removing the benzyl (Bz) and trifluoroacetyl (TFA) protective groups of L-glutamic acid and L-lysine using KOH in THF. The copolymer is a zwitterionic diblock copolymer with only about 15 repeating units in each block [29]. The synthesis of a hybrid DHBC, containing both a polypeptide block and a polyacrylamide block, has been also presented [30]. The synthesis was realized by using an amine terminated PNIPAM as the macroinitiator in the ring-opening polymerization of γbenzyl-L-glutamate, followed by deprotection of the benzyl groups of the polypeptide block, Figure 5. Interestingly, samples with high enough molecular weights were obtained following the aforementioned method; however, the polydispersity of the copolymers was also relatively high. The synthesis of a novel, primary amine containing, DHBC has been described by Liu and coworkers [31]. The synthesis of the amine-containing monomer was performed by a two step click reaction. The preparation of the block copolymer, namely poly(Nisopropylacrylamide)-b-1-(3-aminopropyl)-4-acrylamido-1,2,3-triazole hydrochloride) (PNIPAM-PAPAT), was realized by RAFT. In the first step, the polymerization of PNIPAM was achieved and the as synthesized polymer was used as the macroinitiator for the polymerization of APAT. However, in order to polymerize primary amine containing monomers, acidic media are needed in order to eliminate some side reactions (the primary amine moieties have to be protonated in order to suppress the side reactions).
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Figure 5. Synthesis of a polypeptide containing DHBC by ring opening polymerization. Reproduced from [30] by permission of the American Chemical Society.
The synthesis of a well defined poly(vinyl alcohol)-b-poly(acrylic acid) (PVA-PAA) DHBC has been recently reported in the literature [32] by a two step synthetic scheme. First the synthesis of a poly(acrylonitrile) (PAN) block was realized via cobalt-mediated radical polymerization, using a poly(vinyl acetate) (PVAc) macroinitiator, followed by hydrolysis of both blocks. The polymerization was performed in DMF, a very good solvent for PAN, and at low temperature, where block copolymers with low polydispersity were obtained. The polymerization procedure led to well defined macromolecules with relatively high molecular weights. The obtained copolymers were transformed to the desired DHBCs by hydrolysis, using large excess of potassium hydroxide in a water/ethanol mixture. The successful completion of the hydrolysis reaction was monitored by NMR and IR spectroscopy. An additional macroscopical indication of the DHBC formation was the aqueous solubility of the reaction product. An interesting approach for the synthesis of DHBCs has been presented by Shi and coworkers [33]. In particular, end functionalized homopolymers, namely P4VP and PNIPAM, were synthesized by conventional RAFT polymerization, using the appropriate initiators. Mixing equimolar amounts of the above polymers led to the formation of well defined noncovalent bonded DHBCs through inclusion complexation between the β-cyclodextrin and the adamantyl end groups, Figure 6. The copolymer seems to behave as a typical diblock copolymer, i.e. forms supramolecular self-assembled structures under appropriate solution conditions. Furthermore, the synthesis of a “covalent analogous” polymer via ATRP methodologies has been also described in another work [34].
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AD-PNIPAM70
-CD-P4VP30
Complex
Figure 6. Schematic illustration of DHBCs formation through inclusion complexation. Reproduced from [33] by permission of The Royal Society of Chemistry.
A similar approach has been also used for the synthesis of “covalently linked” DHBCs. Polysaccharide based DHBCs were prepared by end-to-end coupling of two readily available biocompatible water-soluble homopolymers [35]. The synthesis was a two step reaction where a) a terminal aldehyde group of a dextran homopolymer was oxidized and b) a monoamine end functionalized PEG reacted with the oxidized dextran, via a lactone aminolysis reaction. Interestingly, the obtained polymer could be chemically modified, in a controlled way, in order to produce neutral-cationic or neutral-anionic DHBCs. Finally, the controlled aqueous polymerization of various monomers, via nitroxide mediated polymerization has been recently demonstrated by Phan and coworkers [36]. The polymerization was realized by using a chemically modified nitroxide, which bears a carboxylic acid function. The introduction of carboxylic groups offers water solubility, when the groups are in the ionized form, allowing the controlled polymerization of water soluble monomers. The controlled (co)polymerization of nonionic, anionic and cationic monomers has been demonstrated under mild conditions. The polymerization scheme presents the characteristics of controlled polymerizations, such as first-order kinetics up to high conversions, linear increase of the molecular weight with monomer conversion and good agreement between experimentally obtained and theoretically expected molecular weights. However, the relatively high polydispersity index values of the products discourage the use of the above polymerization scheme for the preparation of well defined block copolymers for structure-properties relationship investigations. Additionally, the synthesis of block copolymers was accompanied by the presence of unreacted homopolymer, due to some termination reactions.
3. SYNTHESIS OF DHBCS WITH OTHER ARCHITECTURES 3.1. Linear Multiblock DHBCs The synthesis of an ABA triblock copolymer, where A is a P2VP block and B is a PEO block, was described in the literature [6]. The synthesis of the copolymer was performed via anionic polymerization high vacuum techniques. Initially a diblock copolymer was synthesized by sequential polymerization of vinyl pyridine and ethylene oxide monomers. Subsequently, the as synthesized diblock copolymer was reacted with p-dibromoxylene. A portion of the diblock copolymer reacts with the difunctional reagent leading to the formation of a triblock, namely P2VP-PEO-P2VP, having double the molecular weight of the precursor
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diblock. Fractionation of the crude reaction product took place at elevated temperature in order to remove the unreacted excess diblock. Anionic polymerization has been also used for the synthesis of another ABA DHBC, where A is PAA and B is P2VP [37,38]. The synthesis of a precursor polymer was realized by using a bifunctional initiator and by sequential addition of 2VP and tert-butyl acrylate monomers (tBA). The obtained SEC data indicate the formation of well defined triblock chains, without the presence of neither P2VP homopolymer, nor P2VP-PtBA diblock copolymer. The formation of the desired DHBC triblock was finally achieved by acidic hydrolysis of the PtBA blocks in order to remove the tert-butyl protecting group. A similar procedure has been used by the same group for the preparation of another ABA block copolymer, namely PEO-P2VP-PEO. However, in this case the deprotection step is not necessary, since the PEO block is already hydrophilic [39]. The synthesis of an ABA triblock double hydrophilic copolymer, where A= PEO and B= PSCI has been also reported via the utilization of chlorosulfonylisocyanate functionalization reaction on an anionically synthesized poly(ethylene oxide-b-isoprene-b-ethylene oxide) triblock precursor [11]. Finally, the synthesis of a BAB triblock copolymer, where A is a PDMA block and B is a PNIPAM block, has been realized via RAFT polymerization using a symmetrical bistrithiocarbonate as the bifunctional chain transfer agent for the polymerization of the middle block [40]. The polydispersity indices for the synthesized triblocks, determined by SEC, were in the range 1.19-1.31. The high Mw/Mn values could be also correlated with the possible interaction of the copolymers with the column material, as well as to the association of block copolymer chains in the carrier solvent. A series of copolymers with an identical middle block and outer blocks with different molecular weight have been synthesized. However, the presence of diblocks or unreacted homopolymers in the final product could not be excluded. An ABC triblock terpolymer was successfully synthesized by a sequential three-step RAFT polymerization process of N-n-propylacrylamide, N-isopropylacrylamide and N,Nethylmethylacrylamide monomers [41]. The different cloud points of the respective blocks, present in the terpolymer, are responsible for the rich temperature depended solution selfassembly of the sample in aqueous media. In the concept of water soluble ABC block copolymers one has also to mention a huge amount of work that has been presented on the synthesis and solution behavior of copolymers with two hydrophilic blocks and one hydrophobic. However, the presence of a permanently hydrophobic block in this type of block polymers makes difficult their categorization as DHBCs. The synthesis of ABC triblock terpolymers with at least two hydrophilic blocks has been realized via a number of polymerization methodologies, like GTP and cationic polymerization, and has been studied in detail [42,43,44,45,46,47,48,49]. In most of the cases, the terpolymers were based on suitably functionalized methacrylate monomers and have been produced by the sequential monomer addition method. Finally, the synthesis of multiblock DHBCs has been also reported. The desired multiblocks have been obtained via RAFT polymerization, using polytrithiocarbonate as the chain transfer agent [50]. The synthesis of two multiblocks with different molecular characteristics was achieved. Both block copolymers were consisted of PDMA and PNIPAM sequences. The molecular characteristics of the synthesized macromolecules were studied
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both by NMR and SEC. Polymers with quite high molecular weight were obtained. However, it has to be noted that the polydispersity of the samples was about 1.5.
3.2. Non Linear Double Hydrophilic Block Copolymers One of the first syntheses of non-linear DHBCs has been described by Armes and coworkers [51]. They have prepared a series of A2B miktoarm star copolymers via ATRP of several hydrophilic methacrylate monomers by utilizing Jeffamine macroinitiators. The macroinitiators (which were the B blocks of the copolymers) were synthesized by reaction of the terminal amino group of the polyalkyleneoxide chain with two equivalents of 2-hydroxyethylmethacrylate, followed by esterification with 2-bromoisobutyryl bromide. The A blocks carried pH responsive substituted amine groups (water soluble and positively charged at low pH) or zwitterionic phosphatidylamine groups. The expected chemical structures for the macroinitiators and the resulting star copolymer where confirmed by NMR, MALDI-TOF-MS and SEC techniques. Liu and coworkers [52] have described the synthesis of two different types of branched block copolymers, namely A2BA2 and A4BA4. In both cases, the B block was poly(propylene oxide) (PPO) while the A block was PDEAEMA. The synthesis was realized via ATRP using a commercially available diamine terminated PPO as the backbone of the copolymer. The initial PPO central block was functionalized in order to introduce multi-ATRP initiator functionalities at both free ends of the polymer. Subsequently the polymerization of DEAEMA monomer was performed following typical ATRP procedures. The followed synthesis scheme is shown in Figure 7.
Figure 7. Synthesis of an A2BA2 DHBC by ATRP. Reproduced from [52] by permission of the American Chemical Society.
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ATRP has been also utilized for the synthesis of an AB4 miktoarm star copolymer, where A is a PNIPAM block and B is a PDEAEMA block [53]. At first, a mono-amino terminated PNIPAM homopolymer was synthesized. Subsequently, an addition reaction on the amino group using glycidol was realized, followed by esterification with excess 2-bromoisobutyryl bromide. The end functionalized PNIPAM formed in this way could be used as a tetrafunctional macroinitiator for the polymerization of DEAEMA monomers via ATRP. The formation of pure AB4 miktoarm star copolymer was assumed by the authors based on results from size exclusion chromatography. However, the polydispersity index was quite high (Mw/Mn= 1.23), therefore, SEC alone could not unambiguously confirm the level of purity of the miktoarm stars. The synthesis of a miktoarm star copolymer of the type AnBn has been also demonstrated. The synthesis was performed via ATRP using divinylbenzene, as the core cross-liking agent. PEO macroinitiator chains were utilized for the polymerization of divinylbenzene forming a star polymer, with a random number of branches. The above star polymer was used as a multi-functional initiator for the polymerization of methacrylate monomers. Therefore, the synthesis of an amphiphilic miktoarm star copolymer was realized [54]. Finally, the hydrolysis of the protected methacrylate block led to the preparation of the desired DHBCs, namely the PEOn-PMAAn stars. SEC analysis of the precursor PEOn-PMMAn copolymer revealed a relatively broad molecular weight distribution. Nevertheless, this is a good example for the synthesis of AnBn double hydrophilic star copolymers. The synthesis of a series of star copolymers using GTP has been described by Patrickios and coworkers [55]. The general route for the synthesis of the copolymers was the combination of the conventional polymerization of the desired monomer by GTP and the use of a bi-functional coupling agent, as has been described before. The synthesis of four different types of star copolymers, composed of DMAEMA and methoxy hexa(ethylene glycol) methacrylate (HEGMA) was presented. In particular, isomeric star copolymer, heteroarm, star block and statistical star copolymers were realized by varying the sequences of the monomers and coupling agent additions. In all cases a constant ratio of the two monomers (DMAEMA / HEGMA = 9/1) was utilized. The presence of linear (co)polymer precursors was evident by SEC analysis of the samples, indicating the incomplete formation of star copolymers. Finally, the synthesis of a double hydrophilic linear-hyperbranched block copolymer has been realized by a four step reaction [56]. Initially a polyethylene oxide macroiniator was synthesized either by anionic polymerization of ethylene oxide using Cs as the counterion or by deprotonation of a commercially available monohydroxyl terminated PEO via interaction with cesium hydroxide. Subsequently, the anionic polymerization of ethoxyethyl glycidyl ether monomer (EEGE) was performed under argon and at elevated temperature. The obtained diblock copolymer was subjected to acidic hydrolysis in order to recover the hydroxyl groups, by removing the acetal protecting groups on the PEEGE block. Finally, the desired linear-hyperbranched copolymer was synthesized by initiating the polymerization of glycidol from the hydroxyl groups of the diblock, using cesium hydroxide as the activating agent. The resulting copolymers exhibited low polydispersities, while the molecular weight was up to almost 1.6x104 g/mL. It has to be noted that the presented methodology allows for variation of the molecular weight in both the linear PEO block and of the hyperbranced block.
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4. SOLUTION SELF-ASSEMBLY OF DHBCS Several intriguing and interesting self-assembled nanostructures can be produced in aqueous solutions of DHBC, due to the stimuli responsive character of the blocks comprising the copolymer that in turn determines the amphiphilicity of the system. The solution properties of DHBCs can be tuned via the chemical structure of the copolymer and external parameters including solution pH, ionic strength, temperature, as well as the addition of oppositively charged chemical entities. These features open possibilities for understanding the self-assembly motifs in natural occurring systems and facilitates the utilization of DHBCs in a number of advanced nano(bio)technological applications. In the following sections we focus on selective studies concerning the aqueous solution behavior of this type of copolymers.
4.1. Temperature Responsive Self-Assembly One of the most interesting examples of DHBCs which form self-assembled structures upon changes of solution temperature has been presented by Laschewsky and coworkers [19]. They have developed a system where one of the blocks presents a LCST and the other a UCST. Therefore the copolymer, namely PNIPAM-PSPP, could either be molecularly soluble or associate into aggregates at low and high temperature, Figure 8. The nature of the hydrophobic core of the formed aggregates depends on the solution temperature. In particular the PSPP block is soluble for temperatures higher than 20oC (depending on the molecular weight), while the PNIPAM block is soluble for temperatures lower than 32-34oC (almost independent of the chain molecular weight). In this way, by controlling the molecular characteristics of the copolymer, the desired aggregation/disaggregation profile could be achieved. The studied copolymer is soluble in the whole temperature range under investigation. However, at the low temperature aggregation limit, the insoluble microdomains are relatively polar and probably they have low solubilization efficiency for hydrophobic molecules, a property that would have been useful in the field of nanosized drug carriers.
Figure 8. Micellization behavior of PNIPAM-PSPP upon changes in the solution temperature. Reproduced from [19] by permission of the American Chemical Society.
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An interesting work on the temperature induced self-assembly of DHBCs has been presented by Pasparakis and Alexander. They have proposed the formation of vesicles by copolymers consisting of a highly hydrophilic poly(2-glucosyloxyethyl methacrylate) block and a water-soluble poly(diethyleneglycol methacrylate) block [57]. The above copolymer is molecularly dissolved at 15oC but forms aggregates at higher temperatures. The size of the aggregates depends on the solution temperature, due to the increased hydrophobicity of poly(2-glucosyloxyethyl methacrylate) upon increasing the temperature of the solution. Interestingly, the aforementioned aggregates were found to be vesicles, as has been demonstrated by transmission electron microscopy. Schubert and coworkers observed a temperature induced cylinder-to-vesicles transition in aqueous solutions of P2VP-PEO copolymers [58]. By cooling the polymer solution down to 4oC the initial vesicles are transformed into wormlike micelles, through intermediate formation of basket-like aggregates. A subsequent increase in solution temperature allows for the appearance of vesicles through intermediate discoid and octopus-like structures. The preparation of unilamellar and nearly monodisperse vesicles with controlled sizes, in the range 60 to 500 nm, is possible in these systems by appropriate variation of heating rates, polymer concentration, ionic strength, as well as the incubation time at the limits of the transition temperatures. The authors also demonstrated that several model drugs could be encapsulated in the nanostructures formed in each case. The formation of core-crosslinked, temperature responsive DHBCs micelles have been presented in the literature. The micelles have been prepared by the self assembly of PDMA-bP(NIPAM-co-AzPAM) chains followed by crosslinking, utilizing a difunctional propargyl ether. The formation of the crosslinked micelles can be performed by following two different pathways. In the first one, the micelles were formed upon increasing the solution temperature, since the PNIPAM block becomes hydrophobic at temperatures higher than 32oC, and the rich PNIPAM cores were subsequently crosslinked via click chemistry. In the second approach, the copolymer was molecularly dissolved in DMF, a good solvent for both blocks, and the crosslinking reagent was added leading to the formation of supramolecular structures. After dialysis, of the DMF solution against water, stable polymeric micelles were observed. Both of the above methods had as a result the formation of polymeric micelles, which were not destroyed even at room temperature where both blocks are soluble, Figure 9. In contrast, the micellar core tents to swell at low temperature, leading to relatively larger micelles, indicating the possible use of core-crosslinked DHBCs micelles in drug delivery applications [21]. An insightful work on the temperature induced micellization kinetics has been presented by Zhang et al. [20]. The study was performed by utilizing a PNIPAM-b-PDEAEMA copolymer upon jumps of the solution temperature from 20 to 45 oC, where micelles with a PNIPAM core were formed. The solution pH was set at 4, a pH value where the PDEAEMA block is soluble in the whole experimental temperature range. The experimental results indicate the existence of two relaxation processes. The first, fast process was attributed to association of unimers into a large amount of small micelles and the formation of quasi-equilibrium micelles. The second, slow process was almost independent of the polymer concentration, indicating that unimer insertion/expulsion is the main mechanism for the slow process. The authors attribute the above unimer insertion/expulsion mechanism to electrostatic interactions, due to the charged PDEAEMA block. However, the influence of copolymer molecular weight has to be taken into account, as has been demonstrated by the same group later on [59].
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Figure 9. Formation of thermoresponsive crosslinked micelles following two different pathways. Reproduced from [21] by permission of Wiley.
The temperature induced micellization has been also studied for a linear triblock BAB, where A= PDMA and B= PNIPAM [40]. It is well known that the PNIPAM block is temperature responsive, i.e. is soluble for temperatures lower than the LCST. Therefore, the formation of supramolecular structures was observed at elevated temperatures. Interestingly, the aggregation process was not confined to a small temperature interval, but stretches over a range of 10 to 20oC, or even more. Moreover, the micellization temperature was strongly dependent on the molecular characteristics of the copolymer chains, like the composition and the molecular weight of the B block. Strong kinetic effects have been recorded during the micellization process, since larger aggregates and broader size distributions were observed for slower heating rates. Finally, the solution behavior of thermoresponsive multiblock copolymers has been reported [50]. Multiblocks of PDMA and PNIPAM with sequences of different molecular weights were studied upon increasing the solution temperature. At elevated temperatures the PDMA block is water soluble, while the PNIPAM block becomes insoluble. Depending on the molecular weight of the sequences, the multiblock copolymer can adopt unimolecular flower-like micelle conformation or can aggregate through intermolecular interactions.
4.2. pH Responsive Self-Assembly One of the first and more characteristic examples of pH responsive DHBCs is the solution behavior of P2VP-PEO [8]. The above copolymer is molecularly dissolved at low pH values due to the protonation of the P2VP block, considering that the PEO block is a neutral, water soluble polymer in the whole pH range. However, upon increasing the solution pH, deprotonation of the P2VP block leads to the formation of micelles at pH higher than 4. This behavior, i.e. the formation of micelles upon changes in the solution pH, is characteristic for a number of polymers, as it will be demonstrated below. The micellization of a similar system, namely PHEGMA-PDEAEMA, upon changes in the protonation degree of the amine containing block has been studied in detail by light scattering and NMR [60]. The above polymer was molecularly dissolved when the degree of
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protonation was higher than 30%, although a small fraction of aggregates was also observed, even at protonation degrees higher than 30%. However, the tendency for aggregation of the copolymer chains was increased by decreasing the percentage of the ionized amine groups to 20%. A further decrease of the protonation degree led to the formation of precursor micelles, while the equilibrium micelles were formed only upon complete deprotonation of the PDEAEMA block. The above, step-by-step investigation of the micelle formation reveals a detailed picture of the copolymer solution properties. Changes of solution pH can induce micellization not only because of a decrease in the ionization degree of one block, but also because of the development of secondary interactions, like hydrogen bonds. An example of the above situation is the solution behavior of the PVA-PAA block copolymer [32]. The particular copolymer was almost molecularly dissolved in aqueous media at neutral or alkaline environments. However, the formation of aggregates was observed at low pH values, not only because of deprotonation of the PAA block but also due to the development of intermolecular hydrogen bonds between the PAA and PVA blocks. In a similar context the aggregation behavior of PHOS-PMAA block copolymers was found to be pH dependent [10]. In this particular system both blocks can be considered as acidic, but the pKa values of PHOS and PMAA differ substantially. Loose polydisperse aggregates were observed at pH>9 where phenolate and carboxylate groups are dissociated. At 4< pH4. In contrast, under basic conditions (pH >9), the PGA block was charged, while the PLys block was transformed into a neutral and insoluble block. Therefore aggregates with either the one or the other block as the core forming block could be obtained depending on the solution pH. The formed aggregates were determined to be vesicles. This was the first time that this schizophrenic behavior was observed in vesicle forming block copolymers, Figure 12. The vesicle formation was tentatively explained, according to the authors, by the systematic presence of a polypeptide in a rodlike conformation, in the hydrophobic part of the membrane, which induces a low interfacial curvature and as a result a hollow supramolecular structure.
4.3. Self-Assembly via Complexation with Other Building Blocks The complexation of two DHBCs has been recently described in the literature. The complex formation between a miktoarm star copolymer PEOn-PMAAn and a quaternized PEO-PDMA diblock was studied upon changes in solution pH [54]. At elevated pH values, where PMAA blocks were ionized, the formation of electrostatic complexes was recorded between the oppositely charged PMAA and quaternized PDMAEMA blocks. However, the aggregates were soluble due to the presence of the PEO uncomplexed blocks originating from both of the copolymers. The situation was dramatically different at low pH values, where the PMAA block was not ionized. At the aforementioned pH values the development of hydrogen bonds between the PMAA and PEO blocks took place. In the later case, the aggregates were electrostatically stabilized by the quaternized PDMAEMA polyelectrolyte block. The micellization/complexation behavior is schematically illustrated in Figure 13. Complexation induced micellization can occur not only by using an oppositely charged polyelectrolyte but also by using a salt with divalent ions. Tam and coworkers have described in detail the mechanism of salt induced micellization [64] of a PEO-PAA diblock upon addition of CaCl2. The formation of supramolecular aggregates was strongly dependent on the Ca2+/COO- ratio. When the ratio was 0.5, the formation of coil-like globules was observed due to charge neutralization. An increase of the above ratio to values higher than 2.5, led to the creation of aggregates with a mean diameter of ca. 50 nm. Interestingly, the data reveal that the aggregate formation was not caused by electrostatic interaction between the ions but it was the disruption of the water structure, due to the solvation of CaCl2 excess. It was concluded that the copolymer chains formed clusters in order to compensate the entropic changes, caused by changes in the structure of water.
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Figure 12. Schizophrenic behavior in the formation of vesicles by a polypeptide containing DHBC. Reproduced from [29] by permission of the American Chemical Society.
Figure 13. Complexation induced micellization of a star DHBC. Reproduced from [54] by permission of Wiley.
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An example of electrostatically assembled complexes formed between a PEO-PMAA DHBC and poly(amidoamine) dendrimers of different generations was presented very recently [65]. An increase in the size of the complexes was observed by increasing the amount of G4 dendrimer present in the solutions. The structure of the core of the aggregates was dependent on the dendrimer concentration, while the overall structure of the assemblies could not be described as a micellar-like structure. At low dendrimer concentration a close aggregation of the PMAA chains and G4 dendrimer was evident, while at higher G4 concentration the PMAA chains efficiently fill the aggregate core. The behavior was the opposite for the G0 dendrimer. The size distribution of the aggregates was narrow and the structure of the assemblies was dependent on the solution pH as a result of the different ionization degree of the components. A large amount of work, concerning the interaction and nanostructure formation in DHBC/surfactant and DHBC/polyelectrolytes mixed solutions has been published [66,67,68,69,70,71,72,73,74,75,76,77,78,79, 80,81]. The structural characteristics of the aggregates in such systems can be tuned and controlled by the chemical structure of the block copolymer, the surfactant and the polyelectrolyte utilized. A very important parameter that determines the structure of the nanosystems is the charge ratio between the two components. Electrostatic interactions are the main reason for structure formation, but secondary interactions, like hydrophobic interactions and hydrogen bonding, play also a decisive role. Various morphologies including micellar core-shell like aggregates, cylindrical superstructures and vesicles have been observed. The mixed structures can be also influenced or be responsive to changes in the physicochemical parameters of the system (pH, salinity, temperature). The great versatility of DHBC/surfactant and DHBC/polyelectrolytes systems, concerning structural diversity and properties, makes them a very active field of current research with great potential towards nanoapplications, including drug and gene delivery and surface modification. A special and very interesting class of complexes containing suitably designed and synthesized DHBCs, especially in terms of potential biotechnological applications, are the complexes formed in the presence of oppositely charged proteins and nucleotides [82,83,84,85,86,87,88,89,90,91]. The use of such complexes for delivery of protein drugs, separation and purification of proteins and for gene delivery has been proposed. Several mixed systems have been reported and the fundamental properties of the superstructures in aqueous solutions have been elucidated. Structural characteristics are depending on the structure of the DHBC and a number of physicochemical parameters. Resposiveness of the created nanostructures to stimuli that are primarily encountered under physiological conditions in living organisms, is of primary interest due to the desired functionality of the ensembles. Hydrophobic interactions in conjunction to electrostatic ones are determining the stability and structural and functional tunability of the nanoaggregates.
4.4. Self-Assembly via Combination of Stimuli An interesting example of a DHBC system that can self assemble under the influence of two different stimuli is the PVBA-PMEMA copolymer, presented by Liu and Armes [14]. The particular system formed small well defined micelles with a PVBA core at pH values lower than 5.5 at room temperature and in the absence of salt. No precipitation was observed at the isoelectric
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point. However, in the presence of a sufficient amount of Na2SO4 (0.80 M) or at elevated temperatures (more than 80 oC), well defined micelles with PMEMA cores were formed in alkaline media, because of salting out of the PMEMA block. By choosing the appropriate values of pH, salinity and temperature the observation of micelles with PVBA cores, PMEMA cores micelles or precipitation (at the isoelectric point) was possible. Moreover, the micellization temperature of the system was altered upon changes in the salinity. The micellization kinetics, of the above rather complicated system, has been also studied [92]. A system analogous to the PVBA-PMEMA copolymer, where a PDEAEMA block has been used instead of the PVBA block, has been also presented by the same group [26]. The particular copolymer could form micelles with either PDEAEMA or PMEMA cores depending on the solution pH and salinity. At low ionic strength the PMEMA block was soluble in the whole pH range, in contrast to the PDEAEMA block which is not soluble at pH higher than 7-8. Therefore, at zero salt concentration and pH=8, PDEAEMA core micelles were observed. However, when the solution pH was carefully adjusted to 6.7 and the salinity was increased by addition of NaSO4, then the PMEMA block was no longer soluble in water and PMEMA-core micelles were observed, Figure 14. The temperature of the solution plays a crucial role on the stability and polydispersity of the formed micelles. Another example of a DHBC, which responds to two different stimuli, is the PNIPAPSPP copolymer [93]. This copolymer presents both UCST (PSPP block) and LCST (PNIPAM block) in aqueous solutions. However, the addition of salt increases the hydrophilicity of the PSPP block leading to a disappearance of the UCST. In contrast, the hydrophilicity of the PNIPAM block, is decreased upon addition of salt. Therefore, the size of the supramolecular aggregates formed is altered not only upon changes in solution temperature but also upon changes in the salinity. Copolymer concentration was also found to play a crucial role on the dimensions of the self-assembled structures. The micellization of a polypeptide containing DHBC has been also studied under variable solution pH and temperature [30]. A schizophrenic solution behavior was observed for a PNIPAM-PGA block copolymer. At room temperature and low pH values micelles having polypeptide cores were formed, while at elevated temperatures and increased pH values micelles with PNIPAM cores were observed. It has to be noted that the formation of PGA core micelles was accompanied by a coil-to-helix transition of the polypeptide sequence. The above transition had as a result a different micellization kinetic profile for the system in comparison with other conventional pH responsive systems.
Figure 14. Micellization of MEMA-DEAEMA DHBC upon changes of more than one stimulus. Reproduced from [26] by permission of the American Chemical Society.
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Figure 15. Schematic illustration of the combination of stimuli for producing different types of DHBC aggregates. Reproduced from [9] by permission of Wiley.
Another example of schizophrenic behavior has been observed in the case of A-b-(B-coC) DHBCs. The copolymers P(EGMA-co-MMA)-PDEA (with various contents of MAA) associated in water either at elevated temperature and low pH values or at low temperature and increased pH values [28]. Inverse micelles were observed in each case. Moreover, both the solution salinity and the content of MAA had a great influence on the critical micellization temperature, as was revealed by the experimental observations. The PAA-PDEA block copolymer has a pH responsive block (PAA) and a temperature responsive block (PDEA) [9]. The particular copolymer could be molecularly dissolved in alkaline aqueous solutions at room temperatures. However, upon increasing the solution temperature the formation of crew-cut micelles with a PDEA core were formed, mainly due to the high asymmetry in the lengths of the blocks. This rather effective strategy allows the formation of crew-cut micelles in water without using a cosolvent. Moreover, the formation of inverse micelles can be also achieved at room temperature and at low pH values. In the latter case, star-like core shell micelles are observed, Figure 15. Temperature and pH responsiveness has been also recorded for a non covalently bonded DHBC inclusion complex, namely P4VP-PNIPAM [33]. The non-covalently connected copolymer tends to create micelles with PNIPAM cores at low pH values and at elevated temperatures. However, at room temperature and high pH values, the polymer formed vesicles instead of core-shell micelles. The formation of vesicles was confirmed by a number
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of complementary techniques, including TEM and light scattering. The pH induced vesicle formation by a non-covalently connected DHBC is an attractive approach for the facile synthesis of drug and gene delivery systems. Moreover, the solution behavior of the analogous covalently connected PNIPAM-P4VP copolymer has been also reported [34]. This copolymer was molecularly dissolved at low temperature and low pH. However, increasing of either the temperature or the pH led to the formation of core-shell micelles with PNIPAM or P4VP cores respectively. The formation of core-shell micelles instead of vesicles is probably caused by the differences in the molecular characteristics of the two polymers. Additionally, the nature of the non-covalent connection between the constituting blocks may allow for a more efficient reorganization of the polymer chains under appropriate conditions. The solution behavior of an ABA block copolymer, namely PEO-P2VP-PEO, has been also studied under variable pH, temperature and salinity conditions [39]. In the present case, the polymer tended to form star like micelles with P2VP core at high pH values and room temperature. However, the situation was different at low pH values, increased salinity and elevated temperature. Under those conditions the formation of flower like micelles was observed, since the outer blocks became hydrophobic. Block copolymers with non-linear structure have been also studied under the concept of formation of multi-stimuli responsive DHBCs supramolecular structures [52]. The solution behavior of A4BA4 and A2BA2 copolymers, where A is the pH responsive PDEAEMA block and B the temperature responsive PPO block, has been studied upon changes in solution pH and temperature. According to the authors, the experimental results indicate that the copolymers tend to form large, flower-like aggregates at low temperature and in a basic environment, where the water soluble PPO block forms loops surrounding the PDEAEMA cores of the aggregates. In contrast, unimolecular micelles are formed at higher temperatures and at pH 6.4, where the PPO is insoluble. Finally, the solution behavior of another schizophrenic non linear DHBC has been also investigated. An AB4 miktoarm star copolymer, where A is a temperature responsive PNIPAM block and B is a pH responsive PDEAEMA block, has been studied under varying solution pH and temperature [53]. The copolymer forms PNIPAM core micelles at high temperatures and at low pH values, and the reverse PDEAEMA core micelles at room temperature and high pH values. The micelles with a PDEAEMA core have a much smaller size in comparison with the micelles formed by a linear diblock copolymer with similar composition and molecular weight. Finally, different micellization kinetics has been recorded for the miktoarm star and the linear block copolymer upon pH jumps from 4 to 10, using a stop flow light scattering technique.
5. CURRENT AND POTENTIAL APPLICATIONS The versatile synthetic procedures for the preparation of a wide variety of DHBCs, as well as their intriguing solution properties have promoted and suggested the utilization of such polymers in several demanding and “smart” technological applications. Some of them are discussed in the following sections.
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5.1. Mineralization of Inorganic Compounds The use of double hydrophilic block copolymers in biomimetic mineralization processes has been investigated in recent years. In contrast to rigid templates (like carbon nanotubes and porous aluminum templates which predefine the final structure) water soluble polymers could be used as soluble species at various hierarchy levels. Usually, in the case of DHBCs, one of the block acts as scaffold for the development of the crystal, while the other acts as a solublestabilizing matrix. Therefore, both of the blocks play a crucial role on the development of the crystals. There is a plethora of reports on the emerging bio-inspired mineralization field. Various crystal structures have been presented during the last years, following versatile synthetic routes. A very detailed and illustrious review has been recently given by Colfen [3]. The above review describes in detail all aspects of the specific field. Herein, we present just a few selected examples. Antonietti and coworkers have demonstrated the designed synthesis of barium sulfate crystallites using a large gamut of DHBCs with different functional groups [94]. In particular, the growth of BaSO4 into novel morphologies has been achieved by using block copolymers with varying binding and solvating blocks. Morphologies like rods, peanuts, peaches, nanofibers and flowers have been observed using different polymeric stabilizers and morphology promoters/modifiers. The development of CaCO3 tablet-like arrays was achieved at the air/water interface through the cooperative mineralization regulated by a polypeptide and a DHBC, namely PEO-PMAA [95]. The experimental data indicate that the role of the block copolymer is focused on the regulation of the arrangement and orientation of the CaCO3 tablets. The cooperative action of a polypeptide is essential for the formation of CaCO3 tablet at the air/water interface. In the absence of the polypeptide, the formation of calcite CaCO3 particles in the water phase was observed.
Figure 16. Schematic demonstration of the formation of single crystals, mesocrystals, and polycrystalline aggregates in the presence of DHBCs. Reproduced from [96] by permission of the American Chemical Society.
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Experimental evidence for a unifying model of copolymer-directed crystallization has been recently presented by Colfen and coworkers [96]. According to the proposed model a continuous transition in particle structures and crystallization mechanisms occurs between polycrystalline aggregates, mesocrystals, and single crystals, Figure 16. Particularly, a number of crystals with different shapes could be obtained by aggregation, and the observed aggregation (with low or high degree of orientation) depends on the solution supersaturation. Therefore, a single crystal, a mesocrystal or a polycrystal could be obtained by changing the solution conditions.
5.2. Nanomedicine The utilization of DHBCs as key-playing component in potential nanomedicinal applications has been demonstrated by Kataoka and coworkers [4, 82-90]. An interesting example of using DHBCs for encapsulation and targeted release of oligonucleotides has been demonstrated some years ago [97]. Partially thiolated PEO-PLys block copolymer has been used for the formation of polyion complex (PIC) micelles with a specific oligonucleotide. The formed micelles are stabilized in aqueous media because of the presence of the polyethylene oxide block, while the nucleotide is located at the micellar core in the form of a complex with PLys. After the formation of the PIC micelles, crosslinking of the core took place, by creation of disulfide bonds due to the oxidation of thiol groups preattached on the PLys chain. The resulting core-crosslinked PIC micelles are an effective carrier for the transportation and delivery of the oligonucleotide, since the stability against nuclease was appreciably increased, compared to that of free oligonucleotide and that in the micelles without crosslinking. Selective release of the oligonucleotide in the intracellular environment could be achieved. The increased concentrations of glutathione, an agent that could cleave the disulfide bonds, inside the cells leads to the located release of the oligonucleotide. The present example is featuring the potential of thiolated block copolymers in nucleotide delivery systems. The formation of a model system for targeted drug delivery and smart release has been proposed by Liu and coworkers [98]. The model system is a shell crosslinked micelle formed by a triblock copolymer, namely Ald-PEGMA-PDMAEMA-PDEAEMA where Ald is an aldehyde end-group. The above polymer forms micelles in acidic solutions with a PDEAEMA core, a PDMAEMA inner shell and a Ald-PEGMA outer corona. The PDMAEMA block can be crosslinked, leading to stable structures. In parallel, the core is pH responsive, thus a tunable swelling/shrinkage, for controlled release of encapsulated guest molecules, can be achieved. The formed micelles are biocompatible due to the PEGMA outer corona. Finally, the micelles are surface functionalized with aldehyde groups, which can be used for the controlled conjugation with a number of biomolecules. The authors have already demonstrated the conjugation of the above micelles with the model protein lysozyme, Figure 17. An outstanding approach for the development of drug delivery systems has been recently presented by Zhang and Ma [99]. In this work, the use of a β-cyclodextrin (β-CD) containing DHBC has been proposed for the inclusion of many hydrophobic substances. In their work, the synthesis of a block copolymer with a PEO block and a polyaspartamide block carrying some β-CD units has been described. The β-CD units are used as host sites for a plethora of hydrophobic small molecules, like pyrene and coumarine, as well as for hydrophobic polymers. However, the formation of inclusion complexes leads to the creation of located hydrophobic areas within the copolymer domain, which, in turn, has as a result the formation
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of multi-chain, core-shell supramolecular structures. Moreover, the inclusion of charged molecules has been also achieved leading to the formation of micelles after complexation with oppositely charged polymers. The novelty in the use of CD containing block copolymers is the versatility in choosing various guest molecules, since the solubilization effect of CD toward a wide range of hydrophobic compounds has been well documented. Another approach for the stabilization, transport and delivery of the hydrophobic nonsteroidal anti-inflammatory drug indomethacin (IND) has been presented by Giacomelli et al [100]. They have utilized the covalent attachment of the drug on DHBCs by an esterification reaction. The above reaction leads to an amphiphilic block copolymer/drug conjugate. The block copolymer/drug conjugate forms supramolecular structures in water with variable size and morphology (micelles or vesicles) depending on the molecular characteristics of the DHBCs and the content of IND in the conjugate. Moreover, unbounded IND moieties can be also encapsulated in the polymer aggregates, leading to a further increase of the drug load in the conjugate (up to 58% w/w), Figure 18. However, it has been found that high drug loading has as a result the formation of vesicles with an accompanying undesired increase of the aggregate size. It has been also observed that the drug release is a pH-dependent process. At neutral pH the release of charged unbounded IND is favored, while at acidic conditions, where the ester bond between drug and polymer is no longer stable, the slow release of both free and chemically attached IND was possible. The interaction of aggregates of DHBCs with bacteria has been also recently demonstrated [57]. The interaction of a glucose containing copolymer, which forms vesicles at temperatures higher than 15oC, with E. coli has been studied in detail. The obtained data demonstrate that specific interactions between synthetic vesicles and cells can be developed. However, the most interesting finding was the controllable nature of the interactions according to the size of the polymeric vesicle. The authors assumed that by changing the vesicles’ size it is possible to change the interactions, from bulk aggregation to individual associations. The incorporation of ether phospholipid drugs, used for the treatment of leishmania, into supramolecular structures, formed by chemically modified DHBCs has been also studied [101]. A PEO-PDMAEMA block copolymer, where a portion of the tertiary amine groups of the PDMAEMA block was transformed into phosphorozwitterions, was used for the incorporation of the drug. The above copolymer can form both micelles and/or vesicles in aqueous solutions, depending on solution pH, temperature and the molecular characteristics of the copolymer chains. The phospholipid drug could be encapsulated in both micelles and vesicles. However, the introduction of an increased amount of drug favors the formation of vesicles. Moreover, the vesicles formed have a reduced size, probably due to the better organization of the aggregates in the presence of the drug. A series of star DHBCs were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40, bearing the enhanced green fluorescent protein as the reporter gene [55]. The copolymers utilized were composed of PDMAEMA and PHEGMA blocks (where PDMAEMA is an ionizable block, while PHEGMA is a non-ionic water soluble block). The experimental data indicate a decreased toxicity for the star copolymer, compared to a reference PDMAEMA star homopolymer, for the same amounts of star polymer tested. Moreover, it has been found that the architecture of the star copolymer, i.e. star block, miktoarm star etc, plays a decisive role on the transfection efficiency. The best performance, for all star copolymers tested, was observed for a star block copolymer with
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PDMAEMA as the outer block. Interestingly, the transfection efficiency of the particular copolymer is comparable with that of the commercially available transfection reagent SuperFect.
Figure 17. Schematic demonstration of the micelle formation by an Ald-PEGMA-PDMAEMAPDEAEMA DHBC and interaction of the micelles with lysozyme. Reproduced from [98] by permission of the American Chemical Society.
Figure 18. Inclusion and release of a drug in a micelle formed by a PEO-(PG2MA-IND) DHBC. Reproduced from [100] by permission of the American Chemical Society.
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5.3. Other Nanotechnological Applications Liu and coworkers have presented the synthesis of gold nanoparticles in crosslinked micelles. They have succeeded in selectively crosslinking either the core or the shell of the micelles, formed by a primary amine containing block copolymer, namely PNIPAM-PAPAT [31]. The gold nanoparticles have been synthesized by addition of HAuCl4, which is selectively located at the crosslinked, primary amine containing block. Subsequently, reduction of the gold ions leads directly to the formation of gold nanoparticles at either the core or the shell of the polymeric aggregate. The synthesis of Pt nanoparticles has been also reported in the literature [102]. Three different types of DHBCs, namely PEO-PV2P, PHEGMA-b-PDEAEMA, and PEO-bPDEAEMA, have been used as templates for the development of Pt nanoparticles. All the copolymers tent to self-assemble in solution at high pH values due to the deprotonation of the P2VP or PDEAEMA blocks. Therefore, in both cases, micelles with cores containing amine moieties were formed. The amine groups could be used as coordination sites for Pt ions leading to the preferential location of the ions in the micellar core. In another approach, Pt ions could be used in order to induce micellization in solutions containing molecularly dissolved block copolymers. Pt ions could electrostatically interact with the positively charged block copolymer at acidic solutions. The electrostatic attractions lead to the formation of supramolecular structures. In both cases, addition of Pt ions in polymeric micelles or molecularly dissolved copolymers, leads to core-shell micelles with Pt ions preferentially located in the cores. In these micellar nanoreactors, metal nanoparticles nucleate and grow, upon reduction, with sizes in the range of a few nanometers.
5.4. Miscellaneous Properties and Applications of DHBC Systems The adsorption of DHBCs on solid surfaces has attracted considerable scientific interest due to the possibilities that offers for the construction of smart/responsive surfaces with tunable properties. Biggs and coworkers have studied the behavior of PDMAEMA-PDEAEMA micelles, adsorbed onto mica surfaces, upon changes in solution pH using an in situ AFM instrumentation set-up [103]. In aqueous solutions, the copolymers tend to be molecularly dissolved in acidic environment and to form micelles at elevated pH values [103]. The absorbed polymer chains were found also to respond to changes of the solution pH. However, unlike micelles in bulk solution, the adsorbed copolymer micelles do not dissociate at low pH, but remain attached to the solid/solution interface. Furthermore, the micellar core can open or close upon changes in the solution pH – due to this behavior the term “nanoanemones” was coined in order to describe the specific nanosystem. The above behavior leads to smart surfaces with tunable lubricity, which can be used in a number of emerging technologies. However, the situation is dramatically different in the case where the PDMAEMA block has been quaternized. Increasing the number of permanent charges, in the copolymer, leads to the formation of random surface structures which are irreversibly damaged at low pH values [25]. Gohy and coworkers have studied the adsorption of PMAA-PDMAEMA copolymers on silicon surfaces under varying solution pH [104]. A series of copolymers with different
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molecular weights and composition have been utilized in order to elucidate the influence of polymer molecular characteristics on the adsorbed amount. The experimental results indicate that all the parameters (copolymer molecular weight, composition and pH) play their role on the final adsorbed amount and other characteristics of the adsorbed polymer layer. The investigation of another system, namely P2VP-PDMAEMA, by the same group has led to similar results [105]. The latter copolymer formed micelles at pH higher than 5, while it precipitated at pH higher than 8. The adsorption profile upon changes in the solution pH indicates two adsorption maxima: at the begging of the micelle formation and at the precipitation regime. The great influence of pH on the adsorption was correlated with the ionization degree of the polyelectrolyte chains. Preliminary results have been also presented by Armes and coworkers on the adsorption of zwitterionic block copolymers, both on silica and mica surfaces [17]. The copolymer forms negatively and positively charged micelles depending on the solution pH. Positively charged micelles can easily be adsorbed on oppositely charged silica particles and also on planar mica surfaces. However, the adsorption of even negatively charged micelles on mica has been also observed. The later observation was assigned to the relatively weak hydrophobic interactions between the micelle cores and the mica surface, rather than to strong electrostatic interactions. Nevertheless, the ability of pH responsive zwitterionic micelles to be adsorbed on surfaces has been demonstrated. In a detailed study of the adsorbed DHBCs chain conformation, Hamley et al. have presented experimental results on adsorbed Pluronic triblock copolymers onto both silica and mica substrates [106]. The experimental data were obtained for a wide range of bulk polymer concentrations, even below the critical micelle concentration. The results indicate that, depending on the hydrophilicity of the substrate, the copolymer chains are adsorbed in a completely different way. Moreover, it was concluded that the polymer concentration plays a significant role on the conformation of the adsorbed polymeric layer. The catalytic activity of PVim containing DHBCs has been also demonstrated [23]. A PNIPAM-PVim copolymer was found to form micelles with PNIPAM cores at elevated temperatures. The above micelles showed catalytic activity toward the hydrolysis of pnitrophenyl acetate. Interestingly, molecular dissolved copolymer chains do not show the same catalytic activity. The Arrhenius plot for the PVim-based DHBCs exhibited a pronounced upward curvature above the critical micellization temperature. The ability of DHBCs to encapsulate metalloporphyrins under different pH environments has been recently studied [107]. The polymer utilized was a PAA-P4VP block copolymer which forms micelles both in acidic (PAA core) and alkaline (P4VP core) solutions. The encapsulation of porphyrin molecules in both types of micelles has been observed. However, interactions between the P4VP block and metalloporphyrins leads to higher encapsulation ability for the P4VP core micelles. Depending on the initial micellar type, release of the metalloporphyrins can occur either by increasing or decreasing the solution pH. A similar system, more specifically designed for bioapplications, has been reported by Kataoka and coworkers some years earlier. The formation of core-shell micelles of dendrimer metalloporphyrin with DHBCs has been reported by these authors [108]. The micelle formation took place due to electrostatic interaction of charges at the periphery of the dendrimeric metalloporphyrins with opposite charged block copolymers. Depending on the porphyrin/DHBC system, the formed micelles were stable against NaCl solutions (due to the formation of hydrogen bonds) or dissociated at increased salt concentration. Such kind of
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systems could be used in the battle against cancer, as photosensitizers in photodynamic therapies. Kataoka and coworkers have illustrated the potential of mixed porphyrin/DHBCs systems on this kind of therapies [109]. Particularly, the formation of PIC between dendrimer metalloporphyrins and DHBCs can effectively deliver the porphyrin moieties into cancer cells. Importantly, no toxicity effects were recorded, at least for the concentration range utilized.
6. SOME REMARKS ON FUTURE PERSPECTIVES ON DHBC RESEARCH The existing capabilities on sophisticated synthetic procedures for the designed preparation of DHBCs are enormous and they are expanding rapidly. The knowledge on DHBC self-assembly behavior under varying conditions is continuously enriched with new behavioral motifs, tuned by the chemistry of the systems. However, novel and demanding technological applications require the further development of new DHBC systems. The synthesis of new DHBCs has to be realized by utilizing novel monomers, efficient polymerization schemes or/and post polymerization functionalization methodologies. A future goal could be the synthesis of multicomponent DHBCs with linear and non-linear macromolecular architectures. Surely the development of novel polymers will help to establish more precise structure–properties relationships that will aid the application potential of such systems. A number of aspects have to be elucidated in more detail, in order to improve the design and properties of the block copolymers. Theoretical calculations may help substantially and to some extent orient the experimental work. The application spectrum of DHBCs is extended into a wide range of nanomedicine and nanotechnology oriented fields. Considering, the increasing interest for water compatible systems, further increase of DHBCs utilization in emerging technological applications is expected. The outstanding aqueous solution properties of DHBCs have already started to be utilized in a number of applications and these trends will be enhanced in the future.
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[76] Solomatin, SV; Bronich, TK; Eisenberg, A; Kabanov, VA; Kabanov, AV. Langmuir, 2004, 20, 2066-2068. [77] Solomatin, SV; Bronich, TK; Eisenberg, A; Kabanov, VA; Kabanov, AV. J. Phys. Chem. B, 2005, 109, 4303-4308. [78] Thu1nemann, AF; Beyermann, J; Kukula, H. Macromolecules, 2000, 33, 5906-5911. [79] Berret, JF; Oberdisse, J. Physica B, 2004, 350, 204-206. [80] Berret, JF; Herv, P; Aguerre-Chariol, O; Oberdisse, J. J. Phys. Chem. B, 2003, 107, 8111-8118. [81] Pispas, S. J. Phys. Chem. B, 2007, 111, 8351-8359. [82] Harada, A; Kataoka, K. Macromolecules, 1998, 31, 288-294. [83] Harada, A; Kataoka, K. Langmuir, 1999, 15, 4208-4212. [84] Harada, A; Kataoka, K. J. Am. Chem. Soc., 1999, 121, 9241-9242. [85] Harada, A; Kataoka, K. J. Am. Chem. Soc., 2003, 125, 15306-15307. [86] Harada, A; Kataoka, K. J. Controlled Release, 2001, 72, 85-91. [87] Aoyagi, T; Sugi, KI; Sakurai, Y; Okano, T; Kataoka, K. Colloids Surf. B, 1999, 16, 237-242. [88] Jaturanpinyo, M; Harada, A; Yuan, X; Kataoka, K. Bioconjugate Chem., 2004, 15, 344348. [89] Yuan, X; Yamasaki, Y; Harada, A; Kataoka, K. Polymer, 2005, 46, 7749-7758. [90] Yuan, X; Harada, A; Yamasaki, Y; Kataoka, K. Langmuir, 2005, 21, 2668-2674. [91] Pispas, S. J. Polym. Sci., Part A: Polym. Chem., 2007, 45, 509-520. [92] Wang, D; Yin, J; Zhu, Z; Ge, Z; Liu, H; Armes, S. P; Liu, S. Macromolecules, 2006, 39, 7378-7385. [93] Virtanen, J; Arotarna, M; Heise, B; Ishaya, S; Laschewsky, A; Tenhu, H. Langmuir, 2002, 18, 5360-5365. [94] Qi, L; Colfen, H; Antonietti, M. Angew. Chem. Int. Ed., 2000, 39, 604-607. [95] Liu, X; Liu, B; Wang, Z; Zhang, B; Zhang, Z. J. Phys. Chem. C, 2008, 112, 9632-9636. [96] Kulak, AN; Iddon, P; Li, Y; Armes, SP; Colfen, H; Paris, O; Wilson, RM; Meldrum, FC. J. Am. Chem. Soc., 2007, 129, 3729-3736. [97] Kakizawa, Y; Harada, A; Kataoka, K. Biomacromolecules, 2001, 2, 491-497. [98] Liu, H; Jiang, X; Fan, J; Wang, G; Liu, S. Macromolecules, 2007, 40, 9074-9083. [99] Zhang, J; Ma, PX. Angew. Chem. Int. Ed., 2009, 48, 964-968. [100] Ciacomelli, C; Schmidt, V; Borsali, R. Macromolecules, 2007, 40, 2148-2157. [101] Karanikolopoulos, N; Pitsikalis, M; Hadjichristidis, N; Georgikopoulou, K; Calogeropoulou, T; Dunlap, JR. Langmuir, 2007, 23, 4214-4224. [102] Vamvakaki, M; Papoutsakis, L; Katsamanis, V; Afchoudia, T; Fragouli, PG; Iatrou, H; Hadjichristidis, N; Armes, SP; Sidorov, S; Zhirov, D; Zhirov, V; Kostylev, M; Bronsteinfg, LM; Anastasiadis, SH. Faraday Discuss., 2005, 128, 129-147. [103] Webber, GB; Wanless, EJ; Armes, SP; Tang, Y; Li, Y; Biggs, S. Adv. Mater., 2004, 16, 1794-1798. [104] Mahltig, B; Gohy, JF; Jerome, R; Stamm, M. J. Polym. Sci., Part B: Polym. Phys., 2001, 39, 709-718. [105] Mahltig, B; Gohy, JF; Antoun, S; Jerome, R; Stamm, M. Colloid. Polym., Sci., 2002, 280, 495-502. [106] Hamley, IW; Connell, SD; Collins, S. Macromolecules, 2004, 37, 5337-5351. [107] Bo, Q; Zhao, Y. J. Polym. Sci., Part A: Polym. Chem., 2006, 44, 1734-1744.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 10
THERMO-OXIDATION STABILITY OF POLY (BUTYLENE TEREPHTHALATE) AND CATALYST COMPOSITION Antonio Massaa*, Valeria Bugattib, Arrigo Scettria and Socrate Contessac a
Dipartimento di Chimica, Università di Salerno, Via Ponte Don Melillo 84084 Fisciano, Salerno, Italy. b Dipartimento di Ingegneria Chimica ed Alimentare, Università di Salerno, Via Ponte Don Melillo, Fisciano Salerno. c NGP S.p.A.,R&D, Contrada Pagliarone, Acerra, Napoli.
1. SYNTHESIS AND THE STABILIZATION OF POLYESTERS Polyesters are heterochain macromolecular substances characterized by the presence of carboxylate ester groups in the repeating units of their chains. Predominant in terms of volume and products value are those based on poly(ethylene terephthalate) (PET), long established as basis of fibers, films, molding plastics and containers for liquids, and poly(butylene terephthalate) (PBT) largely used to produce fibers as well as for special applications in motor and electric industry.[1-3] PET and PBT can be conveniently synthesized, both at laboratory and industrial scale, in the presence of metal catalysts in a two-step-polyesterification of the diols, ethylene glycol or 1,4-butanediol respectively, and dimethyl terephthatate (DMT) or terephthalic acid (TPA). The first step is basically a transesterification or esterification process depending on the use of DMT or TPA respectively, while the second step is the polycondensation of the resulting oligomers. The polycondensation step occurs at higher temperature and at reduced pressure to facilitate the distillation of the diols and consequently the polymerisation of the oligomers.
*
Corresponding author: e-mail:
[email protected]; tel.: +39 089 969565.
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There are important differences in the synthesis of these two polymers, depending also on the choice of dimethyl terephthatate (DMT) or terephthalic acid (TPA) as starting materials. A single catalyst, usually a titanium(IV) alcolate, is used in the synthesis of PBT for both ester-interchange and polycondensation steps. Two different catalytic systems are necessary in PET synthesis if DMT is used as starting material: Mn(II), Co(II), Mg(II) as acetate salts are the common catalysts for the transesterification step and Sb(III), usually Sb2O3, is the catalyst for the polycondensation step. Titanium (IV) alcolates are also good polycondensation catalysts for PET synthesis but they are not often used because of the yellowing of the obtained polymer. One of the most accredited hypothesis for the explanation of this phenomenon is the formation of coloured titanium-based complexes during the high temperature polycondensation stage. For these reasons only small amounts of a titanium(IV) alcolates are usually used as co-catalyst in addition to Sb(III) catalyst. However the exploitation of Ti(IV) based compounds for PET synthesis is a hot subject and several questions are still open in order to solve these problems.[4] Direct esterification of TPA is catalysed by COOH terminal groups and ester-interchange metal-catalysts are not necessary. The synthesis from TPA is particularly convenient for the production of PET because the use of the metals is limited only to polycondensation catalytic system. For PBT synthesis, TPA is less convenient because the presence of acidic groups leads to the decomposition of 1,4-butanediol to the formation of tetrahydrofurane. A typical example of the two-step synthesis of PBT is reported in Scheme 1. O
O
H3COC
2 HOCH2CH2CH2CH2OH
COCH3 +
Ti(IV) catalyst T = 150 - 210°C
DMT
Transesterification
O
O
HO(CH2)4OC
Ti(IV) catalyst
CO(CH2)4OH
+ 2 CH3OH
T = 240 - 250° C P = 0.1-1.0 mbar Polycondensation
BHBT
O
O
C
CO(CH2)4O
+
HOCH2CH2CH2CH2OH
n PBT
Scheme 1.
Since the early application of polyesters, great importance was attributed to the stability and shelf-life of these materials.[5-19] The stabilization for poly(ethylene terephthalate) (PET) is mostly related to the stabilization of ester-interchange catalysts. When these metals are not stabilized, a strong degradation and yellowing of the PET occurs at higher temperature in the following polycondensation stage. In this case the addition of phosphates or phosphonates at the end of transesterification stage suppresses the undesired processes and
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increases the stability of the PET. These additives probably form inert complexes or salts with the ester-interchange catalysts.[12-19] Even if several studies demonstrate that PBT is less stable than PET, less attention has been paid to the improvement of the stability of poly(butylene terephthalate).[7,8] The usual strategy to increase PBT stability is the addition, during the synthesis or the manufacturing, of anti-oxidizers, like Ultranox and Irganox the typical phenol-type radical scavengers.[20] However, during the last years, a good number of new catalytic systems are claimed to catalyse efficiently PBT synthesis, but in these studies very few information are available about possible relationships between catalyst composition and polymer stability.[21-23] Only recently Colonna et al. reported that in PBT synthesis, the use of mixtures of titanium(IV) tetrabutoxide and Hafnium(IV) acetylacetonate as catalysts in the presence of metal phosphates as co-catalysts improved the thermal stability of the polymers.[24] Unfortunately these interesting results have not received significant attention and no hypothesis has been formulated about the relationships between the improved stability and the catalyst composition. It is worthy to note that at the end of the typical synthesis of polyesters, the catalytic system is a residual dispersed in the polymer matrix. But what are the effects of these metal impurities? Is this metal residual still active? Can these metal impurities catalyse the degradation processes and reduce the life of the material? For example, solid state polycondensation (SSP), a way to increase the molecular weight directly on pellets of solid PET, is the evidence that Sb(III) residual is still active in solid polymer.[25-28] On the contrary less information are available about the effect of Ti(IV) residual on SSP for PBT. It is a matter of fact that in all the studies about thermal and thermooxidation degradation, the effects of the catalytic system are usually ignored because the catalyst is always present as residual in every PBT sample. It is simply a background effect.
2. RELATIONSHIPS BETWEEN CATALYST COMPOSITION AND PBT STABILITY: THE CASE OF Ti (acac)2(O-iPr)2 Following the idea that the decomposition processes in PBT can be related to catalyst residual and that degradation can be inhibited by “metal deactivators”, we started to test a series of new potential catalytic systems and additives with specific characteristics for polyester synthesis. A “metal deactivator” can be defined like a kind of chelating agent which forms inactive complexes with residuals of metal catalysts or impurities.[20,29] In our investigations the chelating agents, the additives are introduced in the synthesis as complexes with the metal catalysts. For this reason the catalysts must be effective in the synthesis of the polymer and at the same time should reduce the degradation reactions. As a result of these considerations and after a wide screening of several chelating agents and Ti(IV) complexes, we found that commercially available Ti(acac)2(O-iPr)2 (titanium bisacetylacetonate diisopropylate) can be conveniently used in the synthesis of poly(butylene terephthalate) (PBT 1) both at pilot plant and industrial scale.[30] This new catalyst showed higher activity than standard Ti(O-nBu)4 and very interestingly the obtained polymers showed higher thermo-oxidation stability than PBT 2 synthesized in the presence of Ti(O-nBu)4. Furthermore, PBT 3 stabilized with Ultranox 626 and synthesised with Ti(O-nBu)4 was less stable than new PBT 1, but more stable than that synthesized only in the presence of Ti(O-nBu)4 (PBT 2).[30]
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Even the mixture of acetylacetone and Ti(O-nBu)4 was effective in PBT synthesis and the obtained polymer 4 showed again improved thermo-oxidation stability, similar to PBT 1.[31] OiPr O
O
Ti O
O OiPr
Ti(acac)2(O-iPr)2 Figure 1.
Table 1. characteristics of different PBTs PBT polymer
Catalyst
EI stage Time(min.) 72 86 85 67
PC Stage Time(min.) 78 87 85 83
I.V. (dl/g)a
Ti(acac)2(O-iPr)2b 0.889 Ti(O-nBu)4b 0.851 Ti(O-nBu)4 + U626 0.829 Ti(O-nBu)4 + 0.851 acetylacetoned a Determined on a solution of PBT in o-chlorophenol with an auto viscosimeter Shotte-Gerate b The complex was supplied from Aldrich. c The stabilizer U626 was added to the reaction mixture at the end of the transesterification step at a concentration of 0.1% w/w. d The catalytic system was prepared according to the procedure reported in the experimental section. 1 2 3c 4
Regarding the experimental details, these four different types of PBTs were synthesized in a 20 L pilot plant under standardized conditions and they were submitted to standard oven testing for the evaluation of thermo-oxidation stability. In particular, experimental data about reaction time for ester-interchange and polycondensation steps and the values of the intrinsic viscosity (I.V.) of the PBTs are summarized in table 1. It is worthy to note that both Ti(acac)2(O-iPr)2 and the mixture acetylacetone/Ti(OnBu)4 were more effective for both EI and PC stages and the resulting polymers were obtained with a higher I.V. and consequently with a higher molecular weight. This is very important from economical point of view because in the presence of a more active catalyst there is a significant reduction of the manufacturing costs of the materials. Moreover a shorter reaction time involves a reduced permanence of the polymer at high temperature with the consequent reduction of the thermal decomposition processes. The effects of thermo-oxidation were evaluated by means of the standard method of ageing a large quantity of pellets of the four PBTs in forced circulating air oven at 160°C.[32] This is considered the most reliable method for ageing acceleration tests.[33] Then at different time, we measured colour-L (Figure 2), colour-b (Figure 3), intrinsic viscosity (I.V.; Figure 4) and carboxyl end groups (C.E.G.; Figure 5) for the treated samples. All these parameters are reasonably related to the “level” of degradation of macromolecules. Degradation causes the cleavage of the polymer chains and consequently the decrease of molecular weight, the decrease of the related I.V. and the increase of C.E.G. The colour, the
Thermo-Oxidation Stability of Poly (Butylene Terephthalate) and Catalyst…
331
appearance is also important and in the practical experience of everyone the yellowing is commonly related to the ageing. The colour of polymers can be numerically measured in terms of L-value and b-value directly on the pellets, according to Cielab scale. L-Value is related to the degree of whiteness with a greater numerical value showing a higher (desirable) whiteness. The whiteness and consequently colour L is usually related to the level of crystallinity. The b-value is related to the degree of yellowness, and a higher numerical value shows a higher (undesirable) degree of yellowness. A particular importance is attributed to colour b for the relationship with the level of degradation and oxidation of macromolecules.[13] 92
90
"PBT 1: Ti(acac)2(O-iPr)2 88
"PBT 2: Ti(O-nBu)4 86
"PBT 3: Ti(O-nBu)4 + U626 84
"PBT 4: Ti(O-nBu)4 + acetylacetone
82
80
78
0
10
20
30
40
50
Ageing time (h)
Figure 2. Colour L of PBTs aged at 160°C 25
20
Colour b
Ti(acac)2(O-iPr)2 Ti(O-nBu)4
15
Ti(O-nBu)4 + U626 10
Ti(O-nBu)4 + acetylacetone 5
0 0
10
20
30
Time (h)
Figure 3. Colour b of PBTs aged at 160°C
40
50
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Antonio Massa, Valeria Bugatti, Arrigo Scettri et al.
The comparison of the obtained data shows that, after prolonged ageing time, PBTs 1 and 4 keep their properties practically intact. PBTs 1 and 4 suffer a very slight discolouring with a constant degree of whiteness (Colour-L, Figure 2), and a very reduced increase of yellowness (colour-b, Figure 3); a constant value of C.E.G. (carboxyl end groups, Figure 4) and a slight increase of I.V. (Figure 5). However PBT 3, stabilised with U626, is more stable than 2 and less stable than 1 and 4. In this case both the discolouring, the decrease of the intrinsic viscosity (I.V.) and the increase of C.E.G. are not as strong as in 2. Therefore the use of the stabilizer has a positive effect on the stability of the polymer obtained in the presence of Ti(O-nBu)4, but it is useless when Ti(acac)2(O-iPr)2 is employed as catalyst. 40
35
C.E.G. (eq/ton)
30 Ti(acac)2(O-iPr)2 Ti(O-nBu)4
25
Ti(O-nBu)4 + U626 Ti(O-nBu)4 + acetylacetone
20
15
10 0
10
20
30
40
50
Ageing time (h)
Figure 4. C.E.G. of PBTs aged at 160°C.
0,92 0,9
Ti(acac)2(O-iPr)2
0,88
Ti(O-nBu)4
0,86 0,84
Ti(O-nBu)4 + U626 0,82
Ti(O-nBu)4 + acetylacetone
0,8 0,78 0,76 0
10
20
30 T ime ( h)
Figure 5. I.V. of PBTs aged at 160°C.
40
50
Thermo-Oxidation Stability of Poly (Butylene Terephthalate) and Catalyst…
333
Moreover the analysis of the data shows some peculiar features for PBTs 1 and 4 that have to be noted. The significant improvement of colour L (and of the related level of whiteness), after the first 7 h of heating, can be related to the annealing that crystallizes the otherwise crystallinity-retarded polymer (Figure 1). This behaviour as well as the lower value of colour L at t=0 of freshly synthesised PBT 1 and 4, can be explained through the formation and incorporation of a small amount of an additional co-monomer as di(butanediol). The trend of I.V. in PBT 1 and 4, since it cannot be related to the accuracy of the method (0.002 dL/g), is very interesting. If on one hand the slight decrease from 7 to 47 h can be attributed to a slight degradation, the increase after the first 7 h can be related to a small increase of molecular weight. This effect is very intriguing and can be related both to the possibility of a solid state polycondensation on PBT (SSP) and reticulation processes. In addition to acetylacetone other additives and well-known complexing molecules, like those depicted in Figure 6, were tested in the synthesis of PBT. The new potential catalytic systems were obtained as a reaction mixture of the additives with Ti(O-nBu)4 in 1,4-butanediol. This mixtures were used in PBT synthesis without any modification or purification.[31,32] In particular, among the tested additives, it is worthy to note that commercially available titanium complexes are reported for ethylacetoacetate;[34] EDTA is one of the most universally employed chelating agent. A different approach was considered in the choice of TEPF and TEPA. TEPA as a phosphonate based compound, can be used as stabilizer for transesterification catalysts in PET synthesis.[35-37] In this case the aim was to verify if this type of compounds have the same effect on the stability of PBT as widely described for PET. O O
O
P
EtO
P
EtO
O
OEt EtO
EtO
OEt
triethyphosphonformiate (TEPF)
triethyphosphonacetate (TEPA)
NaOOC
O
O
HOOC
N N
COOH
OCH2CH3 COONa
ethylacetoacetate (EAA)
ethylenediamminotetraacetic disodium salt (Na2EDTA)
Figure 6.
Comparing the experimental data about reaction time and the intrinsic viscosity (I.V.) of these new PBTs (Table 2), the Ti(O-nBu)4/acetylacetone catalyst showed again a significant higher activity than the other systems both for ester-interchange and polycondensation steps (Table 2). Only in the case of Ti(O-nBu)4/TEPF the polymer was obtained with a higher I.V., even if in longer reaction time. Then thermo-oxidation stability of the PBTs was tested in the usual way by oven ageing, and the results are summarized in the following figures 7-9.
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Antonio Massa, Valeria Bugatti, Arrigo Scettri et al.
Entry
Table 2. characteristics of different PBT EI stage PC Stage Time(min.) Time(min.) Ti(O-nBu)4 86 87 Ti(O-nBu)4 + acetylacetone 67 83 Ti(O-nBu)4 + EDTA 75 90 Ti(O-nBu)4 + EAA 84 91 Ti(O-nBu)4 + TEPF 85 91 Ti(O-nBu)4 TEPA 81 89
Catalyst
I.V. (dl/g)a
2 0.855 4 0.851 5 0.843 6 0.828 7 0.875 8 0.849 a I.V.s were measured on a solution of PBT in o-chlorophenol employing an auto viscosimeter ShotteGerate. b According to Cielab scale, colours were determined employing a colorimeter BYK Gardner. 92 90
Colour L
88
Ti(O-nBu)4 Ti(O-nBu)4 + acetylacetone
86
Ti(O-nBu)4 + EDTA Ti(O-nBu)4 + EAA
84
Ti(O-nBu)4 + TEPF Ti(O-nBu)4 + TEPA
82 80 78 0
10
20
30
40
50
Tme (h)
Figure 7. Colour L of PBTs aged at 160°C. 25
20
Colour b
Ti(O-nBu)4 Ti(O-nBu)4 + acetylacetone
15
Ti(O-nBu)4 + EDTA Ti(O-nBu)4 + EAA
10
Ti(O-nBu)4 + TEPF Ti(O-nBu)4 + TEPA
5
0 0
10
20
30 Time (h)
Figure 8. colour b of PBTs aged at 160°C.
40
50
Thermo-Oxidation Stability of Poly (Butylene Terephthalate) and Catalyst…
335
The comparison of the reported data for colour L, colour b and I.V. shows again that PBT 4, synthesized in the presence of Ti(O-nBu)4 / acetylacetone, is the most stable. All the other catalytic systems influenced negatively the stability of PBT even if Ti(O-nBu)4 is the worse. However, there are significant differences among the several catalysts on PBT properties. These effects are probably related to the strength of the complexes formed between Ti(IV) and the complexing agents. In particular it should be noted that Ti(O-nBu)4 / ethylacetoacetate is less deleterious: in this case we noted a constant level of colour L, and the decrease of I.V. and color b was less pronounced than in the other cases. 0,9
0,88
0,86 Ti(O-nBu)4
I.V. (dl/g)
0,84
Ti(O-nBu)4 + acetylacetone Ti(O-nBu)4 + EDTA
0,82
Ti(O-nBu)4 + EAA Ti(O-nBu)4 + TEPF
0,8
Ti(O-nBu)4 + TEPA
0,78
0,76
0,74 0
10
20
30
40
50
Time (h)
Figure 9. I.V. of PBTs aged at 160°C. However, it is very difficult to propose a mechanistic explanation of the obtained results, but a look to the general features of the accepted thermo-oxidation mechanism of organic materials and polymers can be helpful to have an idea about the processes involved during the degradation. This subject will be explored in the next sections. In conclusion it is important to say that the use of Ti(acac)2(O-iPr)2 at industrial scale led to a higher quality and cheaper PBT. This polymer has been commercialised for several years until the bankruptcy of our old company. The main characteristic of this product was of course the high stability, achieved without the use of any stabilizer.
3. THERMO-OXIDATION OF ORGANIC MATERIALS AND POLYMERS The study of degradation processes of polymers and organic materials is a fundamental topic both from academic and industrial point of view and it can be expected that thermal and thermo-oxidative decomposition mechanisms are reasonably well established.[20,38] These complex reactions proceed via a typical free-radical chain as outlined in scheme 2.
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Antonio Massa, Valeria Bugatti, Arrigo Scettri et al.
However the details of the initiating process (step 1), the very early formation of radicals, . are not yet fully understood. Usually it is assumed that the oxidation initiating radical R is formed by heat, light, by mechanical stress or by reactions with radicals originating from foreign sources. Then, in the presence of oxygen the radicals lead to the formation of hydroperoxides (step 3). These are the key intermediates of the thermo-oxidation process because the thermally induced homolytic decomposition of -OOH groups into different radicals renews oxidation chains (stage 4).[39] Since homolytic decomposition of hydroperoxides into free radicals (stage 4) requires relatively high activation energies, this process is rather slow and becomes effective only at temperature of about 120°C or higher.[20] However, in the presence of catalytic amounts of certain metal ions, hydroperoxides decompose at a lower temperature and/or at higher rate. This degradation mechanism involves a series of redox processes in which the most active catalysts are those derived by metals easy to oxidize or to reduce by one-electron, such as a series of transition metals like Fe, Co, Mn, Cu, Ce, V. Then several competitive termination couplings between different radicals (step 5) or the decompositions of peroxy radicals (step 6) lead to radical destruction. INITIATION
1)
R.
RH
PROPAGATION
2)
R.
O2
+
ROO .
HYDROPEROXIDE FORMATION
3)
ROO . + RH
ROOH
+
R.
CHAIN BRANCHING
4)
RO
ROOH
.
+
. OH
TERMINATION
5)
R.
6)
ROO .
+
ROO .
ROOR Inactive products
Scheme 2.
The best way to contrast these degradation reactions is the use of an antioxidant. The effectiveness of antioxidants is based on the fact that they are able to stop the reactions described in scheme 2. The majority of antioxidants belong to the class of sterically hindered phenols like those known with the trade names Ultranox or Irganox. These compounds are peroxy-radicals decomposers, according to the reaction reported in scheme 3. The
Thermo-Oxidation Stability of Poly (Butylene Terephthalate) and Catalyst…
337
stabilization is achieved by the fact that the reaction of scheme 3 competes with reaction 3 of scheme 2, transforming the reactive peroxy radical into the much less reactive phenoxy radical. In this way the chain branching in scheme 2 is inhibited and the radical chain process is terminated. The phenoxy radical is also capable to react with another peroxy radical to give a peroxycyclohexanedione as widely established in literature (Scheme 4).[33, 40, 41]. OH t-Bu
. ROO
O t-Bu
.
t-Bu
t-Bu
+
ROOH
+
phenoxy radical
Scheme 3. O t-Bu
.
O
O t-Bu
t-Bu
t-Bu
t-Bu
.
+
ROO
t-Bu
. OOR
phenoxy radical
peroxycyclohexanedienone
Scheme 4.
O O
O
P
P O
O
O
U626: bis-(2,4-ditertbutylphenyl)-pentaerythryl-diphosphite
Figure 10.
P(OR)3
+
R'OOH
OP(OR)3
+
R'OH
Scheme 5.
The thermal stability of peroxycyclohexanedienones is limited. Their decomposition leads to new reaction chains at temperatures below 150°C. Also phenoxy radicals can initiate new radical chains at high temperature. Of course these features do not affect the performances of stabilizers during the normal life of stabilized materials, but these effects should be taken into account especially in high temperature application and testing of antioxidants-added materials. Otherwise this means that the effectiveness of sterically
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hindered phenols decreases with increasing the temperature of application and manufacturing of the stabilized materials. Even within the limits of the testing conditions, the positive effect of a typical antioxidant, Ultranox U626, for the stabilization of PBT synthesised in the presence of Ti(O-nBu)4 was previously highlighted in section 2. The structure of U626 is reported in Figure 10. U626 is a rather complex molecule. Very interestingly the phosphite moieties in U626 can have also an important role in the stabilization. Phosphites are well-known stabilizers and they are called secondary antioxidants, while hindered phenol-based stabilizers are known as primary antioxidants. Phosphites have the ability to react with hydroperoxides to yield phosphates according to scheme 5.[20,38] U626 combines primary and secondary stabilizers in the same molecule.
4. THERMO-OXIDATIVE DEGRADATION OF PBT: THE ROLE OF TITANIUM (IV) RESIDUAL An experimental investigation directly on polymers in order to find out the degradation products is difficult to realize. However, both the study of degradation of model compounds like butylene dibenzoate [5-7], and the general mechanism of thermo-oxidation of organic molecules (scheme 2), can be very helpful to propose a specific mechanism for the thermooxidative degradation of PBT (scheme 6). In order to simplify the discussion, the contribution of thermal degradation has not been considered. The first step of thermo-oxidative degradation is believed to be the homolytic cleavage of α−CH bonds, a process that leads to the formation of α-radicals (step 1).[5-7] Then in the presence of oxygen the α−radicals react to form hydroperoxides that are the key intermediates of the degradation mechanism (step 2). In effect the decomposition of the hydroperoxides (step 4) is the beginning of complicated radical chains that lead to the formation of a great number of products and functionalities that are difficult to isolate and characterize (step 5).[511,42,43] However the results of the these degradation reactions are mainly C-C and C-O cleavages of the bonds adjacent to radical moieties. One of the consequences of these cleavages is the decrease of the molecular weight and the increase of both aliphatic and aromatic carboxyl end groups. As emphasized in section 3, the decomposition of hydroperoxides, the key step of the degradation mechanism, is rather slow and becomes effective only at temperature of about 120°C or higher. However in the presence of catalytic amounts of certain metal ions, such as a series of transition metals like Fe, Co, Mn, Cu, Ce, V, hydroperoxides decompose at a lower temperature and/or at higher rate. According to this concept, the catalyst residual in PBT can have an important role to catalyze the degradation of hydroperoxides (step 4 of scheme 6). In fact it is well-known the capability of titanium(IV) to catalyze important oxidation reactions, such as epoxidation of allylic alcohols and sulfoxidation, in which hydroperoxides are involved as oxidants.[44,45] The role of titanium center in these reactions is the coordination and activation of the hydroperoxides to favor oxygen atom transfer. Ti(IV) compounds are also able to catalyze hydroperoxides decomposition into ketonealcohol mixtures, a well-known industrial process for acetone and phenol production.[46-48] In particular, a kinetic study reported by Sukin et al. showed that, in titanium(IV) catalysed
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radical decomposition of peroxides, the decomposition increases in the order diacyl peroxides ≤ peroxy esters < peroxy ketones = hydroperoxides. In these reactions, sterically hindered and chelated titanium(IV) compounds catalyse hydroperoxide decomposition only very slowly.[46] Moreover, even Ti(III) based compounds play an important role in the radical decomposition of t-butyl hydroperoxide.[48] Therefore the possibility of the different redox species, Ti(IV)/Ti(III), can also affect radicals formation and polymer decomposition. On the basis of these considerations we can confidently assume a relationship between titanium residual and PBT degradation. The different effect of Ti(O-nBu)4 and the chelated Ti(acac)2(O-iPr)2 on PBT stability could be explained with a different capability of the two metal residuals to coordinate the OOH groups and/or a different ability to catalyse hydroperoxides decomposition. Therefore Ti(O-nBu)4 and the chelated Ti(acac)2(O-iPr)2 should affect in a different way the key step 4 of the degradation mechanism (scheme 6) and consequently the rates of the entire degradation process are very different in the two cases. Then, the decrease of I.V., the increase of CEG and the yellowing of standard PBT 1 (see section 2) are related of the further decomposition the alkoxyl radicals (step 5). The observed yellowing could be correlated both to the formation of products with conjugated functional groups and to new coloured titanium species formed during high temperature oven testing. Of course it is difficult to determine experimentally the catalytic species and to establish the role of different Ti(IV) compounds on the degradation process in solid PBT. Probably the thermooxidation of the model compounds in the presence of different titanium based complexes can be useful. In conclusion, even if a specific mechanistic model is not available yet for the obtained results, the improved stability of PBT in the presence of Ti(acac)2(O-iPr)2 can be attributed to the inhibition of the degradation of hydroperoxides by means of metal deactivation using a chelating agent.
CONCLUSION AND OUTLOOK Excellent progresses have been made over past decades in the development of new efficient catalysts for polyesters synthesis, and in the understanding of the properties of these materials. Even if an impressive quantity of information are available, several challenges are still open. One of the most important purpose in PET research is, without no doubt, the substitution of the toxic Sb(III) based catalyst with more friendly compounds. One of these possibilities, as described in section 1, is related to titanium (IV) based compounds. In effect Ti(O-nBu)4 is an active catalyst in PET synthesis but it is not often used because of the yellowing of the obtained polymers. However, many studies are evaluating the use of different additives and/or several Ti(IV) based complexes in PET synthesis in order to solve these problems and to understand the reasons of the discoloration. The different effect of titanium catalysts on PET and PBT colour could be related to the thermal degradation. Polycondensation temperature for PET is significantly higher than that of PBT, and titanium(IV) can affect their thermal decomposition in different ways. However, the role of titanium catalysts on PET properties is not clear yet, but, all these problems, the stability, the discoloration, are correlated directly or indirectly to the catalyst composition.
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Antonio Massa, Valeria Bugatti, Arrigo Scettri et al. O
O
O
.
O
O
C
COCHCH2CH2CH2CO
*
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COCH2CH2CH2CH2CO
*
*
1
2
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O
.
+H
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.
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.
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COCHCH2CH2CH2CO
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Further oxidation products; formation of different radicals; C-C C-O cleavage products, etc. etc.
Scheme 6.
REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
Mark, HF; Ed., Encyclopedia of Polymer Science and Engineering, Vol. 12, 2nd ed.; Wiley-Interscience: New York, 1988. Margolis, JM; Ed.; Engineering Thermoplastics: Properties and Applications; Marcel Dekker: New York, 1985. Brozenick, NJ. Modern Plastics Encyclopedia; McGraw-Hill: New York, 1986-1987. Klausner, E; Santostefano, A; Monti, R; Barchielli, G. European Patent, n°, 1998, 1, 035 916. Buxbaum, LH. Angew Chem Int Ed Engl, 1968, 7, 182. Allen, NS; Edge, M; Mohammadian, M; Jones, K. Polym Degrad Stab, 1993, 41,191. Botelho, G; Queiros, A; Liberal, S; Gijsman, P. Polymer Degradation and Stability, 2001, 74, 39-48. Carroccio, S; Rizzarelli, P; Scaltro, G; Pugliesi C. Polymer, 2008, 49, 3371–3381. Nealy, DL; Adams, LJ. J Polym Sci, 1971, 9, 2063. Goodings E.P. Soc Chem Ind Monograph No., 1961, 13, London, 211. Lum R.M; J Polym Sci Polym Chem Ed, 1979, 17, 203. Dzieciol, M; Trzeszczynski, J. J Appl Polym Sci, 1998, 69, 2377. Edge, M; Allen, NS; Wiles, R; McDonald, W; Mortlock, SV. Polymer, 1995, 36, 227. Zimmerman, H; Kim, NT;. Polym Eng Sci, 1980, 20, 680. Zimmerman, H. Faserforsch u Textiltechn, 1962, 13, 481. The Resinous Products and Chem Co. Brit. Pat., 1947, 5,888,834. Zimmerman, H. Faserforsch u Textiltechn 1968, 19, 372. E. I. Du Pont de Nemours Co. U.S. Pat. 3,406,153 (1968).
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[19] Celanese Corporation, U.S. Pat., 3, 784,507, 1974. [20] Gachter, R; Muller, H. Plastics Additives Handbook; Hanser/ Gardner Publishers: Munchen–New York, 1988. [21] Walter, M; Boerner, F; Rafler, G; Thiele, UK; Stibal, W; Hagen, R. (Inventa-Fischer G.m.b.H. & Co. KG., Germany). Ger. Offen. 2005, 11, Application: DE, 200310339742 20030828. [22] Banach, TE; Fiorini, M; Patel, BR; Pilati, F; Berti, C; Marianucci, E; Messori, M; Colonna, M; Toselli, M. (General Electric Company, USA). PCT Int. Appl. (2000), 24 pp. Application: WO 99-US25685. [23] Putzig, DE. (USA). U.S. Pat. Appl. Publ. (2005), 8 pp. Application: US 2004-810403. [24] Colonna, M; Banach, TE; Berti, C; Fiorini, M; Marianucci, E; Messori, M; Pilati, F; Toselli, M. Polymer, 2003, 44, 4773. [25] Pilati, F; Gostoli, C; Sarti, GC. Polym Process Eng, 1986, 3, 303. [26] Fortunato, B; Pilati, F; Manaresi, P. Polymer, 1981, 22, 655. [27] Guo, B; Chan, CM. J Appl Polym Sci, 1999, 71, 1827. [28] Duh, B; Tallmadge, O. J Appl Polym Sci, 2003, 89, 3188. [29] Typically, metal deactivators are used in the stabilization of polyolefins in contact with copper (see ref. 19). [30] Massa, A; Scettri, A; Contessa, S; Bugatti, V; Concilio, S; Iannelli, P. Journal of Applied Polymer Science, 2007, 104, 3071. [31] Massa, A; Bugatti, V; Scettri, A; Contessa, S. Macromolecules: An Indian Journal, 2008, 4 (1), 45-50. [32] All the samples for ageing tests refers to PBTs synthesized in 20 L pilot plant. All the catalytic systems were tested twice. For experimental details about polymers and catalysts synthesis, and for ageing tests see ref 29. [33] Pospisil, J; Horak, Z; Pilar, J; Billingham, NC; Zweifel, H; Nespurek, S. Polym Degrad Stab, 2003, 82, 145. [34] Puchberger, M; Rupp, W; Bauer, U; Schubert U. New J . Chem., 2004, 28,1289. [35] Hiroshi, K; Shinichi, T. (Teijin Ltd., Japan). Jpn. Kokai Tokkyo Koho (2004) Application: JP, 2003-5564 20030114. [36] Takase, T; Tsukamoto, R. (Teijin Fibers Ltd., Japan). Application: JP, 2003-284767 20030801-2005. [37] Katamani, H; Konagaya, S; Nakamura, Y. Polymer, 1980, 12, 125. [38] Zhang, X. M; Mantzaris, J. Recent Res Dev Org Chem, 1998, 2, 453. [39] Bolland, JL; Gee, G. Trans Faraday Soc, 1946, 42, 236. [40] Pospisil, J, Adv. in Pol. Sci., 1980, 36, 69. [41] Koch, J. Angew. Makromol. Chem., 1971, 20, 7. [42] Lum, R.M. J Polym Sci Polym Chem Ed, 1979, 17, 203. [43] Passalacqua, V; Pilati, F; Zamboni, V; Fortunato, B; Manaresi, P. Polymer, 1976,17,1044. [44] Johnson, RA; Sharpless, KB. In Catalytic Asymmetric Synthesis; Ojima, I., Ed; VCH: New York, 1993, 227. [45] Kagan, HB; Luukaas, T. In Transition Metals for Organic Synthesis; Bolm, C., Beller, Eds; Wiley/VCH: Weinheim, 1999, Vol. 2, p 361.
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[46] Sukin, AV; Bulatov, MA; Spasskii, SS; Suvorov, AL; Khrustaleva, EA; Kochneva, MA. Zhurnal Fizicheskoi Khimii, 1977, 51, 2093. [47] Stepovik, LP; Gulenova, MV; Martynova IM. Russian Journal of General Chemistry, 2005, 75(4), 507 Translated from Zhurnal Obshchei Khimii, 2005, 75 (4), 545. [48] Citterio, A; Arnoldi, A; Griffini, A. Tetrahedron, 1982, 38(3), 393.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 11
HINDERED AMINE STABILIZERS AS SOURCES OF MARKERS OF THE HETEROGENEOUS PHOTOOXIDATION / PHOTOSTABILIZATION OF CARBON CHAIN POLYMERS J. Pilař and J. Pospíšil Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v. v. i.; 162 06 Prague 6, Czech Republic.
ABSTRACT Application of Hindered Amine Stabilizers (HAS) is the state-of-the-art approach to protection of carbon-chain polymers such as polyolefins and polystyrene or blends containing these against weathering. During outdoor exposure, the polymers loose their material properties due to solar radiation-triggered photooxidation. The complex mechanism of the stabilization involving cyclic oxidation-triggered transformation of HAS is outlined. Monitoring of the formation of the HAS-developed key transformation products, HAS-related nitroxides, responsible within the regenerative mechanism for the effective stabilization was used to confirm the heterogeneous character of photooxidation of two carbon-chain polymers, polypropylene and a specific polyethylene copolymer. Depth profiles of nitroxides were monitored in a long-term photooxidation regime using Electron Spin Resonance Imaging (ESRI) technique. The shape of concentration profiles of the nitroxides accumulated in the equilibrium state upon filtered Xenon lamp-equipped Weather-Ometer exposure was interpreted in terms of the oxygen diffusion limited oxidation and radiation penetration in oxidation-stressed polymer surfaces. The data indicate differences in the character of the heterogeneous process in dependence on the polymer matrix and on the used stabilizer system based on secondary HAS and Oalkylhydroxylamine HAS and/or HAS combination with UV absorbers. Imaging of nitroxides is a precise tool for marking heterogeneous oxidation of polyolefins.
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INTRODUCTION AND BASIC CONSIDERATION Carbon-chain commodity and engineering polymers (PH) suffer during all phases of their lifetime in the earth atmosphere from oxidative degradation, either thermal or phototriggered. Mechanism of oxidation was studied in detail [1, 2]. The oversimplified free radical oxidation scheme involves initiation (1), propagation (2 and 3) and termination steps (4) followed by reactions of primary intermediates and products.
⎯→ P• PH ⎯⎯ ⎯
(1)
P• + O2 → POO•
(2)
POO• + PH → POOH + P•
(3)
P•, POO• → Products
(4)
initiation
The last two steps are rather well understood. There exist some uncertainties encountering initiation steps due to the character and distribution of initiation centers in the polymer mass such as sensitizing impurities, charge transfer complexes (CTC) and/or participation of active environmental pollutants [3-5]. Generally this does not affect however the structure of the oxidation products having oxidation properties formed in the consecutive steps to initiation [alkylhydroperoxides POOH, peroxyacids PC(O)OOH] and their oxygen-centered free radical precursors [POO•, PC(O)OO•] that play an important role in the fate of polymer additives, stabilizers in particular. Phototriggered reactions such as photolysis and photooxidation play a decisive role in outdoor exposure (weathering) of polymers [1, 4-6]. Degradation of real polymer systems was shown to be a heterogeneous process strongly affected by the sample morphology and geometry. Analysis of the physical chemistry of polymer oxidation and stabilization confirmed [7] differences between oxidation of amorphous and crystalline parts of the material. This results in degradation heterogeneity due to the oxygen impermeability of the crystalline areas. The heterogeneity on molecular and supramolecular level is primarily affected by the random localization of initiation and chromophoric centers in the polymer mass followed by infectious oxidative spreading from the centers [8]. Concentration of activated centers depends on the intensity of UV radiation penetrating through the polymer that depends on polymer transparency. Moreover the oxidation rate and localization of oxidation products are dependent on the availability of oxygen within the system. Consequently rate of oxygen diffusion into the material, rate of oxygen consumption and the possibility to supplement the consumed oxygen by diffusion are decisive factors affecting the depth of oxidation of thick walled materials. Diffusion limited oxidation (DLO) results in differences in oxidative changes between the heavily damaged surface layers and central parts of the materials [5]. The differences are dependent on the thickness of the material and are observed as concentration gradients from the surface to the bulk of the material using step-bystep analyses [5, 9-11]. DLO is a process characteristic of both thermal and photooxidation of thick-walled materials and results mostly in U-shape formed spatial distribution of changes [5, 9, 11].
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Oxygen insufficiency, such as in the central parts of the thick samples of the material accounts for accumulation and subsequently for autoreactions of polymeric alkyl radicals generated according to (1) from the less oriented taut tie molecules located between crystallites. Autoreactions of alkyls, disproportionation and recombination result in chain scission and crosslinking of polyolefins. The ratio between scission and crosslinking depends on the basic structure of polyolefins and has a mostly U-shaped gradient character similarly as concentration gradients of oxygenated products or polymer unsaturation [10, 12, 13]. Macroalkyls remaining in the polymer bulk are not only examples of the heterogeneity of polymer oxidation. Their reactivity with suitable additives accounting for alkyl annihilation may contribute to the polymer stabilization process differing from that based on deactivation of oxygenated structures. For thermal stabilization of carbon-chain polymer, chain-breaking donor antioxidants such as sterically hindered phenols are generally used. They scavenge alkylperoxy radicals generated in the propagation step (2), but are unable to deactivate polymeric alkyls. Chain breaking antioxidants are conventionally used with hydroperoxide deactivating antioxidants, e.g., organic phosphites or sulfides [14]. Both mentioned classes of antioxidants are rather inactive against photooxidation. Compounds acting by other mechanisms are used as photostabilizers. They consist either of additives with preventive function, such as UV absorbers competing with the polymer in preferential absorption of the actinic radiation, preventing thus polymer degradation, or are photoantioxidants or quenchers of excited states having the function of curative stabilizers deactivating products of the photochemistry/photophysics of the polymer matrix and blocking thus their participation in the propagation steps of the chain oxidation [4, 14]. Hindered amine stabilizers (HAS) are the most common class of the curative additives and their application is the state-of-the-art in photoprotection of carbon-chain polymers, polyolefins in particular. HAS shape future polymer development, promote their consumption in new areas and expand material performance by increasing its lifetime. Application of HAS is based on a long-term effective development and is connected with commercial benefits for polymers. An optimized technical application of HAS required explanation of their chemistry and activity mechanisms in different phases of the oxidative degradation of polyolefins [1417]. There is not a full consensus what everything is involved in the HAS mechanism because it is difficult to confirm some of the proposed mechanistic steps in the polymer matrix. Formation of HAS-derived nitroxides (>NO•) (5) is the assumed primary step of the stabilizing activity connected with the sacrificial transformation as shown for a secondary HAS (having active function >NH) [15, 16].
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Identification of nitroxides in the oxidized hydrocarbon substrate doped with HAS is considered as a proof of the photoantioxidant activity of HAS. The latter involves unique mechanistic features in polymer stabilization differing thus from the activity of conventional chain-braking antioxidants. The developed nitroxides are able to scavenge polymeric alkyls formed not only in the surface layers but also in areas of oxygen insufficiency in deeper polymer layers where they may be formed due to presence of residual oxygen. Participation of the polymeric alkyls in oxidative transformation into alkylperoxyls POO• (2) is thus disabled. Resulting O-alkylhydroxylamines >NOP are formed not only from >NH but also from various N-substituted HAS (>NX, X=alkyl, aryl, alkoxyl) and are considered as active reservoirs of HAS efficiency (6). (6) Nitroxide oxidative regeneration (Klemchuk-Gande mechanism) from >NOP based on its interaction with oxygen-centered radical oxidation products of polyolefins (polymeric alkylperoxyls and acylperoxyls) was proposed (7) where symbol CHR1R2 represents “P” in >NOP [18]. According to this scheme no peroxidic by-products with pro-oxidative character are formed within the resulting products.
(7) Formation and location of nitroxides, the key HAS-developed transformation products, was monitored using electron spin resonance imaging (ESRI) technique that confirms the heterogeneous character of photooxidation of polyolefins.
EXPERIMENTAL Materials Polymer plaques containing stabilizers were prepared using additive-free polypropylene (PP) PH6 (Polymer Institute, Brno), Mw = 308 000, Mn = 62 500, of ca. 50 % crystallinity or recently developed amorphous polyolefin poly(ethylene-co-norbornene) Topas® 8007 (TP, TOPAS Advanced Polymers GmbH, Frankfurt am Main, Germany), volume flow index at 260 oC/2.16 kg: 30 ml/10 min, density 1.02 g/cm3, norbornene content ca 17 weight %. HASs Tinuvin®770 and Tinuvin®123, UVAbs Tinuvin®327 as preventive light stabilizer acting by an excited state intramolecular proton transfer (ESIPT) mechanism and benzoate photoantioxidant Tinuvin®120 were used as additives. All photostabilizers were supplied by Ciba Specialty Chemicals, Basel, Switzerland.
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Polymer-stabilizer mixtures were homogenized in the W 50 EH chamber of a Brabender Plasti-Corder laboratory mixer at 190 °C and 60 rpm for 10 min. After removing the material from the chamber, 6 mm thick plaques 8 x 8 cm were molded in a laboratory hot press at 200 °C for 2 min under 50 kN for deairation plus another 2 min under 100 kN followed by water cooling to 70 °C for ca. 15 min under 150 kN.
Accelerated Photooxidation and Oven Test The plaques were exposed to accelerated photooxidation in an Atlas Ci 3000+ Weather-Ometer under the following test conditions: filtered Xenon light wavelength bandpass 295-800 nm (inner and outer filtercombination Type S Boro/Type S Boro), irradiance 0.5 Wm-2nm-1 at 340 nm, black panel temperature (bpt) 60 °C (used PP and TP were non-pigmented, accordingly the surface sample temperature was lower), dry bulb temperature (dbt) 30 °C, RH 20%. Oven tests were performed in a heating box at temperature 60 °C. Cylindrical samples (diameter 3 mm, length 6 mm) were bored out from the plaques after appropriate time periods of exposure. Between periods of exposure in Weather-Ometer or in oven the plaques were stored in the dark at room temperature. Tests proved a negligible change of concentration profiles in the samples bored out from the plaques during the storage.
Electron Spin Resonance Imaging ESRI experiments were performed with a commercial Bruker ELEXSYS E540 X-band spectrometer equipped with a pair of eight-shaped Lewis gradient coils delivered by George Associates (Berkeley, USA) that are able to produce vertical gradient perpendicular to the external magnetic field Gmax~320 G/cm. Concentration profiles of nitroxides in the samples along the axes of the cylinders coinciding with the direction of irradiation incident on the plaque in the Weather-Ometer were calculated using the 1D ESRI and deconvolution procedure described previously [19]. Matlab software tool was used for the data manipulation and programs targeting the deconvolution procedure in 1D experiment and filtered back projection procedure in 2D experiment based on the Matlab scripting language were developed. Spatial dependence of ESR spectral line shapes was excluded by checking spatial-
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spectral images calculated using back projection algorithm from 2D ESRI data. Experimental scheme is given in Fig. 1. In order to obtain intensity of images in absolute units, usual spectrometer calibration for a given sample setup with the standard of known concentration was performed. In the 1D experiment we calibrated each sample spectrum (taken with gradient coils off) used for calculation of the particular one-dimensional profile using the spectrum of the standard taken under similar conditions. As the second integrals of the EPR spectra are directly proportional to the number of spins present in the measured sample, we were able to assess the total concentration of spins in the given sample and ascribe this number to the first integral of the resulting 1D profile. Anticipating axial symmetry of nitroxide distribution in the cylindrical sample we obtained the true concentration profile in number of spins per volume unit in particular depth of the sample after normalization of the calibrated 1D profile to the sample cross-section.
Figure 1. Experimental arrangement.
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Nitroxide Formation and Location in Oxidized Polypropylene In spite of some uncertainties in the individual steps of the HAS mechanism in polymer stabilization due to the specific effects of the polymer matrix and the environmental stress, the HAS-based nitroxides are considered the key intermediate in the HAS reactivity mechanism. Detection and quantification of the formed nitroxides using ESRI spectroscopic technique has been exploited for confirmation of the primary transformation step in HAS mechanism [15, 16, 20], as a consequence of interactions of HAS with oxygenated radical and molecular products of polyolefins (5). Monitoring of the nitroxide development enables tracing of the oxidation process within the polymer matrix. Consequently it is also a tool for marking the heterogeneity of the oxidative transformation of semicrystalline carbon chain polymers [polypropylene (PP), polyethylenes (PE)] or amorphous polymers [copolymers of ethylene with norbornene, polystyrene (PS), high impart polystyrene (HIPS), acrylonitrilebutadiene-styrene polymer (ABS)]. Most of the experimental studies dealing with tracing of nitroxide formation were performed using thin sectioned polymers, films of PP in particular [20, 21]. The determined nitroxide level is generally low [15]. Time-dependent ESR analysis of photooxidized and HAS-doped PP films revealed an increase in the amount of formed nitroxides from the beginning of the exposure [20, 21]. After reaching a maximum, a gradual decrease approaching slowly the steady state concentration characterizes the in-polymer behavior of nitroxides in consecutive steps, such as scavenging of polymeric alkyl radicals P•, formation of >NOP (5) and nitroxide regeneration (7). The process is affected by the structure of the used HAS and by the external stress [4, 20]. Differences between accelerated and natural aging in PE doped with oligomeric HAS [22] or differences between thermal and photooxidation in ABS [23] were observed. Increase of the concentration of the HAS originally added to the polymer results in an increase of the concentration maximum of the nitroxide in the initial phase of the process [22] and prolongs the period of the effective material protection. The trend in the change of the nitroxide concentration indicating an approach to the equilibrium concentration is analogous at various in practice used concentrations between 0.5 and 3.0 % and confirms generally the overall cyclic HAS stabilization mechanism. Development trends in the nitroxide level analogous to that in plastics were found in HAS doped automotive clearcoats [24]. Determination of the heterogeneity of oxidative damages increases the insight into the material properties of aged polymers because the damaged regions influence seriously the overall properties of the material. For example the heavily degraded polymer surface layer deteriorates seriously tensile fracture properties of the whole material or infects components of polymer blends containing recyclates. Monitoring of nitroxides in thick materials (plates several mm thick) after oxidative stress is a sensitive method to localize the degradation within the material depth. Using HAS of various structures in different polymer materials provides moreover important information about specific mechanistic features of the process. In this paper we concentrate on secondary HAS Tinuvin® 770, O-alkylhydroxylamine-type HAS Tinuvin® 123, a combination of Tinuvin® 770 with UV absorber Tinuvin® 327 and photoantioxidant Tinuvin® 120 in semicrystalline additive-free PP. For matter of comparison, experiments with an amorphous polyethylene, poly(ethylene-co-norbornene) copolymer and a HAS-developed nitroxide additive were performed.
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Several studies of the spatial resolution of oxidation processes in HAS stabilized polymer appeared since the pioneering paper [25] and deal with events in PP [26 - 28] or commercial poly(acrylonitrile-co-butadiene-co-styrene) (ABS) copolymer plaques with thickness from 2 to 6 mm [27, 29]. We preferred for our experiments plaques of 6 mm thickness providing more detailed information on the spatial distribution of nitroxides in both thermal and photooxidative stress situations [28]. Results of experiments performed with oxidized PP doped with secondary HAS Tinuvin® 770 show high concentration of nitroxides in the vicinity of both surfaces due to the DLO. This was observed after continuous exposure to radiation in the Weather-Ometer on the irradiated (front) and non-irradiated (back) surfaces as well as on the both surfaces of the samples exposed thermally in hot air oven. Very low concentration of nitroxides was present inside of the samples. The concentration profiles are of characteristic U-shape and indicate preferential surface oxidation of PP, with a specific response to thermal and photochemical stress (Fig. 2). The assumed complex HAS mechanism is thus more explicitly confirmed in thick samples than by monitoring nitroxide concentration in PP films. It is consistent with surface consumption of oxygen in thick plaques and lower availability of oxygenated products in the depth of the PP matrix necessary for a direct development of nitroxides from HAS as well as for nitroxide regeneration from O-alkylhydroxylamine >NOP within the regenerative cycle (7). The comparison of the content of nitroxides generated in PP plaques containing 1 % of Tinuvin® 770 as a sole additive measured after 73 days exposure indicates faster degradation (monitored by nitroxide formation) during the phototriggered process (Fig. 2a) in comparison with oven aging (Fig. 2b). Generation of nitroxides in the oven aged samples confirms at the same time a heat stabilizing contribution of HAS in PP at used conditions (60 °C).
Figure 2. Dependence of nitroxide concentration profiles measured in PP plaques stabilized by 1 % of Tinuvin®770 as a sole additive and exposed to accelerated photodegradation (a) or oven test (b) on the net exposure time. The arrow indicates direction of the incident light.
The nitroxide level in samples containing from 0.5 to 2.0 % of Tinuvin® 770 increased with increasing HAS concentration. Approximately symmetrical U-shape of the nitroxide concentration profile shown in Fig. 2a was confirmed by comparison of shapes of the nitroxide concentration profiles from the front and back surfaces of PP plaques oxidized for overall net exposure 87 days in the presence of different amounts of HAS (Fig. 3). A
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significant decrease in the nitroxide concentration with exposure time in samples doped with the lowest concentration (0.5 %) of HAS was observed indicating consumption of considerable portion of the stabilizer during the degradation process.
Figure 3. Dependence of concentration of nitroxides generated in the irradiated (full symbols and solid lines) and unexposed (empty symbols and dashed lines) surface layers of the PP plaques stabilized by 0.5 % (!, ∀), 1 % (Β, Χ), and 2 % (,, −) of Tinuvin®770 as a sole additive and exposed to accelerated photodegradation on the net exposure time.
A simplified model explains the observed shapes of nitroxide profiles in the thick polymer plates exposed to the environmental oxidative stress characteristic of DLO and radiation penetration. Oxygen is able to diffuse from both sides of the sample into the polymer mass and is consumed into radical and molecular oxygenated species. The latter are concentrated predominantly in the near surface areas as confirmed experimentally by formation of surface-centered concentration profiles of carbonyl species, accounting for transformations of the primary peroxidic species [2, 5, 12]. Within these areas the secondary HAS Tinuvin® 770 (>NH) is transformed into nitroxide (>NO•). Formation of Oalkylhydroxylamine (>NOP) after trapping polymeric alkyl radicals and regeneration within the cyclic mechanism (7) [18] follow in the next steps and result in equilibrium nitroxide concentration. The maximum equilibrium nitroxide concentration found in irradiated surface layer of the PP plaque stabilized by 1 % of Tinuvin® 770 equals approximately to 1 % of concentration of the stabilizer added. Intensity of the UV radiation penetrating through the
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polymer and participating in formation of the polymeric alkyl radical depends on polymer transparency. Due to the high transparency of PP there is sufficient intensity of light to trigger oxidation also in the (rich in oxygen) back surface layer of the sample where the concentration of nitroxides similar as in the irradiated surface layer is generated. Due to the preferential surface oxygen consumption, the central part of the sample suffers from oxygen deficiency. The HAS developed >NOP, if present, has a limited possibility for nitroxide regeneration. The U-shape nitroxide concentration profile arises consequently in the PP plaque (Fig. 2a). We found in an earlier study [28] and confirmed in new experiments that the shapes and time-dependent change of the nitroxide concentration profiles inside polymer plaques subjected to accelerated aging as measured by ESRI depend on the polymer matrix (comparison between PP and polystyrene) and on the responses of the HAS structure to oxidizing counterparts of the matrix. Nitroxide concentration profiles measured in TP plaque stabilized with 1.0 % of Tinuvin® 770 oxidized for overall net exposure 147 days shows slower increase of nitroxide concentration and lower concentration of nitroxides in the back surface layer when compared with PP plaque. Both observations are similar to the results obtained in polystyrene [28], but even more pronounced (Fig. 4). It indicates the lower transparency of TP in comparison with PP and even with PS which is confirmed by optical measurements.
Figure 4. Dependence of nitroxide concentration profiles measured in TP plaques stabilized by 1 % of Tinuvin®770 as a sole additive and exposed to accelerated photodegradation on the net exposure time. The arrow indicates direction of the incident light.
Generation of nitroxides in the PP matrix is dependent on the intensity of the incident UV radiation. By using effective UV absorbers (light stabilizers with preventive mechanism) in combination with HAS [4] the efficiency of the oxidative phototriggered process decreases with depth in the sample due to the light filtration effect. We studied two light stabilizers with
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different activity mechanisms (Fig. 5), the benzotriazole-based UV absorber Tinuvin® 327 and the phenolic photoantioxidant Tinuvin® 120 for overall net exposure 73 days. Decrease of efficiency of the oxidative phototriggered process in the back sides of the exposed plaques is markedly shown by the changed shape of the nitroxide concentration profile. The gradually reduced penetration of the radiation through the sample results in reduced oxidation of the non-irradiated side of the sample shown by formation of lower concentration of nitroxides in the back side surface layer. The photostabilizing effect depends on the structure of the light stabilizer, i.e., on its activity mechanism and efficiency. The benzotriazole-based UV absorber Tinuvin® 327 is comparatively more effective (Fig. 5a) than the phenolic photoantioxidant Tinuvin® 120 (Fig. 5b) in decreasing nitroxide concentration in the irradiated surface layer.
Figure 5. Dependence of nitroxide concentration profiles measured in the PP plaques stabilized by (a) 1 % of Tinuvin®770 + 0.5 % of Tinuvin®327 and (b) 1 % of Tinuvin®770 + 0.5 % of Tinuvin®120 and exposed to accelerated photodegradation on the net exposure time. The arrow indicates direction of the incident light.
Participation of the protective mechanism affected by the modified structure of HAS was evidenced by comparison of nitroxides developed in PP plaques doped with secondary HAS Tinuvin® 770 (Fig. 2) and NOR-HAS Tinuvin® 123 (Fig. 6). The latter was developed for polymer photostabilization in acid environment [15, 16]. Very high concentration of nitroxides was found in thermally pressed Tinuvin® 123 containing plaques prepared for oxidation experiments. Nitroxides were distributed almost homogeneously within the mass of the plaque. The high nitroxide concentration remained in thermally oxidized PP (Fig. 6b). A fully different progress in the development of the nitroxide distribution within the sample was observed in the phototriggered process (Fig. 6a, 7). The initial concentration decreased with increasing exposure time. At the same time the shape of the nitroxide profile changed from the nearly homogeneous distribution to the asymmetric U-shaped profile characterized by a lower amount of nitroxides in the irradiated front surface in comparison with the back surface and a low amount of nitroxides in the central part of the sample.
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Figure 6. Dependence of nitroxide concentration profiles measured in the PP plaques stabilized by 1 % of Tinuvin®123 as a sole additive and exposed to accelerated photodegradation (a) or oven test (b) on the net exposure time. The arrow indicates direction of the incident light.
Figure 7. Dependence of the amount of nitroxides generated in normalized volume of the cylinder (1 mm2 cross-section) bored out from the PP plaques stabilized by: 1 % of Tinuvin®770 (!,∀), 1 % of Tinuvin®770 + 0.5 % of Tinuvin®327 (,,−), 1 % of Tinuvin®123 as a sole additive (Β,Χ), and exposed to accelerated photodegradation (full symbols and solid lines) or to the oven test (empty symbols and dashed lines) on the net exposure time.
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Development of the high nitroxide level generated from Tinuvin® 123 in the sample from the beginning of the experiment is explained by cooperation of thermochemical and photochemical degradation of NOR-HAS. The thermolysis participates even in the phase of the sample preparation and accounts for formation of nitroxides from the NOR-HAS according to (8) [15, 30]. Δ
>NOR ⎯ ⎯→ >NO• + olefin
(8)
Differences in the shape of nitroxide concentration profiles during photo and thermal degradation of the sample containing Tinuvin® 123 are most probably due to presence of polymer alkyl radicals generated during photodegradation proces in the whole volume of the PP sample. Nitroxide concentration in the central part of the sample subjected to photodegradation is reduced by the reaction of the nitroxides with polymer alkyl radicals (6) and cannot be renewed by reaction (5) due to the oxygen deficiency. Negligible concentration of polymer alkyl radicals are generated during thermodegradation process when practically no change of the nitroxide concentration profiles is observed.
CONCLUSION In PP plaques, 6 mm thick, stabilized with secondary HAS Tinuvin® 770 as the sole additive, with the combination of Tinuvin® 770 with UV absorber Tinuvin® 327 or with photoantioxidant Tinuvin® 120, U-shaped concentration profiles of nitroxides were determined during exposure to accelerated photooxidation in a Weather-Ometer already after the first day of irradiation. This type of the nitroxide distribution results from the DLO and penetration of the incident radiation that initiates degradation of PP through the plaques and reaches the unexposed side of the plaque with sufficient intensity. Due to the DLO, radical and molecular oxidation products are formed from the alkyls predominantly near to the front and back surfaces of the PP plaques indicating the depth of oxygen diffusion. These oxidation products participate in transformation of the added HAS marking thus clearly the heterogeneous character of oxidation. Oxygen insufficiency in the central part of the plaques (accounting not only for limited oxygen diffusion but also for the effective and preferential oxygen consumption in the surface layers) allows polymeric alkyls to survive. Consequently, oxidation of the originally added HAS or oxidative regeneration of its transformation product >NOP into nitroxide are practically hindered. Moreover, small amounts of nitroxides are effectively scavenged by the alkyls. Low concentration of nitroxides confirms unequivocally limited degradation in the central part of the plaques. Application of a combination of HAS with UV absorber indicates efficient cooperation between the two types of light stabilizers acting by different stabilization mechanisms as well as the heterogeneity of the degradation process. The latter is affected in the presence of UV absorbers by the lower radiation intensity of the UV light penetrating into the back (not directly irradiated) side of the plaque in comparison with the intensity of the incident light. A lower level of nitroxides than in photooxidized PP plaques characterized by the concentration profile confirming slightly heterogeneous oxidation was detected in plaques
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doped with Tinuvin® 770 and thermally treated in oven. This is a proof of the long term heating stabilization influence of HAS at experimental conditions. Monitoring concentration of nitroxides in PP stabilized with the NOR-HAS Tinuvin® 123 reveals their high concentration already from the beginning of the oxidative stress. This is due to the nitroxides generated during thermal pressing of the plaques. Concentration of nitroxides decreased during the photooxidative stress. Scavenging of nitroxides by polymeric alkyls generated by the phototriggered degradation is assumed. Performed experiments proved the heterogeneous character of oxidation of the PP plaques containing HAS by profiling nitroxide formation using ESRI method.
ACKNOWLEDGMENT The financial support from the Grant Agency of the Academy of Sciences of the Czech Republic (project IAA400500804) and gifts from Ciba Specialty Chemicals (stabilizers) and from TOPAS (Topas®) are acknowledged. We thank Dr. Jiří Pfleger for characterization transparency of the plaques by diffusion transmission measurements and Dana Michálková, MS (both IMC Prague), for preparation of the plaques and Weather-Ometer operation.
REFERENCES [1] [2] [3] [4] [5]
[6]
[7]
[8]
[9]
Rabek, J. F.. Polymer Photodegradation: Mechanisms and Experimental Methods. Chapman & Hall, London, England, 1995. Gillen, K. T.; Wise J.; Clough R. L. Polym Degrad Stab. 1995, 47 (1), 149-161. Gijsman P.; Meijers G.; Vitarelli G. Polym Degrad Stab. 1999, 65 (1), 433-441. Pospíšil J.; Nešpůrek S. Prog Polym Sci. 2000, 25 (9), 1261-1335. Pospíšil J.; Nešpůrek S.; Pilař J. Effect of environmental stress and polymer microenvironment on efficiency trials and fate of stabilizers. In Service Life Prediction of Polymeric Materials; Martin, J. W.; Ryntz R. A.; Chin J.; Dickie R. A.; Eds.; Springer, New York, USA, 2009, pp 493-520. Pospíšil J.; Nešpůrek S.; Kruliš Z.; Pilař J. Effect of atmospheric impurities on polymer weathering mechanism and longevity. In Natural and Artificial Ageing of Polymers; Reichert, T.; Ed.; Gesellschaft fűr Umweltsimulation: Pfinztal, Grmany, 2007; pp 125139. Billingham, N. C. The physical chemistry of polymer oxidation and stabilization. In Atmospheric Oxidation and Antioxidants; Vol. IV; Scott, G.; Ed.; Elsevier: Amsterdam, The Netherlands, 1993; pp 219-277. George, G. A.; Celina, M. Homogeneous and heterogeneous oxidation of polypropylene. In Handbook of Polymer Degradation, 2nd Edition; Halim Hamid, S.; Ed.; Dekker, M.: New York, USA, 2000; pp 277-314. Gillen K. T.; Clough, R. L. Techniques for monitoring heterogeneous oxidation of polymers. In Handbook of Polymer Science and Technology, Vol. 2, Performance Properties of Plastics and Elastomers; Cheremisinoff, N. P.; Ed.; Dekker M.: New York, USA, 1989; pp 167-202.
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[10] White, J. R. Plast Rubber Compos Process Appl. 1998, 27 (3), 124-131. [11] Pospíšil, J.; Pilař, J.; Billingham, N. C.; Horák, Z.; Nešpůrek, S. Polym Degrad Stab. 2006, 91 (3), 417-422. [12] White, J. R.; Shyichuk, A. V. Polym Degrad Stab. 2007, 92 (11), 2095-2101. [13] Shyichuk A. V.; White, J. R. Scission and crosslinking rates during photooxidation of thick-sectioned polyolefins: depth profiles for different exposures. In Natural and Artificial Ageing of Polymers; Reichert, T.; Ed.; Gesellschaft fűr Umweltsimulation: Pfinztal, Germany, 2007; pp 175-184. [14] Pospíšil, J.; Nešpůrek, S. Highlights in the inherent chemical activity of polymer stabilizers. In Handbook of Polymer Degradation, 2nd Edition; Halim Hamid, S.; Ed.; Dekker, M.: New York, USA, 2000; pp 191-276. [15] Pospíšil, J. Adv Polym Sci. 1995, 124, 87- 189. [16] Pospíšil; J.; Pilař, J.; Nešpůrek, S. J Vinyl Addit Technol. 2007, 13 (3), 119-132. [17] Pfaendner, R. C R Chimie. 2006, 9 (11-12), 1338-1344. [18] Klemchuk P.; Gande, M. E.; Cordola, E. Polym Degrad Stab. 1990, 27 (1), 65-74. [19] Marek, A.; Labský, J.; Koňák, Č.; Pilař, J.; Schlick, S. Macromolecules 2002, 35 (14), 5517-5528. [20] Wiles, D. M.; Jensen, J. P.; Carlsson, D. J. Pure Appl Chem. 1983. 55 (10), 1651-1659. [21] Gugumus, F. 3rd International Conference on Polymer Photochemistry, Sestri Levante, Italy, September 5-10,1993. [22] Scoponi, N.; Simmino C.; Kaci, M. Polymer 2000, 41 (22), 7969-7980. [23] Motyakin, M. V.; Schlick, S. Macromolecules 2001, 34 (9), 2854-2864. [24] Gerlock, J. L.; Kucherov, A. V.; Smith, C. A. Polym Degrad Stab. 2001, 74 (2), 201210. [25] Lucarini, M.; Pedulli, G. F.; Borzatta, V.; Lelli N. Polym Degrad Stab. 1996, 53 (1), 917. [26] Franchi, P.; Lucarini, M.; Pedulli, G. F.; Borzatta, M.; Vitali M. Macromol Chem Phys. 2001, 202 (7), 1246-1256. [27] Lucarini, M.; Pedulli, G. F.; Motyakin, M. V.; Schlick S. Prog Polym Sci. 2003, 28 (2), 331-340. [28] Marek, A.; Kaprálková, L.; Schmidt, P.; Pfleger, J.; Humlíček, J.; Pospíšil, J.; Pilař, J. Polym Degrad Stab. 2006, 91 (3). 444-458. [29] Motyakin, M. V.; Gerlock, J. L.; Schlick, S. Macromolecules. 1999, 33 (16), 54635467. [30] Berger, H.; Bolsman, T. A. B. M.; Brouwer, D. M. Catalytic inhibition of hydrocarbon autoxidation by secondary amines and nitroxides. In Development in Polymer Stabilization – 6; Scott, G.;Ed.; Applied Science Publishers: London, England, 1983; pp 1-78.
Reviewed by Dr. Jacques Sampers, DSM, Geleen, The Nederlands.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 12
A REVIEW OF THE HAZARDOUS EFFECTS OF POLYCYCLIC AROMATIC HYDROCARBONS ON LIVING ORGANISMS Maria Aparecida Marin-Morales1,2∗; Daniela Morais Leme1,2,3; Dânia Elisa Christofoletti Mazzeo1,2 1
Department of Biology, Institute of Biosciences, São Paulo State University, Rio Claro, SP, Brazil 2 Researcher of the Programa de Formação de Recursos Humanos em Geologia e Ciências Ambientais do Setor de Petróleo, Gás and Biocombustíveis – PRH05 (UNESP/MCT/FINEP/ANP) 3 Researcher of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are one of the most important, abundant and dangerous class of persistent organic contaminants found in air, water, food and waste sites. The great concern with this class of pollutants is due to the fact that these chemicals cause various deleterious effects, which could jeopardize the genome of exposed organisms or even lead them to death. Many studies have been conducted in several laboratories distributed around the world and with different organisms to quantify the impact of those pollutants and to diagnose their action mechanisms. Thus, there is a real necessity to compile the data already described in the literature, obtained from tests in situ, in vivo and in vitro, derived from simulation experiments, occupational exposure or environmental disasters, in order to produce a report with the main effects already described for that class of pollutant. This review article intends to address the following issues: toxicity, cytotoxicity, genotoxicity, mutagenicity, teratogenicity and carcinogenicity of PAHs, besides information about estrogenicity and metabolism of these substances. In this context, it will be also presented the ∗ Tel: +55 19 3526 4143; Fax: +55 19 3534 0009. E-mail address:
[email protected] (M.A. Marin-Morales).
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test systems that betteresponded to the assessments of the PAHs effects, in an attempt to show the most sensitive and efficient organisms to detect the contamination of these agents.
1. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are characterized by a diversified group of organic compounds constituted of two or more condensed rings. These substances present a wide distribution in the environment, having their levels already determined in soils, drinking water, rivers, lakes, marine sediments and also in air analyses (Harvey, 1997). PAHs are originated from several sources, being possible find them, mainly, in cigarette smoke, incomplete combustion of organic matter during industrial activities and other human activities such oil refining, fossil fuel burning resulted from vehicle traffic and coal production (Stanley, 1992; Thirman et al., 1994; Mitchell et al., 1999; Annas et al., 2000; Bihari et al., 2006). Composition and complexity of PAHs mixtures depends on the emission sources, which makes available a great variety of these substances, in different concentration levels (Pereira Netto et al., 2000). PAHs present low solubility in water, due to its lipophilic nature and, in consequence of this characteristic, concentrations of these chemicals in sediments can be much higher than from those found in water. However, the low bioavailability of the PAHs present in sediments, reduces, significantly, the toxic potential in this environment. In the air, PAHs tend to react with the particulate matter, which can be the contamination vehicle of other sources such as soil and food, by deposition of these particles in crops, fruits and vegetables (Albers, 2002; WHO, 2000). Due to its wide distribution in the environment, great part of the living beings is exposed to these pollutants, having some of the adverse effects already detected. Detection of PAHs hazardousness to the exposed organisms have characterized these chemicals as one of the most dangerous classes of environmental pollutants (Aina et al., 2006), being also considered as one of the most toxic among all the hydrocarbons (Catoggio, 1991).
2. EFFECTS ON THE LIVING BEINGS Toxic environmental pollutants can cause several alterations in the normal physiologic process of the living beings. To a pollutant exercise its deleterious effects in one exposed organism it must first enter in the individual to after reach its target site (Landis and Yu, 1998). Thus, assessment of the damages caused in the organisms must consider, besides the substance physico-chemical properties, the magnitude, duration and frequency of the exposition, introduction via and the susceptibility of the biological systems, intra and interspecies (Stanley, 1992, 1994; Landis and Yu, 1998; Pedrozo et al., 2002). PAHs are characterized by their high lipophilic affinity, which allows their fast absorption by the biological membranes and, consequently, their accumulation in the tissues (Tuvikene, 1995). Biological membranes are characterized as barriers between the cell and the environment, regulating the passage of toxic agents into the cellular interior and exterior (Stanley, 1992, 1994). Independently from the introduction via used, the chemical compound
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must cross several cellular membranes, until reaching their target site and produce its harmful effect (Pedrozo et al., 2002). Comprehension of the distribution and the storage places of these chemicals inside the organism is another important issue. According to Landis and Yu (1998), organs such as liver, lungs, kidney, bone and adipose tissues are considered as storage deposits. Nevertheless, the relation between the target site of the substance is not always true since the agent can be stored in one determined tissue/organ, but act in another place. In case of the PAHs, due to its lipophilic nature these chemicals are easily stored in adipose tissues (Ballantyne and Sullivan, 1992). Living beings present certain mechanisms that help in the elimination of the toxic substances introduced in their organic system such as the biotransformation process (Guecheva and Henriques, 2003). However, for some pollutants classes such as PAHs, biotransformation can result in a more drastic effect since the reactions conjunct involved in this process lead to a formation of metabolites highly reactive, which become responsible for the toxic effect of the original substances.
2.1. Biotransformation: PAHs Bioactivation Most living beings have the capacity to metabolize xenobiotics by the process denominated biotransformation (Tuvikene, 1995). Biotransformation is characterized as a conjunct of enzymatic reactions, responsible for the conversion of the liposoluble substances in hydrosoluble facilitating, thus, their excretion process. However, although the purpose of the xenobiotics biotransformation is detoxification not always the originated metabolite is less toxic than the own chemical. Thus, xenobiotics biotransformation can increase the toxicity of the chemical products by the formation of electrophilic metabolites, extremely reactive, which can present potential to bind, covalently, with macromolecules inside the cells with DNA, RNA and proteins, which can result in several alterations such as disturbance in the immune system, mutations and even the organism death (Stanley, 1992, 1994; Landis and Yu, 1998; Guecheva and Henriques, 2003). PAHs constitute a group of chemical substances that characterize, perfectly, the situation exposed above. These agents per si not only confer toxic effects to the living beings, but, after the oxidative metabolization process, they become bioactivated, exerting then their deleterious effects (Tuvikene, 1995; Luczyński et al., 2005). The main PAHs metabolization via involves the cytochrome P-450 (CYP) system, which is constituted of mixed function oxidases (MFO). These enzymes are bound to the membrane and present a wide distribution in the organism, with elevated concentration in the endoplasmic reticulum of the liver, kidney, lungs, nasal vias and intestine. Additionally, CYP enzymes are characterized by an ancient proteic family, possibly the bigger ever found in nature, with a wide distribution among bacteria, fungi, plants and animals. It is known, nowadays, that there are about 215 different families of the CYP enzymes being 72 in bacteria, 29 in lower eukaryote, 50 in plants and 67 in higher animals (Guecheva and Henriques, 2003). According to Stegeman and Hahn (1994) and Luczyński et al. (2005), isoenzymes CYP1A1 and CYP1A2 of the subfamily CYP1A are the main responsible for the biotransformation process of the PAHs.
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Biotransformation process, in general occurs in two phases: phase I and phase II reactions. In the phase I, one reactive polar group such as OH, NH2, COOH or SH, is introduced in the molecule of the chemical agent by oxidation, reduction or hydrolysis reactions, generating metabolites highly reactive (bioactivation). In phase II, occurs the detoxification process, where the metabolites generated in phase I are conjugated with endogenous substrates like glycine, cisteine, glutathione, glicuronic acid and sulphates by transferases enzymes, making them hydrophilic and passible of being excreted (Stanley, 1992, 1994; Tuvikene, 1995; Landis and Yu, 1998; Pereira Netto et al., 2000; Pedrozo et al., 2002) (Figure 1).
Figure 1. Representative diagram of the xenobiotic biotransformation.
Monooxigenases of the CYP1A family are the responsible for the PAHs enzymatic oxidation. Firstly, the PAHs are activated in arene oxides, in specific regions by the monooxigenases CYP action. Arene oxides can be both spontaneously isomerized in phenols or converted, enzymatically, to dihydrodiols by the epoxide hydrolases action. Dihydrodiols can suffer new oxidation forming metabolites highly reactive. Thus, this last stage plays an important role in the formation of the carcinogens, which can react together with other metabolites, with DNA, and initiate the malign transformation process of the cell (Jacob, 1996). Among the PAHs known until now, benzo[a]pyrene is, undoubtedly, the most studied, having its metabolic via already reported. In vitro experiments with B[a]P have indicated that dihydrodiols is capable of binding to DNA characterizing more than75% of the metabolites formed by the CYP via (Stegman and Klopper-Sams, 1987; Jimenez and Stegeman, 1990). These experiments have still shown that an increase in the monooxygenases activity, due to exposition to environmental pollutants, can rise the quantity of reactive potentially chemicals that, consequently, can react with DNA, originating the DNA adducts. Identification of the formed metabolites from the PAHs exposition in different groups of living beings is an important issue, since it allows a correlation between the test-system and extrapolation of the obtained results to other organisms. According to Ahokas (1979) and Ronis et al. (1992) microsomes of the trout (Salmo trutta lacustris), starry flounder (Platychthys stellatus) and coho salmon (Oncorhynchus kisutch) liver, metabolize B[a]P in metabolites similar to those produced by rat liver microsome. The main metabolites produced
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by fish microsome are 7,8 and 9,10-dihydrodiols, which are precursor of the main B[a]P carcinogenic metabolites. Many are the metabolites originated by the PAHs biotransformation. As already exposed, some of these metabolites can be excreted by the organism or even not be reactive to the organisms not conferring, therefore, hazard to the exposed organism. However, many of them, when transformed can cause several alterations in the biological system and put in danger individuals, populations or even entire ecosystems exposed to their action.
2.2. Toxicity Toxicity tests are reliable techniques to predict the impacts of contaminant agents over several communities of various environments. With these tests it is possible to determine in which fractions each substance can be available to exercise toxic effects on the organisms. Chemical compounds of anthropogenic origin suffer several interactions and can act in many ways on the exposed communities. Toxicity tests can be developed in laboratories (in vivo or in vitro) or in field (in situ) and use several methodologies since the more basic such as the dose answer curve, observation of behavior, mental, physiologic and lethal changes, even more sophisticated methods such as biochemical, cytological, genetic and teratogenic alterations. The observed effects also vary according to the characteristic of the used methodology, the biology of the specie and the type of the used compound. The PAHs most common action mechanism that result in toxicity for the organisms is related with the interference in the cellular membranes function by the bond in the membrane hydrophobic sites, causing deformity and molecular perturbance, besides alterations in the enzymatic system (Tuvikene, 1995). As a consequence of these bonds, the membrane permeability is altered and the substances transport in and out the cells is damaged. Moreover, as many enzymatic systems are associated with structure of the membrane, other essential process for the cells can be damaged and the normal energy balance becomes debilitated (Knutzen, 1995). Although the PAHs can result in a direct toxic effect, the major concern resides in the formation of reactive metabolites such as epoxides and dihydrodiols, which can bind to the genetic material and to the cellular proteins (Albers, 2002). PAHs with high molecular weight do not result in high toxicity for the organisms, but the effect can be potentialized by exposition to the ultraviolet solar radiation (Arfsten et al., 1996), which leads to the formation of new photoproducts with toxicological properties different from the original molecule (Huang et al., 1993; Krylov et al., 1997). The absorption of light energy lead the PAHs molecules to an excited state, whose energy can be transferred to biological molecules, generating with this, the reactive oxygen species (ROS), intermediary reactive, free radicals or photo-metabolite that can damage these cellular constituents, resulting in toxicity (Betowski et al., 2002; Yu, 2002). Chemicals potentially dangerous such as the PAHs and their metabolites, endanger not only human health, but also aquatic and terrestrial ecosystems by affecting, directly, the soil biota or, after lixiviation and percolation, the aquatic biota (Bispo et al., 1999). PAHs affect the organisms in different ways, since some species are more sensitive to these contaminants than others. Thus, for a better comprehension of the PAHs toxic potentiality it must be considered their effects over several species, in different trophic levels,
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and in all life stages. Besides this, an important factor to be considered ís the investigation of the movement of these molecules in the environment and their action mode on the organisms. The PAHs toxic effect and action mode on plants are not very known and seem to vary according to the biologic specie and the PAH own chemical constitution (Wittig et al., 2003; Baek et al., 2004). Smith et al. (2006) studied the toxic effects of soils contaminated with PAHs on the germination and growth of four graminaceous species and on three leguminous species. No significative effect was observed for the germination index of the studied species during the ten days of exposition, however, the subsequent growth was significantly reduced being this effect more pronounced in the leguminous. The toxicity of soils contaminated with PAHs was evaluated by laboratory assays (in vivo test), using four different plant species (Zea mays L., Hordeum vulgare L., Sinapis alba L. and Helianthus annus L.). The authors considered that the root length was the parameter that best answered to the toxicity tests, being barley (Hordeum vulgare L.) the most sensitive specie among all the species tested, which indicates that this specie can be characterized as an efficient test-organism for PAHs toxicity assay (Smreczak and Maliszewska-Kordybach, 2003). Studies carried out with Brassica napus (canola), to assess the toxic and phototoxic effects of 3 PAHs, have shown that these compounds present direct toxic action on the roots growth, not being transported to the other organs of the plant (Ren et al., 1996). The authors have also concluded that, between the tested PAHs, anthracene was the most toxic, followed by B[a]P and fluoranthene. This same toxicity order was maintained when these hydrocarbons were exposed to light. In order to detect the toxicity of sediment samples of the Rovinj marine coast, in Croatia, contaminated with PAHs, Bihari et al. (2006) used the Microtox® test with the marine bacteria Vibrio fisheri. All the tested sediments were potentially toxic, nevertheless the PAHs were not considered the most toxic chemicals present. The toxicity of lixiviate extracts of soils contaminated with PAHs extracted by water and methanol was evaluated by assays with the Vibrio fischeri bacteria (Microtox® test), with the alga Pseudokirchneriella subcapitata and with micro crustaceans Daphnia magna and Thamnocephalus platyurus. The toxicity of the lixiviated soils with water was much lower than the extracts lixiviated with methanol, mainly, for the tests carried out with bacteria and alga, which were considered by the authors as the organisms more suitable for the detection of the toxicity of soils contaminated by PAHs (Bispo et al., 1999). Erstfeld and Snow-Aihbrook (1999) assessed the chronic effect of soil contaminated with low PAHs levels on the soil microfauna (represented by the nematode), on the mesofauna, (by the microarthropod of the Collembola and Acarina orders) and on macrofauna (by the earthworm Eisenia fetida andrei). The observed PAHs contamination levels (5.28 to 80.46 mg/Kg) seem to have a stimulatory effect on the growth of the invertebrates population present in the soil. According to the authors, this answer can be due to the increase in the microbial population that is capable of metabolizing the PAHs, breaking into smaller molecules that can be easily assimilated by the soil biota. Saint-Denis et al. (1999) studied the effect of different concentrations and different exposition times to PAHs, on the earthworm Eisenia fetida andrei. According to the authors, this chemical was metabolized, both by the CYP via, and by the free radicals oxidation via,
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promoting the generation of free radicals that can stimulate the lipids peroxidation, resulting in toxicity for this organism. PAHs tend to accumulate in the bottom sediments, which can become a constant source of toxic contaminants putting in danger, continuously, the biota health and viability, mainly, of the benthic organisms that suffer constant expositions to PAHs by the direct contact with the soil (Bihari et al., 2006; Boscolo et al., 2007). Due to the fast accumulation and to the small metabolization capacity the bivalve mollusks have been used, frequently, as sentinel organisms for the PAHs retention (Baussant et al., 2001). According to Boscolo et al. (2007), the benthic mollusk specie Tapes philippinarum is a good indicator of the PAH contamination, because it concentrates and tolerates high levels of these chemicals in its tissues. PAHs sub-lethal toxic effects were registered for the mollusk Tapes semidecussatus, that were exposed to sediments containing high levels of these chemical substances (Byrne and O’Halloran, 2004). Toxicity tests are very useful tools to identify the possible impacts of the PAHs on aquatic organisms. Lethality tests are considered good indicators of the toxicity of different chemicals and for different species, while the sub-lethality tests are interesting since they consider the impacts on the population by evaluating the effects on the organisms reproduction and growth (Wake, 2005). Individual toxicity of the PAHs present in aquatic environments, for plants and animals, increase when the molecular weight of these substances are next to 202 (e.g. fluoranthene, pyrene). Values superior to this weight configure a less soluble molecule, which promotes a reduction in the PAHs concentration to lower levels than of the considered lethal (Albers, 2002). However, exposition to PAHs with high molecular weight even in small concentrations, can lead to sub-lethal effects to the organisms (Neff, 1985). Kim et al. (2007) analyzed the PAHs toxic action on the genic expresion of the genes recA (related with DNA repair), KatG (that responds to oxidative stress), grpE (that is expressed in response to damages in the proteins) and fabA (that responds to damages in the membrane) of Escherichia coli, using in real time PCR (RT-PCR). The analyses showed that naphthalene and B[a]P caused damages in the E. coli DNA, while phenanthrene did not present any significative effect on the expression of these genes. Reece and Burks (1985) observed that the 11 PAHs present in petroleum refinery effluents caused lethal acute effects in Daphnia magna and that these substances were acting in an additive or synergistic way. Holst and Giesy (1989) studied the chronic effect of the exposition to anthracene and its photoproducts on the cladoceran Daphnia magna reproduction. In the experiments carried out both in the absence and in the presence of the UVR light, there was a reduction in the neonate number and an increase in the mortality number but these effects were more conspicuous when there was exposition to the light. An important result was observed by Allred and Giesy (1985), in studies about the anthracene phototoxicity on Daphnia pulex. The exposition of these organisms to anthraquinone, which is the first and more stable photoproduct of the anthracene, did not result in toxicity but in the experiments where the animals were previously treated with anthracene in the presence of light and after were transferred to water, the toxicity was significative. By the results, the authors suggest that the toxicity was due to the activation, by the light energy, of the own anthracene molecule that binds to the molecules that are
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constituents of the D. pulex cells, and not of the metabolites of this substance present in the medium. The contaminants bond to specific receptors alters the metabolic and functional capacities of the tissues, by the induction or suppression of important proteins, causing harmful alterations to the organism. Thus, the induction or suppression of proteins can be used as biomarkers of effect and/or exposition (Roos et al., 2004). Bellas et al. (2008) studied the toxic and phototoxic potentials of 6 PAHs, individually and in mixtures, on embryos and larvae of mussels, sea urchin and ascidian. All the PAHs were toxic in the tested concentrations for sea urchin embryos and all, with the exception of fluoranthene, were toxic for mussels embryos. In relation to the ascidian embryos, only naphthalene presented significative toxic effect. However, exposition to fluorescent light increased the toxicity of phenanthrene, fluoranthene, pyrene and hydroxypyrene but not of the naphthalene and fluorene. The PAHs mixture did not present additive effect of the toxicity. B[a]P and anthracene effects, on the activity of several enzymes of the fish Pomatoschistus microps, was evaluated by Vieira et al. (2008). After 96h of exposition to several concentrations, it was observed that these substances inhibited acetylcholinesterase, leading to an interruption of the cholinergic transmission and induced the activity of lactate dehydrogenase, catalase, superoxide dismutase, gluthatione peroxidase and gluthationereductase. It was observed an induction of the glutathione S-transferase activity in fishes exposed to B[a]P and an inhibition of this enzyme after exposition to anthracene, suggesting an involvement of glutathione S-transferase in the B[a]P detoxification. Studies carried out by McCloskey and Oris (1993) showed an increase of the hematocrit, evidences of hemolysis and a decrease in the presence of hemoglobin in the blood, besides a significative enzymatic inhibition of Na, K-ATPase and Mg-ATPase in the gills of the bluegill fish, after 96 hours of exposition to anthracene and ultraviolet solar radiation. The authors concluded that this toxicity is associated with a perturbation in the cellular membrane functions, promoted by anthracene. It was evaluated the PAHs effects on the neotropical fish Oreochromis mossambicus, by enzymatic bioindicators. Expositions carried out with low concentrations of PAH ( 12) are almost entirely particle associated, and compounds with PL (log KOA) between these values can be considered as mixed phase atmospheric constituents (Lohmann et al., 2001). PAHs with two or three rings, which have relatively low log- KOA values, are mainly associated with the vapor phase, 4-ring PAHs occur in both the gas and particle phase, whereas PAHs with five rings or more, which have high log KOA values, are mostly bound to particles. However, a higher temperature probably increases the gas partitioning of the PAHs with four rings or more (Soderstrom et al., 2005). Some high molecular weight (more than four fused rings) PAHs are considered to be carcinogenic, mutagenic and teratogenic even at low concentrations (IARC, 1983; Bostrom et al., 2002; Yu et al., 2006). However, lighter PAHs are most abundant in the urban atmosphere and react with other pollutants to form more toxic derivatives (Chang et al., 2006). Because of their hydrophobic nature and low solubility, PAHs are more resistant to biodegradation and can bioaccumulate in the environment through the food web (Ohura et al., 2004a; Conde et al., 2005; Motelay-Massei et al., 2005; Bartkow et al., 2006; Cupr et al., 2006; Huckins et al., 2006; de Souza Pereira et al., 2007; Zhu et al., 2008). Therefore, PAHs in the environment represent a long-term threat to human health and have received more and more environmental concern. Regulation of PAH emission and reliable monitoring of PAH concentration in ambient air is thus of paramount importance for public health (Lehndorff and Schwark, 2004).
LEVELS OF PAHS IN THE ENVIRONMENTAL MEDIA ATMOSPHERIC AIR No specific guideline value can be recommended for PAHs as such in air. These compounds are typically constituents of complex mixtures. Some PAHs are also potent carcinogens, which may interact with a number of other compounds. In addition, PAHs in air are attached to particles, which may also play a role in their carcinogenicity. Although food is thought to be the major source of human exposure to PAHs, part of this contamination may arise from air pollution with PAHs. The levels of PAHs in air should therefore be kept as low as possible. In view of the difficulties in dealing with guidelines for PAH mixtures, the advantages and disadvantages of using a single indicator carcinogen to represent the carcinogenic potential of a fraction of PAH in air were considered. Evaluation of, for example, BaP alone will probably underestimate the carcinogenic potential of airborne PAH mixtures, since co-occurring substances are also carcinogenic. Nevertheless, the well-studied common constituent of PAH mixtures, BaP, was chosen as an indicator, although the limitations and uncertainties in such an approach were recognized The determination of atmospheric PAHs is hindered by many problems, the most common derived from sampling. Therefore, it is difficult to integrate the existing information into a coherent data set. PAHs concentrations have been reported in dry deposition in a number of urban areas as well as in remote places, including the open Mediterranean Sea and developing countries Table 3. Despite the variety of components analysed, some seasonal and spatial trends are apparent. Concentrations are consistently higher in winter than in summer,
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and in urban than in rural areas. The higher values are found in particular places like stack gases, tunnels of motorways, open burning sites, etc. To compare between PAHs concentration in developed countries with other developing ones Table 4 showing data of estimated indoor and outdoor air concentrations for PAHs (ng/m3) at each concerned area using sampling rates of 2.5 m3/day (indoor) and 3.5 m3/day (outdoor) (Bohlin et al., 2008). Using sampling rates suggested in the literature, the sums of 13 PAHs in the different sites were estimated to be 6.1–180 ng/m3, with phenanthrene as the predominant compound. Indoor PAH levels tended to be higher in Gothenburg and outdoor levels higher in Mexico City. Table 3. Concentration of PAHs in aerosol samples from different cities*. Location Algeria Algiers
Sampling period
PAHs
Conc. (ng/m3)
Wint./sum. 1999
Oued Smar landfill
Wint./sum. 1999
∑18 PAHs BaP ∑18 PAHs BaP
59.9/13.7 0.4-2.3 136.5/24.9 0.3-3.9
Egypt Cairo(urban area)
Wint/sum
Alexandria
Summer 97-98
∑18 PAHs BaP ∑18 PAHs BaP
14.8/7.5 0.1-0.2 32 0.95
Greece Thessaloniki
1987-1988
∑9 PAHs BaP ∑13 PAHs ∑10 PAHs BaP
22-55 2-6 5-60/0.4-8.1 11.3(V), 0.8(P) 0.003(V),0.025(P)
∑7 PAHs BaP BaP PAHs BaP ∑4 PAHs BaP
5.96-7.33 1.2 0-174/(av.=22.7) 2-130 0.31-3.8 0.8-19.1 1.1-2.0
North Greece town Crete inokalia Italy Rome
wint/sum 96-97 2000-2001
Bolgan Naples
1991-1993 1996-1997
Genoa (traffic site)
1997-1999 1994-1999
Syria Damascus
1999-2000
∑18 PAHs BaP
14.8-45.7 0.9-2.7
FR Yugoslavia Belgrade
2000
BaP
0.2-0.6
1993-1998
*(modified from UNEP, 2002)
Phenanthrene was the predominant PAH compound and the 3–4 ring PAHs which are mainly in the gas phase contributed to >96% of ∑13PAH. The indoor levels in Mexico City ranged from 6.1 to 92 ng/m3, with median levels being 28 ng/m3 at the urban area, and 25 ng/m3 at the semi-rural area. The indoor levels of PAHs tended to be higher in Gothenburg (median 37 ng/m3) than in Mexico City and Lancaster (median 28 ng/m3). The estimated levels of indoor air in Kuwait, both in terms of the composition profile and ∑PAHs (1.3–16 ng/m3). The levels herein are also in agreement with estimated indoor air
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levels of PAHs from an area in Sweden where domestic wood burning is widespread (20–250 ng/m3) (Strandberg et al., 2006). The outdoor levels in Mexico City ranged from 32 to 92 ng/m3. Highest median levels (82 ng/m3) were found in the semi-rural area where both point sources of PAHs (i.e. main roads, backyard burning and forest fires), and wind drift from the urban plume occur. As expected, outdoor levels tended to be lower in Gothenburg and Lancaster compared to Mexico City (Coleman et al., 1997; Motelay-Massei et al., 2005). Table 4. Indoor and outdoor concentrations of PAHs in different areas
Mexico City urban In Out Mexico City semi-rural In Out Gottenburg In Out Lancaster In Out
Range
Mean
∑13 PAHs Median
6.1-92 32-64
34 47
28 44
12-37 72-92
25 82
25 82
14-180 7.7-68
69 37
37 33
8.5-60 6.8
30 6.8
28 6.8
The levels of PAHs released into the environment from various human activities especially from industrial sources in the Niger Delta area of Nigeria shows that the most polluted medium is the air followed by the soil and water media. Comparing the study locations there is an indication that Eleme (the most industrialized) as compared to Ahoada East (one of the least industrialized) recorded PAH levels 7- million fold higher in the air. Levels of PAHs in Eleme were 3-fold higher in surface water and 7 -fold higher in soil than the levels found in Ahoada East as clearly shown in Fig. 1.
Figure 1. Environmental levels of PAH in the Niger Delta Area.
PAHs occurrence and situation in atmosphere of Egypt is well shown in the following section which represent a case study of developing country. Since the 1960s, Egypt has witnessed rapid development and industrialization, a by-product of which has been the generation of large amounts of air pollutants. As a result, air quality has decreased drastically.
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In Egypt as an example of developing countries, most air toxics originate from human-made sources, including; mobile sources (e.g., cars, trucks, buses) and stationary sources (e.g., factories, refineries, power plants); as well as indoor sources (e.g., building materials and activities such as cleaning). Toxic air pollutants are emitted from many industrial and nonindustrial sources; fuel combustion, open burning of organic material, solvent uses, storage and transport of petroleum products, volatilization from the use of consumer products, and various manufacturing and commercial processes. These sources are categorized as point, area or non-point, non-road mobile, and on-road mobile sources. Several new industrial areas were established such as 6th of October, 10th of Ramadan and Sadat City. These new industrial cities were founded to divert industrial development from the Nile Valley and Delta towards the adjacent desert areas. PAHs are at the present time one of the most documented class of organic pollutants in the environment due to their toxicity carcinogenicity, and mutagenicity, PAHs continue to attract the curiosity of researchers. PAH concentrations found in the atmosphere are highly dependent of the size of particles and the highest concentrations are found in breathable particle. Certain PAHs are suspected of being carcinogens and some are associated with acute and chronic health problems. The main sources of PAHs in Egypt are diesel exhausts, the petroleum and printing industries, and road transport. However, there are few available studies on PAHs in ambient air in Cairo city. PAH concentrations in Sadat city as an example of new industrial area were measured, beside old industrial areas in Egypt. The seasonal and annual mean concentrations of ∑9PAHs in Sadat City during the year 2003 were 403.79 and 143.82 ng/m³, respectively at two measuring sites. The ∑9PAH reached the maximum during winter season (786.69 ng/m³), while the minimum value was recorded during autumn season (33.09 ng/m³). The annual average concentration of benzo(a)pyrene was 18.464 ng/m3 at the industrial area in Sadat city during the period under study. The mean concentrations of benzo(a)pyrene in the present study were 6 times higher than the annual limit set by Italy (2.5 ng/m3), and the current guideline limit of the Federal Environmental Agency of Germany (1 ng/m3) (Ezzo, 2006). In the same way, the concentration of PAHs were measured in the industrial area in Greater Cairo, the obtained values were much higher than recorded for the industrial areas for many countries such as New Jersey, Canada and Belgium. This may be due to the higher local emission of PAHs from the coke factory, iron and steel factory and other industries in and around this area. PAHs are normally associated with small particles that generally have long residence times in atmosphere and, for that reason have the potential to be transported to quite long distances (Hassan, 2006). The annual mean concentrations of atmospheric PAH over the 4 investigated sites in Cairo from autumn 2001 to autumn 2003 were illustrated in Table 5 (Hassan, 2006). It can be noted that the highest levels were recorded in Shoubra Elkhiem (industrial area) followed by El-Tebeen (industrial area), then City center, and finally Faysal (residential area). Worldwide, regulations and standards regarding permissible levels of PAHs in ambient air vary somewhat. The maximum permissible concentration in ambient air is 1 ng/m3 in Soviet Union former and Hungary (Hassanien et al, 1996). In the US, no national ambient air quality standard exists. However, several states have established ambient standards. For B(a)P, these range from 0 to 100 ng/m3 (8-hour average). In the Netherlands, the Environmental Programme 1988-1991 recommended a draft tolerable B(a)P concentration of 5 ng/m3 (annual average) (RIVM, 1989) see Table 6.
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Table 5. Seasonal and annual mean concentrations of atmospheric PAH in different sites in Egypt Location
PAHs
Sampling year
Cairo
∑12 PAHs
Sadat City New Indus. city Greater Cairo Soubra ElKheima (old indus. Area)
∑9 PAHs
1997 Wint/Sum 2003 Win./sum
Recorded value (ng/m3) 22.32 (annual) 14.79/7.53 273.8 (annual) 786.7/145.2
2001-2002 Wint/Sum
4460 (annual) 7040/1771
2001-2002 Wint/Sum
3552 (annual) 5416/1378
Elattaba
2001-2002 Wint/Sum
3477 (annual) 248/134
Faysal (resid..area)
2002-2003 Wint/Sum 2005
1965 2274/1789 37.3 (outdoor) 29.7 (indoor) 20.6 (outdoor 8.8 (indoor)
City Center ∑16 PAHs
∑12 PAHs Cairo (traffic
∑carcino.PAHs
References Hassanien et al, 2001 Ezzo, 2006
Hassan, 2006
Hassanien, 2006
An average of 32 ng/m3 of total PAHs in the ambient air found in a study in heavy traffic areas in Alexandria City (Barakat, 2002). A summary of the data obtained from analysis of polycyclic aromatic hydrocarbons in suspended dust collected during 1997 in Cairo Egypt (Hassanien et al., 2001 and recently Hassanien, 2006) is given in Table 5. It can be seen from the analysis of the 12 components that the sum of PAH concentrations at the sampling site was higher during the winter season 1997 (14.79 ng/m3) than in summer during 1997 (7.53 ng/m3). It was found that the carcinogenic materials constitute about 20%; 33% and 25% of the total PAH for the winter, summer and annual periods, respectively. A highly significant difference between the estimated concentrations of PAHs of winter and summer was observed. It was found that PAHs concentrations in outdoor air generally exceeded that in the corresponding indoor air. The sum of PAHs concentrations in outdoor air was 37.3 ng/m3; the corresponding concentrations in indoor air was 29.7 ng/m3. Carcinogenic compounds measured in b-oth outdoor and indoor represent 20.6 and 8.8 ng/m3, respectively with a contribution 55.2% and 29.6% from the total of PAHs. In addition, from the eighteen PAHs, fluoranthene, pyrene, chrysen, benzo(b)fluroanthene, dibenzo(a,h)anthracene, benzo(ghi)perylene, and benzo(a)pyrene were the most abundant PAHs found in indoor and outdoor air. They contributed more than 55-65%, respectively to the sum of PAHs (Hassanien, 2006).
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Table 6. Ambient air quality standard for the PAHs*.
Netherlands PAH WHO-AQG PAH
Limit 5 ng/m3
Guide value 5 ng/m3 1.0 ng/m3
Measuring period Years as ng/BaP/m3 Years as ng/BaP/m3
*(Hassanien, 2006)
INDOOR VERSUS OUTDOOR PAHS LEVELS IN SOME CITIES In recent decades, there has been a tendency toward atmospheric pollution in the developing countries due to fast urbanization and industrialization. In past years, many studies have stressed the characterization of inorganic components of aerosols in developing cities in Asia and Africa because the desert land profile for these countries which considered the largest source of particulate matter in the world (Baek et al., 1991; MaliszewskaKordybach, 1999; Mastral and Callen, 2000; Ohura et al., 2004b). Indoor emission sources of PAHs include smoking, cooking, heating, and furniture (Liu et al., 2001; Ohura et al., 2002). Ratios between indoor and outdoor air concentrations (I/O ratios) may be used as a tool to indicate whether there are indoor sources (I/O> 1) or outdoor sources (I/O1 in Gothenburg, and generally close to 1 in Mexico City. The high I/O ratios for ∑PAH in Gothenburg together with somewhat elevated indoor levels may indicate that PAH emissions in these homes are mainly from indoor sources while the opposite pattern was observed in Mexico City (Motelay-Massei et al., 2005, Strandberg et al., 2006). An interesting observation was that low molecular weight PAHs in Gothenburg in general show an I/O ratio well above 1 while the 5–7 ring PAHs are found in more samples outdoors than indoors, giving an I/O < 1. Lower I/O ratios for the 5–7 ring PAHs were also seen in Mexico City. This may indicate the presence of emission sources of mainly low molecular weight PAHs (vapour phase PAHs) indoors. PAH levels in the PM2.5 fraction of Beijing urban outdoor air were lowest in summer and highest in winter (He et al., 2006). The size distributions of ambient particles showed no significant difference between summer and winter, either outdoors or indoors. This suggested that the distributions were strongly affected also by the sources. On the other hand, ∑PAH associated with particles was predominantly (>80%) found in PM2.5 fractions. The proportion of ∑PAH in PM2.5 fractions was about one order of magnitude higher than in coarse particles (PM>10 and PM2.5–10) (Ohura et al., 2004a). The concentrations of indoor ∑PAH associated with PM2.5 (∑PAH– PM2.5) measured in his study ranged from 1.6 to 23.7 ng/m3). In outdoors, the ∑PAH– PM2.5 concentrations ranged from 1.1 to 29.5 ng/m3. The concentrations of indoor ∑PAH associated with PM>10 (∑PAH–PM>10) and PM2.5–10 (∑PAH– PM2.5–10) ranged from 0.13 to 1.8 and 0.18 to 2.05 ng/m3, respectively. Outdoors, ∑PAH–PM>10 and ∑PAH– PM2.5– 3 10concentrations ranged in 0.10–1.64 and 0.17–2.48 ng/m , respectively. The variability of outdoor ∑PAH associated with particles was considerably larger than that of the corresponding particles. This may be due to the difference of emission sources, lifetime, and physical properties, between PAH and particles. For light molecular PAHs, concentrations were strongly affected by seasonal factors because of their high vapor pressures.
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Smoking is a major emission source of indoor pollutants (Liu et al., 2001; Ohura et al., 2002). Humans spend about 90% of their daily lives indoors; thus, estimating indoor pollution is critical to ensuring a healthy environment. BaP is widely used as an indicator of human health risks and pollution levels because of its strong carcinogenicity (Finlayson-Pitts and Pitts, 2000). In Guanzhou (China), the seasonal concentration for ∑PAH ranged from 8.11 to 106.26 ng/m3. The average ∑PAH measured were highest in winter and lowest in summer. 5-6 ring PAHs were the abundant compounds, which accounted for 65-90% of ∑PAH and benzo[b+]fluoranthene dominated in the four seasons. Moreover, the total PAHs in Guangzhou were significantly higher than those in Hong Kong, Naples, Rom and Seoul; however, the distribution patterns of PAHs of PAHs in Guangzhou showed some similarities. In these cities high molecular weight (HMW) compounds dominated with Bbkf as maxima (except Naples) and Ant had the lowest concentration in all PAHs (Tan et al., 2005). Fourteen polycyclic aromatic hydrocarbons were identified and quantified in PM10 rang in Guangzhou during the period from March 2002 and June 2003. The ∑PAH ranged from 8.11 to 106 ng/m3. A strong seasonal trend of PAHs abundance was observed. The highest level was in winter and the lowest was in summer. The average ∑PAH in winter was 3.8 times higher than that in summer; the Asian monsoon had a strong influence on the seasonal variation. The distribution pattern of PAHs varied with the season. PM10-bound PAHs were dominated by high molecular weight compounds. LMW PAHs tend top have a higher contribution in the winter while HMW PAHs tend to have a higher contribution in the summer. Based on the diagnostic ratios, it is concluded that vehicular were the predominant source of airborne PAHs in Guangzhou (Tan et al., 2005).
OCCURRENCE OF PAHS IN DUST Settled house dust can be a source of human exposure to toxic PAHs through non-dietary ingestion and dermal contact. Information regarding the concentrations of various contaminants in house dust would be useful in estimating the risk associated with exposure to these compounds. PAHs in settled house dust were collected from homes in three locations: Sumgayit, Azerbaijan; Shanxi Province, China; and southern Texas, United States. The highest PAH floor surface loadings were observed in China, followed by Azerbaijan and Texas. Median concentrations of high molecular weight (four ring and larger) PAHs ranged from a low of 0.11 μg/m2 in Texas, to 2.9 μg/m2 in Azerbaijan and 162 μg/m2 in China as listed in Table 7. These trends in total surface loading and relative carcinogenicity indicate that the risk of health effects from exposure to PAHs in house dust is highest in the Chinese population and lowest in the Texas population (Naspinski et al., 2008). PAHs in house dust may originate from both outdoor and indoor sources. Typical indoor sources include combustion of fossil fuels and biomass for heating and cooking; burning of tobacco products, candles and incense; and cooking fumes. Maertens et al. (2004) estimated the excess lifetime cancer risk from non-dietary ingestion of PAH in settled house dust to exceed the acceptable range for pre-school children in homes with dust PAH loading at the 95th percentile. Chuang et al. (1999) reported that ingestion of house dust and soil contributed 24% of the estimated daily dose of B2 PAH in a study of children in low-income
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households. The floor dust collection procedure used in this study is quick, inexpensive, easily portable, and requires no specialized equipment. Although surface loading cannot be used directly in an exposure calculation, it is valuable for the hazard identification step of risk assessment. Carcinogenic PAHs in the house dust collected from different places was found in general, that the heavier PAHs are more toxic. All of the carcinogenic PAHs (cPAHs) contain 4 or more rings and are therefore in the HMW fraction. The percentage of HMW PAHs was highest in China and lowest in Texas (Naspinski et al., 2008). Table 7. Concentrations of total and high molecular weight PAHs of dust samples collected from houses.
Total PAHs Median Min Max High molecular weight PAHs Median Min Max EPA B2 carcinogen PAHs Median Min Max
Azerbaijan
China
µg/m2 Texas
6.1 0.7 49.6
232 42 6720
1.34 0.24 35.3
2.9 0.5 20.0
162 30 4440
0.11 0.03 2.90
1.0 0.2 5.4
53 12 1620
BD BD 0.27
In Egypt, road dust PAHs was received relatively little attention to date and studies focused on heavy metals almost. Consequently, background level of PAHs in dust of Egyptian roads is not available. Recently, a study carried out by Hassanien and Abdel-Latif (2008) on the road dust collected from 17 sites over greater Cairo (Fig. 2). The concentration of PAHs was determined by using HPLC technique. Twelve common environmental PAHs were found to be distributed. The data illustrated that the total average of PAHs over the investigated sites was ranged from 0.045 mg/kg to 2.6 mg/kg. On individual scale, the highest concentrations were 1.031 and 1.028 mg/kg for pyrene and phenanthrene, meanwhile the lowest was benzo(a)pyrene with value 0.0001 mg/kg. The obtained results showed that the carcinogenic content of PAHs (benzo(a)anthracene, chrysene, benzo(a)pyrene, and benzo(k)fluoranthene) ranged from 0.2 % to 23.9% of total PAHs. The highest average concentrations for PAH species in Cairo's road dust were for PHE (phenanthrene) of 0.404 mg/kg followed by PYR (pyrene) and FLA (flouoranthene) with average levels of 0.166 and 0.068 mg/kg, respectively, in consistent with those results found in Beijing (Li et la., 2006). Moreover, Bostrom et al. (2002) found PHE (phenanthrene) to be the most abundant of 14 different PAHs compounds monitored at the street-level site in Stockholm. Meanwhile the lowest measurable species in dust were BaP (benzo(a)pyrene), ACE (acenaphthene), BaA (benzo(a)anthracene), and BbF (benzo(b)flouranthene) with average concentrations of 0.0024, 0.0027, 0.0028, and 0.0035 mg/kg, respectively.
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Figure 2. Map of the Greater Cairo with location of road dust sampling sites (solid circles).
The spatial variability in road dust PAHs concentration in Cairo was presented in Figure 3. It involves 17 representative locations, with site-specific information gathered at each location Distribution patterns of PAH in Cairo's road dust is obtained by processing data of species concentrations rang between 0.045 and 2.6 mg/kg, as shown in Fig. 3. Spatial distribution of PAHs highlighted sites of high concentration, and as it has revealed from results, these hot-spot-sites are distributed through out whole Cairo, i.e. sites 4 and 5 in the north, site 6 in the down town, site 11 in the west and site 13 in the south. These sites are heavily traffic areas, in addition to the industrial activities at sites 4 and 13. A moderate pollution was recorded also in sites 5, 15 and 16. This is the first comprehensive information on PAH spatial distribution pattern in different district areas of Greater Cairo, Egypt. This work revealed the road dust contamination with PAHs residues in different district areas of Cairo. It extends our understanding of the current PAHs contamination status in this mega city. The average concentration of 12 PAHs was 0.704 mg/kg with carcinogenic content of 17.1% of total PAHs. The present study confirms the contribution of vehicular traffic, industrial emissions and the incomplete combustion of open waste burning to road dust PAHs. Plotting PAH results on a map of city can be used to advantage in investigating road dust pollution and its potential deleterious effects. This study suggested that the pollution of PAHs in Cairo should emphasize on controlling the industrial emissions and traffic exhausts.
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Concentration (mg/kg) 3 2.5 2 1.5 1 0.5
Total PAH (mg/kg)
Carcinogenic % of total PAH
Carcinogenic % 50 45 40 35 30 25 20 15 10 5 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Invesrigated Sites
Figure 3. Total concentrations of PAH and carcinogenic percent at investigated sites.
PAHS IN FOOD MATERIALS Although many PAHs are included in the “priority pollutant” listings, there are many uncertainties over the significance of PAHs in relation to their transfer from the natural environment into the human food chain. There are very few available studies concerned with PAHs in food materials in developing countries. Therefore, it should be important to see the experiences from developing countries. Based on the premise that the plant leaf contents can provide a ‘time integral’ of the airborne concentration (Carberg et al., 1983, Eriksson et al., 1989, Bacci et al., 1991, Franzaring et al., 1992) the paddy leaves were selected as passive samplers to observe contemporary levels of PAHs in the atmosphere. PAHs reach the surface of plant leaves primarily from the atmosphere (Nash and Beall, 1970, Bacci and Gaggi, 1986, Trapp et al., 1990) PAHs in the atmosphere exist in either the gas or the particulate phase or both. Thus, the mechanism of transfer to plant leaves may be through gaseous dry deposition, particulate dry deposition, or wet deposition (including rain, snow and fog). Gaseous dry deposition has been shown to be the main process for many organic contaminants (Umlauf et al., 1994, McLachlan et al., 1995) PAHs with a vapor phase component in air are regarded as being subject to an air-plant exchange process, moving toward equilibrium over time. In addition, the paddy root was used for observing the adsorption of PAHs from soil and water because of root uptake, bioconcentration and translocation. In Japan, a study was carried out to estimate the PAHs content in rice as component of food chain. The total PAHs content in rice ranged from 46 to 77 µg/kg dry weight with an arithmetic mean value of 58 µg/kg dry weight. Phenanthrene was the most abundant in most cases. For analyzed PAHs, the arithmetic mean value of acenaphthylene, fluorene, anthracene, pyrene, weight (152–202) to heavier molecular weight (228– 252) PAHs in paddy leaf (73 : 27) was similar to that in rice (70 : 30) (Liu et al., 2001). The mobility of light molecular weight PAHs through the atmosphere was greater than that of the heavy molecular
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weight PAHs because of the greater gas phase component. Conversely, heavy molecular weight PAHs were considered to be only particle-bound and will be subject to deposition processes that effectively remove them from the atmosphere as air masses move away from source regions (Eriksson et al., 1989, Bacci et al., 1991, Franzaring et al., 1992).
POLYCYCLIC AROMATIC HYDROCARBONS IN MARINE BIOTA Concentrations of PAHs in marine biota indicate a high degree of contamination of certain areas of the region. Data given for the Ligurian Sea for Mytilus sp. range from 14 to 571 ng/g dw. In other studies, the 14 priority PAHs in mussel tissues (Mytilus sp.) and fish livers (Serranus and Mullus sp.) of specimens collected in the French and Spanish Mediterranean coasts and found values of 25-82, 27-87 and 15-49 ng/g dw, respectively, n values up to 390 ng/g dw were found in mussels collected inside harbours. Similar determinations carried out in deep sea fish from the NW Mediterranean basin (Mora sp.) showed liver concentrations of 7-16 ng/g ww (UNEP, 2002). A large survey of mussels carried out during 1995-99 in the French Mediterranean and Atlantic coasts gave mean values of 590 and 411 ng/g dw (Σ16 PAHs), respecively. A survey carried out recently in bivalves (Perna perna) from 18 stations along the Atlantic coast of Morocco gave results from 3.4- 34.6 ng/g (Σ14 PAHs) with values up to 87-109 ng/g ww in the vicinities of the cities of Agadir and El Jadida. Values of 170-750 ng/g dw were found in mussels from the Thermaikos Gulf (Greece). The concentrations of 16 PAHs in mussels collected in 1999 from Izmit Bay (Turkey) were found in the range 1.25-34.0 ng/g. Blubber samples were collected in 1993 and 1996 from live specimens of fin whales (Balenoptera physalus) and stripped dolphins (Stenella coeruleoalba) from the Ligurian and Ionian Seas. In whales, the median value of total PAHs was 350 ng/g ww while median carcinogenic PAH values were 114 ng/g ww, in dolphins. The median values of total and carcinogenic PAHs were 938 and 670 ng/g ww, respectively (UNEP, 2002).
HEALTH AND ENVIRONMENTAL IMPLICATIONS The hazards caused by toxic chemicals are devastatingly dangerous in the developing countries because:
People and governments are unaware of the inherent dangers of pesticides and other chemicals; The necessary financial resources are either scarce or do not exist; Facilities either for containing the wastes or for its destruction are not available Appropriate legal measures do not exist or, if they do, are either impossible or difficult to implement; Environmental activists are either absent or too weak to stage public demonstration, and Expertise or skilled manpower is not available. However, some of the adverse health and environmental impacts, include
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Direct exposure of workers and farmers, local residents, to unsecured pesticides stocks Indirect exposure through contaminated food and water, thus endangering public health Poisoning of farm lands and other workers Pollution of ground and surface water sources, which impacts on fauna and flora Impacts on land use resulting in limiting the potential of development of the area surrounding any chemical site Destruction of beneficial insects Risks and costs of destruction and incineration.
The effects associated with exposure to PAHs are enormous. They could be classified into environmental and health effects. While the environmental effects are associated with the pollution of the air, water and soil ecosystems, the later being the most dominant is associated with disturbances and disorders ranging from the disorders of the nervous or immune system, to increases in the risk of certain cancers. They persist in the environment, and may bioaccumulate and magnify in the food web due tof the tendency of these chemicals to accumulate in fatty tissue. There may be special windows of vulnerability in the development of fetuses when these chemicals can have longterm, irreversible effects on the reproductive and neurological systems.
CARCINOGENIC ASSESSMENT OF PAHS TOXIC EFFECT OF PAHS Short-term studies showed adverse hematological effects. Systemic effects caused by long-term treatment with PAHs have been described only rarely, because the end-point of most studies has been carcinogenicity. Significant toxic effects are manifested at doses at which carcinogenic responses are also triggered. In studies of adverse effects on the skin after dermal application, non- or weakly carcinogenic PAHs were inactive, whereas carcinogenic compounds caused hyperkeratosis. Benz[a]anthracene, benzo[a]pyrene, dibenz[ah]anthracene, and naphthalene are embryotoxic to mice and rats. Benzo[a]pyrene also have teratogenic and reproductive effects (Ravindra et al., 2008). PAHs have also been studied extensively in assays for genotoxicity and cell transformation; most of the PAHs are genotoxic or probably genotoxic. The only compounds for which negative results were found in all assays were anthracene, fluorene, and naphthalene. Owing to inconsistent results, phenanthrene and pyrene could not be reliably classified for genotoxicity. PAHs have generally been reported to have immunosuppressive effects (Ravindra et al., 2001). The toxicity of PAHs represents a risk to human health, and there are varied risk assessment approaches to quantifying the risk posed by PAHs based on exposure routes and scenarios. PAHs are not carcinogenic until they are metabolically activated as the body attempts to break them down and forms reactive metabolites that bind to the DNA causing subsequent replication in the cells. Fundamental to assessing risk posed by PAHs is understanding metabolism of PAHs. Since exposure to PAHs is never to single PAHs,
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understanding what differences may occur in mixtures of PAHs gives accurate assessment of the dangers of PAHs. Understanding the dynamics of complex metabolism vis-à-vis single metabolism of PAHs and possible effects on the toxicity expression of PAHs is a necessary advancement to accurately impact and guide remediation strategies. The bioaccumulation of PAHs and their bioamgnification along successive trophic levels is another means of exposure to human and animal life. PAHs can cause mutations, malignant and benign tumors, among a host of genotoxic effects. The link between PAHs and harmful effects in humans was made since 1775 by Sir. Percival Pot, surgeon to St. Bartholomew’s Hospital in London. He observed the increased incidence of scrotal cancer in chimney sweepers originated from their exposure to soot; (Cerniglia 1980). This observation provided an important link into studying occupational related diseases and their causes. Increasingly, links were made between tumors and occupation that had PAH exposure, in workers such as miners, chemical plant workers, tar-producing industry workers.
HUMAN EXPOSURE Air pollution is often suggested as being partly accountable for an increased incidence of lung cancer in cities. Over the past decade, an increasing body of scientific evidence has accrued associating outdoor air pollution with certain types of cancer. In many researching scientists have found out that exposure to air pollution, even at low levels, can lead to a wide range of adverse health effects including lung cancer. An individual’s total exposure to air pollution depends on indoor as well as outdoor exposures. Indoor air quality has large potential health implications because people may spend considerable amounts of time indoors. Indoor air pollution may stem from incoming outdoor air or may originate indoors from tobacco smoking, building materials, soil gases, household products, and combustion from heating and cooking. The exposure of human populations to carcinogens in outdoor air may be the result of proximity to more localized sources such as industrial facilities, small businesses, municipal facilities, or areas with high vehicular traffic. PAHs are lipophilic compounds and can be absorbed by the lung and gastrointestinal tract tissues and by the skin. Several studies have been carried out to determine the levels of exposure of humans to PAHs (Ciganek et al., 2002). The dietary sources of PAHs are cereals and vegetables, rather than meat, excepting populations with a high consumption of meat cooked over an open flame. The recently developed biomonitoring procedures allow the assessment of human exposure to PAHs, as well as identification of diet as the major exposure source. The U.S. Environment Protection Agency published “The Exposure Factors Handbook” in which available statistical data on various factors used in assessing human exposure are summarized (http://www.epa.gov/ncea/exposfac.htm). The major steps in exposure assessment include: (1) determination of exposure pathways, (2) identification of environmental media conveying the contaminant, (3) determination of the contaminant concentration, (4) determination of exposure time, frequency, and duration, and (5) identification of the exposed population.
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EXPOSURE AND ROUTES OF EXPOSURE Exposure to chemical hazards may occur via all types of exposure: inhalation, oral ingestion, absorption through the skin, absorption through the eyes, and placental transfer from pregnant woman to the fetus, inoculation and direct penetration to target organs and from mother to child through breastfeeding. In the non occupational environment, ingestion of substances containing chemicals is the most common route of exposure. In the workplace, because of the nature of exposure, duration of the workday, and character of the compounds; inhalation is the most significant route of entry, followed by skin absorption and ingestion. Health effects, of course, derive from exposure and thus it can be expected that global illhealth from air pollution is dominated by indoor exposures in developing countries as well. The precise extent of impact, unfortunately, is not known yet, but enough information is available to make reasoned estimates for India, where more data are available than in most other developing countries (Smith, 2000). The main sources of general population exposure are: vehicular traffic, residential heating, smoke from open fireplaces and cooking, tobacco smoke, contaminated food and drinking-water, and the use of PAHs contaminated products. The calculated total daily intake of PAHs from food has been estimated to be 3 µg/day (1.4 µg/day carcinogenic PAHs) per person in Italy (UNEP, 2002). In addition to a lack of studies on the risks of exposures, there is a dearth of studies on the distribution and magnitude of exposure to PAHs in developing countries itself. Such information is needed both to estimate the total burden of disease and to identify the regions and populations where interventions need to be focused. Compared with the thousands of studies on outdoor and indoor air pollution in developed countries, only few studies have been carried out in the developing world (African countries). Although striking because of the high exposures to major pollutants that have been demonstrated, the available studies have not been conducted in a fashion that allows the results to be confidentially extrapolated to large populations.
PAH POTENCIES PAH potencies are used to determine quantitative health risks posed by PAH exposure. The risks posed by a mixture of PAHs are based on an assumption of additivity of the individual risks posed by the PAHs. The IPCS monogram on PAHs (IPCS, 1998) describes three approaches used to calculate PAH potencies: 1) toxicity equivalence factors approach is based on expressing of individual potencies relative to benzo(a)pyrene, 2) comparative potency approach, which does not identify or quantify the individual compounds but determines the potency of the mixture of compounds and 3) benzo(a)pyrene surrogate approach assumes that benzo(a)pyrene is an indicator of all the PAHs. As the toxicities (carcinogenicity and mutagencity) of individual PAHs differ considerably, toxicity assessments of PAHs are complex. A number of approaches have been developed for evaluating the potencies of the various PAHs with regard to the possible inhalation cancer risk to humans (IARC, 1983). One approach is to calculate the inhalation unit risk for excess lung cancer over the risk posed by BaP for each of its copollutant carcinogenic PAH in the polluted ambient air divided the particular PAH’s risk by the risk
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for BaP to obtain their individual potency equivalence factors (PEFs); thus, the PEF for BaP would be, by definition, 1.00. These PEFs can be used to estimate the relative carcinogenicity of ambient air with a known distribution of PAHs (Ohura et al., 2004a,b). The risk associated with the inhalation of particulate PAHs indoors showed that the contribution of BaP to the total carcinogenic potential was dominant in the range of 51% to 64%. Human activity patterns indicate that people tend to spend 90% or more their time indoors. In addition, indoor BaP concentrations are strongly affected by the corresponding outdoor ones. Thus, the risk assessment for inhalation of PAH could be roughly estimated by only the outdoor BaP concentrations. PAHs half-lives in humans are in fact in the range of days/hours, and metabolism is responsible of the formation of toxicologically active (carcinogenic) metabolites. Because of the lack of persistence and to the complexity of PAH mixtures, human exposure is usually characterised in terms of “internal dose” rather than of “body-burden”, by the use of biomarkers of exposure, mainly 1-OH pyrene in urine and DNA adducts in peripheral blood lymphocytes.
RISK ASSESSMENT OF PAHS RISK RESEARCH NEEDS Hundreds of urban developed-country epidemiological studies have been carried out in recent decades, allowing in some cases large-scale meta-analyses and reviews for important pollutants like small particulates (e.g. WHO, 1999; Kreweski et al., 2000; Samet et al., 2000). Extrapolating these relatively well-characterized results to the populations using solid fuels indoors is difficult, however, because of different pollutant mixtures, health conditions, agedistributions, exposure patterns and so on, as well as the scarcity of actual exposure measurements. There is clearly need to find ways to make these extrapolations with more confidence (McMichael and Smith, 1999). In addition, however, there are needs to conduct additional research directly within the communities of interest. Given what is known about the relative importance of the major air-pollution-related diseases in developing countries, the risks from existing studies, and the relative difficulties of measuring effects in studies of reasonable duration, the following kinds of studies would seem to have the highest priority. Although many people associate public exposures to air pollution primarily with urban outdoor settings, readers of this chapter are well aware that indoor environments can also be contaminated, both from pollution penetrating from outside and from indoor sources. General population exposure to PAHs mainly occurs from atmospheric pollution and diet, this latter accounting for about 90% of the total exposure (Hassanien et al., 2001 ). The largest exposures to health-damaging air pollution probably occur in the developing world. As a result, much of the health impacts from air pollution worldwide seem to occur among the poorest and most vulnerable populations, largely women and young children who are most exposed to the indoor pollution sources of importance in poor countries. For many PAHs, toxicological effects are still unknown or not fully clarified, and mechanistic information is lacking. Moreover, even for PAHs whose human health hazard has been characterised, no risk assessment is actually feasible due to the lack of exposure data in most countries. Food being the main exposure route, data on PAHs daily intakes are of major relevance and represent the necessary basis to develop an adequate risk assessment.
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These high individual and population exposures are the result of a set of factors:
About half the world’s households use unprocessed solid fuels for cooking, ranging roughly from near zero in developed countries to more than 80% in China, India, and Sub-Saharan Africa (Holdren et al., 2000). The individual peak and mean exposures experienced in such settings are large by comparison with WHO guidelines and national standards.
BaP is one of the polycyclic aromatic hydrocarbons formed when gasoline, garbage or any animal or plant materials burn incompletely. It was first isolated from coal tar. People may be exposed to BaP from air, water, soil, cigarette and other plant product smoke, food and some work environments through inhalation, ingestion and skin contact. Exposure to PAHs may cause harmful health effects. When humans are exposed to BaP at high concentrations for relatively short periods of time, BaP may cause red blood cell damage leading to anemia and suppressed immune system. Mixtures of PAHs including BaP such as coal tar were shown to be dermal carcinogens in animals as early as 1918. BaP has caused tumors in laboratory animals when administered in the diet, when applied to their skin or when inhaled for a long period of time. Humans exposed to mixtures of PAHs and other compounds at high concentrations over long periods of time can also develop cancer. To set priorities with respect to control, an excess lifetime cancer risk, expressed in terms of the BaP concentration and based on observations in coke oven workers exposed to mixtures of PAHs, is presented here. It must be emphasized that the composition of PAHs to which coke-oven workers are exposed may not be similar to that in ambient air, although it was noted that similar risks have been derived from studies of individuals exposed to other mixtures containing PAHs. Having also taken into consideration some recent animal data from which a unit risk of the same order of magnitude can be derived, it was concluded that the occupational epidemiology data should serve as the basis for the risk estimate. Based upon epidemiological data from studies in coke-oven workers, a unit risk for BaP as an indicator in air constituent is estimated to be 8.7 × 10-5 (ng/m3)-1 which is the same as that established by WHO (1987). The corresponding concentrations of BaP producing excess lifetime cancer risks of 1/10 000, 1/100 000 and 1/1 000 000 are 1.2, 0.12 and 0.012 ng/m3, respectively. To assess health-risks associated with PAH exposures, Lin et al. (2008) reported that it is important to know the total carcinogenic potency arising from the exposures of various PAH compounds. In principle, the carcinogenic potency of a given PAH compound can be assessed according to its benzo[a]pyrene equivalent concentration (BaPeq). Calculating the BaPeq concentration for a given PAH compound requires the use of its toxic equivalent factor (TEF; using benzo[a]pyrene as a reference compound) to adjust its original concentration (Chu and Shen, 1984; Thorslund and Farrer, 1991; and Nisbet and LaGoy, 1992). Among the available TEFs lists, the one established by Nisbet and LaGoy has been demonstrated to best reflect the actual state of knowledge of the toxic potency of each individual PAH species (Nisbet and LaGoy, 1992). By using the TEF list the carcinogenic potency of total PAHs (total BaPeq) can be determined as the sum of BaPeq concentrations of the 21 selected PAH compounds. For estimating the lung cancer risk associated with inhalatory PAH exposures, the World Health Organization (WHO) has suggested a unit risk of 8.7×10−2 (µg/m3)−1 for the lifetime (70 years) PAH exposure, assuming one was exposed to BaP concentration of 1 µg/m3
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(WHO, 1987). It is worth noting that the above unit risk was proposed for lifetime exposure, therefore, it has been adopted for assessing the exposure of general adults to the ambient atmospheric PAHs (Zmirou et al., 2000). For occupational exposure, Pott established a relationship between BaP exposure and lung cancer risk (Pott, 1985), based on a data bank provided by an epidemiological study conducted by Redmond et al. (1976). He suggested the unit risk of 7.0×10−2 (µg/m3)−1 for a 25-year occupational PAHs exposure with the averaged BaP concentration of 1 µg/m3. By using the same data bank, the US Environmental Protection Administration (EPA, 1984), however, suggested a different unit risk of 6.4×10−4 (µg/m3)−1 for PAHs exposure based on its total PAH content (expressed as the benzene soluble fractions). Since another study has indicated BaP could be a better indicator than total PAH content on characterizing the carcinogenic potency of PAHs (Petry et al., 1996).
INTERVENTIONS AND RISK RESEARCH NEEDS In spite of the clear need to conduct more research on risks and exposures, the current, if imperfect, knowledge of the health burden imposed by air pollution in developing countries argues that action is warranted now. Unfortunately, however, there also is a severe lack of good information on the interventions that might be best applied to effectively reduce the risks. Better ventilation, better stoves, better fuels, and behavioral changes would seem to encompass the range of potential interventions, but remarkably little systematic work has been performed on any of these, considering again the potential scale of the problem and consequent potential benefit. There is research needed not only on the technologies and their acceptance at the household level, but also on the policy tools needed to promote exposure-reducing changes in household fuels and stoves.
How to develop confounding for exposure-reduction efforts among developing sectors (health, energy, housing, and environment) in the worst affected regions. Conduct research on ways that different sectors can implement programs with exposure reduction potential, for example, fuel pricing in the energy sector. Conduct systematic program of research to evaluate the effectiveness of various policy measures
CONCLUSION AND FUTURE RESEARCH The environmental pollution mixtures in major population centers should be characterized both in terms of physical and chemical constituents and in terms of sources of major constituents. If possible, retrospective characterization of levels of certain constituents could be accomplished. Although a program of risk, exposure, and intervention studies as outlined above would not be inexpensive by some measures, it is likely to be highly costeffective as a means to address the large exposures now being experienced widely in developing countries.
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Better estimates of the amount of effect will need additional epidemiologic studies. Large numbers of cases will be essential to measure the effects of environmental pollution by PAHs and to measure combined effects of pollutants and factors such as occupation, smoking and such studies will probably require pooling data from multiple locales. New studies will need to develop and apply improved methods and to compare the effect of exposure to the pollutant mixtures on lung cancer in different cities while effectively controlling confounding factors including different exposure sources. Also we need methods for the retrospective estimation of lifetime exposure to environmental pollutants.
ACKNOWLEDGMENT The author would like to report that this chapter is the result of the work of many dedicated professionals who helped him in design, concept, and research. The author wishes heartily to thank his colleagues in Air Pollution Department, National Research Center for their sincere support and encouragement. He would like also to thank Prof. Alia A. Shakoiur, Dr. Mamdouh El melegy, Dr. Nasser M. Abdel-Latif and Aliaa A. Ezoo for supplying him with important data and information. Finally, the author thanks his sons for editorial assistance and their patience.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 15
ADIPOSE TISSUE AS A KEY CONTRIBUTOR TO POLYCYCLIC AROMATIC HYDROCARBON-INDUCED CARCINOGENESIS P. Irigaray1∗ and D. Belpomme2 1
Cancer Research Center, Association for Research and Treatments Against Cancer (ARTAC), Paris, France 2 Department of Medical Oncology, European Hospital Georges Pompidou (HEGP), University of Paris, Paris, France
ABSTRACT We have previously shown that benzo[a]pyrene used at extremely low dose can cause weight gain in mice and that the increase in adipose tissue mass is due to inhibition of β-adrenergic stimulation of lipolysis. Moreover we have suggested that adipose tissue is a reservoir for many carcinogens including Polycyclic Aromatic Hydrocarbons (PAHs).In this paper we demonstrate that the two C4 PAHs pyrene and phenanthrene can accumulate into adipocytes, but at the difference of benzo[a]pyrene have no impact on lipolysis.Since benzo[a]pyrene has been proved to be mutagenic while C4 PAHs are tumor promoters, we consider that a mixture of the three PAHs may act as a complete carcinogen. Furthermore, because adipose tissue can be induced by benzo[a]pyrene, is a reservoir for many PAHs and other carcinogens and can stimulate proliferation of normal or cancer cells, through the secretion of adipokines by adipocytes, we therefore propose that adipose tissue is a key contributor to chemical carcinogenesis by acting not only upon initiation and promotion but also upon progression.
Low molecular weight polycyclic aromatic hydrocarbons (l-PAHs) (3 to 4 rings) such as the C4 PAHs, pyrene and phenanthrene have been shown to be non genotoxic tumor ∗
P. Irigaray, PhD,ARTAC ,57-59 rue,de la convention,75015 Paris, Phone: +33 (0)1 45 78 53 53,Fax: +33 (0)1 45 78 53 50,E-mail:
[email protected].
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promoters, while high molecular weight PAHs (h-PAHs) (5 to 7 rings), such as the C5 PAH benzo[a]pyrene (B[a]P) are mutagenic though DNA adduction and may act as tumor initiators and/or tumor promoters [1]. We have previously shown that in addition to its genotoxic properties, B[a]P can inhibit adipocyte lipolysis and contribute to the hypertrophy/hyperplasia of adipose tissue [2], and have consequently hypothesized that in humans, B[a]P can generate both obesity and cancer [3]. Furthermore we have proposed the concept that the adipose tissue in addition to its endocrine function acts as a reservoir for many lipophilic liposoluble exogenous carcinogens and that these carcinogens can be permanently released in the blood circulation and target peripheral tissues for carcinogenesis. [3, 4]. In this paper, we attempt to further elaborate the concept that adipose tissue act as a reservoir for chemical carcinogens by analysing the ex vivo incorporation of PAH into adipocytes and by testing whether the C4 PAHs pyrene and phenanthrene can inhibit lipolysis as the C5 PAH B[a]P does. In this study, we confirm that adipocytes can incorporate h-PAH and l-PAH in a similar manner and that B[a]P in addition to its mutagenic carcinogenic properties can significantly inhibit lipolysis. So because adipocytes can incorporate both mutagenic and promoting PAHs, we conclude that PAHs cocktails may be equivalent to complete carcinogens and that the adipose tissue may play a key role in PAH-induced carcinogenesis.
MATERIALS AND METHODS Isolated Adipocyte Preparation Adipocytes were isolated from mice adipose tissue using Rodbell’s method as previously described [2].
Lipogenesis Assays Lipogenesis assays were performed as previously described [5]. Primary adipocytes freshly isolated from murine white adipose tissue were incubated with insulin (90 nM) and HAPs. This insulin concentration permits to obtain good lipogenesis [5]. More precisely, cell suspensions were aliquoted in Eppendorf tubes containing Krebs Ringer. Bicarbonate Buffer (KRBB) supplemented with 4% (w ⁄ v) bovine serum albumin (BSA), 1mM glucose and 0,278 µCi of 14C-phenanthrene (0.0066 µmol/l) or 0.296 µCi of 14 C-pyrene (0.007 µmol/l) or 0,272 µCi of 14C-benzo[a]pyrene (0,0065 µmol/l). After different incubation time at 37°C under gentle agitation (40 r.p.m) with or without insulin (90 nM), aliquots of the media were removed for radioactivity determination by liquid scintillation (Ultima Gold) counting in a Packard Tricarb in order to determine the percent of extra cellular radioactivity.
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Lipolysis Assays Lipolysis assays were performed as previously described [2]. Primary adipocytes freshly isolated from murine white adipose tissue were incubated with increasing concentrations of HAPs and with a dose of 10µM of epinephrine. This process was followed by measurement of the Free Fatty Acids (FFA) released in the media. Preliminary experiments using different concentrations of epinephrine (Figure 1A) and incubation times with epinephrine (Figure 1B) allowed us to determine the epinephrine concentration of 10 µM and incubation time of 45 minutes that permit obtaining good lipolysis.
Figure 1. Effect of different concentrations and incubation times of epinephrine on the release of FFA from mice adipocytes. Freshly isolated mice adipocytes were incubated (A) for 60 min at different concentrations of epinephrine and (B) for different times with a previously determined concentration of 1.8 mg/l (10 µM) of epinephrine. Each point represents the mean ± SEM of 4 experiments during which FFA levels were measured in duplicate.
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Figure 2. Kinetics of incorporation into adipocytes of B[a]P (■), pyrene (○) and phenanthrene (▲) in the presence of 90 nM of insulin. Each point represents the mean ± SEM of 4 experiments during which FFA levels were measured in duplicate. Data are expressed as the percent of molecules incorporated into mice adipocytes.
Cell suspensions were aliquoted in Eppendorf tubes containing KRBB supplemented with 4% (w⁄v) BSA, 5 mM glucose and one of the three PAHs, i.e. B[a]P, pyrene and phenanthrene at different concentrations during 45 min. After this preincubation period of 45 min, an epinephrine solution was added to the medium at a final concentration of 10 µM for 45 minutes, before measurements of the FFA released in the media were made.
RESULTS 1. Incoporation study As reported in figure 2, the kinetics of incorporation was identical for the three PAHs. PAHs incorporate into adipocytes uniquely during the first ten minutes (p = 0.001). This rapid incorporation suggests that PAHs incorporation is not insulino-dependant [5]. In addition, as indicated in tables 1 and 2 incorporation appears to depend upon the lipophilicity of molecules. Table 1. Incorporation rates of PAHs after 60 minutes of incubation at a concentration of 90 nM of insulin. Values indicated are the mean ± SEM of 4 experiments during which FFA levels were measured in duplicate. Compounds Benzo[a]pyrene Pyrene Phenanthrene
Percent of PAHs introduced in or adsorbed on adipocyte 35.04±2.28 20.62±2.55 23.08±2.89
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Table 2. Physico-chemical properties of the three PAHs tested. Compounds
Benzo[a]pyrene Pyrene Phenanthrene
Number benzenoïd rings 5 4 3
of
Molecular weight (g/mol) 252.3 202.3 178.2
Water solubility (mg/l at 25°C)
Lipophilicity Kow)
3.80.10-3 1.30.10-1 1.21
6.31 4.88 4.6
(log
2. Lipolysis Inhibition A significant inhibitory effect on epinephrine-induced FFA release (P < 0.01) was achieved with 1 mg/l B[a]P concentration (Figure 3A). The estimated Kd value for B[a]P inhibition of lipolysis is 0.9 mg/l (3.5 µM). This value is in the same range as that measured for the known β blocker atenolol (Kd = 0.4 mg/l or 1.5 µM for β1-adrenoreceptor and 2.3 mg/l or 8.6 µM for β2-adrenoreceptor) [6]. The inhibitory effect of 1.8 mg/l of B[a]P on epinephrine-induced lipolysis was detectable within 5 min of preincubation (Figure 3B). This suggests that B[a]P directly affects the cellular signal transduction pathway rather than affecting gene expression. However, for the two C4 PAHs (pyrene and phenanthrene), no effect was observed on the epinephrine-induced FFA release (Figures 4A and 4B).
Figure 3. Effect of B[a]P on the release of FFA from mice adipocytes. Freshly isolated mice adipocytes were incubated (A) for 45 min with different concentrations of B[a]P and (B) during different preincubation periods with a predetermined concentration of 2.5 mg/l (10 µM) of B[a]P. Epinephrine (1.8 mg/l i.e. 10 µM) was then added and the incubation continued for 45 min. Each point represents the mean ± SEM of the 4 experiments during which FFA levels were measured in duplicate.
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Figure 4. Effect of the two C4 PAHs, pyrene and phenanthrene on the release of FFA from mice adipocytes. Freshly isolated mice adipocytes were incubated for 45 min with different concentrations of Pyrene (A) or Phenanthrene (B). Epinephrine (1.8 mg/l i.e. 10 µM) was then added and the incubation continued for 45 min. Each point represents the mean ± SEM of 4 experiment during which FFA levels were measured in duplicate.
DISCUSSION B[a]P is a well-known ubiquitous carcinogen and consumption of B[a]P-rich foods have been suggested to contribute to the overall cancer burden affecting human populations [7]. The carcinogenicity of B[a]P is well-documented. This pollutant is metabolized via the cytochrome P450 system into reactive dihydrodiol epoxide derivatives (e.g. B[a]P-7,8dihydrodiol-9,10-epoxide, BPDE). These metabolites bind covalently to DNA and form BPDE adducts, that may lead to mutations and consequently to uncontrolled cell growth and tumour formation in various tissues (lung adenocarcinoma, mammary adenocarcinoma, lymphoma and hepatocellular carcinoma) [8]. B[a]P has also been shown to display immunotoxic properties that affect macrophage function [9] and increase local inflammatory response which may contribute to increased atherosclerotic lesion size [10]. In this paper, we show that in ex vivo experiments, B[a]P can inhibit epinephrine-induced FFA release whereas the two C4 PAHs pyrene and phenanthrene cannot. This confirms our previous observation that the administration of a single dose of B[a]P (0.5 mg/kg in IP) in male C57BL6/J mice induces a significant reduction of the epinephrine-induced lipolysis. This phenomenon occurs in less than 30 minutes with maximum inhibition 24 hours later before returning to the basal lipolytic capacity levels 72 hours after the injection. In addition, we have shown that repeated intraperitoneal injections of B[a]P (0.5 mg/kg/48h) for 14 days induce a significant body weight gain (+43%, p>0.1) in mice without detectable changes in food consumption [2]. We previously proposed that the inhibition of lipolysis by B[a]P most likely results from physical perturbations of the plasma membrane phospholipid bilayer. Our interpretation is that B[a]P strongly and rapidly inhibits the signalling capacity of the β1-, β2-, β3-adrenergic receptors and of the ACTH receptors. These receptors share common features: all contain
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seven transmembrane spanning domains and are coupled to G-proteins which are themselves anchored to the inner leaflet of the plasma membrane. In contrast atrial natriuretic peptideinduced lipolysis via the stimulation of natriuretic peptide receptor A, that contains a single transmembrane-spanning domain, was not affected by B[a]P [2] Yet, physicochemical studies using differential scanning calorimetry, infrared spectroscopy and small-angle X-ray diffraction have shown that B[a]P incorporates into phospholipid bilayers and localizes in the most apolar region of the phospholipid matrix. This phenomenon may account for the observation of an expanded and swollen membrane [11]. We have therefore, proposed that distortion of the physiochemical properties of the adipocyte plasma membrane by B[a]P decreases the signalling capacity of G-coupled receptors intimately linked to the phospholipid bilayer, via their seven transmembrane domains. Our data tends to show that the capacity of PAHs to incorporate into adipocytes depends upon their Coeficient Octanol Water (Kow). We therefore suggest that because the capacity of lipophilic molecules to be incorporated into the phospholipids bilayer depends on their Kow, the lack of lipolysis-inhibiting effect of the two C4 PAH pyrene and phenanthrene might be related to their low Kow. A striking observation in our study is that the concentrations needed to achieve the B[a]Pinduced lipolysis inhibition effect are 2000-fold lower than those causing carcinogenesis in rodents and 20-fold lower than those causing alterations of the epidermal growth factor receptors in cultured human uterine cells, RL95-2 [12]. Another observation is that B[a]P can directly inhibit epinephrine-induced lipolysis, whereas in order to adduct DNA and produce mutagenic and carcinogenic effects, B[a]P needs to be activated through the CYP1A1 cytochrome P450 process. It has been shown that in the ob⁄ob obese mouse model, CYP1A1 levels are decreased [13]. We thus assume that because B[a]P contribute to the hypertrophy/hyperplasia of the adipose tissue, it may simultaneously increase its bioaccumulation in this tissue by reducing its metabolic bioactivation. Since pyrene and phenanthrene are also activated through CYP1A1, a similar process of storage in the adipose tissue may also concern these two C4 PAHs, so B[a]P might in fact generate the storage in the adipose tissue of both types of mutagenic and promoting PAHs. In our study, all three PAHs incorporated adipocytes in a similar manner. Our results are similar to those found by Victor, who obtained a complete incorporation of the 2,3,7,8-TCDD in hepatocytes suspension within 2 minutes [14]. These data reinforce the concept that adipose tissue may act as a reservoir for many different lipophilic exogenous chemicals including PAHs. This concept is opposed to the hypothesis according to which adipose tissue might be a ‘‘protective reservoir’’ [15]. This assumption was based on the opinion that because adipose tissue is able to bioaccumulate chemical pollutants, it might protect the organism against their deleterious effects [15-18] Indeed, we believe that this concept needs to be revised. Following environmental exposure to low-dose chemical pollutants during many years, accumulation of carcinogens in the adipose tissue might in fact correspond to a stronger exposure, as it is observed in hot spot polluted area. On the basis of many experimental toxicological data, it is assumed that prolonged chronic exposure to low dose xenochemicals may in fact lead to carcinogenesis, as it is the case for occupational cancers during hotspot exposure [19, 20]. As a result of this new concept, it clearly appears that the carcinogenic effects of environmental xenochemicals, be they mutagens and/or promoters may be the consequence of a chronic exposure to repetitive lower doses than those at which no effect level is observed in classical
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rodent tests. This concept is based on the fact that many exogenous chemical carcinogens are persistent organic pollutants accumulating in fat and that they can be released in the organism from adipose tissue at doses which do not correspond to those observed in the environment. Therefore, during prolonged environmental exposure, cancer may be induced by repetitive lower doses than it is usually expected [20, 21]. Furthermore, it has been shown that mixtures of pollutants may have synergic effects and that some types of ‘‘cocktail effects’’ that occur in adipose tissue may contribute to carcinogenesis [22] and/or cancer cell proliferation [23]. Since our study shows that C4 as well as C5 PAHs can incorporate into adipocytes, we hypothesize that PAH cocktails that bioaccumulate in the adipose tissue may be equivalent to complete carcinogens (Figure 5). As summarized in figure 5, we believe that the concept of chronic toxicity must also be totally revised in the light of the role of reservoir of the adipose tissue for lipophilic carcinogens. Even in lean people, white adipose tissue (WAT) represents about 15 to 25 % of body weight, and this percentage can increase by more than 50 % in the case of obese patients. Environmental lipophilic pollutants can enter the organism via food, water and air, bioaccumulate and often concentrate in the adipose tissue, to be permanently released in the blood circulation and elicit their effect in peripheral tissues at convenient doses according to their potential biologic effects. The amount of pollutants that bioaccumulate in WAT may depend on dietary exposure and on individual susceptibility [24]. The postnatal stock of innate preadipocytes, the volume of WAT and the capacity of the organism to assure energy homeostasis might be risk factors, inter alia. Also weight loss may result in an increased concentration of pollutants in reduced WAT [25] and in their decreased elimination rate [26]. Finally we conclude that in addition to its endocrine properties, the adipose tissue can act as a reservoir for many exogenous chemical molecules including PAHs and that B[a]P may play a pivotal role in carcinogenesis not only by inducing cancer through its mutagenic carcinogenic effects but also by increasing the bioaccumulation capacity of the adipose tissue mass.
Figure 5. Adipose tissue as a reservoir for PAHs. Hypothetic mechanism according to which repeated low doses of PAHs can induce carcinogenesis.
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[16] Geyer, HJ; Schramm, KW; Scheunert, I; Schughart, K; Buters, J; Wurst, W; et al. (1997). Considerations on genetic and environmental factors that contribute to resistance or sensitivity of mammals including humans to toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Part 1: Genetic factors affecting the toxicity of TCDD. Ecotoxicol Environ Saf , 36, 213-230. [17] Geyer, HJ; Scheuntert, I; Rapp, K; Kettrup, A; Korte, F; Greim, H; et al. (1990). Correlation between acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and total body fat content in mammals. Toxicology, 65, 97-107. [18] Geyer, HJ; Scheunert, I; Rapp, K; Gebefugi, I; Steinberg, C; Kettrup, A. (1993). The relevance of fat content in toxicity of lipophilic chemicals to terrestrial animals with special reference to dieldrin and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ecotoxicol Environ Saf, 26, 45-60. [19] Belpomme, D; Irigaray, P; Hardell, L; Clapp, R; Montagnier, L; Epstein, S; Sasco, AJ. (2007). The multitude and diversity of environmental carcinogens. Environ Res, 105, 414-429. [20] Howard, CV; Newby, JA. (2004). Could the increase in cancer incidence be related to recent environmental changes?. In: Nicolopoulou-Stamati P, Hens L, Howard CV, Van Larenbeke L, editors. Cancer as an environmental disease. Dordrecht, The Netherlands: Kluwer Academic Publishers; 2004. p. 39-56. [21] Hutcheon, DE; Kantrowitz, J; Van Gelder, RN; Flynn, E. (1983). Factors affecting plasma benzo[a]pyrene levels in environmental studies. Environ Res, 32, 104-110. [22] Knerr, S; Schrenk, D. (2006). Carcinogenicity of ‘‘non-dioxinlike’’ polychlorinated biphenyls. Crit Rev Toxicol, 36, 663-694. [23] Payne, J; Scholze, M; Kortenkamp, A. (2001). Mixtures of four organochlorines enhance human breast cancer cell proliferation. Environ Health Perspect, 109, 391-397. [24] Smeds, A; Saukko, P. (2001). Identification and quantification of polychlorinated biphenyls (PCBs) and some endocrine disrupting pesticides in human adipose tissue from Finland. Chemosphere, 44, 1463-1471. [25] Jandacek, RJ; Anderson, N; Liu, M; Zheng, S; Yang, Q; Tso, P. (2005). Effects of yoyo diet, caloric restriction, and olestra on tissue distribution of hexachlorobenzene. Am J Physiol, 288, G292-G299. [26] MichalekK, JE; Tripathi, RC. (1999). Pharmacokinetics of TCDD in veterans of operation ranch hand: 15-year follow-up. J Toxicol Environ Health, 57, 369-378.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 16
CHLORINATE POLYCYCLIC AROMATIC HYDROCARBONS: A NEW CLASS OF ENVIRONMENTAL TOXIN? Takeshi Ohura∗ and Hiroyuki Sakakibara Institute for Environmental Sciences, University of Shizuoka, Japan
ABSTRACT A review has provided the environmental sources and behavior of chlorinated 3- to 5-ring polycyclic aromatic hydrocarbons (ClPAHs). ClPAHs have been known to be ubiquitous contaminants, which can be found in urban air, vehicle exhaust gas, snow, tap water and sediments. The concentrations in the each environment are generally higher than those of chlorinated dioxins, although which are significantly lower than the concentrations of parent compounds, PAHs. Based on the environmental data and emission sources analysis for the ClPAHs, the dominant production process is by incineration of organic materials including chlorine like as the case of dioxins. On the other hand, treatment of PAHs with chlorine in water also produced ClPAHs. Indeed, such secondary reaction for ClPAHs generation could occur in the gaseous phase. For the biological effects, the toxicities such as mutagenicity and aryl hydrocarbon receptor activity of certain ClPAHs are emphasized than those of parent PAH, in which the toxicities of exposure to ClPAHs based on AhR activity were estimated to be approximately 30-50 times higher than that of dioxins. To investigate the sources and environmental behavior of ClPAHs is of great importance to assess human health risks.
∗ Address correspondence to T. Ohura,Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.Telephone: +81 54 264 5789. Fax: +81 54 264 5798.E-mail:
[email protected] Takeshi Ohura and Hiroyuki Sakakibara
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INTRODUCTION To date, the studies on the environmental behaviors and biological effects of PAHs have been carried out in the world, and the numbers of papers are more than 10,000. On the other hand, those studies on chlorinated congeners, ClPAHs, are very limited [1]. One of the reasons will be due to the lack of purified, individual ClPAH analytical standard. Recently, Ohura et al. succeeded to synthesize 12 individual ClPAHs with 3 to 5 ring as analytical standards (Figure 1) [2]. It has been advanced the studies on environmental behaviors and biological effects of ClPAHs. This section outlines the latest advance of ClPAHs study. Cln Cln
Cln Cln
chloroanthracen e (n=1−2)
chlorophenanthrene (n=1−3)
chlorofluoranthene (n=1−2)
chlorochrysene (n=1−2)
Cl
Cln 1-chloropyrene
Cl
chlorobenz[a]anthracene 6-chlorobenzo[a]pyrene (n=1−2)
Figure 1. Structures of ClPAH derivatives.
Occurrences of Clpahs in the Environment The first report of environmental ClPAHs will be on the survey of tap water performed by Shiraishi et al., which were performed as long ago as 1980s [3]. In this paper, relatively low molecular weight ClPAHs (< 4-ring) such as chlorophenanthrene and chloropyrene derivatives have been detected, which have been suggested to produce during chlorine treatment of water as the possible production mechanism. Indeed, Oyler et al. have confirmed to be caused production of low molecular weight ClPAHs by process of chlorine disinfection in water [4, 5]. Some time later, Nilsson et al. have reported that ClPAHs with 3 to 5-ring were present in both gas and particle phase in urban street and road tunnel air [6]. Based on the profile analysis, the authors suggested that the ClPAHs were formed in secondary reactions with PAH and leaded gasoline scavenger containing dichloroethane. In the mid2000s, Ohura and co-workers have been carrying on the studies on atmospheric behaviors and sources of ClPAHs [2, 7-9]. In a Japanese urban air, 26 species of ClPAHs have been detected in either the gaseous or particulate phase. The ambient (gaseous + particulate) concentration of total ClPAH ranged from 18 to 330 pg/m3 [8]. Those ClPAHs levels tended to be approximately ~1000 times lower than PAHs. A seasonal variability of both gaseous and particulate ClPAHs concentrations was observed, with higher concentrations in winter
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than in summer, which was different profile from PAH. Only under the study, the natural logarithm of total concentrations of particulate ClPAHs were significantly correlated (p < 0.05) with the reciprocal mean temperatures, but not for gaseous ClPAHs. It implies that the gaseous ClPAHs, rather than particulate ClPAHs, would be affected by various factors such as long-range transport, photodecay reactions, and sources. According to factor analysis using particulate ClPAH in addition to particulate PAH concentrations, the predominant source of particulate ClPAHs was suggested to be incineration. Indeed, Ohura and Kannan group have been vigorously carried out the analysis of possible emission sources for ClPAHs, and detected much of ClPAHs in fly ash and bottom ash from waste incinerators [10, 11]. With regard to the occurrences of ClPAHs in other environments such as soil and sediment, there is not report, yet.
Biological effects of ClPAHs AhR activity A majority of the adverse effects of xenobiotics such as PAHs and dioxins is thought to be mediated through AhR activation. Therefore, to investigate AhR-mediated activity of ClPAHs will be important to assess the exposure risks. Ohura et al. have investigated the AhR activities of 18 ClPAHs with 3 to 5 rings using the yeast assay system and compared the potencies to the corresponding PAHs [12]. All ClPAHs tested showed appreciable dosedependent increases in AhR-mediated reporter gene expression. Among the ClPAHs, 3,8dichlorofluoranthere was the most potent AhR ligand, followed by 6-chlorochrysene and 7chlorobenz[a]anthracene, 3,9,10-trichlorophenanthrene, having activities that were about 6, 2, and 0.8 times higher than that of benzo[a]pyrene.
AhR-mediated cross-talk In the addition of AhR activation described above, some xenobiotics such as dioxins and 3-methylcholanthrene have been recently reported to stimulate estrogen response elements (EREs)-mediated transcription without binding directly to estrogen receptors (ERs) or affecting expression levels of ERs, and consequently exert estrogen-related effects [13]. These activations were verified to be exerted via directly association of ligand-binding AhR/Arnt heterodimer with ERs, so-called AhR-ER cross-talk. One of our recent topics is whether ClPAHs affect AhR-ER cross-talk pathway or not. As reported by many researchers including our group [12, 14], phenanthrene itself have vastly weak effect on AhR activation. On the other hand, mono-, di-, and tri-chlorination increased the AhR activation and consequently activate the transcription of genes related to the drug-metabolizing enzymes such as cytochrome P-450s with the number of chlorine atom on the phenanthrene skelton [12]. However, these chlorinated phenanthrene derivatives did not affect the gene expression of cathepsin D, which is one of the target genes expressed via ER pathway, in spite of there ligand activity to AhR. These results suggest that estrogenic action mediated ER signaling through AhR activation could not necessarily occur in every AhR activated ligands. We are now under investigation on AhR-ER cross-talk activity of other chlorinated PAHs, such as chlorinated benzo[a]pyrene, and also other halogenated PAHs.
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Risk of Exposure to Clpahs To evaluate the risk of human exposure to ClPAHs, it is important to demonstrate the relationship between residue levels in the environment and toxicity. Most of the adverse effects of halogenated aromatic compounds are thought to be mediated through AhR activation. Therefore, the toxic potencies of these compounds can be expressed in terms of relationship between congener-specific concentrations and a relative potency (REP), which is specific to an experiment, or a TCDD equivalency factor (TEF), which is consensus-derived from many relative potency values. Ohura et al. established the REP values of each ClPAH relative to BaP (REPBaP) in a yeast assay system [12]. In a similar system, the intensity of the AhR-associated toxicity of TCDD was 60-fold higher than that of BaP [15]. Therefore, the overall toxicity or toxic equivalents (TEQs) of a mixture of ClPAHs relative to TCDD can be defined as follows: TEQ =∑[Ci] × REPBaPi / 60
(1)
where Ci represents the concentration of an individual ClPAH. For air samples collected from a Japanese urban city, the calculated TEQ concentration of total ClPAHs based on equation 1 was 1.18 pg-TEQ/m3, whereas the TEQ concentrations of dioxins ranged from 0.024 to 0.043 pg-TEQ/m3 in 2005 [12]. Thus, it shows that the toxicity of exposure to ClPAHs would be approximately 30 to 50 times higher than that of dioxins. On the other hand, the exposure risk of ClPAHs and PAHs in the air was also calculated using the individual REPBaP and corresponding mean concentrations obtained from ambient particles in Japanese urban air [16]. The calculated TEQ concentrations of total ClPAHs and PAHs were 2.95 and 650 pg-TEQ/m3, respectively, indicated that the contribution of ClPAHs to PAHs was less than 1 %.
Genotoxicity of ClPAHs The study on genotoxicity of ClPAHs had long been performed in comparison to other biological effects as well as the environmental surveys. The first report will be to investigate the mutagenicity of ClPAHs using Salmonella typhimurium TA98 and TA100 in the presence or absence of S9 activation enzyme system [17]. In the study, some ClPAHs such as 1chloropyrene showed the direct mutagenicity, which was apparently different pattern from parent PAH, pyrene. The reason of such toxic activation occurs remain unclear. Fu et al. have investigated the tumogenicity of ClPAH, especially focused on 7-chlorobenz[a]anthracene (7ClBaA), indicated that 7-ClBaA are potent carcinogen and that bay-region diol epoxides are the ultimate metabolites that lead to DNA adduct formation and tumor initiation [18, 19]. Conversely, 6-chlorobenzo[a]pyrene (6-ClBaP) reduced the level of mammary epithelial tumors of female Sprague-Dawley rats, compared to parent PAH, BaP [20]. It was suggested that the weak activity of 6-ClBaP could be due to poor substrate for metabolism by cytochrome P-450.
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CONCLUSION The risk assessments based on relative potencies of each AhR activity showed that ambient ClPAHs was extremely low contributors, as well as order of the ambient concentrations. In general, the significant contributions of PAHs to AhR activities in the environmental samples have been often observed in the assessment of persistent organics from sediments, which were 5~50 times order higher than that of polychlorinated dibenzo-pdioxins and dibenzofurans (PCDD/Fs). Similarly, the contribution of PCBs to the total TEQ in sediment was less than 1%, PAHs occupied the considerable portion, whereas PCBs accounted for a greater proportion (> 95%) of total TEQs in coastal and tidal flatfishes. PCDD/Fs and PCBs are extremely persistent and bioaccumulative compared to PAHs. Although metabolic characteristics of halogenated PAHs remain unknown, they are expected to be more lipophilicity and be difficult to metabolize rather than origin PAHs. Thus, such ClPAHs may be accumulated at high level in biological tissues, and exert the toxic action. In order to reveal the toxicities of ClPAHs, further studies concerning the metabolic dispositions will be needed.
REFERENCES [1] [2]
[3] [4]
[5]
[6]
[7]
[8]
Ohura, T. (2007) Environmental behavior, sources, and effects of chlorinated polycyclic aromatic hydrocarbons. ScientificWorldJournal. 7, 372-380. Ohura, T.; Kitazawa, A.; Amagai, T. & Makino, M. (2005) Occurrence, profiles, and photostabilities of chlorinated polycyclic aromatic hydrocarbons associated with particulates in urban air. Environ. Sci. Technol. 39, 85-91. Shiraishi, H.; Pilkington, NH.; Otsuki, A. & Fuwa, K. (1985) Occurrence of chlorinated polynuclear aromatic hydrocarbons in tap water. Environ. Sci. Technol. 19, 585-590. Oyler, AR.; Liukkonen, RJ.; Lukasewycz, MK.; Cox, DA.;Peake, DA. & Carlson, RM. (1982) Implications of treating water containing polynuclear aromatic hydrocarbons with chlorine: A gas chromatographic-mass spectrometric study. Environ. Health Perspect. 46, 73-86. Oyler, AR.; Liukkonen, RJ.; Lukasewycz, MT.; Helkklla, KE.; Cox, DA. & Carison, RM. (1983) Chlorine "disinfection" chemistry of aromatic compounds. Polynuclear aromatic hydrocarbons: Rates, Products, and Mechanisms. Environ. Sci. Technol. 17, 334-342. Nilsson, UL. & Ostman, CE. (1993) Chlorinated polycyclic aromatic hydrocarbons: Method of analysis and their occurrence in urban air. Environ. Sci. Technol. 27, 18261831. Kitazawa, A.; Amagai, T. & Ohura, T. (2006) Temporal trends and relationships of particulate chlorinated polycyclic aromatic hydrocarbons and their parent compounds in urban air. Environ. Sci. Technol. 40, 4592-4598. Ohura, T.; Fujima, S.; Amagai, T. & Shinomiya, M. (2008) Chlorinated Polycyclic Aromatic Hydrocarbons in the Atmosphere: Seasonal Levels, Gas-Particle Partitioning, and Origin. Environ. Sci. Technol. 42, 3296-3302.
468 [9] [10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
Takeshi Ohura and Hiroyuki Sakakibara Ohura, T.; Kitazawa, A. & Amagai, T. (2004) Seasonal variability of 1-chloropyrene on atmospheric particles and photostability in toluene. Chemosphere. 57, 831-837. Horii, Y.; Ok, G.; Ohura, T. & Kannan, K. (2008) Occurrence and Profiles of Chlorinated and Brominated Polycyclic Aromatic Hydrocarbons in Waste Incinerators. Environ. Sci. Technol. 42, 1904-1909. Ohura, T.; Kitazawa, A.; Amagai, T. & Shinimiya, M. (2007) Relationships between chlorinated polycyclic aromatic hydrocarbons and dioxins in urban air and incinerators. Organohalogen Compd. 69, 2902-2905. Ohura, T.; Morita, M.; Makino, M.; Amagai, T. & Shimoi, K. (2007) Aryl hydrocarbon receptor-mediated effects of chlorinated polycyclic aromatic hydrocarbons. Chem. Res. Toxicol. 20, 1237-1241. Ohtake, F.; Takeyama, K.; Matsumoto, T.; Kitagawa, H.; Yamamoto, Y.; Nohara, K.; Tohyama, C.; Krust, A.; Mimura, J.; Chambon, P.; Yanagisawa, J.; Fujii-Kuriyama, Y. & Kato, S. (2003) Modulation of oestrogen receptor signalling by association with the activated dioxin receptor. Nature. 423, 545-550. Lampen, A.; Ebert, B.; Stumkat, L.; Jacob, J. & Seidel, A. (2004) Induction of gene expression of xenobiotic metabolism enzymes and ABC-transport proteins by PAH and a reconstituted PAH mixture in human Caco-2 cells. Biochim. Biophys. Acta. 1681, 3846. Kawanishi, M.; Sakamoto, M.; Ito, A.; Kishi, K. & Yagi, T. (2003) Construction of reporter yeasts for mouse aryl hydrocarbon receptor ligand activity. Mutat. Res. 540, 99-105. Ohura, T.; Sawada, K.; Amagai, T. & Shinomiya, M. (2009) Atmospheric behaviors and exposure risks of novel halogenated polycyclic aromatic hydrocarbons associated with particulates. submitted. Colmsjo, A.; Rannug, A. & Rannug, U. (1984) Some chloro derivatives of polynuclear aromatic hydrocarbons are potent mutagens in Salmonella typhimurium. Mutat. Res. 135, 21-29. Fu, PP.; Von Tungeln, LS.; Zhan, D-J. & Bucci, T. (1996) Potent tumorigenicity of 7chlorobenz[a]anthracene and 7-bromobenz[a]anthracene in the neonatal B6C3F1 male mouse. Cancer Lett. 101, 37-42. Xia, Q.; Yi, P.; Zhan, D-J.; Von Tungeln, LS.; Hart, RW.; Heflich, RH. & Fu, PP. (1998) Liver tumors induced in B6C3F1 mice by 7-chlorobenz[a]anthracene and 7bromobenz[a]anthracene contain K-ras protooncogene mutations. Cancer Lett. 123, 2125. Cavalieri, E.; Rogan, E.; Cremonesi, P.; Higginbotham, S. & Salmasi, S. (1988) Tumorigenicity of 6-haloganated derivatives of benzo[a]pyrene in mouse skin and rat mammary gland. J. Cancer Res. Clin. Oncol. 114, 10-15.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN: 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 17
RECENT TRENDS IN ANALYTICAL METHODS TO CHEMICALLY MONITOR PAH POLLUTION Verónica Pino Department of Analytical Chemistry, Nutrition and Food Science, University of La Laguna, Spain
ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic compounds with two or more fused aromatic rings. They have received increased attention in recent years in pollution studies because some of these compounds are highly carcinogenic or mutagenic. Some, while not carcinogenic, may act as synergists. These compounds are widespread in the environment. Their occurrence is largely a result of anthropogenic and natural emissions such as fossil fuel burning, motor vehicles, waste incinerators, oil refining, coke and asphalt production, aluminum production, etc. PAHs have relatively low solubility in water but are highly lipophilic. Most of the PAHs with low vapor pressure in the air are adsorbed on particles. When dissolved in water or adsorbed on particulate matter, PAHs can undergo photodecomposition when exposed to UV light. In the atmosphere, PAHs can react with many other pollutants such as ozone, nitrogen oxides and sulfur dioxide, yielding diones, nitro- and dinitro-PAHs, and sulfonic acids, respectively. PAHs may also be degraded by some microorganisms in soils. PAHs in soils/sediments can be taken up by biota, and some bio-magnification of these toxic compounds may occur as they pass through the food chain. The US Environmental Protection Agency (EPA) has identified 16 un-substituted PAHs as priority pollutants. The European Food Security Authority (EFSA) has classified 15 PAHs by their food toxicity. Thus, potential exposure to PAHs must be assessed by testing contaminated soils, sediments, air, water and foods, and estimating the degree of intake of each of these media into the human body, as well as the overall toxicity.There are several main problems associated with the determination of PAHs in environmental and food samples: their low concentrations, the complexity of the samples, and the necessity for fast and reliable analytical methods. To achieve successful results, many extraction-separation techniques have been developed depending on the nature of the sample. An in-depth description of the most commonly-used techniques, together with recent trends in analytical methods to monitor PAHs levels in different kind of samples, will be the main
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Verónica Pino focus of this chapter. Among those that will be cited are Soxhlet extraction, liquid-liquid extraction (LLE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), pressurized fluid extraction (PFE), supercritical fluid extraction (SFE), solidphase extraction (SPE), and solid-phase microextraction (SPME), mainly in combination with chromatographic techniques (HPLC and GC). Moreover, the utilization of active or passive samplers in pollution monitoring, as well as the increased utilization of semipermeable membrane passive samplers (SPMDs), will be described.
1. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) refers to a group of a few hundred organic compounds consisting of two or more condensed benzene rings that can incorporate rings with an odd number of carbon atoms, sharing at least two atoms in each pair of contiguous rings. Table 1 shows several of the most significant PAHs. In general, PAHs are originated during the incomplete combustion of organic matter in natural processes (i.e., forest fires, volcanoes, and hydrothermal processes) [1,2] and in anthropogenic activities (fossil fuel burning, motor vehicles, waste incinerators, oil refining, coke and asphalt production, aluminum production, heat, food processing, etc.) [3-5]. The formation mechanism of PAHs involves several steps, not well established, but probably related with pyrolysis and pyrosynthesis reactions. It has been established that the rupture of organic matter at high temperatures in combustion processes forms radicals of low molecular weight, which later will recombine by pyrosynthesis to form PAHs [6]. In 1973, Crittenden et al. [7] proposed a PAH formation mechanism in which the acetylene molecules react to form unsaturated radicals C4, C6, C8 and C2n. These species can interact to form polyacetylenes, or can suffer a ring closure reaction to form radicals constituted by an aromatic ring attached to a 2-carbon chain (phenyl-acetylene or styrene). PAHs are then formed by reactions involving the radicals phenyl-acetylene and styrene. In 1983, Bockhorn et al. [8] established a similar mechanism, but proposed that the phenyl-acetylene is formed by the reaction between acetylene and 1,3-butadiene, as can be observed from Figure 1. The authors described in depth the different formation reactions of PAHs starting with phenyl-acetylene. Other authors have suggested the formation of PAHs starting with cyclopentadienyl radicals [9,10]. In spite of the difficulty in establishing the formation mechanism of PAHs [3], it can be ensured that, in general, the first PAH to be formed is naphthalene, mainly due to its high volatility and stability. Heavier PAHs, especially the ones with higher thermodynamic stability, are formed by addition of acetylene or by intra-molecular ring closure of lighter PAHs when the combustion temperature is increased [9-12].
Table 1. Names and structures of several significant PAHs. PAH (Abbreviation)
Structure
PAH (Abbreviation)
Structure
PAH (Abbreviation)
Structure
Naphthalene (N)
5-Methylchrysene (5-MeChy)
Anthracene (A)
Me
Fluoranthene (Ft)
Acenaphthene (Ace)
Acenaphthylene (Acy)
Benzo(b)fluoranthene (B(b)Ft)
Benzo(k)fluoranthene (B(k)Ft)
Pyrene (Py)
Fluorene (Fl)
Benzo(j)fluoranthene (B(j)Ft)
Benzo(a)anthracene (B(a)A)
Phenanthrene (Phe)
Chrysene (Chy)
Perylene (Per)
Table 1 (Continued) Names and structures of several significant PAHs. PAH (Abbreviation)
Structure
PAH (Abbreviation)
Benzo(e)pyrene (B(e)Py)
Structure
PAH (Abbreviation)
Structure
Dibenzo(a,c)anthracene (diB(a,c)A)
Dibenzo(a,l)pyrene (diB(a,l)Py)
Benzo(a)pyrene (B(a)Py)
Dibenzo(a,e)pyrene (diB(a,e)Py)
Dibenzo(a,h)anthracene (diB(a,h)A)
Dibenzo(a,h)pyrene (diB(a,h)Py)
Indeno(1,2,3-cd)pyrene (I(1,2,3-cd)Py)
Dibenzo(a,i)pyrene (diB(a,i)Py)
1-Hydroxypyrene (1-OHPy)
Cyclopenta(c,d)pyrene (cyPent(c,d)Py)
Benzo(ghi)perylene (B(ghi)Per)
OH
Recent Trends in Analytical Methods to Chemically Monitor…
· C 2H
CH
H
CH
·C
C
C
C
C
CH
CH
+ C 2 H2
+
CH
H C
C
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CH
C
C
C
HC ·
C
C
·
CH
· CH CH C
CH
CH + C 2 H2
·
·
C
·
+ C 2 H2
CH
C H -· C 2 H
- H·
PAHs of higher molecular weight
Figure 1. Formation mechanism of PAHs proposed by Bockhorn et al. [8].
1.1. Physicochemical Properties PAHs are compounds of high chemical stability, due to the conjugated systems of π orbital. Table 2 shows some physical properties of several significant PAHs. PAHs are in general solids at room temperature. Many of them can be classified as semivolatile organic compounds. They are soluble in many different organic solvents, but practically nonsoluble, or slightly soluble, in water. Nevertheless, the aqueous solubility can be increased by the presence of detergents, salts, fatty acids, or organic solvents [13]. In the same manner, the aqueous solubility can be three times higher when increasing the temperature. Their physicochemical properties, especially nonpolarity and hydrophobicity, are responsible for their persistence in the environment. Their lipophilicity justifies the bioaccumulation of these compounds in the lipid tissues of living organisms. Table 2. Physicochemical properties of several PAHs PAHs Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene
Vapor pressure (Pa) 10.4 0.90 0.30 0.09 0.02 1.0·10-3 1.2·10-3
Boiling point (ºC) 218.0 270.0 277.5 295.0 339.0 340.0 375.0
Solubility (mg·L-1) 31.0 16.1 3.80 1.90 4.57 0.05 0.26
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Table 2 (Continued) Physicochemical properties of several PAHs PAHs Pyrene Benz(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Dibenzo(a,h)anthracene Benzo(g,h,i)perylene Benzo(j)fluoranthene Cyclopenta(c,d)pyrene Dibenzo(a,e)pyrene Dibenzo(a,h)pyrene Dibenzo(a,i)pyrene Dibenzo(a,l)pyrene 5-Methylchrysene
Vapor pressure (Pa) 6.0·10-4 2.8·10-5 5.7·10-7 6.7·10-8 5.2·10-8 7.0·10-7 1.4 10-8 3.7·10-10 1.3·10-11 2.4·10-6 2.4·10-5 1.5·10-9 1.5·10-9 1.5·10-9 1.5·10-9 1.0·10-5
Boiling point (ºC) 360.0 435.0 407.0 481.0 481.0 495.0 536.0 524.0 525.0 480.0 438.3 552.3 552.1 552.1 552.1 449.4
Solubility (mg·L-1) 0.13 0.01 9.6·10-3 1.5·10-3 8.0·10-4 3.8·10-3 1.9·10-3 6.0·10-4 2.6·10-4 5.0·10-3 0.014 2.7·10-4 2.7·10-4 2.7·10-4 2.7·10-4 9.7·10-3
1.2. Carcinogenic Effects The first evidence of the carcinogenic effect of PAHs was reported in 1775, when the British medical doctor Percival Pott (1714–1788) pointed out the relationship between the scrotum cancer in chimney sweeps and their long-term exposure to tar and soot. In 1930 it was demonstrated that the PAH benzo(a)pyrene, contained in the soot, was carcinogenic [14]. Up to the present, several studies have evaluated the risk for cancer development in humans exposed to PAHs via the lungs or dermis [15-17]. Recent epidemiological studies suggest that PAH-DNA adduct formation in blood and breast tissue may play an important role in breast cancer development [18]. Given their carcinogenic characteristics, several agencies have included PAHs in their list of priority pollutants. The US Environmental Protection Agency (EPA) has selected 16 PAHs as contaminants of high priority. In the same fashion, the Food Scientific Committee of the European Union (FSC), now named the European Food Security Authority (EFSA), has selected 15 PAHs by their potential toxicity in relation to foods. In 2002, the former European Commission’s Scientific Committee on Food (SCF) considered the study of the 33 PAHs included in an Environmental Health Criteria document on PAHs [19]. Not all PAHs have been demonstrated to be carcinogenic. In many instances, the carcinogenic effect is in fact due to the presence of more than one member of the PAH family, as well as several of their derivatives. Hence, some PAHs, while not carcinogenic, may act as synergists. Thus, the implication of human exposure to mixtures of PAHs, rather than to individual substances, is important. The toxicity equivalent factor (TEF) is a useful tool to quantitatively estimate the overall carcinogenic effect of an environmental sample containing several PAHs. Based on toxicological studies, a toxicity value is given to each PAH by comparison with a well-known carcinogenic PAH, benzo(a)pyrene, which has a
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reference TEF value of 1. The overall carcinogenic effect of a sample is then determined considering both the amount of each PAH in the sample and their respective TEF values. Table 3 shows several TEF values for a group of PAHs, proposed by different authors and/or agencies. It can be observed that hydrocarbons with a high number of condensed rings in the structures (between 5 and 6) are the ones with higher TEF values. The potential toxicity of a sample, expressed as equivalents of benzo(a)pyrene (B(a)Py), depends on the TEF values selected for each hydrocarbon. The carcinogenic and mutagenic activities of PAHs are closely related with their chemical structures. In general, planar PAHs are more stable and less toxic than non-planar PAHs. In addition to this, the introduction of an alkyl group into a PAH moiety generates a structural distortion of the hydrocarbon molecule, which can provoke activation or deactivation effects [20]. PAHs are apparently not carcinogenic by themselves, but they are converted into active carcinogens when metabolized by a living organism. The induction process of cancer in mammals involves a group of enzymes able to convert PAHs in water soluble compounds [21]. These oxydase enzymes of multiple functions belong to the group of cytochrome P450. As shown in Figure 2, PAHs are transformed into diol-epoxide adducts, followed by a rapid transformation in carbocations, able to covalently bind with DNA macromolecules, and therefore modifying the genetic message during the formation of new cells [22,23]. Similar reactions have also been observed with other macromolecules such as albumin and hemoglobin [20]. The presence of PAHs-DNA adducts in human tissues has been conclusively established [24]. A recent study has shown that chromosomal aberrations in cord blood are associated with prenatal exposure to carcinogenic polycyclic aromatic hydrocarbons [25]. There are various industrial workplaces which present a significant risk of certain cancer diseases. This may be attributed to an unusually high exposure to PAH [17]. Furthermore, as highly exposed occupational groups, chimney sweeps, roadmen (pavement-tarring) and roofers (roof-tarring) are also under increased risk [23]. OH O
OH
OH
OH
OH
OH
OH
OH
OH O
Macromolecule
Figure 2. Schematic representation of the metabolic activation of benzo(a)pyrene.
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Table 3. Toxicity equivalent factors proposed by different authors for the 16 PAHs listed by the EPA (numbered from 1 to 16) and for the 15 PAHs listed by the EFSA (numbered from 9 to 23). PAHs 1. Naphthalene 2. Acenaphthylene 3. Acenaphthene 4. Fluorene 5. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene 9. Benz(a)anthracene 10. Chrysene 11. Benzo(b)fluoranthene 12. Benzo(k)fluoranthene 13. Benzo(a)pyrene 14. Indeno(1,2,3-cd)pyrene 15. Dibenzo(a,h)anthracene 16. Benzo(g,h,i)perylene 17. Benzo(j)fluoranthene 18. Cyclopenta(c,d)pyrene 19. Dibenzo(a,e)pyrene 20. Dibenzo(a,h)pyrene 21. Dibenzo(a,i)pyrene 22. Dibenzo(a,l)pyrene 23. 5-Methylchrysene
Nisbet [26] 0.001 0.001 0.001 0.001 0.001 0.01 0.001 0.001 0.1 0.01 0.1 0.1 1 0.1 1 0.01 — 0.1 — — — — —
EPA [27] — — 0 — — — — — 0.1 0.001 0.1 0.01 1 0.1 1 — — — — — — — —
Kalberlah [28] — 0.01 0.001 0 0 0.01 0.01 0.001 0.1 0.01 0.1 0.1 1 0.1 1 0.01 0.1 — — — — — —
Cecinato [29] — — — — — — — — 0.06 — 0.07 0.07 1 0.08 0.6 — — — — — — — —
Larsen [30] — — — — 0.0005 0.0005 0.05 0.001 0.005 0.03 0.05 0.1 1 0.1 1.1 0.02 0.05 0.02 0.2 1 0.1 1 —
1.3. Environmental Distribution of PAHs Once PAHs are formed in the atmosphere, many of them can suffer photo-oxidation reactions with nitrogen oxides, ozone, and hydroxyl radicals, with reaction rates varying between hours and days [31]. PAHs, as semivolatile organic compounds, exist in both the gaseous and the particulate phase in air. The distribution of PAHs between the gas phase and the particulate matter (PM) is a complex process which depends on the temperature, the relative humidity, the composition of the particulate matter, and the nature of the PAH, mainly, the molecular weight and vapor pressure. In general, it has been demonstrated that PAHs of lower molecular weight tend to stay in the gas phase whereas PAHs of higher molecular weight tend to exist associated to the particulate matter, especially with particulate matter having diameters lower than 10 μm (PM10) [32,33]. In general terms, heavy PAHs are associated with the particulate matters by adsorption and absorption mechanism [34]. Independently on the sorption process that is taken place, the distribution constant is inversely dependent on the vapor pressure of the hydrocarbon.
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The presence of PAHs in the aquatic ecosystems is mainly as a result of anthropogenic inputs [35]. PAHs can be transported by direct effluents, lixiviation of soils, dry atmospheric deposition, and, mainly, humid atmospheric deposition (rain). For some watersheds, atmospheric deposition represents a significant input to the total surface water PAH burden, and may be independent of adjacent land use [36]. Once PAHs are in the aqueous medium, they can associate or bind to colloids, and so being transported in water. This justify the presence of PAHs in living organisms that are located in areas far away of an intensive industrial activity, and justify the fact that sediments are much richer in PAHs than the surrounding aqueous media [37-39]. Atmospherically derived PAHs in marine sediments have been linked to industrial and athropogenic sources [40,41]. The degradation of PAHs in sediments is in general a very slow process [42,43]. PAHs can accumulate in aquatic living organisms [44], entering into their biologic processes and affecting physiological lethal and sub-lethal levels [44,45]. Evidently, this effect is not isolated, because everything belongs to the ecological equilibrium. PAHs are metabolized by fishes [46] and into a lesser extent by mollusks or crustaceans, generating polar metabolites that can be excreted. In addition to this, the presence of PAHs may change the mechanisms of metal uptake by several plants, thus influencing kinetics and extent of metal phytoextraction [47]. It results clear that the biomagnification of PAHs into higher trophic levels across the food chains results into dispersal of pollutants far from their point of origin and from areas of significant human activity. Figure 3 illustrates a general scheme of the environmental distribution of PAHs. Atmosphere Particulate matter - Gas phase
Volatilization / Aerosolization
Dry/Wet deposition
Water
Soils
Vegetal
Colloids
Sediments
Biota
Human life
Figure 3. Environmental distribution of PAHs.
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1.4. Monitoring PAHs Given all these precedents, the wide environmental distribution and the carcinogenic risk associated to PAHs, it results necessary to monitor these contaminants in all kind of environmental samples, as well as to establish their impact in living organisms [48]. They must therefore be measured in the atmosphere for air quality assessment, in biological tissues for health-effects monitoring, in waters, sediments and mollusks/fishes for environmental monitoring, and in foodstuffs for safety reasons. To understand the extent of human exposure to PAHs, reliable sampling and analytical methods are necessary. The monitoring of PAHs in environmental samples presents several problems. One of the main limitations is related with the low concentration levels of PAHs to be monitored in the samples, with the consequent difficulty for being detected and quantified. Another limitation is related with the inadvertent errors during the sampling. In addition to this, the complexity of the samples, the undoubtedly presence of interfering substances, and the necessity of accurate and fast analysis, do the monitoring of PAHs an analytical challenge. In the analysis of PAHs, chromatographic techniques, mainly gas chromatography (GC) and liquid chromatography (LC), are often the preferred approach for separation, identification and quantification. Spectrofluorimetric techniques have also been used with screening purposes. In general, capillary GC is preferred over LC for the determination of PAHs mainly because of favorable combination of greater selectivity and resolution [49-51]. On the other hand, HPLC with fluorescence detection (FD) presents the best detection limits for PAHs [15]. Nevertheless, to reach the adequate sensitivity limits in gas chromatography or liquid chromatography, independently on the detection technique used (mass-spectrometry or fluorescence, respectively), it results necessary to carry out preconcentration procedures as well as clean-up steps [52]. The utilization of clean-up and preconcentration procedures introduces more sources of error in the analytical method, as well as increases the analysis time. The most common extraction-preconcentration procedures are Soxhlet extraction, liquid-liquid extraction (LLE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), pressurized fluid extraction (PFE), dialysis, solid-phase extraction (SPE), and solid-phase microextraction (SPME). The sampling is a critical step that must be carried out carefully, with the appropriate sampling design and sampling device. The increased utilization of semi-permeable membrane passive samplers (SPMDs) during the sampling stage of PAHs should be highlighted. The main aim of this chapter is to describe the most common procedures to monitor PAHs in environmental samples, with emphasis to the analytical methods used in extraction and determination procedures. This chapter is divided in analytical monitoring depending on the nature of the sample, though some methods turn out to be common for different samples.
2. AIR MONITORING The highest concentrations of atmospheric PAHs can be found in the urban environment, due to the traffic and the small dispersion of the atmospheric pollutants [15,53]. Hence, the cancer risk development in humans is much higher in the cities, considering the population
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density [54]. Sources of PAHs in urban atmosphere also include re-suspended soils, refineries and power plants [55-57]. Lighter PAHs, which have weaker carcinogenic effects by themselves, are the most abundant in the urban atmosphere; however, their presence is quite risky because they can react with other air pollutants to form more toxic derivatives [58], i.e., they can react with ozone, nitrogen oxides and sulfur dioxide, yielding diones, nitro- and dinitro-PAHs, and sulfonic acids, respectively [31]. Despite the efforts conducted to reduce urban particulate pollution in cities, the traffic still contributes to increase urban particulate pollution. Besides, particles produced by cars are much smaller than coal particles and can be found in the breathable size fraction [59]. Heavier PAHs, with higher carcinogenic effects, are usually associated to particulate matter in the atmosphere. Particles of concern to human health are those known as inhalable particles (PM2.5, PM with aerodynamic diameter less than 2.5 μm). Most particulate-phase PAH are adsorbed onto fine particles, which are deposited slowly and, depending on atmospheric conditions and chemical reactivity, may be transported over long distances, being placed even in remote areas. The European Union requires continuous air-quality monitoring with longterm assessment of benzo(a)pyrene (B(a)Py) and other relevant PAHs [60]. The Council Directive 2004/107/EC establishes a target value for B(a)Py of 1 ng·m−3, and a long-term objective of 0.1 ng·m−3, applying to the B(a)Py content in the particulate matter fraction with an aerodynamic diameter lower than 10 μm (PM10) [60]. Other sources of PAHs are the incomplete biomass combustion which can take place in both natural processes (as fires or volcanic eruptions) and anthropogenic processes (as elimination of residues, cooking or smoking of foods). Smokes coming from biomass combustion not only increases the amount of PAHs in the atmosphere but also constitute an important source of particulate matter in the atmosphere [61], contributing to increase the risk of sorption of PAHs. The composition of PAHs in association with airborne particles depends significantly on the emission sources. PAH concentration ratios can be used to identify possible emission sources: combustion of vegetal biomass or combustion of fossil fuels. [62]. These ratios are usually restricted to PAHs with equal molecular weights, which are normally present in combustion processes due to their chemical stability [63-65]. Table 4 shows several isomeric ratios of PAHs used to distinguish among combustion sources. Table 4. Isomeric ratios of PAHs used to distinguish among different combustion sources. Isomeric ratio Ft / (Ft + Py) = Ft/202 A / (A + Phe) = A/178 1,7-diMePhe / (1,7-diMePhe + 2,6-diMePhe) = 1,7-diMePhe/206 I(1,2,3-cd)Py / (I(1,2,3-cd)Py + B(ghi)Per) = I(1,2,3-cd)Py/276
Combustion source Petroleum Biomass 0.40-0.50
> 0.50
< 0.10
> 0.10
< 0.45
> 0.70
0.20–0.50
> 0.50
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Indoor air quality is also of great interest in the world, especially if considering that most people spend approximately 90% of their time indoors (in their residences, at work, or elsewhere), where scarce ventilation usually helps in increasing PAHs contents. Indoor air in houses is polluted by PAHs that come from not only outdoor air but also indoor emission sources such as cooking [66] and gas home appliances [67,68]. Additional contributions to ambient air levels arise from tobacco smoking [69-71]. To monitor air quality, it results therefore important not only to determine the PAHs contents in the gas phase but also the content associated to the particulate matter. In addition to this, the monitoring methods for airborne PAHs maybe classified in those concerned with either direct occupational exposures or with ambient exposures (outdoor) [72]. Occupational environment is usually monitored by active sampling, whereas ambient exposure is usually monitored by passive sampling, but not exclusively. Active sampling methods require pumping the air stream through a medium (e.g., filter, sorbent, impinger, cryogenic trap) which will retain the PAHs. The analysis of PAHs from air samples in active sampling is followed by an extraction step, clean-up procedures and/or exchange of solvents, and then chromatographic separation and quantification. Sample preparation is considered to be the bottleneck of most analytical methodologies, as it constitutes the principal source of error in the analytical process and remains one of the most time-consuming steps [73]. Figure 4 shows a general scheme of the usual procedure for the monitoring of PAHs in air. The active sampling methods commonly used to determine and quantify PAHs in air are combination of fiber filters, to retain the particle-bound PAH fraction, whereas impregnated filters, impingers, solid sorbents like polyurethane foam plugs (PUF) and XAD resins, and cryogenic traps are used to trap the semivolatile and volatile PAH fractions, respectively [74-76]. After sampling, the most common extraction and preconcentration methods are Soxhlet [77] or sonication [78] of the systems used to trap the PAHs. These routine methods are time and solvent consuming. Apart of these common extraction procedures, many times other clean-up techniques are necessary given the complexity of the samples, such as a cleaning using a chromatographic column (filled with silica gel, Florisil or alumina, as sorbent phases), or a solid-phase extraction (SPE) step. Passive sampling techniques rely on the controlled transport of the analyte along a concentration gradient towards a surface which acts as a sink for the PAHs. Passive air samplers are preferred to conventional active air samplers since they do not require electricity or maintenance and are less expensive. The main disadvantage is the long sampling time required due to the low PAH levels and limited analytical sensitivity [79-81]. Followed passive sampling, extraction-preconcentration, clean-up, and chromatographic procedures, identical to the ones used in active sampling approaches, are conducted.
2.1. Sampling Systems The most critical step to ensure an accurate monitoring of PAHs in air is the choice of the sampling strategy. The main problems associated to the sampling systems of PAHs in air are related to insufficient sorption on the sampling devices, the re-evaporation of already retained PAHs, and even the chemical destruction of the PAHs already retained. Then, PAHs losses
Passive sampling
General scheme
Common procedures
Other procedures
PAHs in air Pump. Active sampling
·Fibre filters ·Impinger solutions ·Adsorbents (PUF or XAD resins)
Sampling
Extraction
·Soxhlet ·Ultrasound-assisted extraction
·Dialysis (passive sampling with SPMDs) ·Microwave-assisted extraction ·Pressurized-fluid extraction ·Supercritical-fluid extraction
Clean-up
·Chromatographic column (i.e. silica gel) ·Solid-phase extraction ·Solvent exchange
·Solid-phase microextraction
GC or HPLC Figure 4. Common steps of the analytical method to monitor PAHs in air.
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are practically unavoidable due to their chemical reactivity and volatility. The importance of the sampling is such that, indeed, the posterior utilization of any extraction and/or chromatographic method does not turn out to be so critical in the analysis of PAHs for air monitoring.
2.1.1. Active Sampling While diffusive passive sampling offers advantages in terms of cost and simplicity, active-pumped sampling remains the most versatile option for simultaneous monitoring of multiple target PAHs. The major drawbacks of active sampling are the high cost of the equipment and requirement of power and training; these restrict its application to widespread locations especially to remote regions. Given that large air volumes (>100 L) may be drawn through the tubes in many cases, pumped active sampling also offers better detection limits than other air monitoring techniques. There are multiple pump models on the market, from a wide variety of brands, designed to fulfill different needs depending on the specific sampling. High/low volume pumps designed for active sampling typically operate at flow rates ranging from 0.5-1400 L min-1. The sampling time is normally a few hours up to one day. Sampling of airborne PAHs requires special equipment since PAHs with a lower molecular weight are found mainly as vapor, while those with a higher molecular weight are to be found in the condensed phase. Consequently, an adequate sampling method usually combines a filter for the collection of particulate, and one or more sorbents such as XAD-2 and/or polyurethane foam (PUF) for the collection of the vapor phase. Table 5. Sampling systems recommended by standard methods. Standard method ASTM D 6209-98 (2004) ISO 11338-2 (2003)
ISO 12884 (2000)
ISO 16362 (2005)
Application Standard test method for determination of gaseous and particulate PAHs in ambient air Stationary source emissions— determination of gas and particlephase PAHs—Part 2: sample preparation, clean-up, and determination Ambient air—Determination of total (gas and particle phase) PAHs—Collection on sorbentbacked filters with gas chromatographic–mass spectrometric analyses Ambient air—Determination of particle-PAHs by HPLC
Sampling system Fine particulate filter followed by a vapor trap containing PUF or resin XAD-2. Filters to collect the particulate phase whereas gas phase is trapped on an adsorbent (XAD-2 or PUF). Fine-particle filter followed by a vapor trap containing PUF or XAD-2.
Fine-particle filters.
Historically, two types of filters, glass fiber (GFF) and silver membrane have been utilized most often in the sampling of airborne PAHs in both active and passive sampling. Glass filters have mainly been used in active sampling whereas silver membranes have mainly been utilized in passive sampling [72].
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New materials have recently been developed for filters [82], like silica quartz (QFF), teflon-coated glass (TGFF), reagents-coated glass [83], PTFE membrane bonded to polyethylene net, and PTFE member supported by PTFE fibers, among others. Improvements in sampling methods designed to minimize the losses of PAHs volatized during sampling have also relied on addition of solid sorbent back-up cartridges and cryogenic traps. For the sampling of PAHs, the sorbents most commonly used include Tenax, XAD-2, polyurethane foam (PUF), Florisil, Chromosorb 102, Chromosorb 106 and SepPakC18 on Porasil [84]. The procedures UNE-77250 and EPA TO-13A described a sampling system to determine PAHs in ambient air based on the utilization of a particulate fiber filter followed by a PUF cartridge or a XAD-2 resin [85,86]. Table 5 shows several sampling devices utilized in standard methods. The most common sampling devices utilized in recent years for the trapping of PAHs in air monitoring are XAD-2 resins [87], glass-quartz filters [88-90], combinations of filters and PUF [15,91], combination of glass-quartz filters, PUF, and XAD-2 [92], combination of PTFE filters and XAD-2 [93], or combination of denuders and filter pack (XAD-4 impregnated quartz) [94]. With respect to the PAHs determination in biomass smoke, Table 6 includes the most common sampling systems utilized in recent years. It can also be observed that glass-quartz filters, PUF and XAD-2 are the preferred trapping systems. Table 6. Sampling procedures to determine PAHs in biomass smoke. Combustion source Wood, barley, corn, rice and wheat Woods Woods Woods residues Pines wood and needle Agricultural and sylvicultural debris Cork residues Cattle residues Urban residues Medical residues Pine and coal Coal Coal * G-Q: glass or quartz
Sampling procedure G-Q* filter, XAD-2 and impingers G-Q* filter and XAD-2 G-Q* filter, XAD-2 and impingers Impingers (glass wool and CH2Cl2) G-Q* filter and PUF/XAD-2/PUF G-Q* filter and PUF/XAD-2/PUF G-Q* filter and XAD-2 G-Q* filter and cartridge XAD-2/PUF G-Q* filter and PUF G-Q* filter and cartridge XAD-16/PUF G-Q* filter, XAD-2 and active coal XAD-2 impregnated with CH2Cl2 G-Q* filter and PUF
Ref. [95] [5] [96] [97] [98] [99] [100] [101] [102] [4] [74] [103] [104]
A recent method has been reported for the ambient air sampling of the 16 EPA PAHs plus B(e)Py. The method is based on active sampling on sorption tubes consisting of polydimethylsiloxane (PDMS) foam, PDMS particles and a TENAX TA bed [105]. It was demonstrated that both the high molecular weight PAHs and the more volatile PAHs, such as naphthalene, are efficiently trapped and quantitatively desorbed, without breakthrough and chemical reaction artifacts. An important result is that the total toxicity equivalence value (TEQ) for PAHs was ca. two times higher compared to the conventional sampling method, illustrating that the concentrations of PAHs in ambient air have been underestimated until
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now [105]. This important conclusion points out the need to develop accurate and quantitative methods of sampling in order to report authentic values of PAHs contents in the air. In any case, the best exposure and risk assessments for PAHs for the general population and the working population can be done using personal monitors. Ideally, this should consider a sampling time relevant to exposure scenarios, and hence be representative for possible health effects. These personal samplers are usually equipped with a battery to facilitate the pumping, and are available for a variety of brands. The personal sampler is characteristics for being portable and should be carried by a person as close to the breathing zone as possible [16,17,93,106]. In addition to this, people do not need to be excessively trained on the utilization of these systems. These personal samplers are equipped with filters and/or sorbents, as any sampling device above described.
2.1.2. Passive Air Samplers (PAS) Diffusive samplers, also called diffusive monitors, passive samplers or passive monitors, are utilized for sampling without the need for an air mover, that is, without a pump. Manmade diffusive sampling operates by allowing gas or vapor molecules to diffuse through a defined volume of still air or through a polymer membrane, until they reach a sorbent bed. The principles of uptake are to consider that the passive sampling medium is uniform and “porous” and that it traps PAHs from the atmosphere by gaseous diffusion, and sorption. The mass collected is a function of the external concentration and the diffusion coefficient of the molecules. The diffusion coefficient varies in a known manner with temperature and pressure, and so the result can be corrected for these parameters [107]. Passive samplers provide information about long-term contamination. They can be used in a screening method for semi-quantitative comparison of different sites contamination with the advantage of low sensitivity to accidental short-time changes in concentration of pollutants. Other advantages of PAS are the simplicity and easiness of handle. In addition to this, they are cheaper because they do not need a pump. The main disadvantage is the long sampling time required due to the low PAH levels and limited analytical sensitivity. Most of the passive air sampling measurements have been performed using semipermeable membrane devices (SPMDs) [80,108,109], polyurethane foam disks (PUFs) [79,110-112] and XAD resins [113], which can be exposed over the period of several weeks or months. SPMDs were initially developed by Huckins et al. [114]. The importance of SPMDs for air monitoring is expected to increase in the near future. SPMDs were initially designed to mimic the behavior of aquatic animal tissues in bioconcentrating contaminants, that is, the purpose was that only the bioavailable fraction of contaminants is absorbed and not compounds bonded to macromolecules [80]. As pointed out by Esteve-Turrillas et al. [80], semi-permeable membrane devices comprise a low-density polyethylene (LDPE) tube, 70-90 μm wall thickness, filled with triolein (1,2,3-tris-cis-9-octadecenoyl glycerol). Standard, these devices are 106 cm long, 2.54 cm wide, and contain 1 mL of triolein. Such devices are usually located in chambers to avoid external contamination. Due to both their high membrane surface area and their relatively large volume of receiving medium, SPMDs proved to be most effective in their capacity to accumulate lipophilic contaminants such as PAHs. The main disadvantage of the SPMD technique is the complex sample preparation procedure required to recover the accumulated pollutants from the collecting phase (triolein). This is achieved by dialysis using considerable amounts of organic solvents, followed by
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concentration of the extracts and an expensive cleanup before the chromatographic analysis. SPMDs composed of some other materials for the membrane and for the internal solvent have also been described in recent years [80]. Photo-degradation of PAHs has been described in several SPMDs [115], so recent studies have focused in developing the best sorption chamber to avoid such photo-degradation [116]. SPMDs have been used not only to monitor PAHs in air [117], but also to monitor PAHs and nitro-PAHs in a wide survey which includes five European countries [118]. SPMDs must be calibrated in the laboratory, and the equations to relate the extracted concentrations with environmental concentrations must be established. Some other devices for low-flow sampling are the denuders, which consist of sections of capillary gas chromatographic columns in which the vapor-phase of the semi-volatile organic compounds are sorbed by diffusion into the stationary phase, while the particulates pass through and are collected on a filter. Advantages of the method are the more accurate measurement of the vapor-particle partitioning and the more quantitative sampling of the most volatile compounds. Disadvantages are the low sampling flows resulting in very long sampling times (several days) [94,119]. Many studies utilize biomaterials as pollutant sorbents, such as plant samples, e.g. bark [120-123], lichen or pine needles [117,123-125], to monitor ambient air concentrations assuming that their spatial pollution distribution in the plants and the atmosphere will be closely related. However, the concentration capacity and sampling rate of plant tissues vary with the plant species and age, location and season. Therefore, these kinds of plant data are still subject to several uncertainties, which complicate their interpretation and limit the potential of plants as monitoring tools. For this reason, man-made passive air samplers are preferred up to date, despite their additional costs and that they have to be deployed, since the variability of samplers at different locations is low, and the sampling period can be controlled.
2.2. Extraction Procedures After sampling, convenient transport and storage of PAHs must be conducted, to avoid losses by volatilization, sorption on recipients, or photo-degradation. In general, the sampling devices are protected from light until they reach the laboratory. It is also advisable to transport the filters and adsorbents that contain the PAHs in a refrigerated bag or icebox [94,99]. Once in the laboratory, samples are conserved at ≤ 4ºC, in absence of light, during at period not longer than 7 days prior to extraction. The most common extraction methods when monitoring PAHs in air are Soxhlet and ultrasound-assisted extraction (Figure 4).
2.2.1. Soxhlet Extraction Soxhlet has been a standard technique during more than one century and it is still the main reference procedure to which the performance of other analytical methods is compared. The Soxhlet extraction is based on the apparatus invented in 1879 by Franz von Soxhlet, and is mainly used when extracting solid and complex samples. Figure 5 shows a typical scheme of a Soxhlet apparatus. The typical Soxhlet procedure starts by placing the sample in a cellulose thimble, which is loaded into the main chamber of the extractor. The Soxhlet extractor is then placed onto a distillation flask containing the organic extraction solvent, being acetone, toluene, and methylene chloride the most common organic solvents used.
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Once the organic solvent is heated to reflux, the solvent vapor travels up to the distillation arm and floods into the chamber housing the thimble. When the Soxhlet chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle is repeated several times, over hours or days. It is obvious that during each cycle a portion of the PAHs dissolves in the solvent. After many cycles the PAHs are then concentrated in the extracting solvent. After extraction the solvent is removed, typically by means of a rotary evaporator, to decrease the amount of organic solvent and so to preconcentrate the analytes. The non-soluble portion of the extracted sample remains in the thimble, and is usually discarded. Soxhlet extraction is still today the preferred extraction technique to ensure quantitative extraction of complex samples. Table 7 shows significant examples of works which utilize Soxhlet extraction in the determination of PAHs for air monitoring. Soxhlet is applied in the extraction of the solid systems used to trap PAHs in air monitoring. Table 7 also includes the most common organic solvents used, and the extraction time required to complete the extraction step. It should be noted the high extraction times (around 24 h following official procedures) and the high volumes of toxic organic solvents required in this traditional extraction approach, which are not only expensive to dispose off but which cause additional environmental issues. These are undoubtedly the main drawbacks associated with the Soxhlet extraction. Official procedures also recommend the utilization of Soxhlet extraction in PAHs air monitoring, in the methods ISO 11338-2 (2003) “Stationary source emissions — determination of gas and particle-phase PAHs—Part 2: sample preparation, clean-up, and determination”; and ISO 12884 (2000) “Ambient air—Determination of total (gas and particle phase) PAHs—Collection on sorbent-backed filters with gas chromatographic–mass spectrometric analyses”. The ASTM D 6209-98 (2004) “Standard test method for determination of gaseous and particulate PAHs in ambient air” also recommends Soxhlet extraction. It is also recommended by EPA TO-13A (toxic compounds in ambient air). Protection from light is always recommended during Soxhlet extraction performance, to avoid photo-degradation of PAHs. Given the above mentioned disadvantages of Soxhlet, several authors have tried to improve the conventional Soxhlet device, either by modifying its design or the operational procedure [126]. The main updates of this “old-fashioned” technique have been related to the miniaturization (and therefore diminution of the organic solvent consumption), automation, and particularly, the combination with microwaves [127]. Main applications have been found when combining focused microwave-assisted extraction with Soxhlet, which is based on the same principles as conventional Soxhlet extraction but using microwaves as auxiliary energy to accelerate the process. However, up to date there are no reported applications of the focused microwave-assisted-Soxhlet in the analysis of PAHs in air monitoring. There are few works which utilize devices based on automatic Soxhlet in air-monitoring of PAHs [79,112]. It is therefore necessary that the recent developments in analytical procedures are followed by an implementation in monitoring techniques.
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1.- Thimble 2.- Distillation path 3.- Siphon
2
3 1
Figure 5. Typical scheme of a Soxhlet extractor.
Table 7. Recent works which utilize Soxhlet for the monitoring of PAHs in air. Extraction time (hours) 20 18 13 8 (each) 8 24 16 72 18
Organic solvent hexane:acetone (1:1) or toluene diethyl ether:hexane (1:9) hexane:methylene chloride (1:1) methylene chloride methylene chloride methylene chloride methylene chloride methylene chloride diethylether:hexane (1:9)
Organic solvent volume (mL) 150 200 90 1st 250 and 2nd 150 — 200 — — 300
Reference [77] [90] [121] [94] [15] [122] [16] [104] [99]
2.2.2. Ultrasound-Assisted Extraction (UAE) Any sonication waves with a frequency higher than the ones detected by the human hear are called ultrasounds (US), that is, ultrasounds have wave frequencies higher than 16 kHz. The production of ultrasounds is possible due to the discovery of Marie and Pierre Curie in 1880 of the piezoelectric effect and its inverse. Nevertheless, the first commercial equipment of ultrasounds was not developed until 1917. Such equipment was curiously proposed in a contest of ideas in 1912, with the purpose of detecting icebergs right after the Titanic shipwreck. The utilization of ultrasounds in analytical chemistry laboratories did not take place until 1970, which appears to be illogical considering that it is obvious that the
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propagation of the sonication waves in any medium is going to increase the mobility of the molecules present in such medium. The most common ultrasounds devices in an analytical laboratory are the sonication bath and the sonication probe. The ultrasonic bath is so common in the chemical laboratories that it does not need presentation. It is widely used due to their economical feasibility, ensuring at the same time a relatively good energy distribution in the reactor placed in the bath. The sonication probe has a higher power, which can be modulated. The main applications of the ultrasounds in analytical chemistry are the ultrasound-assisted extraction and the ultrasound-assisted digestion, among others [128-130]. The most common procedure to carry out an ultrasound-assisted extraction is to place the sample in a recipient containing the extractant organic solvent, and to activate the ultrasounds during a fixed period of time. Protection from light of recipients-containing PAHs during the sonication extraction is always recommended. EPA Method 3550 C describes the utilization of sonication for solid samples, which can be extended to several samples coming from air monitoring. Recent applications of sonication in the extraction of PAHs from the sampling devices used in air monitoring are summarized in Table 8. The extraction times are lower compared to traditional Soxhlet, and also the amounts of organic solvents used, but higher enough to be an environmental issue. Sonication probes or the most-recent US-devices have not been used up to date in the analysis of PAHs in air monitoring. A paradoxical situation has emerged as most of the analytical methods employed to investigate environmental problems also generate chemical wastes, like high amounts of organic solvents, resulting in an environmental impact. In some circumstances, the chemicals employed are even more toxic than the species being monitored. In this sense, the work of some analytical chemists has focused on the development of methods less harmful to humans and to environment. Great concern over the disposal of the toxic organic solvents employed in the described method and their effect to the environment has led to move towards cleaner extraction methods such as microwave-assisted extraction and pressurized fluid extraction, among others. Improvements in clean-up methods also include solid-phase extraction (SPE) and solid-phase microextraction (SPME). Table 8. Recent works reporting the utilization of ultrasound-assisted extraction to remove PAHs from sampling devices in air monitoring Sampling device/sample Extractant Filters 5 mL cyclohexane XAD-2 sorbents 1 mL acetonitrile Pine needles/bark 30 mL hexane:CH2Cl2 1:1 Glass filter cyclohexane 20 mL hexane:acetone 1:1 SRMa 1648 or 1649a SRMa 1648 or 1649a 20 mL toluene Aerosol 25 mL CH2Cl2 Filters 150 mL CH2Cl2 a Standard reference material (certified)
Extraction time (min) 30 min 30 min 10 min (2 times) 30 min 30 min (2 times) 30 min (2 times) 10 min (4 times) 30 min (3 times)
Ref. [93] [93] [123] [88] [77] [77] [89] [104]
2.2.3. Microwave-Assisted Extraction (MAE) Microwaves are non-ionizing radiation with a frequency between 300 and 300000 MHz. Molecules under microwaves are moved by ion migration and dipole rotation. Commercial
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microwaves operate at 2450 MHz, which generate the alignment of molecules under the radiation followed by return to disorder 4.9·109 times per second. This quick movement-relax is consequently followed by a fast heating. The only condition for a molecule to suffer such heating is to be a dielectric compound. Fastest heating are obtained with analytes of high dielectric constants. The first analytical application of microwaves was carried out by Abu-Samra in 1975 [131]. The utilization of microwaves to extract organic compounds from a number of samples (soil, seeds, foods and feeds) was carried out at the end of the 80s utilizing a domestic microwave [132]. The analytical applications in extraction procedures are based on the heating of the extractant by microwaves. The extractant is usually an organic solvent characteristic for being a dielectric compound. The use of solvent mixtures with and without dipoles opens up a variety of potential solvent mixtures. The extractant is placed in a reactor altogether with the sample, followed by heating under microwaves during a certain period of time. It should be highlighted that microwave heat the sample and the extractant without the need of heating the reactor, therefore reaching high temperatures with lower extraction times. After the application of the microwaves, the analytes originally bound to the sample are dissolved in the extractant solvent. The efficiency of the microwave extraction process depends on the nature of the sample and the extractant. While domestic microwaves are still used in several analytical laboratories, commercial microwaves specifically-designed for laboratory use are preferred, especially attending to safety reasons. Most analytical applications have been carried out using cavity-diffuse or, to a lesser extent, focused microwave systems. Usually they are referred to as closed- or openvessel systems, respectively, but this terminology is not correct as the so-called open vessels in focused systems are not completely open, and as it is also possible to operate closed vessels in a focused microwave [133]. The utilization of diffuse microwaves needs of a cavity (an oven) where samples are placed in a rotor to be moved for ensuring reproducibility in microwave irradiation, because the electric field is not uniform inside the cavity. Several samples can be placed simultaneously in a cavity-microwave. Focused microwaves are more efficient in terms of homogeneous heating. Figure 6 shows a scheme with the differences between diffuse and focused microwaves devices. The main variables to be optimized in an analytical extraction by microwaves are: the microwave power, the extraction temperature and/or extraction pressure, the extraction time, the nature and volume of the extractant, and the characteristics of the matrix such as amount of water or presence of interferences. The optimization of these variables is time consuming unless an experimental design is used [134]. Microwave-assisted extraction has successfully been used in the extraction of sampling devices utilized for monitoring of PAHs in air, as it can be observed in Table 9. It can be seen that shorter extraction times and lower amounts of organic solvents are needed, especially when compared to Soxhlet extraction. In spite of the advantages of the microwave-assisted extraction method, including high extraction efficiencies, its utilization in samples coming from air monitoring is not widespread. EPA Method 3546 utilizes microwaves to extract solid samples, which can be extended to several samples coming from air monitoring.
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Table 9. Microwave-assisted extraction procedures used in the monitoring of PAHs in air. Sample SRM1648 SRM1649a SRM1649a
Extractant 15 mL hexane:acetone (1:1)
Power-Time Microwave 400 W – 20 min commercial
Ref. [77]
400 W – 20 min commercial 15 mL toluene (10% water) or 15 mL CH2Cl2 XAD-2 resins 10 mL ethyleneglycol:NaCl 1M 120 W – 40 min domestica (7:3) 513 W – 30 min domestica bark / needles 90 mL hexane:CH2Cl2 (1:1) a modified domestic oven
[77] [87] [123]
Microwave oven
Diffuse microwaves
Extraction vessels Extraction vessel
Magnetron Focused microwaves
Wave guide
Focused microwave Rotor
Cavity microwave
Figure 6. Differences between cavity and focused microwave devices.
2.2.4. Pressurized Liquid Extraction (PLE) The technique pressurized liquid extraction (PLE), also named pressurized fluid extraction (PFE), was originally launched by Dionex Inc. in 1996 under the name accelerated solvent extraction (ASETM) [135,136]. PLE is a solid-liquid extraction process performed with organic solvents in stainless steel closed-vessels into which solvent is pumped and brought to relatively elevated temperatures, between 80 and 200ºC, and elevated pressures, between 10 and 20MPa [137]. The sample must be placed in the extraction vessel typically dispersed in a drying or inert sorbent such as sodium sulfate, Hydromatrix (Isco, Lincoln, NE, USA), or diatomaceous earth. During the heating cycle, solvent is pumped in and out of the cell to maintain the pressure and to perform the number of static cycles indicated by the user. In PLE, the organic solvents used as extractant are still below their critical conditions but with enhanced solvation power and with lower viscosities and surface tensions. The combination of high temperature and pressure results in better extraction efficiency, so minimizing solvent use and expediting the extraction process. PLE is like Soxhlet extraction, the only difference being the use of solvents that are raised to the near-supercritical region, where they show better extraction properties.
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Water can also be employed as the extraction solvent in PLE, and so the technique is called pressurized hot water extraction, subcritical water extraction (SWE), super-heated water extraction, or high temperature water extraction [138]. The pressure in PLE is not a significant variable to optimize, because its role is simply to maintain the solvent in its liquid state. The main variables to optimize are extraction time and temperature, which considerably simplifies the experimental design. The main advantages of PLE are: fastness (around 15 min), low amounts of toxic organic solvents are required (between 15 and 40 mL), and several samples can be extracted simultaneously (up to 24). However, the instrumentation is much more expensive than Soxhlet or even microwaves. EPA method 3545 uses PLE in the determination of semivolatile organic compounds from solid samples [139], which can be extended to solid devices used to trap PAHs in air monitoring. Furthermore, PLE has proven to be an efficient method to extract analytes from complex samples, with efficiencies comparable to Soxhlet extraction [140]. Nevertheless, few applications of PLE have been reported for the extraction of sampling devices when monitoring PAHs in air. Table 10 reports several examples of reported applications in the literature for this kind of samples. Table 10. Utilization of PLE in the determination of PAHs for air monitoring. Sample PM10 filters SRM1649a*
Pretreatment diatomaceous earth and cellulose filter
Cell 22 mL
Extractant hexane : acetone (1:1)
Conditions 125ºC - 1500 psi 2 static cycles 5 min hold pine needles dried — hexane : acetone (1:1) 100ºC - 1500 psi 2 cycles 20 min hold XAD-2 dried portions of 30 g 100 mL dichloromethane 100ºC - 1500 psi PUF 1 cycle 15 min hold * Method optimized by experimental design: extraction efficiencies between 96-103%
Ref. [90]
[117]
[92]
2.2.5. Dialysis Separation processes brought about differences in concentration through a semipermeable membrane are generically known as dialysis. Dialysis is a membrane barrier separation process in which differential concentration forces one or more analytes to transfer from the sample solution to another fluid through a semi-permeable membrane, which allows contact between the fluids without mixing. The extraction of PAHs from SPMDs filled with triolein, utilized in passive air monitoring, is widely performed by dialysis using organic solvents such as hexane [80]. In dialysis of SPMDs, PAHs diffuse from the inner triolein solution, where they were initially retained, to the hexane outer side of the LDPE membrane as a result of a concentration gradient (see Figure 7). The extraction is normally carried out in 48 h [107]. The main disadvantages of dialysis are the long times required to perform the extraction, and the high volumes of hexane required (usually between 100 and 200 mL). The main advantage is the fact that there are practically no losses of triolein during the extraction step, and so the SPMDs devices can be reutilized. The conventional dialysis procedure is quite simple because it only requires placing the SPMDs device in a recipient containing hexane, all protected from UV light to avoid PAHs’ photodegradation. It has been suggested the improvement of the conventional dialysis by utilizing ASE [141] in order to decrease
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both, extraction time and organic solvent consumption. Other extraction techniques have been proposed for desorption of SPMDs [80], however, they have not been applied yet to the analysis of PAHs in air monitoring. Covered with aluminum foil LDPE membrane
Hexane diffusion Triolein solution
PAHs
Triolein solution
Hexane
Figure 7. Dialysis extraction of PAHs contained in SPMDs devices.
Table 11 summarizes the main applications of conventional dialysis in the extraction of SPMDs devices used in air monitoring of PAHs. It can be observed that, in all cases, extraction times of 48 h, and organic solvent volumes higher than 200 mL, are required. Table 11. Dialysis extraction reports of SPMD used in the air monitoring of PAHs. Organic solvent Hexane Hexane Hexane 95:5 cyclopentane:CH2Cl2 Hexane
Volume of extractant — 180 mL × 2 180 mL × 2 1st 100 mL 2nd 180 mL 130 mL × 2
Time 2 × 24 h 2 × 24 h 2 × 24 h 2 × 24 h 2 × 24 h
Reference [117] [115] [116] [118] [141]
2.2.6. Supercritical Fluid Extraction (SFE) Supercritical fluid extraction (SFE) is an extraction procedure which utilizes solvents in their supercritical state as extractive agents. These supercritical fluids have similar densities to liquids, but lower viscosities and so higher solvation power. Supercritical fluids constitute an acceptable alternative to conventional liquid solvents in the analytical extraction of environmental samples [137]. Supercritical fluids are obtained in commercially available instruments, applying high temperatures and pressures to the solvent to ensure conditions above the critical point. The sample is located in an inert extraction cell, where the supercritical fluid is pumped. The most common fluid to date has been supercritical CO2 because of its reasonable critical properties (relatively low critical pressure and temperature, i.e., 7 MPa and 31.3ºC, respectively), low toxicity, and chemical inertness. In addition, supercritical CO2 has Lewis base characteristics, induced dipole interactions, and quadrupole interactions that allow it to solvate different analytes ranging in polarity from nonpolar to moderately polar. While CO2 is
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a relatively good solvent, these solvating interactions are weak and it has been shown that pure CO2 often cannot extract environmentally persistent pollutants [142]. The problem is usually overcome using modifiers to adjust the overall polarity, such as acetone, methanol and dichloromethane, among others. Extraction can be performed in static, dynamic or recirculation mode. In static mode, the cell containing the sample is filled with the supercritical fluid, pressurized and allowed to equilibrate. In dynamic mode, the supercritical fluid is continuously passed through the extraction cell. In the recirculation mode, the same supercritical fluid is repeatedly pumped through the sample until it is finally pumped out to the collection system. After depressurization, the extracted analytes can be collected in a small volume of organic solvent or on a solid-phase cartridge (adsorbent trap) [137]. An important advantage of SFE is the fact that the extracts are relatively quite clean, and only moderate additional clean-up is required. In fact, combined with solid adsorbent traps, the extracts are practically ready for the chromatographic determination. Several parameters must be optimized in order to achieve successful extraction efficiency, such as supercritical fluid nature, working temperature and pressure, extraction time, shape and size of the extraction cell, sample particle size, moisture content of the matrix and the analyte collection system. Due to the high number of parameters affecting the overall performance, the optimization in SFE is often tedious and difficult, which has avoided a wider applicability of the technique. Other disadvantages of the SFE technique include: limited sample size and high cost of the equipment. Since 1980s, several applications of SFE in the analysis of environmental samples have been reported [143-145]. In addition, SFE has been adopted by the EPA as a method for extracting PAHs (Method 3561) from solid environmental matrices, which can be extended to several samples coming from air monitoring. Table 12 lists several reported applications of SFE in the analysis of PAHs for air monitoring. The reported optimum extraction conditions are also listed. In aerosol analysis, the most abundant compounds are nonpolar mixture of branched, cyclic and unsaturated hydrocarbons, generally characterized as an unsolved complex mixture (UCM) [149]. The UCM generates a large hump in the gas chromatogram, obscuring the peaks of other trace compounds. Several works have suggested the online coupling of SFELC-GC-MS to determine PAHs in aerosols and so to avoid the UCM problems [146,148]. The coupling of multidimensional techniques to sample extraction methods further decreases the manual sample pretreatment steps, which results convenient.
2.3. Clean-up Procedures After the sampling and the extraction procedure, independently on the technique used for extraction, various matrix components are frequently present in the extract altogether with the PAHs extracted. These non-desirable components must be eliminated to ensure an accurate identification and quantification of PAHs, which are present at low levels of concentration in the extract. In addition, several matrix components present in the extract can later damage the chromatographic system. Clean-up steps are therefore necessary to remove not only the bulk of the co-extracted material, but also those compounds closely related to the analytes that could potentially interfere in the final determination (i.e., aliphatic hydrocarbons when trying
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to determine PAHs). Furthermore, many times a solvent exchange is necessary, right after the extraction step, to allow the injection of the extracted PAHs in a chromatographic system. Table 12. Applications of SFE for the monitoring of PAHs in air. Sample-PAHs SRM 1649 urban air PM (0.3 g)
Technique off-line SFE GC-MS
Optimum SFE conditions CO2 + different modifiers. 400 atm. 2.5 mL extraction cell 1) 5 min static 2) 10 min dynamic Elution on CH2Cl2 GFF on-line SFE CO2. 400 atm. 150ºC 16 EPA PAHs LC-GC-MS 45 min Elution with n-pentane:ethyl acetate 97:3 v/v GFF off-line SFE CO2 + toluene* 95:5 v/v. 350 6 nitro-PAHs GC-ECD atm. 90ºC 7 oxy-PAHs 5 mL extraction cell. 1) 5 min static 2) 30 min dynamic 1 mL·min-1 Elution: a) 2 mL hexane for aliphatic, b) 3 mL hexane:CH2Cl2 4:1 for PAHs, and c) 6 mL CH2Cl2 for nitroand oxy-PAHs QFF on-line SFE CO2 + CH2Cl2 (400 μL). 400 17 PAHs LC-GC-MS atm. 150ºC 3 mL extraction cell. 1) 10 min static 2) 45 min dynamic 1 mL·min-1 Solid phase trap, Elution with n-hexane:ethyl acetate 95:5 v/v *A first step with pure CO2 (150 atm, 45ºC) is carried out to remove other components.
Reference [142]
[146]
[147]
[148]
2.3.1. Liquid adsorption chromatography in columns Several procedures have been developed to achieve a successful clean-up. Among them, classical liquid adsorption chromatography in columns is still the dominant technique for purification of extracts. This technique involves passing the extract through an adsorbent column prepared in the laboratory. The interferences remain retained in the sorbent whereas the analytes are eluted from the sorbent using a convenient organic solvent. The operating procedure is simple, and once the sample is loaded into the glass column already filled with the sorbent, an adequate organic solvent will elute PAHs from the sorbent. The flow of the eluent must be carefully controlled to avoid insufficient elution of PAHs or elution of matrix interferences. Flows between 1 and 2 mL·min-1 are usually employed for sorbent amounts of 10 g. The most common sorbents used in these columns are alumina, silica gel, and Florisil, with different mesh sizes. These sorbents must be carefully cleaned and conditioned before being used, being Soxhlet the most common pre-cleaning step [85]. In many occasions, the columns are also loaded with 1 cm of anhydrous Na2SO4, located into the upper side of the sorbent, to remove any water content of the samples, especially if a GC procedure is going to be used. EPA methods 3630C, 3610B and 3620B, which use silica gel, alumina and Florisil,
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respectively, have been approved for the purification of organic extracts from solid environmental samples. The official method UNE-77250 and the method EPA TO-13A also recommend the utilization of a silica gel column as a clean-up step for PAHs [85,86]. Table 13 includes common clean-up procedures with sorbents-columns for PAHs samples in air monitoring. It can be observed the complexity of the step and the timing required to carry out a successful clean-up. Several reports in air monitoring of PAHs also utilize silica-gel columns [79,91,95,104,112,117,118] or alumina columns [88,95], but without specifying the conditions in which the clean-up procedure is conducted.
2.3.2. Solid Phase Extraction (SPE) Solid-phase extraction (SPE) is a widely used sorbent extraction technique which involves the use of sorbent materials to trap analytes and separate them from the bulk solution. As the sample solution passes through the activated sorbent bed of the device, analytes concentrates together with some other matrix components. To remove the analytes of interest, an adequate organic solvent is employed in the final elution step. The most widely used trapping mechanism for SPE is hydrophobic interaction. The most common sorbent material is alkyl-bonded silica (C18 silica, C8 silica, and C2 silica), because it is reactive enough to permit its surface to be chemically modified and yet stable enough to allow its use with a wide range of solutions. Copolymer sorbents such as cross-linked polystyrene divinylbenzene and hydrophilic lipophilic balanced polymers are very popular too [150]. Most recently, molecularly imprinted polymers (MIPs), which are synthetic materials with artificially generated recognition sites able to specifically rebind a target molecule in preference to other closely related compounds, start to be quite common [151]. Today, there is a wide variety of SPE sorbent materials commercially available, which are valid for PAHs clean-up [152]. The size of the sorbent particles usually ranges from 10 to 60 μm. The stationary phase in SPE comes in the form of a packed syringe-shaped cartridge, a 96-well plate [153], or a flat disk. These devices can be mounted on commercially available extraction manifold which allow many samples to be processed simultaneously. Up to 24 cartridges can be placed in a typical cartridge SPE manifold, while 6 disks can be placed in a typical disk SPE manifold. These SPE manifolds are usually equipped with a vacuum port to speed up the extraction process. The analytes are collected in sample tubes inside or below the manifold after they pass through the sorbent. The SPE extraction is commonly performed in four steps: conditioning-wetting (that is, a solvation of the functional groups of the sorbent bed to make them interact with the sample), retention-loading (that is, the sample is loaded into the cartridge to make the analytes bound to the sorbent), selective washing (that is, the elution of undesired matrix components by adequate choosing of the eluting agent), and elution (the analytes are desorbed using an adequate solvent and collected to be further analyzed). Sometimes, column regeneration is accomplished. Figure 8 shows a scheme with the common steps followed in SPE. Method development in SPE is accomplished optimizing pH, ionic strength, polarity and flow-rate of the eluent, and physicochemical characteristics of the sorbent bed. The main advantages of this clean-up technique are the lower amount of organic solvents required and the shorter times needed. In addition to this, the technique is cheap when compared to other extraction and/or clean-up procedures, and quite efficient in terms of extraction recoveries.
Table 13. Specific conditions for a successful clean-up of PAHs samples coming from air monitoring utilizing sorbent columns. Sorbent Silica gel Alumina
Pre-conditioning 130ºC (silica) 350ºC (alumina)
Silica gel 8 g glass column 1 cm i.d. × 30 cm Alumina 1 g Silica 2 g Na2SO4 1 cm Silica gel 10 g Silica gel 10 g Alumina 5 g 8 mm i.d.
130ºC 16 h
180ºC (alumina) 140ºC (silica) 450ºC (Na2SO4) — 16 h 150ºC 12 h 160ºC
Conditioning Deactivated silica. Soxhlet – alumina: 1) CH2Cl2:MeOH 2:1 (12 h) 2) CH2Cl2:hexane 30:70 (12 h) Cleaning with 50 mL of hexane prior to sample loading
Interferences elimination Hexane (aliphatic)
Elution of PAHs CH2Cl2:hexane (30:70)
Reference [77]
20 mL hexane (aliphatic)
30 mL CH2Cl2:hexane (3:7)
[121]
Deactivated silica
Hexane
25 mL CH2Cl2:hexane (1:1)
[116]
Cleaning with 25 mL of hexane, 2 mL·min-1, prior to sample loading Soxhlet 6 h. Cleaning with 40 mL of pentane prior to sample loading Cleaning with cyclohexane
25 mL n-pentane
25 mL pentane:CH2Cl2 (2:3). 1 mL sample 25 mL pentane:CH2Cl2 (6:4). 1.5 mL·min-1 30 mL toluene
[16]
25 mL pentane (aliphatic) 20 mL cyclohexane
[5,98,99] [74]
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Several attempts have been conducted to improve SPE devices in order to decrease the consumption of organic solvent or to minimize the human handling. The main step forward has been the introduction of on-line (and sometimes automated) SPE–LC [154,155] or SPEGC [156]. The SPE procedures are essentially the same as off-line ones, the major difference being that the elution volumes are usually smaller and often different adsorbent materials are used for the extraction. 2. Loading
1. Wetting Organic solvent
SPE cartridge
Sample
Naphthalene (representative PAH) Other matrix components
Syringe body
Frits SPE packing
Tip
3. Selective washing Organic solvent
4. Elution Organic solvent
Figure 8. Common steps to carry out a SPE clean-up procedure.
Recent applications of SPE in the clean-up of PAHs extracts obtained in air monitoring have been carried out by off-line SPE. Ratola et al. [123] used SPE cartridges which were conditioned with 50 mL of hexane, and eluted with 50 mL of hexane:dichloromethane (1:1) followed by 50 mL of dichloromethane. Xie et al. utilized a 10 port vacuum SPE manifold to carry out the clean-up procedure. The authors paid special attention to the drying steps of the procedure. The sample was successfully eluted with 3 mL of a solution composed of 20% of dichloromethane in hexane (v/v) at a flow rate of 1 mL·min-1 [89].
2.3.3. Solid Phase Microextraction (SPME) Solid-phase microextraction (SPME) is a relatively novel extraction technique developed by Pawliszyn in the nineties [157,158]. The working procedure of this non-exhaustive extraction technique always involves two steps. In the first step, the analytes contained in an aqueous matrix are extracted by contact during a certain period of time with a fiber coated by an active phase. This fiber coating is linked to a syringe to facilitate its manipulation. This
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sorption step can be achieved by direct immersion or in the head space of the matrix solution. The second step implies desorption of the analytes retained in the fiber into an analytical system. Figure 9 shows a general scheme of the SPME process. Desorption in a GC system is simple, because it only requires thermal desorption in the hot injection port of the GC. Online desorption in CE or HPLC needs a specific interface device to accomplish such step [159]. The most common commercial SPME fiber coatings are the polyacrylate (PA) and the polydimethylsiloxane (PDMS). Nevertheless, many new polymeric sorbents are currently being developed with specific purposes [151,160]. The main advantage of SPME is the fact that is a solvent-free technique, with undoubtedly environmental interest. Furthermore, the technique is simple, fast, easy to automate, portable, not expensive, and require low amounts of sample (sample volumes lower than 20 mL). The main disadvantage of the technique when used in direct immersion is the fact that analytes must be dissolved in water, which undoubtedly limits its utilization to polar analytes and/or to water samples. If intending to extract solid samples, headspace mode or utilization of hollow-fiber membranes is required, but the latter is not always quite successful with hydrophobic analytes such as PAHs. In addition, the technique is limited to volatile compounds when used in the headspace mode. One important feature that must be kept in mind is the fact that SPME is a non-exhaustive extraction procedure. In fact, only a small fraction of analytes are extracted from the sample. This non-exhaustive characteristic is usually compensated by the high-preconcentration factors achieved, by the avoiding of co-extracting matrix components, and by the utilization of an external calibration, with standards subjected to the whole SPME step [161]. Coating retracted Coating exposed (CR) (CE) Plunger Needle Syringe body
Needle Fiber coating
Step1: sorption
Step2: desorption
The coating will be exposed in the injection port of a GC, in a convenient interface device, or in an adequate solution for off-line desorption.
OR CR
Sample vial with PAHs
CR
CE in headspace
CE in direct immersion
CR after the extraction time
Figure 9. General scheme of the SPME procedure.
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The main parameters to optimize in a SPME extraction procedure are the selection of the optimum fiber coating and width (wider coatings are preferred to ensure higher extraction efficiencies), extraction time (equilibrium conditions are usually preferred), extraction temperature (mainly for headspace mode, it should be noted however that the sorption process on the fiber coating is not favored at high temperatures), selection of modifiers to increase the extraction efficiency (such as small amounts of organic solvents or high contents of salts), pH, stirring, and sample volume. The main application of SPME as clean-up procedure for PAHs monitored in air has been its headspace (HS) mode combined with microwaves for the analysis of XAD-2 resins [87]. Three different SPME fibers were used in the study: 65 μm PDMS-DVB, 85 μm PA, and 100 μm PDMS. The microwave power was 120 W during 40 min, while the HS-SPME mode was simultaneously carried out. Both, SPE and SPME can be used as extraction techniques. However, when dealing with complex samples such as the ones obtained in air PAHs monitoring, the techniques are not totally successful as extraction procedures, and therefore their utilization is in general restricted to clean-up steps, or used in combination with other extraction procedures.
2.3.4. Gel-permeation chromatography (GPC) Gel-permeation chromatography (GPC) is a technique also referred to as size exclusion chromatography (SEC), and based on molecular size separation. This size exclusion cleanup procedure utilizes hydrophobic gels in the separation of macromolecules and organic solvents to elute retained analytes. The packing gel is porous and is characterized by the range or uniformity (exclusion range) of that pore size. The GPC column retains molecules that are small enough to enter the pores of the gel. Larger molecules are un-retained and therefore eluted from the column first. In the choice of gels, the exclusion range must be larger than the molecular size of the molecules to be separated. The most common sorbent is a cross-linked divinylbenzene-styrene copolymer (SX-3 Bio Beads or equivalent). The main advantage of GPC is its more applicability than adsorption chromatography to unknown contaminants isolation when little information on the sample is available. Furthermore, it can be fully automated, with pumps, detector and fractions collector. Another advantage is the lifetime of the GPC column, up to several months. An EPA method (Method 3640A GPC Cleanup) has been approved for the purification of organic extracts from solid environmental samples. The main disadvantage of GPC is that, mainly for quite complex samples, is necessary to combine it with other clean-up procedures, such as classical liquid adsorption chromatography in columns. Nevertheless, it should be highlighted herein that many of the already described clean-up procedures are not always used isolated. In fact, it is very common to combine some of them in order to ensure an exhaustive purification of the PAHs’ extract. Several works of PAHs monitoring in air utilize GPC as a clean-up step [90,116,118]. These reported applications utilize an automated GPC instrument, eluting PAHs with dichloromethane at flow rates between 4.5 and 5 mL·min-1, and collecting the hydrocarbons fractions at around 20 min. 2.3.5. Other clean-up and preconcentration steps Clean-up steps must be always followed by a preconcentration procedure. The injection of the final extract into a chromatographic system requires the reduction of the final amount
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of organic solvent volume to reach the adequate limits of sensitivity. In addition to this, many times is also necessary to exchange the solvent, because the solvent used in the extraction/clean-up step is not compatible with the chromatograph. Such exchange is preceded by a reduction and/or elimination of the original solvent volume. The most common systems to carry out such reduction of the solvents volume are the rotary evaporator (rotavap) and the TurboVap® evaporation system. A rotary evaporation is in general used to separate solvents such as n-hexane or ethyl acetate (with relatively low boiling points) from compounds which are solid at room temperature and pressure. A rotavap consists of a motor unit which rotates an evaporation flask containing the sample extract. The flask is partially submerged in a water bath, with a temperature not exceeding 35 - 40ºC. A condenser is also connected to the system to produce the condensation of the solvent that is gathered in a collector. The whole system is under vacuum, to reduce the pressure within the evaporator system. It is well-known that lowering the pressure above a bulk liquid lowers the boiling points of the component liquids, and this is the basis of the rotavap. The TurboVap® is an evaporation system which uses a patented gas vortex shearing action and optical sensors to provide fast and efficient evaporation of organic solvents. The time required to carry out this solvent reduction depends on the amount of solvent to evaporate. The main advantage of the rotavap is that there are no losses of analytes by evaporation, whereas the TurboVap® needs to be optimized to avoid such losses. On the other hand, only one sample can be placed in the rotavap whereas several samples can be placed in a TurboVap®. Table 14 includes a summary of the most common clean-up procedures utilized in the monitoring of PAHs in air. In all cases, it can be observed the complexity of the overall procedure, and the long time and human handling required. Sample preparation is still the bottleneck of any analytical method.
2.3.6. Mechanisms control during sampling Given the complexity and labor-intense of the extraction and clean-up procedures, it is clearly necessary to utilize mechanisms to control and quantify possible losses of PAHs during these steps. In this sense, it results quite useful the utilization of internal standards (IS) and recovery standards (RS). An internal standard, by definition, is a compound, different from the analyte to be measured, which is added with a perfectly known concentration to the unknown sample (or to the standards). The calibrations plots are then constructed by plotting the ratio of the signal from the analyte to the signal from the internal standard versus the analyte concentration. The internal standard must be added right before conducting the quantification, that is, right after the last clean-up step. It is quite difficult to select the most adequate internal standard, which must be a compound that matches as closely, but not completely, the analytes to be monitored. The utilization of deuterated standards is quite common if a GC procedure is going to be accomplished, because a deuterated analyte is not going to be present in the sample, and at the same time present quite similar characteristics with the analytes to be monitored. Recovery standards take into account the whole extraction/clean-up procedure, and so they are added to the sample since the first beginning of the process, in order to quantify the efficiency of the extraction-preconcentration procedure developed in the laboratory. Decafluorobiphenyl (DFB) as recovery standard for PAHs is recommended in the regulations UNE-77250 and US EPA-TO13 [85,86].
Table 14. Clean-up procedures most frequently used in the monitoring of PAHs in air. Sample SRM1648 SRM1649a PM10 (filters) SRM1649a GFF and PUF Bark QFF and XAD GFF and PUF Pinus bark SPMDs SPMDs + pine needles SPMDs Biomass Pinus needles and bark
Clean-up steps right after the extraction procedure 1) Filtration-Centrifugation. 2) Redissolve in hexane. 3) Silica-gel:alumina column. 4) Rotavap (up to 0.2 mL or dryness). 5) Redissolve in hexane (1 mL) for GC and in acetonitrile (0.5 mL) for HPLC. 1) N2. 2) Redissolve in 700 μL of CH2Cl2. 3) GPC (elution with CH2Cl2). 4) TurboVap®-N2. 5) Redissolve in hexane (0.2 mL) for GC. 1) Rotavap. 2) Silica-gel:alumina column. 3) Rotavap (up to 0.1-0.5 mL). 1) Rotavap. 2) Redissolve in 1 mL of hexane. 3) Sillica-gel column. 4) Rotavap. 5) Redissolve in hexane (1 mL). 1) Rotavap (up to 1 mL). 2) Ultrapure N2 (up to 0.5 mL). 1) Rotavap. 2) N2 (up to dryness). 3) Redissolve in acetonitrile (2 mL) for HPLC. 1) Glass wool. 2) Anhydrous Na2SO4. 3) Rotavap (up to 10 mL). 4) N2 (up to 0.4 mL). 1) Solvent exchange to CH2CL2. 2) GPC (elution with CH2Cl2). 3) Silica-gel:alumina column + anhydrous Na2SO4 (eluted with hexane:CH2Cl2 (1:1)). 4) N2 (up to 50 μL). 1) Silica-gel:alumina column. 2) GPC. 1) Rotavap (up to 0.5 mL). 2) GPC. 3) Silica-gel:alumina column. 4) Rotavap. 5) N2 (up to 100 μL). 1) Solvent exchange to cyclohexane. 2) Silica-gel:alumina column. 3) Rotavap (up to 1 mL). 4) 1 mL acetonitrile. 1) Rotavap. 2) SPE. 3) N2 at room temperature. 4) Solvent exchange to 1 mL hexane.
Reference [77] [90] [104] [121] [94] [15] [122] [116] [117] [118] [16] [123]
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The performance of SPMDs, which implies longer exposition and so higher risk of losses of PAHs during sampling, needs to be evaluated too. It results advisable to use reference compounds to validate them, the so-called performance reference compounds (PRCs) for SPMDs. These standards act like RS, but are only referred to the sampling stage. Table 15 shows the most common IS, RS and PRCs, employed in several analytical methods used to monitor PAHs in air. Table 15. IS, RS, and PRCs used in analytical methods to monitor PAHs in air. IS — N-d8 Me6Benzene A-d10
Chy-d12, Per-d12 A-d10, Py-d10 Me6Benzene — p-terphenyl-d Ace-d10, Phe-d10, Chy-d12, Per-d12 B(e)Py-d12 terfenyl Ace-d10, Phe-d10, Chy-d12, Per-d12 —
RS Ace-d10, Fl-d10, Phe-d10, Ft-d10, B(a)A-d12, Chy-d12, B(b)Ftd12, B(a)Py-d12, B(ghi)Per-d12, I(1,2,3-cd)Py-d12 — — N-d8, Ft-d10, Py-d10, Acy-d10, Fl-d10, Phe-d10, 2-MeN-d10, Biphenyl-d10, Ace-d10, B(a)A-d12, Chry-d12, B(b)Ft-d12, B(k)Ft-d12, B(a)Py-d12, B(e)Py-d12, Per-d12, I(12,3-cd)Pyd12, diB(a,h)A-d12, B(ghi)Per-d12 Py-d10, B(k)Ft-d10 — N-d8, Phe-d10, Ace-d10, Chy-d12, Per-d12 N-d8, Phe-d10, Ace-d10, Py-d10, Chy-d12, B(a)Py-d12 N-d8, Ace-d10, Py-d10, B(a)Py-d12, B(ghi)Per-d12 —
PRCs A-d10, Py-d10
Fl-d10, Phe-d10, Ace-d10, Phe-d10, B(a)A-d12, Chry-d12, B(b)Ft-d12, B(a)Py-d12, I(12,3-cd)Py-d12, B(ghi)Per-d12 N-d8, Phe-d10, Per-d12 DFB
A-d12, Py-d12
N-d8, Phe-d10, Ace-d10, Py-d10, Chy-d12, Per-d12
Ref. [115] [95] [89] [92]
[90] [162] [104] [121] [94] [122] [116] [79] [98] [117]
2.4. Chromatographic Procedures Gas chromatography and high-performance liquid chromatography are the preferred techniques to carry out the final determination and quantification of PAHs. It is not the purpose of this book chapter to deeply describe and revise the principles and applications of both well-known chromatographic procedures [49,163]. Furthermore, several reviews have focused on the utilization of GC [51] and HPLC [164] for PAHs. Traditional PAHs separations by reverse phase (RP)-HPLC is conducted using gradient elution, mainly with acetonitrile:water mixtures and sometimes with methanol:water mixtures. RP-HPLC on chemically bonded C18 phases has been widely used in the environmental analysis of PAHs. Up to date, a wide variety of brands of HPLC columns is available for PAHs’ determination. Packed columns are rarely used in contemporary GC methods for PAHs, being practically 100% of applications carried out with capillary columns. Many commercial brands of GC and HPLC include in their web pages information related to best columns to work with in a specific application, advices to optimize the best gradient elution, and even suggestions for troubleshooting. The most common detectors used in HPLC for the determination of PAHs are ultraviolet (UV), fluorescence (FD), diode-array (DAD) and electrochemical (ECD). In GC the most
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utilized detector is flame ionization (FID). Enhancements to GC (large-volume injection, thermal desorption and fast GC) have also been applied in the analysis of PAHs [51]. Massspectrometry (MS) is the preferred detection system GC to identify and determine PAHs. Advantages of HPLC are good selectivity and extremely good sensitivity when fluorescence detection is used. On the other hand, GC is the method with the highest resolution power when a PAH mixture analysis is needed, and with good selectivity. Both, GC and HPLC are good choices for the determination of PAHs in air monitoring. It must be taken into account that the main problems in the monitoring of PAHs are encountered during the sampling, and also during the extraction and preconcentration of PAHs, and so the final choice of the chromatographic procedure is not that critical. Nevertheless, there is an increased tendency to use GC over LC in recent years [51]. The coupling of liquid chromatography with gas chromatography (LC-GC) results in a powerful technique that combines the best characteristics of both techniques. To perform LCGC, the whole sample is injected into the LC and then subsequently analyzed by GC. Christensen et al. have reported a LC-GC/MS method for the determination of PAHs in particulate matter from ambient air [165]. Several attempts have also been conducted to develop portable GC for PAHs monitoring. Several sorbents can also be used as selective concentrators for sample air as part of the analysis protocol for air taken from canisters or air sampled directly by automated gas chromatographs (autoGCs), which is essentially equivalent to ambient air monitoring. However, these autoGCs have not been quite successful up to date for PAHs in air monitoring. The utilization of GC-MS is recommended by EPA TO-13A. The official procedures ISO 12884 (2000) and ISO 16362 (2005) include the utilization of GC-MS and HPLC-FD (or – DAD), respectively, for the determination of PAHs in air. Table 16 summarizes the most recent extraction techniques combined with GC or HPLC to determine PAHs for air monitoring.
3. WATER MONITORING Waters are important receptors for PAHs coming from several sources. The contamination of watercourses is clearly undesirable if considering that rivers, lakes and ponds are frequently used for potable water supply [166]. Waters can be divided in drinking waters, surface waters, groundwater and coastal waters, each one with different implications with regards to human health. PAHs enter surface waters mainly via atmospheric fallout (wet and dry deposition), urban run-off, municipal effluents, industrial effluents and oil spillage or leakage. Urban run-off contains PAHs deposited on surfaces, as well as mobile-related PAHs from gasoline and oil drips or spills, exhaust products, and bitumen from road surfaces [167]. Although specific sources are known to be responsible for the presence of PAHs in surface waters, their occurrence cannot always be related to a particular source [166].
Table 16. Extraction techniques combined with chromatographic procedures for the monitoring of PAHs in air. Sample / sampling
PAHs determined
SPMDs GFF and XAD-2 GFF and XAD-2 FF and XAD-2 GFF QFF, PUF+XAD-2
16 EPA, B(e)Py, Per
SRM 1649a PUF bark pine QFF and PUF + XAD-2 QFF and XAD-4 FF + PUF
XAD-2 Mixed bed Pinus bark SPMDs SPMDs GFF + PUF pine needles SPMDs GFF GFF *
16 EPA, B(e)Py 16 EPA, 2-MeN, B(e)Py, Per 16 EPA 16 EPA, 1-MeN, 2-MeN, 2,6-DMeN, Biphenyl, 2,3,5-TMeN, 1-MePhe, B(j)Ft, B(e)Py, Per Ft, B(a)A, B(b)Ft, B(j)Ft, B(k)Ft, B(a)Py, I(1,2,3-cd)Py, diB(ah)A 16 EPA, B(e)Py, Coronene 16 EPA 16 EPA 16 EPA 16 EPA 16 EPA except N, B(e)Py, diB(a,e)Py, diB(a,c)A, diB(a,e)Py, diB(a,e)Ft, diB(a,h)Py, diB(a,i)Py N, Ace, Acy, Fl, Phe, A, Ft, Py 16 EPA 16 EPA except N, 2-MeA, 9-MeA, 1-MePy, Per 16 EPA, B(e)Py, Per 16 EPA
16 EPA Ft, B(a)A, Py, B(a)Py, B(b)Ft B(a)A, Chy, B(b)Ft, B(k)Ft, B(a)Py, diB(a,h)A, I(1,2,3-cd)Py
Clean-up procedures are not listed
Extraction technique*— Chromatographic procedure dialysis-GC-MS Soxhlet-GC-MS UAE-HPLC-UV Soxhlet-GC-MS UAE-SPE-GC-MS ASE-GC-MS
Extraction efficiency— Detection limits (DLs)
Reference
45-105% (PRCs) 93-122% DLs around 0.3 ng DLs: 2-50 ng 83-97% from aerosols DLs around 0.1 ng·m-3
[115] [5] [95] [89] [92]
ASE-GC-MS
DLs: 0.26-0.30 pg·m-3
[90]
Soxhlet or UAE –GC-MS Soxhlet-GC-MS MAE or UAE –GC-MS
[104] [121] [123]
Soxhlet-GC-MS Soxhlet-GC-MS Soxhlet-HPLC-UV or FD
74-98% DLs: 0.03-0.5 ng·g-1 70-130%. LODs around 0.2 ng·g-1 45-105% DLs down to 0.002 ng·m-3 83–110%
MW-HS-SPME-GC-MS GC-MS Soxhlet-GC-MS
80%. DLs: 0.02-1.0 ng DLs: 13 pg·m-3 88%
[87] [105] [122]
dialysis-GC-MS dialysis-GC-MS Soxhlet-GC-MS ASE-GC-MS dialysis-GC-MS UAE-GC-FID Soxhlet-HPLC-UV
32–68% (PRCs) 95–120% 72–112% 80–110% — — 82-94% DLs: 0.5-5 ng·mL-1
[116] [117]
[99] [94] [15]
[118] [88] [16]
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PAHs in groundwater may originate from polluted surface waters, agricultural irrigation with effluents, leaching from solid wastes disposal sites or contaminated soils. Groundwater is naturally filtered as it flows through various soil matrices, and PAHs adsorb well to organic soil [166]. Coastal waters are mainly characterized for its salinity and high particulate organic matter content. The quality of coastal waters has social, economic and environment impacts [168]. It is important to consider that PAH concentrations at off-shore sites are usually at low or undetectable levels, while higher concentrations are observed in many coastal and estuary sites [169]. According to the World Health Organization study in 1997, the concentration of individual PAHs in surface and coastal waters is generally around 0.05 μg·L-1 and concentration above this point indicates some contamination. The presence of PAHs in drinking water may be due to the contamination of surface or groundwater used as raw water sources [166]. The chlorination of drinking water which contains PAHs has been related to the formation of oxygenated and chlorinated PAHs, compounds that are even more toxic. The Water Framework Directive (WFD) 2000/60/EC [170] intends to achieve and preserve the good ecological status of the waters of the European Union member states until 2015. For achieving such purposes, it is required to monitor the waters quality. The new European Drinking Water Guideline (98/83/EC) [171] gives special attention to benzo(a)pyrene by setting a threshold limit of 10 ng·L-1 for this single compound in drinking waters. The guideline also sets a limit of 0.10 μg L-1 for the sum of benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(ghi)pyrene, and indeno(1,2,3-cd)pyrene. Moreover, the European Union has fixed very restrictive limits for these compounds in different kinds of superficial waters in the proposal for a new Directive 2006/0129EC [172], which was approved in 2007/C 97/02 [173]. The limits for benzo(a)pyrene were fixed on 0.05 μg L-1 as annual medium value and 0.1 μg L-1 as maximum admissible concentration for different kinds of superficial waters. The limits for the sum of benzo(b)fluoranthene-benzo(k)fluoranthene and the sum of benzo(ghi)perylene-indeno(1,2,3-cd)pyrene were fixed to 0.03 and 0.002 μg·L-1, respectively, as annual medium value and as maximum admissible concentration. Naphthalene, fluoranthene, and anthracene were also included in this directive. All the above limits must be achieved in 2015. Furthermore, analytical methods used for the assessment of compliance with the requirements of the WFD should demonstrably meet stated minimum performance criteria and be implemented taking into account approved guidelines on quality assurance and quality control (QA/QC) [174]. Some other countries in the world have fixed low admissible limits for benzo(a)pyrene in driking waters, i.e. the maximum contaminant level (MCL) is 25 ng·L-1 in Japan [175]. Health Canada and the Ontario Ministry of the Environment (MOE) have established a maximum acceptable concentration (MAC) of 10 ng·L-1 for benzo(a)pyrene in drinking waters [176], whereas the US-EPA establish a MCL of 200 ng·L-1 for benoz(a)pyrene. The World Health Organization has recommended the maximum permissible concentration of six representative PAHs: fluoranthene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene and indeno(1,2,3c,d)pyrene, not to collectively exceed 200 ng·L-1 in drinking waters [who2]. The solubility of PAHs in water is low and decreases with increasing molecular weight, which is mainly due to their hydrophobic nature (log Ko/w = 3-8). Nowadays, the analysis of natural waters for PAHs, given the direct implications with human health, has been developed
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to high standards. Nevertheless, the low concentration levels of PAHs in waters continue to promote research interest, often directed towards achieving more convenient, cost-effective, and environmental-friendly methods. Main problems with water samples are found during the sampling stage. In addition to this, PAHs in water, due to their low solubility, present adsorption problems in containers. Methanol, acetonitrile, 2-propanol or surfactants are added during sampling and storage to avoid these adsorption problems [178,179]. For the extraction of water samples, most methods use liquid-liquid extraction [180] and solid-phase extraction [178]. Other extraction methods, such as supercritical fluid extraction (SFE) [181], solid-phase microextraction (SPME) [182], stir-bar sorptive extraction (SBSE) [183], single-drop microextraction (SDME) [184], and cloud-point extraction (CPE) [185], have been developed more recently. Figure 10 shows a typical scheme of the steps usually involved in the determination of PAHs for water monitoring. It should be highlighted the important decrease in the clean-up steps when dealing with water samples, mainly with drinking water samples, due to the lower complexity of the matrixes, especially when compared to samples obtained for air monitoring.
3.1. Sampling Strategies The nature of the water sample has an important influence on the sampling strategy. Drinking waters generally have low suspended solids content, and are therefore relatively straightforward to sample. In contrast, river, lake, coastal waters and groundwater exhibit much greater temporal and spatial heterogeneity [186]. Disturbances of water composition during sampling as well as alterations during transport and storage, all can irreversibly affect the outcome and subsequent interpretation of water analysis results, and so the sampling strategy must be carefully carried out. The sampling strategy should also reflect the known or expected variability of the system. The changes in concentration with sampling position at a site are important where point source inputs are found. Moreover, when samples are needed to measure discharge loads in rivers or fluxes in lakes, it is important to plan the sampling so as to cover periods when the concentrations are likely to differ from baseline values, e.g. during spates or storm periods [186]. The higher molecular-weight PAHs, due to their hydrophobic nature, tend to be particleor colloid associated, not truly dissolved in water [187,188]. These species have limited potential for off-site transport unless through facilitated transport, i.e., while associated with mobile colloids [187,189]. Thus, if sampling methods tend to increase the amount of suspended solids in water samples; reported PAH levels may be biased upward.
Passive sampling
General scheme
Common procedures
Other procedures
PAHs in water Active sampling
·Bailers ·Amber glass bottles ·Automated systems (low flow)
Sampling
Extraction
·Liquid-liquid extraction ·Solid-phase extraction
Clean-up
·Solid-phase extraction ·Solvent exchange
·Dialysis (passive sampling with SPMDs) ·Supercritical-fluid extraction ·Single-drop microextraction ·Stir-bar sorptive extraction ·Cloud-point extraction ·Solid-phase microextraction
GC or HPLC Figure 10. Scheme of the analytical steps employed in the determination of PAHs for water monitoring.
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3.1.1. Active Sampling The conventional sampling technique is the active removal, so-called snap-shot sampling, of a defined volume of water at a given time mainly using amber glass bottles [190], bailers or pumps [191]. Amber borosilicate glass containers are used for in-situ measurements, normally capped with PTFE-lined tops. They must be conveniently rinsed prior to sampling. Usually, apart from washing of the glassware with detergent and water, it includes rinsing with an organic solvent [192]. Alternatively, non-volumetric borosilicate glassware may be heated to 400ºC for 1 h [186]. The glass containers are usually completely filled with water during sampling, to avoid an excessive amount of dissolved oxygen in the aqueous sample. Ballasted bottles, which are opened at the desired water depth to carry out the sampling, are also commonly used. Bailers are sampling systems composed of two caps, which are closed at the desired water depth to carry out the sampling. They are commonly used when sampling deep waters (rivers, seawaters, ponds, etc.). Hand bailers are commonly used to collect samples due to their simplicity, low-cost, and universality but can impact chemical constituents due to the turbulence associated with their use. The automatic water samplers have two variants. They can be time-dependent, that is, the automatic samplers take sub-aliquots of waters every fixed period of time, without accounting for flow variations, whereas other automatic samplers are caudal dependent. These automated system usually accomplish the process using low flow pumping (> Tcloud point OR Equilibration time Clear aqueous solution of a nonionic surfactant C > CMC
Turbid
Addition of surfactants at C > CMC
Surfactant-rich phase
Equilibration
Analytes in an aqueous solution
Analytes associated to the micelles
Analytes concentrated in the surfactantrich phase volume after CPE
Figure 11. Steps involved in the CPE phenomena.
The coacervation extraction has been employed for the phase separation of ionic surfactants, and it is induced by other conditions such as the presence of HCl (pH induced coacervation) for anionic surfactants [231] or the presence of saturated sodium chloride and 1-octanol for cationic surfactants [232]. The separation of anionic surfactants into two isotropic phases in an acid medium takes place at room temperature. The main advantages of this approach are the absence of UV chromophore groups in alkylsulfate or alkylsulfonate molecules of ionic surfactants, the lack of time and temperature dependence in the extractionpreconcentration step, the speed of extraction, and the ability to extract thermally labile and polar compounds. An important disadvantage is the utilization of quite acidic conditions,
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which must be controlled before injection in HPLC. More recently, the coacervation extraction has also been conducted with reverse micelles [228], and its operating procedure is quite simple: addition of the extractant colloids followed by centrifugation. Main applications of CPE for the analysis of PAHs in water samples are included in Table 22, together with the operational conditions and the method performance (recoveries and detection limits).
3.2.8. Dialysis The utilization of SPMDs (filled with triolein) in water monitoring is commonly followed by dialysis to carry out desorption of retained PAHs [107,191,197,198]. This process is in general accomplished using hexane for 48 h, as described in air monitoring. 3.2.9. Capillary Microextraction (CME) and Solid-Phase Nanoextraction (SPNE) Two novel extraction approaches have been proposed for the monitoring of PAHs in water. Capillary microextraction (CME) uses a chromatographic capillary column as both extractor and preconcentrator to enrich analytes from aqueous samples. The sorption of the analytes on the thin stationary phase PDMS film of a gas chromatographic capillary column helps to extract and preconcentrate the analytes. The analytes were then desorbed with either suitable solvent or thermal desorption. The sorption area in the capillary microextractor is much higher than SPME, resulting in high recovery and high sample capacity. Peeraprasompong et al. have reported the application of a CME-GC-MS procedure to determine B(a)Py, B(b)Ft and diB(a,h)A from tap waters and raw waters, with average recoveries of 90.5% and detection limits in the ng·L-1 region using 7 mL of sample [233]. Another novel alternative is an approach named solid-phase nanoextraction (SPNE), which takes advantage of the strong affinity that exists between PAH and gold nanoparticles [234]. The novel extraction method has been applied to the determination of PAHs in waters. Among other features, it must be highlighted the small volume of water sample needed (500 μL). Furthermore, the entire extraction procedure consumes less than 100 μL of organic solvents per sample, which makes it environmentally friendly. The limits of detection ranged from 0.9 (anthracene) to 58 ng·L−1 (fluorene), and the analytical recoveries from tap water samples of the six regulated PAH varied from 83.3 to 95.7% [234].
3.3. Clean-up Procedures The lower complexity of the water samples compared to that of samples coming from air monitoring, especially when dealing with drinking waters, justify the lower number of cleanup procedures and the high number of applications that can be carried out on-line. Nevertheless, for contaminated samples it is still necessary to carefully carry out a number of clean up procedures. Among them it should be cited classical liquid adsorption chromatography in columns, solid-phase extraction, gel-permeation chromatography, reduction of solvent volume and solvent exchange, which imply an important timing of the process. Their characteristics and operational procedure have been described in air monitoring, and will not be further detailed here.
Table 22. Application of CPE to the extraction of PAHs from water samples. Sample Seawaters (filtered) 3 mL Seawaters (filtered) 3 mL Seawaters (filtered) 3 mL Wastewater (filtered) 36 mL Tap, river, groundwater (filtered) 35 mL Tap water 25 mL
River, tap (filtered) 15 mL Tap water 3 mL
PAHs 14 EPA
Surfactant polyoxyethylene-10-lauryl-ether (C16E10) 1% w/w
15 EPA
Mixture C16E10 + Brij 30, total surf. 1% (w/w), weight fraction Brij 30 = 0.74 B(a)Py, B(k)Ft Mixture C16E10 + Brij 30, total surf. 1% (w/w), weight fraction Brij 30 = 0.74 B(a)A, B(a)Py, Chy, 70 mg decanoic acid in 4 mL THF, diB(a,h)A, I(1,2,3stirring 5 min, 700 rpm cd)Py pH = 2 15 EPA 0.05 g sodium dodecane sulfonic acid + 15 mL HCl conc. 5 min stirring 15 EPA
1% Triton X-114 (w/w)
16 EPA minus N, 0.5% Triton X-114 (w/w) Aci, Chy, Ace, Fl and Phe Py, Phe, N, Chy, 0.26 g SDS + 3 mL HCl 12 M B(a)A, B(a)Py
Extraction 90 min, 95ºC Vs = 170 μL
Clean-up No 25 μL directly
Technique HPLC-FD
Performance 1–150 ng·L-1 N: 38% rest: 62-107% 23–200 ng·L-1 72–99%
Ref. [227]
60 min, 78ºC Vs = 270 μL
No 20 μL directly
HPLC-FD
60 min, 78ºC Vs = 270 μL
No Vs in the well
Fluorescence (screening)
Cut-off values: μg·L-1 region
[229]
Centrif. 10 min Vs = 115 μL
No Vs in the well
HPLC-FD
0.1–1 ng·L-1 88–95%
[228]
15 mL centrifuge 50 μL acetonitrile to HPLC-FD tubes 10 min 100 μL of Vs: 20 μL Vs = 300 μL directly
0.1–8 ng·L-1 63–106%
[235]
40 min, 40ºC
Vs + cyclohexane, silica gel column
HPLC-FD (20 μL)
[236]
5 min, 40ºC Centrif. 5 min
Vs + 100 μL acetonitrile: 10 μL directly No
HPLC-FD
0.3–12 ng·L-1 N: 0% Ace: 30%, rest: 61– 109% 2–120 ng·L-1 62–110%
Centrif. 5 min
Fluorescence
67-95%
[185]
[237]
[231]
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The utilization of internal standards (IS), recovery standards (RS), and performance reference compounds (PRCs) for SPMDs, described in air monitoring, is also applicable to water samples.
3.4. Chromatographic Procedures The selection of GC and HPLC to carry out the final determination of PAHs has been described in air monitoring. Along this section dedicated to water monitoring, the utilization of GC or HPLC in each extraction technique has been commented (Tables 18 to 22) and will not be further repeated here.
4. SOILS AND SEDIMENTS MONITORING Soils are important long-term repositories for PAHs and so they are used as an indicator of the environmental pollution. Soils are continuously subjected to pollution because of their open-system nature. Together with their capability to accumulate various pollutants, soil serves as a medium to integrate historical and recent pollution status indirectly. Accumulation of PAHs in soils may lead to further potential contamination of vegetables and food chains [238], and then cause direct or indirect exposure to humans. Moreover, leaching, evaporation and migration are possible PAHs sources of atmospheric or groundwater contamination. Therefore, knowledge of soil contamination with PAHs is needed to avoid any food production risk and to reduce the effect of these contaminants. The fate and transport of PAHs in the environment are controlled by sorption and degradation processes [239]. Sorption also plays a key role in controlling transport of PAHs during leaching, as well as in transformation and bio-accumulation processes [240]. Yunker et al. have reported that most PAHs found in soils are derived from oil seeps, bitumen, coal, plant debris, and forest fires [241]. Lichtfouse et al. have reported that soil PAHs are mainly of pyrolytic origin, with a small contribution of uncombusted fossil fuels [242]. With regard to PAHs contents, Wilcke and Amelung reported that the total of 20 PAHs in the soils from North America Great Plain ranges from 60 to 180 μg·kg-1 [243]. PAHs in vegetable soils in China are in higher contents [244], especially when irrigated with wastewaters [245]. Agricultural soils close to highways also present higher PAHs levels [246]. Higher concentrations have also been found in urban soils, with amounts ranging from 400 to 600 μg·kg-1 [247], or up to 3700-3900 μg·kg-1 [248,249]. Average contents for the 16 EPA PAHs of 140 μg·kg-1 have been reported in 138 soils in Hong Kong, with hotspots contents of 19,500 μg·kg-1 in one urban park [250]. In the same fashion than soils, sediments always belong to any control program to locate contamination sources due to their preferential sequester of contaminants and accumulation [251]. Higher weight PAHs deposited in sediments are quite resistant to bacterial degradation and so the determination of sediment concentrations yields valuable information about the PAH load and possible adverse impacts on the marine environment. Sediment retention capacity may be related to physicochemical properties such as grain size and organic matter. PAHs in sediments are mainly associated with organic carbon, and therefore the PAH
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concentrations in sediments also tend to be related to the particle size distribution. Sediments with a smaller particle size accumulate PAHs to a greater degree than do coarse, sandy sediments [252]. The presence of PAHs in sedimentary systems can have a large (negative) impact on sedimentary microbial communities and dynamics [253]. PAHs have been monitored in the marine environment since the 1960s and this continues today in local, regional, and national-scale programs [254,255]. Major long-term monitoring programs, for example the Arctic Monitoring and Assessment Program (AMAP), the Baltic Marine Environment Protection Commission (Helsinki Commission), and the National Status and Trends Program (US), focus on PAHs in marine biota and sediment as part of their strategies to assess marine environmental quality [255]. Sediment contamination evidently affects the surrounding media [256]. Heavy pollutions of halophytic plants, especially Salicornia species, have been reported further to the significant impacts of sediments by fuel oil [257]. With respect to PAHs content is sediments, Sprovieri et al. have reported PAHs contents in highly contaminated surface sediments in Naples harbor varying from 9 to 318,000 μg·kg-1 [258], whereas total content values up to 3766 μg·kg-1 have been found in the Mersey Estuary (UK) [259]. Slater et al. have reported significant temporal variability in PAH deposition to surface sediments both proximate and distal to the industrialized shoreline [253]. The monitoring of PAHs in sewage sludge results also necessary. Sewage sludge samples have been used as fertilizers for agricultural lands over a long time [260]. This is part of a sustainable practice utilizing and recycling the macronutrients back to land but, on the other hand, the negative effect due to the possible presence of contaminants in sludge has to be controlled [261,262]. PAHs concentrations about 10 times higher in urban than in rural sludge samples have also been reported [261]. A draft directive of the European Union limits to 6000 μg/kg the maximum allowable concentration of 10 PAHs in sewage sludge used in agriculture [263]. PAH distributions and concentration ratios can also be used to distinguish PAHs of petrogenic and pyrolytic origin in contaminated soils and sediments, as in air monitoring [264,265]. The monitoring of PAHs in soil and sediment samples utilizes, in general, tedious analytical schemes, due to the complexity of the matrix. Several official methods include procedures to determine PAHs in soil/sediment samples. ISO standard 13877:1998 describes a method for the determination of PAHs in soil by HPLC, whereas the recently published standard ISO 18287:2006 specifies a method for the determination of PAHs in soil by GCMS. Several EPA methods report the utilization of different extraction procedures for solid samples. As examples, the EPA method 8275 A describes the determination of semivolatile organic compounds (PAHs and PCBs) in soils/sludges and solid waste using thermal extraction (TE) followed by GC-MS. EPA method 3540 describes the utilization of Soxhlet extraction for solid samples. EPA method 3545 is related to the utilization of PLE in the determination of semivolatile organic compounds from solid samples [139]. EPA method 3561 adopts the utilization of SFE for the extraction of PAHs from solid environmental matrices, and EPA method 3550 C recommends the utilization of sonication for solid samples. Most common extraction methods for PAHs with soils and sediments are Soxhlet extraction, ultrasound-assisted extraction, microwave-assisted extraction, pressurized liquid
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extraction and supercritical fluid extraction. Figure 12 shows a scheme with the usual steps involved in the monitoring of PAHs in soil and sediment samples. On-line combination of extraction system suitable for sample pre-treatment of solid samples is much more complex than for water samples. The main reason for this is that more exhaustive methods are required for efficient extraction of target analytes from a solid matrix, and this in practice means that the extracts typically contain high amount of co-extracted matrix components, that should not be transferred to a chromatographic system. Moreover, relatively high volumes of organic solvents are required for efficient extraction, making the on-line combination more challenging [156].
4.1. Sampling Strategies Sampling of solids is more difficult than liquids or gases and larger sampling errors may easily arise due to the lack of uniformity of the material. Indeed, literature indicates that up to half of the variability between similar soils may occur within a distance of one meter. The sampling of solid samples is quite complicated, and usually requires an important amount of time to design an adequate sampling plan [264]. Sample spacing, sample collection depth, and exposure period, among others, are key factors for both passive and active approaches. The investigator must decide the number of samples to be taken, the field sample size, and the cost of sampling and analysis. Furthermore, the investigator will have to determine if gravel, cobbles, and other non-soil fractions will have to be sampled and how these are to be handled if they are sampled. General scheme
PAHs in soils or sediments
Common procedures
· Exhaustive sampling plan
Active sampling (mainly)
Sampling
Extraction
· Soxhlet extraction · Ultrasound-assisted extraction · Microwave-assisted extraction · Pressurized liquid extraction · Supercritical fluid extraction
Clean-up
· Filtration · Solid-phase extraction · Chromatographic column · Solvent exchange
GC or HPLC
Figure 12. Common steps followed in the analytical procedure for the monitoring of PAHs in soil, sediment or sludge samples.
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The minimum amount of sample that should be taken related to total size of the bulk material is around 2.5 kg per 0.5 tons of soil if the bulk material is not excessively heterogeneous. Very heterogeneous materials may need to be sampled in great bulk, amounting to 1 percent or more of the total. The resulting sample then needs to be reduced in size by some means that preserves its representative character. “Coning and quartering” is one approach. The original sample is formed into a cone-shaped pile and then flattened into a disk. The disk is divided into four quadrants. Two opposite quadrants are shoveled into a second pile, mixed together, and then coned and quartered again. This sequence continues until the selected material has been reduced to a size small enough for a useful laboratory sample. According to the EPA, there are essentially five major types of sampling situations that the environmental investigator is likely to encounter. These are: -
Large area studies, where the pollution is in the surface or shallow layers, e.g., in support of an ambient monitoring effort.
-
Large area studies, where pollution has moved down into the soil profile, e.g., assessing the impact from a major industrial complex.
-
Localized area studies, where pollution is in the surface layers, e.g., sampling around a recent hazardous chemical spill or capacitor rupture.
-
Localized plume studies, where the major source of contamination is below the surface at some depth, e.g., sampling near a leaking hazardous waste disposal site.
-
And special research studies conducted for litigation, scientific understanding, method development, or source identification.
There are two portions of the soil that are important to the environmental investigator. The surface layer (0–15 cm) reflects the deposition of airborne pollutants, especially recently deposited pollutants and also pollutants that do not move downward because of attachment to soil particles. On the other hand, pollutants which have been deposited by liquid spills, by long-term deposition of water soluble materials, or by burial may be found at considerable depth. The methods of sampling each of these are slightly different, but all make use of basic techniques [266]. The most common sampling approaches are: simple random sampling, stratified sampling and composite sampling. Simple random sampling is carried out locating the sampling points referenced to coordinate axis fixed in the sampling area, and selecting the sampling coordinates points with random numbers. Divisions of 1/100 are considered sufficient. Classically, the sampling is achieved through random sampling schemes. However, such simple random sampling schemes can be very demanding of laboratory effort to analyze the large numbers of samples that are collected, and can therefore be costly to carry out [264]. One of the tools of sampling that can be used to reduce the variability of the sample is stratified sampling. Strata are identified as regions of the site that are expected to be uniform in character. The variance within the strata should be smaller than the variance between strata. Sampling points within the strata are selected systematically or by some random process. In the soil environment, strata are often associated with soil types or as areas of known pollution versus areas where pollutants are not expected to be present.
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Composite sampling is standard practice in the analysis of soils, and is mainly used to estimate mean concentrations for an area at a reduced cost. Composite sampling involves the physical combination of homogenized samples to form a set of new samples (i.e., composite samples). The chemical analyses are then performed on aliquots of the composite samples. Because the compositing physically averages the individual samples, averaging the analytical results of the smaller number of composite samples can theoretically produce an estimated mean that is more precise than, or at least as precise as, one based on many more individual sample results [264]. The main anticipated benefits of composite sampling are that it may achieve better precision of an estimated mean at less cost, and data analysis is usually straightforward. The limitation of composite sampling is that it yields a reduced amount of information on variability; for example, composite sampling loses information on individual samples and on spatial (or temporal) patterns. After the convenient sampling plan, active sampling strategies are mainly conducted [267].
4.1.1. Active Sampling Active sampling relies on taking the soil (or sediment, or sludge) sample, to further carry out the analytical extraction of PAHs in the laboratory. Samples can either be collected with some form of core sampling or auger device, or they may be collected by use of excavations or trenches. In the latter case, the samples are cut from the soil mass with spades or short punches. Drags are usually employed for the sampling of the surface layers, and are composed of two spades (or shovels) with opposite movements. Tubular probes are often used when intending a study of temporal evolution. A tubular probe is formed by a stainless steel cylinder containing a plastic tube, with a valve which closes the whole system when the desired depth is reached. Glass tubes are also used for surface layer sampling. It is obvious that care must be taken to ensure that no fragments of the container contaminate the sample [267]. Active sampling is the main sampling approach when intending to monitor PAHs in soil, sediment or sludge samples. 4.1.2. Passive Sampling Passive sampling relies on trapping the PAHs directly from the soil sample, without the necessity of sampling the soil itself. Passive sampling from solid matrices is in essence a soil gas or soil vapor sampling methodology, and so it is mainly limited to volatile hydrocarbons. In addition to this, the concentration of the volatile hydrocarbons in the vapor phase is dependent on many variables, such as soil particle size and mineralogy, organic and moisture contents, temperature and overall heterogeneity of particle size and composition, leading to difficulties in converting the amount of analyte collected by the sampler to its concentration in the sampled medium. As a result, most passive solid sampling applications are restricted to qualitative or screening purposes only [268]. Typically, soil gas sampling involves collection of vapor occupying the pore space of soils. The vapor is composed of air and water vapor, naturally occurring organic compounds, anthropogenic organic compounds, organic compounds migrating from deep fossil fuel reserves, and other constituents capable of partitioning from liquid and solid materials into the vapor phase under ambient conditions [107].
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Solid-phase microextraction (SPME) devices can be employed with solid samples as passive samplers, but this approach must be conducted in headspace mode and so it is limited to the more volatile PAHs [269]. Furthermore, it appears that SPME is a better approach to extract primarily PAHs present in sediment porewater rather than in the colloidal phase [270]. EPA method 8272 describes the determination of parent and alkyl polycyclic aromatics in sediment pore water by SPME-GC-MS. In a similar way, SPMDs have been mainly used for the determination of PAHs contained in sediment porewater [271].
4.2. Extraction Procedures Chemical preservation of solids is generally not recommended and therefore cooling to 4ºC is usually the best approach to preserve solid samples. Wide mouth glass containers with Teflon lined caps are mainly utilized during transport and storage of the samples. If composite sampling techniques or multiple grabs are employed, equal portions of solid sample from each location are deposited into a stainless steel, plastic, or other appropriate composition (e.g., Teflon) containers [266]. Once in the laboratory, water content must be removed from solids samples, mainly by lyophilization (also called freeze-drying) [46,259,261,264,265]. It is a relatively complex and expensive form of drying, and it usually takes between 24 to 48 h the complete dryness of a sediment sample. Important advantages of the technique are the avoiding of changes in the structural characteristics of soils and sediment samples during the drying process and the avoiding of losses of the most volatile PAHs. Other procedures for water removing are heating [253] and air drying [250,272-274]. PAHs contained in soils and sediments are mainly extracted using Soxhlet extraction [46,250,259,272,275], ultrasound-assisted extraction [46,264,265,270], microwave-assisted extraction [253,276,277], pressurized liquid extraction [258,272,275,278-280] and supercritical fluid extraction [280,281]. These techniques have been widely described in air and water monitoring. Focused-microwave assisted Soxhlet (FMAS), which up to date have not had any applications in air monitoring, is however widely employed with soils and sediments for PAHs monitoring [282-284]. FMAS have demonstrated successful quantitative results, being characteristic for the necessity of short extraction times if compared to Soxhlet. PAHs contained in sludge are mainly extracted by Soxhlet extraction [285], but more recently some alternative techniques have been employed, including ultrasonic-assisted extraction [286], microwave-assisted extraction [287] and pressurized liquid extraction [288]. Table 23 includes main extraction techniques used in the monitoring of PAHs in soil, sediment and sludge samples in recent years, altogether with some other analytical features. An interesting environmental improvement to traditional extraction methods for solid samples, mainly with microwave-assisted extraction and ultrasound-assisted extraction, has been conducted in recent years. The environmental modification implies the avoiding of organic solvents in the extraction step, and the successful substitution of such organic solvents by aqueous micellar media [289-291], as can be observed in Figure 13. The aqueous micellar extract containing PAHs can be further injected in a HPLC after filtration or centrifugation. The micellar microwave-assisted extraction and the micellar ultrasoundassisted extraction procedures have been validated using certified reference material [292]. IL-micelles have been also employed in extraction processes of PAHs bounded to sediments [293]. An important disadvantage is that surfactants usually bind to the C18 stationary phases
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commonly used in HPLC, and thus their co-elution with analytes can be problematic if both, analyte and surfactant, have similar absorbance (or fluorescence) spectra. Another disadvantage of micellar assisted extraction with microwaves or ultrasounds is the fact that applications are limited to the further utilization of HPLC. The combination of the micellar extracts with GC is not possible, unless complicated clean-up steps are carried out to remove the surfactants, and so organic solvents are then again involved in the procedure. Nevertheless, it has recently been proposed the utilization of solid-phase microextraction to link micellar extracts with GC. The technique has been named as micellar solid-phase microextraction (MSPME) [294]. MSPME combines the high extraction efficiency and analyte preconcentration advantages of micellar assisted extraction with the speed and high separation efficiency of GC. MSPME has been applied to the determination of PAHs in sediment samples using traditional surfactant micelles [294] and IL-micelles [295], and the procedures have been validated using certified materials. Several reports have also described the utilization of cloud-point extraction as a preconcentration technique to be conducted after a micellar microwave- or ultrasound-assisted extraction of PAHs from soil/sediment samples [237,296], therefore improving the whole detection limits of the technique. Such preconcentration procedures can be carried out on-line [297]. Several applications which utilize micellar media in microwave-assisted extraction, ultrasound-assisted extraction, MSPME, and micellar assisted extraction followed by CPE, for PAHs contained in solid samples, are included in Table 23, together with other conventional extraction techniques.
Extraction vessel
Addition of an aqueous micellar solution (surfactant concentration above the CMC)
Application of Microwaves or Ultrasounds to accelerate the extraction process
PAHs trapped by the micelle Solid sample PAHs bound to the solid matrix
Micelle
Clean-up steps (i.e. filtration)
Direct injection of the aqueous micellar extracts in a HPLC to perform the quantification of the PAHs
Figure 13. Scheme of steps involved in a micellar microwave- or ultrasound-assisted extraction to determine PAHs in solid samples.
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4.2.1. Matrix Solid-Phase Dispersion (MSPD) Matrix solid-phase dispersion uses bonded-phase solid supports as an abrasive to produce disruption of sample architecture and a bound solvent to aid complete sample disruption during the sample blending process. The sample disperses over the surface of the bonded phase-support material to provide a new mixed phase for isolating analytes from various sample matrices [298]. The solid support is commonly a silica-based material such as derivatized silica, silica gel, sand and florisil, and sometimes graphitic fibers and alumina [52]. The blended mixture should then be packed into a column and a sequential elution is conducted with solvents to collect the analytes by fractionation. The eluate may be directly used for further instrumental analysis, but additional SPE is suggested to remove the coeluted interferences or to clean-up the analytes by further fractionation. MSPD is characteristic for a significant reduction in solvent consumption compared to traditional extraction techniques, without requiring special expensive instrumentation [261]. The method is however fairly labor intensive. Applications of MSPD for the determination of PAHs in soil, sediment or sludge samples are included in Table 23.
4.3. Clean-up Procedures In general, clean-up procedures are necessary for solid samples, due to the common high level of interfering materials (i.e., aliphatic hydrocarbons) present in the extracts. Filtration [253,276,289,290,297], SPE [46,152,261], and classical liquid adsorption chromatography in columns [250,253,265,270,276,278] are the most common clean-up procedures conducted with soil, sediment or sludge samples. Solvent exchange and reduction of solvent volume before injection in the chromatographic system are often conducted [250,253,264,270,274,275]. These procedures have been described in the section of air monitoring in this chapter. The utilization of internal standards (IS) and recovery standards (RS) is a common quality control practice in soil/sediment monitoring, as in air or water monitoring.
4.4. Chromatographic Procedures GC and HPLC (widely described in previous sections of this book chapter) are the common chromatographic techniques to carry out the final determination of PAHs in soil monitoring. Its utilization in combination with each specific extraction procedure is included in Table 23.
5. FOOD MONITORING Food appears to be the main source of PAH intake for humans not occupationally exposed to PAH [48]. Vyskocil et al. have pointed out that food consumption represents the main source of PAH exposure for the children even in big cities that are heavily polluted by PAH from industrial sources [303]. PAH contamination of food arises from two sources,
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environment and food-processing technique. The PAHs present in water and rivers are available to fishes and other biota via different uptake routes, such as diet and/or through direct transport across external membranes [304], and so to human beings by direct consumption. Consumers can also be exposed to PAHs by eating contaminated milk, cereals, flour, bread and vegetables [305,306]. PAHs can enter plant tissues by partitioning from contaminated soil to the roots and translocation into the shoot. However, the extent of PAH uptake by roots is still debated in the literature [305,307,308]. It seems that the main pathway of plant contamination is atmospheric exposure. Heat processing of meat and dairy products, such as charcoal grilling [309], roasting, and smoking [310-312], constitute important sources for contamination with PAHs. Several cooking practices, common round the globe, such as repeated frying of vegetarian and non-vegetarian foods in edible oil, are quite risky. Pandey et al. have studied the cytotoxicity of polycyclic aromatic hydrocarbon residues arising through repeated fish fried oils in humans [313]. In addition, PAHs could arrive in food products through contaminated packaging material [314]. In vivo studies have suggested a transfer in human intestinal epithelium by diffusion, which appears extensively governed by the physicochemical properties of PAHs, particularly lipophilicity [21,315,316]. The PAHs content have been assessed in several kinds of foods. The purpose of this section is not to deeply summarize all the works conducted to estimate PAHs levels in foods rather but to give an overview of the common levels found in different kind of foods. These levels will give an idea about the sensitivity required for the analytical methods to be used in food monitoring. Kishikawa et al. have reported that the average concentrations of total PAHs in commercial milk, infant formula and human milk were 0.99, 2.01 and 0.75 μg·kg-1, respectively [317]. The total PAHs content have also been examined in green tea leaves, with average values ranging from 497 to 517 μg·kg-1 whereas brick tea leaves presented total contents 1,048 to 1,162 μg·kg-1 [318]. The PAHs content in fats and oils has been reviewed by Moret et al. [319]. The determination of PAHs in smoked meat products and smoke flavouring food additives has recently been reviewed by Simko [310], whereas the determination of PAHs is smoked fish has recently been reviewed by Stolyhwo et al. [320]. The highest contents of B(a)Py have been found in smoked fish [310] if compared to other smoked foods. With respect to marine foods, the bioaccumulation of PAHs by various marine organisms has been extensively reviewed by Meador et al. [321]. Boehm et al. have reported total PAHs contents in mussels ranging from 3 to 1200 ng·g-1 (dry weight) [322]. Average contents for the 16 EPA PAHs around 5400 ng·g-1 (wet weight) have been reported in mussels of the Red Sea coast by Nemr et al. [323]. Kong et al. have examined PAHs content in freshwater fishes in Hong Kong [324]. The contents found in fishes purchased from the markets supplied from the mainland showed higher values, around 76 ng·g-1 (wet weight), than those collected from fish ponds, with values around 60.1 ng·g-1.
Table 23. Analytical techniques most commonly used in the monitoring of PAHs in soil, sediment, and sludge samples. Sample sandy soil, industrial soil CRM-524 19 sludge samples and one CRM clay soil clay soil clay soil
PAHs Analytical procedure Py, B(a)A, B(a)Py, B(k)Ft, PLE (hot water + SDS)-on-line-FD B(ghi)Per 27 PAHs MSPD-US-SPE-GC-MS
Analytical features Quantitative extraction, short extraction time DLs: 0.03-0.45 μg·kg-1
Ref. [278,279]
Py, B(a)A, B(a)Py, B(e)Py, FMAS (CH2Cl2)-GC-ECD B(k)Ft Py, B(a)A, B(a)Py, B(e)Py, FMAS (acetonitrile)-on line-FD B(k)Ft, B(ghi)Per nitro-PAHs FMAS (CH2Cl2)-GC-ECD
Recoveries > 94%, overall time: 1 h
[282]
Recoveries > 98%
[283]
[261]
Recoveries > 89.7%, DLs: 0.5-1.5 μg·kg- [284] 1
miniaturized PLE-LVIa-GC-MS
Recoveries > 83% except N (59%), DLs: 0.8-30 μg·kg-1, comparison with Soxhlet sea sediment 16 EPA PAHs minus Ace, PLE (hot water)-LC-GC QLsb: 0.001-0.01 μg·g-1, comparison Aci and diB(a,h)A with SFE-GC-MS sediments, soils and one CRM 16 EPA PAHs PLE (hot water)-DTSc-on line-GC-MS Successful recoveries, extraction times: 35–60 min, comparison with Soxhlet sediments 16 EPA PAHs UAE (CH2Cl2)-GC-MS DLs: 1-4 μg·kg-1 sediments 16 EPA PAHs UAE (CH2Cl2)-GC-MS Recoveries > 80% 2 CRM sediments N, Ace, Aci, Fl, A, Ft, Py HSPME-GC-FID DLs: 0.3-3 pg·g-1 sediments and CRM 16 EPA PAHs MAE (hexane:acetone 1:1)-GC-MS Recoveries > 80% sediments and CRM 16 EPA PAHs MAE (CH2Cl2)-GC-MS — QUASIMEMEd sediments 16 EPA UAE (CH2Cl2:methanol 1:1)-GC-MS soils Ace, Fl, Phe, A, Ft, Py HS-LPME (hollow fiber)-GC-MS drop 3 μL octanol, DLs: 5.9-76 ng·g-1 soils 16 EPA UAE or PLE (hexane)-GC-MS DLs: 0.02-0.75 μg·kg-1, recoveries: 59110% except N (in PLE) 138 soils and CRM 16 EPA Soxhlet (acetone:CH2Cl2 1:1)-GC-MS DLs: 5-9 μg·kg-1, recoveries > 78% clay soil 16 EPA Soxhlet or UAE (acetone:CH2Cl2 1:1)- DLs: 0.5-1 mg·kg-1 GC-FID sediments N, Ace, Aci, Fl, Phe, A, Ft, SPME (100 μm PDMS)-GC-MS Comparison with LLE (CH2Cl2) Py soils Phe, Py, Chy, B(k)Ft, off-line micellar MAE or UAE (1% Overall recoveries > 88.7% B(a)Py tergitol)-on-line CPE-HPLC-FD a b LVI : large volume injection QLs: limits of quantification c DTS: different trapping systems: solid-phase extraction, micro-porous-membrane liquid extraction (hollow fiber or flat sheet). LLE is off-line. d QUASIMEME: quality assurance of information for marine environmental monitoring in Europe organic and sandy soils
16 EPA PAHs
[272] [280] [275] [270] [264] [269] [253] [276] [265] [273] [274] [250] [299] [270] [297]
Table 23 (Continued). Sample soils, sediments, sludges (CRMs) soils, sediment (CRM)
PAHs Py, B(a)A, B(b)Ft, B(k)Ft, B(a)Py, B(e)Py, I(1,2,3-cd)Py 16 EPA minus Aci, Ace, Fl, diB(a,h)A and B(ghi)Per 16 EPA PAHs + B(e)Py
Analytical procedure micellar extraction-CPE (2% SDocSe)off-line HPLC-FD SFE-GC-MS
Analytical features Recoveries: 71-98%, comparison with Soxhlet Recoveries > 88%, comparison with Soxhlet sediments and CRM PLE/ASE (hexane:acetone 80:20 v/v)- Recoveries: 94-107%, DLs ~ 1 ng·g-1 GC-MS sediments and CRM 16 EPA PAHs minus Aci Soxhlet (CH2Cl2)-HPLC-FD — sediments 16 EPA Soxhlet or UAE (CH2Cl2:hexane 1:1)- Recoveries: 47–122% except N GC-MS soils and CRMs 16 EPA PAHs minus Aci Soxhlet (THF) or UAE (THF or Comparison with ELISA. Recoveries: acetonitrile)-HPLC-FD 60–102% Quantitative recoveries CRM sediment 16 EPA PLE/ASE (CH2Cl2)-GC-MS marine sediments 16 EPA minus A, Ace, Aci off-line micellar MAE (1% POLEf)- Recoveries > 87% HPLC-UV CRM sediment Py, B(a)A, B(b)Ft, B(k)Ft, off-line MAE (45mM IL HDBIm-Brg)- Average recoveries 91.1%, DLs: 0.8–53 ng·g-1 B(a)Py, B(e)Py, I(1,2,3-cd)Py HPLC-FD CRM sediment Phe, Fl, Py, B(a)A, Chy, focused MW (CH2Cl2)-GC-MS Quantitative recoveries B(b)Ft, B(a)Py, B(e)Py, Per, diB(a,h)A, B(ghi)Per, I(1,2,3cd)Py CRM sediment Fl, Phe, A, Ft, Py, B(a)A, Chy, off-line MAE (POLE)-SPME-GC-MS R: 58.6-111.5%, DLs: 20-547 ng·g-1 B(b)Ft, B(k)Ft, B(a)Py sludge (30) Ace, Fl, Phe, Ft, Py, B(b)Ft, — UAE (CH2Cl2)-GC-MS B(j)Ft, B(k)Ft, B(a)Py, B(ghi)Per, I(1,2,3-cd)Py marine sediments 16 EPA minus N, Aci, A and micellar UAE or micellar MAE (with R: 58-118%, DLs: 50–1300 ng·g-1 B(k)Ft POLE)-CPE-HPLC-UV sludge 16 EPA PLE/ASE (CH2Cl2:hexane 1:1)-GC- R: 60.6-93.4%, comparison with Soxhlet MS soils 16 EPA ELSh (CH2Cl2:acetone)-HPLC-FD — soils and one CRM 16 EPA UAE (acetone:petroleum ether 1:1)DLs: 2-42 ng·g-1, R: 75–120% GC-MS soils 16 EPA Soxhlet (hexane)-GC-MS R: 32-96%, QLs ~ 0.1–0.2 ng·g-1 e f SDocS: sodium dodecane sulphonate POLE: polyoxyethylene-10-lauryl ether g h HDBIm-Br: 1-hexadecyl-3-methylimidazolium bromide ELS: liquid-solid extraction with mechanical shaking
Ref. [300] [281] [258] [259] [46] [301] [302] [289] [293] [277]
[294] [286]
[296] [288] [245] [249] [247]
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Total PAHs contents ranging from 4 to 69 ng·g-1 (wet weight) in Lipophrys pholis fishes have been reported by Lima et al. [325]. Perugini et al. have reported total PAHs content of 44.163.3 ng·g-1 in fishes taken at pools in the Adriatic Sea [326]. Cheung et al. have reported differences in PAHs content depending on the fish studied (river or marine fishes), but the differences were not that accused among marine fishes [327]. Nevertheless, with respect to the uptake of PAHs by fishes, a major fraction of the PAHs absorbed is rapidly metabolized to hydroxylated derivatives (phase 1 metabolism), then conjugated to polar groups (phase 2 metabolism), and stored in the gall bladder to be excreted. The analysis of PAH metabolite levels in fish bile can be used to assess the actual PAH uptake, rather than the analysis of the non-hydroxylated PAHs content [328]. This aspect will be further discussed in the section of biomarkers within this book chapter. There have been several regulations with respect to PAHs levels in food samples, mainly focused on B(a)Py contents, with maximum contents around 1 μg·kg-1. In 2002, the Scientific Committee on Food of the European Union assessed 33 PAHs and identified 15 PAHs that possess both genotoxic and carcinogenic properties, and concluded that B(a)Py could be used as a marker [329]. The Committee also recommended monitoring B(a)A, B(b)Ft, B(j)Ft, B(k)Ft, B(ghi)Per, Chy, cyPent(c,d)Py, diB(a,h)A, diB(a,e)Py, diB(a,h)Py, diB(a,i)Py, diB(a,l)Py, I(1,2,3-cd)Py, and 5-MeChy in food to enable long-term exposure assessments. In 2005, the European Commission (EC) asked the EU Member States for further investigations on the 15 EU priority PAHs (Table 3) together with B(c)Fl [330], highlighted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2005 [331]. The European union has set maximum levels for PAHs, specifically for B(a)Py, in certain food products. The maximum allowed concentration for B(a)Py in various food products is in the range 1-10 μg·kg-1 [332], and for B(a)Py and B(a)A in liquid smoke flavoring primary products is 10 μg·kg-1 and 20 μg·kg-1, respectively [333]. The PAH on-line analytical database is available on the EFSA web site [334]. Wenzl et al. have recently reviewed the current European Union (EU) legislation on PAHs for official control of food samples [335]. The foods samples most commonly analyzed for the determination of PAHs are marine foods (due to the risk of PAHs content by water contamination), smoked foods (due to the risk of PAHs formed during the smoking process), oils, and in general foods that could be potentially exposed to PAHs formation during their preparation and manufacturing. Foods are quite complex matrixes for any kind of monitoring. Due to the multiplicity of food matrices, it is not possible to use one sample preparation technique, so many methods have been proposed for meeting all the requirements. There are few national or international standard methods related to B(a)Py in food samples, but also little information about collaborative trials has been published so far [335,336]. Furthermore, the majority of the reports of analytical methods to monitor hydrocarbons in foods have been focused on the determination of B(a)Py exclusively or on the 16 EPA PAHs. Several works are starting to show up which includes the determination of the 15 PAHs recommended by the EU in food samples [336-338]. Fidalgo-Used et al. [137] have recently reviewed the main extraction and clean-up procedures, published in the last 5 years, applied to the analytical determination of both conventional and emerging persistent organic pollutants (POPs), not exclusively PAHs, in environmental biota (vegetal and animal) samples. Buldini et al. [339] have also reviewed recent sample preparation methods for food analysis.
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For the analysis of foods, the choice of the sampling strategy depends on the nature of the food sample and on the purpose of the analytical measurement. In general, foods are acquired in supermarkets, or in production lines when performing quality control steps. In any case, main cautions are taken with the recipients used to transport the samples to the laboratory to avoid contamination. The utilization of clean scissors, knifes or any other cutting device when dealing with complex samples such as mussels or fishes is also mandatory. In many occasions, freeze-drying of the samples is also advisable to ensure a better conservation. An important analytical step which is widely conducted with food samples, especially with greasy foods, and has not been described yet in this book chapter, is the saponification [276,309,310,317,323,326,336,340-342]. Saponification is carried out in the analytical laboratory as a clean-up step to break the fatty acid esters (usually triglycerides) of vegetable oils and animal fats, and so to digest the sample releasing the PAHs. The process is conducted by reflux using a strong alkali, normally sodium or potassium hydroxide, in an aqueous alcoholic solution (methanol or ethanol). Traditional saponification procedures require 40 min up to several hours. After saponification, liquid-liquid extraction is usually conducted [276,309,317,326,336,340-342]. The overall procedure results labor intensive and timeconsuming. General scheme
Common procedures
PAHs in foods
Sampling
Extraction
· Saponification–Liquid-liquid extraction · Soxhlet extraction · Ultrasound-assisted extraction · Microwave-assisted extraction · Pressurized liquid extraction · Supercritical fluid extraction · Matrix solid-phase dispersion
Clean-up
· Solid-phase extraction · Chromatographic column · Solvent exchange
GC or HPLC
Figure 14. Common analytical steps carried out in the monitoring of PAHs in food samples.
Other common extraction techniques for the monitoring of polycyclic aromatic hydrocarbons in food samples are Soxhlet extraction [305,318,323,324,327,343], ultrasound-
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assisted extraction [312,338,344], supercritical fluid extraction (SFE) [345-347], microwaveassisted extraction (MAE) [340,348], pressurized liquid extraction (PLE) [46,302,337,349,350], and matrix solid-phase dispersion (MSPD) [351,352]. In some cases, the alkali saponification step is also conducted as a clean-up step right after these extraction techniques (or simultaneously) to ensure an adequate recovery of PAHs from food samples with high fat content [46,312,323,348]. In some cases, the digestion of complex samples such as fish bile requires an enzymatic hydrolysis, in general utilizing β-glucuronidase/sulfatase [328]. Other extraction techniques, such as headspace extraction techniques, have also proven to be successful when dealing with olive oil samples using a triple-quadrupole MS detector (QqQ) [353]. All these extraction techniques have been described in other sections of this book chapter and will not be further discussed here. The complexity of the food samples limits the utilization of on-line procedures in combination with chromatographic techniques. This problem is normally related to any complex solid matrix. Clean-up steps such as SPE, liquid adsorption chromatography in columns, size exclusion chromatography, SPME, solvent exchange, reduction of solvent volume… are absolutely necessary when dealing with food samples. Given the complexity of the food samples, as well as the necessity of a number of clean-up steps to purify the extract, the utilization of IS and/or RS, as well as adequate certified materials, becomes an inherent necessity. These clean-up steps have been described in other sections of this book chapter. Figure 14 shows a scheme of the typical steps used in the monitoring of PAHs in food samples. Table 24 includes a list of several analytical procedures utilized in recent years for the determination of polycyclic aromatic hydrocarbons in several food samples as a summary of the state of the art.
6. BIOMARKERS Several definitions have been given for the term biomarker, which is generally used in a broad sense to include almost any measurement reflecting an interaction between a biological system and a potential hazard, which may be chemical, physical or biological [357]. Van Gestel et al. considered biomarker as any biological response to an environmental chemical at the subindividual level, measured inside an organism or in its products (urine, faeces, hair, feathers, etc.), indicating a deviation from the normal status that cannot be detected in the intact organism [358,359]. A bioindicator is defined as an organism giving information on the environmental conditions of its habitat by its presence or absence or by its behavior. Biomonitoring provides an assessment of the integrated uptake through all exposure routes. This is important since only the bioavailable chemical is likely to be assimilated-concentrated in tissues, body fluids, and excreta.
Table 24. List of analytical methods recently used in the extraction of PAHs from a number of food samples. Food SRM 2974a
trout and sardines mussels tissues, SRM 2977a oils, SRM 2974b smoked and fresh fishes, rape-oils mussels, SRM 2974b
PAHs 16 EPA, 2-MeN, 1-MeN, Biphenyl, 1-MePhe, B(b)Ft, Triphenylene, Per, diB(a,c)A, diB(a,j)A, B(b)Chy Py, B(a)A, B(k)Ft, B(a)Py, B(ghi)Per 16 EPA 15 EU foods + Phe, A, Ft, Py B(a)A, B(a)Py, B(b)Ft, B(k)Ft, B(ghi)Per, I(1,2,3-cd)Py 16 EPA PAHs
Analytical method PLE (CH2Cl2)-GC-MS
Analytical performance Ref. Quantitative extractions, comparison [302] with Soxhlet
PLE (hot water)-HPLC-FD
R > 97.6%
[278]
Soxhlet (acetone:hexane 1:1)-GC-MS
DLs: 0.2–1 ng·g-1
[325]
DLs: 0.008–0.15 μg kg-1 DLs ~ 0.3 μg·L-1 (oil), ~ 0.25 μg·g-1 (fish), R ~ 75% R: 86–108%
[337] [354]
PLE (acetone:hexane 1:1)-GC-MS-MS hydrolysis (digestion)-LLE (CH2Cl2:hexane 1:3)-GC-MS Soxhlet (methanol)-saponification-LLE (hexane)-GC-MS olive oils Ft, Py, B(a)A, Chy, B(b)Ft, B(k)Ft, HS-GC-MS-MS (QqQ) B(a)Py, I(1,2,3-cd)Py, diB(a,h)A, B(ghi)Per pumpkin seed oils, CRM 16 EPA MAE-saponification-LLE (hexane)-GC458 MS smoked meat (beef) Phe, A, Ft, Py, B(a), Chy, B(b)Ft, SFE-simultaneously SPE (C18)-GC-MS B(e)Py, B(a)Py, Per A, Ft, B(a)A, B(b)Ft, B(k)Ft, miniaturized MSPD―in-tube in-valve mussels, tellins, SRM B(a)Py, diB(a,h)A, B(ghi)Per SPME-HPLC-FD 1974bc mussels, SRM 1947ad triphenylene, B(j)Ft,B(a)Ft, Per, saponification-LLE (pentane)-GC-MS B(e)Py, 16 EPA minus N, Aci, Ace, Fl mussels 16 EPA PLE (hexane:acetone 1:1 / hexane:CH2Cl2 1:1)-saponification-GC-MS saponification-LLE (cyclohexane)-HPLCbilvalves, fish, cephalopods, 16 EPA minus N, Aci, Py FD crustaceans fish 16 EPA Soxhlet (acetone:CH2Cl2 1:1)-GC-MS tea 16 EPA Soxhlet (acetone:CH2Cl2 1:1)-GC-MS milk (commercial, infant, 16 EPA minus N, Aci, Ace, Fl saponification-LLE (hexane)-HPLC-FD human) cabbage, carrot, lettuce, B(e)Py, 16 EPA minus Aci Soxhlet (CH2Cl2)-HPLC-FD leek, endive fish 16 EPA Soxhlet (acetone:CH2Cl2 1:1)- GC-MS a b SRM 2977: organics in freeze-dried mussels tissue SRM 2974: mussel tissue c d SRM 1974b: mussel tissue SRM 1947a: mussel tissue
[323]
R: 96–99% DLs: 0.02–0.06 μg·kg-1
[353]
DLs: 0.01–0.12 ng·g-1 R > 83.6% DLs: 2.5–4.1 ng·g-1
[340] [346]
DLs ~ 0.6 ng·g-1
[352]
R > 60% DLs ~ 0.05 ng·g-1
[276]
DLs ~ 0.5-8 μg·kg-1 R: 64-121% R: 63-100% DLs: 0.05-0.25 μg·kg-1 DLs ~ 0.05 ng·g-1 R: 90-105% DL: 1.3-76 ng·kg-1 R: 71-92% except N
[46]
DLs ~ 20 ng·g-1
[326] [327] [318] [317] [305] [324]
Table 24 (Continued) Food smoked cheese
Analytical method UAE (cyclohexane)-saponificationLLE (cyclohexane)-GC-MS
Analytical performance use of IS
Ref. [312]
fish
PAHs B(c)Phe, B(j)Ft, B(e)Py, Per, 16 EPA, diB(a,l)Py, diB(a,i)Py, diB(a,h)Py, 23 alkyl-PAHs 16 EPA
Soxhlet (CH2Cl2)-GC-MS
[343]
mussels
total PAHs content
UAE (1st CH2Cl2, 2nd hexane)spectrofluorimetry MAE (hexane)-saponification-HPLCFD PLE (hexane:acetone 1:1)-HPLC-FD. PLE/ASE (CH2Cl2)-GC-MS
R: 45-84% DLs: 1–87 pg·g-1 R: 98-10% DLs (total PAHs): 21 ng·g-1 R ~ 90% QLs: 0.07–0.53 ng·g-1 Comparison with Soxhlet and UAE DLs for B(a)Py: 1.5–5.8 pg·g-1
fish, SRM 2977a
B(a)A, B(b)Ft, B(k)Ft, B(a)Py, diB(a,h)A, I(1,2,3-cd)Py fish B(e)Py, 16 EPA, 6 alkyl-PAHs mussel, salmon, fish oil, fish 24 PAHs feed, SRM 2977a fish, SRM 2977a B(a)A, B(b)Ft, B(k)Ft, B(a)Py, diB(a,h)A, I(1,2,3-cd)Py trout, sardine Py, B(a)A, B(a)Py, B(k)Ft, B(ghi)Per smoked fish, bovine milk barbecued meat sausages, CRMs 458e – 459e toasted bread
Phe, A, Ft, Py, B(a), Chy, B(b)Ft, B(e)Py, B(a)Py, Per 16 EPA 15 EU foods minus cyPent(c,d)Py, diB(a,e)Py, diB(a,h)Py, diB(a,i)Py 16 EPA
strawberries, apples, tomatoes, lettuce, parsley, potatoes, kale sunflower oils in containers N, 2-MeN, 1-MeN, DiMeNs, 9-H-Fl commercial liquid smoke 34 PAHs flavorings smoke flavorings 15 EU foods marine plant biota vegetable oils
16 EPA
[344] [348] [349] [350]
R ~ 80% DLs: 0.04–0.32 ng·g-1 PLE (hot water + SDS)-on-line-FD Quantitative extraction, short extraction time SFE-simultaneously SPE (C18)-GC- R > 97% MS DLs: 10-14 ng·g-1 (fish) saponification-LLE (cyclohexane)-GC- DLs ~ 0.06 μg·kg-1 MS or HPLC-UV/FD UAE (hexane)-HPLC-FD R > 82% DLs: 0.01-0.70 μg·kg-1 PLE/ASE (hexane:acetone 1:1)-HPLC- — FD
[351]
HS-SPME-GC-MS saponification-LLE (cyclohexane)-GCMS saponification-LLE (cyclohexane)-GCMS saponification-LLE (hexane)-GC-MS
use of IS R > 84%
[356] [341]
R > 69% DLs: 0.1–1.3 μg·kg-1 R: 88-116% DLs: 3.5–18 μg·kg-1 DLs ~ 1.55 μg·kg-1
[336]
MSPD-HPLC-FD
B(a)A, B(e)Py, B(a)Py, B(b)Ft, B(k)Ft, SFE-HPLC-FD diB(a,h)A, B(ghi)Per CRMs 458 and 459: coconut oil reference materials containing six PAHs
[279] [345] [309] [338] [355]
[342] [347]
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Among several classes of biomarkers, the biomarkers of exposure cover the detection and measurement of an exogenous substance or its metabolite that is measured in a compartment within an organism. Therefore, biomarkers of exposure will be the focus of this section, and more specifically, bioaccumulation markers [360]. The bioaccumulation monitoring can be defined as an exposure assessment by measuring contaminant levels in biota. This section will specifically focus on the chemical methods used in the monitoring of PAHs and their metabolites in biota, tissues, body fluids and excreta, as a measure of exposure contamination. Fish bioaccumulation and biomarkers in environmental risk assessment have been reviewed by Oost et al. [360]. Fish bioaccumulation markers may be applied in order to elucidate the aquatic behavior of environmental contaminants and to assess exposure of aquatic organisms. The feasibility of PAH tissue concentrations in marine species as a monitoring parameter for PAH exposure depends on their uptake, biotransformation and excretion rates. Since it remains hard to accurately predict bioaccumulation in marine species, even with highly sophisticated models, analyses of tissue levels are required. The main problem is that PAHs do not tend to accumulate in fish tissues in quantities that reflect the exposure. The analysis of PAH metabolite levels in fish bile can be used to assess the actual PAH uptake, rather than the analysis of the non-hydroxylated PAHs content [328,361]. A number of sentinel fish species have been proposed to asses pollution by PAHs [325,326], as well as several mussels [322,323,326,352]. Several studies have also correlated the high levels of 1-OHPy and B(a)Py metabolites found in the bile of cat-shark with contamination sources such as boat traffic and combustion-based industries present in the sampling area [362]. Various plants such as moss, bryophytes, lichen, leaves, and tree bark have also been employed as bioindicators to monitor airborne PAHs pollution levels, assuming that their spatial pollution distribution in the plants and the atmosphere will be closely related [117,122,123,363,364]. This aspect has already been revised in this chapter within the section passive air samplers. Several studies achieved in environmental conditions have shown that PAHs can be excreted in the milk of ruminants when feeded with PAHs [306]. The authors concluded there was an excretion of PAHs into milk under native forms when rumiants were fed with low PAHs content. However, a slight increase in concentration could induce the metabolism, which should lead to an increase in the excretion of metabolites into the milk. In spite of the absence of a significant transfer of parent PAHs to milk, the appearance of metabolites in milk raises questions of their impact on human health. Urinary 1-hydroxypyrene (1-OHPy) is commonly tested as biomarker for the assessment of low level PAHs exposure of general population. The determination of 1-OHPy in urine as a biomarker was first suggested by Jongeneelen [365]. Several reports have suggested that levels of 1-OHPy reflect the levels of environmental PAH exposure from different sources such as air, food and the environment of the home life [303,366,367] or in certain working activities [368]. Controlled studies using human volunteers have shown a strong association between urinary excretion of 1-OHPy and consumption of PAH contaminated diet [21,303]. Other studies have reported that the urinary excretion of 1-OHPy was not correlated with ingested dose of pyrene under normal feeding conditions [369], and so it was not considered a reliable bioindicator of ingested pyrene. It is also well-known that cigarette smoke contains a variety of potentially toxic components including PAHs. Py from smoking is presumably the
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main source of 1-OHPy detected in the cigarette smokers’ urine, particularly when compared with non-cigarette smokers [370,371]. However, the background urinary 1-OHPy is linked to a number of sources such as diets, ambient air, and second hand smoke. It should be also considered that the concentration of urinary 1-OHPy indicates only recent exposure because the halflife for its urinary excretion ranges from 6 to 35 h. Although 1-OHPy has been utilized as a reasonable surrogate biomarker for exposure to a wide variety of PAHs in general, determination of the urinary profile of PAH metabolites would provide more information on estimation of human exposure, internal doses, and individual variability in metabolism of a wide range of PAHs [372]. Hecht et al. have proposed r-1,t-2,3,c-4tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (trans, anti-PheT) as a better biomarker for assessing PAHs metabolic activations [373]. The analytical methods commonly used to biomonitor PAHs or their metabolites in marine species or plants have been described in other sections of this chapter, in food monitoring and in air monitoring, respectively, and will not be repeated here. The analysis of PAHs metabolite in urine samples is quite complex and so intricate analytical protocols must be used for analyses. The analytical methods used are a challenge to biomonitoring studies since most PAH compounds and their metabolites are predominantly excreted in the faeces rather than urine, and so extremely low concentrations are expected in the latter. Jongeneelen et al. proposed an analytical method in which the urine sample is hydrolyzed by β-glucuronidase, purified by solid-phase extraction (SPE) and analyzed by high-performance liquid chromatography (HPLC) with fluorescence detection [374]. It is therefore quite common to carry out an enzymatic hydrolysis of the urine samples followed by HPLC-FD using off-line sample cleanup by SPE [368,369,372,373,375]. However, because of strong matrix interferences, the isolation of more polar metabolites (e.g., hydroxyphenanthrenes) with conventional SPE techniques is difficult and is subjected to error. In this sense, numerous variants have been proposed to improve the sensitivity, specificity, and speed of the method. Schedl et al. have proposed the utilization of immunosorbent of anti-pyrene antibodies which were encapsulated in a sol-gel glass (SGG) matrix in the extraction of urine samples to monitor hydroxyl PAHs [376]. It has also been proposed the utilization of SPME [377,378] or LLE [371,379] instead of SPE. When determining PAHs or its metabolites in urine, several reports suggest that correction for urinary creatinine can be useful, mainly for substances that are eliminated primarily by glomerular filtration and are not reabsorbed by the tubules; however, such corrections may be inappropriate insituations involving concentration-dependent passive elimination or reabsorption [380]. For other biological samples an alkali digestion is also commonly conducted [381]. Another problem often encountered is the small amount of sample available, which can be the limiting factor of the analysis. Table 25 summarizes the most common analytical methods employed in the determination of PAHs or its metabolites in urine, hair or blood samples. HPLC-FD [368,369,375,376,381] and also HPLC-MS [372,382] are quite helpful chromatographic techniques for the determination of hydroxyl-PAHs. The GC method for hydroxy-PAHs requires the utilization of a time-consuming derivatization step in order to decrease the polarity of the analytes and so reduce the peak-tailing in the chromatograms [371,373,377,378]. The derivatization reaction is commonly a silylation reaction, where active hydrogens of hydroxyl PAHs are replaced by trimethylsilyl (TMS) groups. The most
Table 25. Analytical methods commonly employed in the determination of PAHs or its metabolites in urine, hair or blood samples. Sample PAHs human urine 1-OHPy
Ref. [368]
human hair human urine
[381] [376]
seabird blood human urine human urine human urine
human urine human urine crab urine human urine human urine human urine
human urine human urine human urine
Analytical method and performance enzimatic hydrolysis (β-glucuronidase/arylsulphatase, 37ºC, 16 h)-SPE-HPLC-FD. Comparison with PAHs air levels. 16 EPA minus Aci NaOH digestion-LLE (hexane)-HPLC-FD. Comparison with PAHs air levels. 1-OHPy; 2-,3-,4-,9-OHPhe; 3-OHB(a)Py enzymatic hydrolysis (β-glucuronidase/sulphatase, 37ºC, 16 h)-Inmuno extraction cartridge-HPLC-UV/FD. DLs: 1-16 ng·L-1 15 Prestige oil PAHs MAE (hexane:acetone 1:1)-HPLC-FD. DLs: 0.01-0.05 ng·g-1 1-OHPy enzimatic hydrolysis (β-glucuronidase/arylsulphatase, 37ºC, overnight)-SPE-HPLC-FD. Comparison with PAHs in foods. 1-OHPy enzimatic hydrolysis (glucuronidase/arylphosphatase, 37ºC, 16 h)-SPE-HPLC-MS/MS. R ~ 77%. QL ~ 0.1 μg·L-1. Accuracy: 85-115%. 1-, 2-naphthol; 2-OHFl; 1-OHPy; 3-OHFt; enzimatic hydrolysis (glucuronidase/sulphatase, 37ºC, overnight)-SPE-HPLC-MS/MS 1-OHB(a)A; 3-, 9-phenanthrol; 2OHB(c)Phe; 3-, 6-OHChy; 3-, 9OHB(a)Py 1-OHPy enzimatic hydrolysis (β-glucuronidase/sulphatase, 37ºC, 1 h)-SPE-HPLC-FD. Comparison with PAHs air levels 1-, 2-OHN; 2-, 9-OHFl; 9-OHPhe; 3enzimatic hydrolysis (β-glucuronidase, 37ºC, overnight ~ 18 h)-LLE (pentane)OHB(a)A; 1-OHChy; 3-, 7-, 10-OHB(a)Py Derivatization (MSTFA) by microwaves-GC×GC-FID. R ~ 76%. DLs: 0.03-0.18 μg·L-1 1-OHPy, 9-OHPhe enzyme-linked immunosorbent assay (ELISA)-spectrofluorimetry r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4enzimatic hydrolysis (β-glucuronidase/arylsulphatase, 37ºC, overnight)-SPE-HPLC-FD. tetraOHPhe; 1-OHPy HPLC eluates were derivatizated (MSTFA)-GC-MS. unmetabolized N, Aci, Ace, Fl, Phe, A, Ft, HS-SPME-GC-MS. Comparison with PAHs air levels. Py 1-, 2-OHN; 2-, 3-OHFl; 1-, 2-, 3-, 4-, 9enzimatic hydrolysis (β-glucuronidase/arylsulfatase, 37ºC, 2 h)-SPME-on-fiber OHPhe; 3-OHFt; 1-OHPy; 1-, 2-, 3derivatization (MSTFA)-GC-MS. DLs: 0.78-15.8 ng·L-1 OHB(c)Phe; 1-, 3-B(a)A; 3-, 6-OHChy 1-OHPy enzimatic hydrolysis-SPE-HPLC-FD 1-, 2-, 4-, 9-OHFl; 2-OH-5,6,7,8enzimatic hydrolysis (β-glucuronidase/arylsulphatase, 37ºC, overnight)-SPME-on-fiber tetrahydroPhe derivatization (MSTFA or BSTFA)-GC-MS 1-OHPy; 1-, 2-OHN; 3-, 9-OHPhe enzimatic hydrolysis (β-glucuronidase/arylsulphatase, 37ºC, 17 h)-LLE (ethyl acetate)derivatization (BSTFA)-GC-MS. R > 93%. DLs: 0.28-2.64 μg·L-1
[383] [369] [382] [372]
[375] [379] [384] [373] [385] [377]
[303] [378] [371]
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common derivatizating reagents are N-O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). The derivatization steps, in spite of being quite common in laboratories when dealing with polar analytes in GC, add time to the overall analytical procedure and are labor intensive.With respect to GC methods, the successful utilization of GC×GC approaches have also been reported for the analysis of hydroxyl-PAHs in urine [379].
CONCLUSIONS This chapter has intended to give an overview of the analytical steps more commonly carried out in laboratories when determining PAHs (or their metabolites) in environmental, food or biological samples in monitoring studies. The challenges in the analytical methods are significant, because the samples to be analyzed are quite different and complex, and the PAHs are present at low levels altogether with many interfering substances. In addition to this, the developed methods are required to be fast, environmental-friendly (solventless if possible), accurate and reproducible. There are no universal approaches to carry out an analytical determination for the monitoring of PAHs, and the utilization of a number of clean-up steps is, in general, unavoidable. In spite of the fact that traditional methods to monitor PAHs successfully reach the detection limits imposed by several regulations, the overall procedures are generally time-consuming and not environment-friendly. Today, sampling of PAHs is the most critical step in overall analysis, and efforts must be made to improve the efficiency of this step in both active and passive sampling approaches.
ACKNOWLEDGMENTS Dr. Francisco J. Conde is gratefully acknowledged for his helpful comments and discussion.
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[351] Pensado, L.; Casais, M. C.; Mejuto, M. C.; Cela, R. J. Chromatogr. A 2005, 1077, 103109. [352] Campíns-Falcó, P.; Verdú-Andrés, J.; Sevillano-Cabeza, A.; Molins-Legua, C.; Herráez-Hernández, R. J. Chromatogr. A 2008, 1211, 13-21. [353] Arrebola, F. J.; Frenich, A. G.; Rodríguez, M. J. G.; Bolaños, P. P.; Vidal, J. L. M. J. Mass Spectrom. 2006, 41, 822-829. [354] Yurchenko, S.; Mölder, U. J. Food. Compos. Anal. 2005, 18, 857-869. [355] Wennrich, L.; Popp, P.; Zeibig, M. Int. J. Environ. Anal. Chem. 2002, 82, 677-690. [356] Guillén, M. D.; Goicoechea, E.; Palencia, G.; Cosmes, N. J. Agric. Food Chem. 2008, 56, 2028-2033. [357] WHO International Programme on Chemical Safety (IPCS), 1993. Biomarkers and risk assessment: concepts and principles. Environmental Health Criteria 155, World Health Organization, Geneva. [358] Van Gestel, C. A. M.; Van Brummelen, T. C. Ecotoxicology 1996, 5, 217-225. [359] Sarkar, A.; Ray, D.; Shrivastava, A. N.; Sarker, S. Ecotoxicology 2006, 15, 333-340. [360] van der Oost, R.; Beyer, J.; Vermeulen, N. P. E. Environ. Toxicol. Pharmacol. 2003, 13, 57-149. [361] Neves, R. L. S.; Oliveira, T. F.; Ziolli, R. L. Mar. Pollut. Bull. 2007, 54, 1813-1838. [362] Fuentes-Rios, D.; Orrego, R.; Rudolph, A.; Mendoza, G.; Gavilán, J. F.; Barra, R Chemosphere 2005, 61, 192-199. [363] Krommer, V.; Zechmeister, H. G.; Roder, I.; Scharf, S.; Hanus-Illnar, A. Chemosphere 2007, 67, 1956-1966. [364] Domeño, C.; Blasco, M.; Sánchez, C.; Nerín, C. Anal. Chim. Acta 2006, 569, 103-112. [365] Jongeneelen, F. J.; Anzion, R. B. M.; Leijdekkers, Ch. M.; Bos, R. P.; Henderson, P. Th. Int. Arch. Occup. Environ. Health 1985, 57, 47-55. [366] Kanoh, T.; Fukuda, M.; Onozuka, H.; Kinouchi, T.; Ohnishi, Y. Environ. Res. 1993, 62, 230-245. [367] Jongeneelen, F. J. Toxicol. Lett. 1994, 72, 205-212. [368] Kim, M. K.; Oh, S.; Lee, J. H.; Im, H.; Ryu, Y. M.; Oh, E.; Lee, J.; Lee, E.; Sul, D. Exp. Mol. Med. 2004, 36, 396-410. [369] Viau, C.; Diakité, A.; Ruzgyté, A.; Tuchweber, B.; Blais, C.; Bouchard, M.; Vyskocil, A. J. Chromatogr. B 2002, 778, 165-177. [370] Li, H.; Krieger, R. I.; Li, Q. X. Sci. Total Environ. 2000, 257, 147-153. [371] Serdar, B.; Waidyanatha, S.; Zheng, Y.; Rappaport, S. M. Biomarkers 2003, 8, 93-109. [372] Xu, X.; Zhang, J.; Zhang, L.; Liu, W.; Weisel, C. P. Rapid Commun. Mass Spectrom. 2004, 18, 2299-2308. [373] Hecht, S. S.; Chen, M.; Yagi, H.; Jerina, D. M.; Carmella, S. G. Cancer Epidemiol. Biomarkers Prev. 2003, 12, 1501-1508. [374] Jongeneelen, F. J.; Anzion, R. B. M.; Henderson, P. Th. J. Chromatogr. B 1987, 413, 227-232. [375] Huang, W.; Smith, T. J.; Ngo, L.; Wang, T.; Chen, H.; Wu, F.; Herrick, R. F.; Christiani, D. C.; Ding, H. Environ. Sci. Technol. 2007, 41, 2711-2716. [376] Schedl, M.; Wilharm, G.; Achatz, S.; Kettrup, A.; Niessner, R.; Knopp, D. Anal. Chem. 2001, 73, 5669-5676. [377] Smith, C. J.; Walcott, C. J.; Huang, W.; Maggio, V.; Grainger, J.; Patterson Jr, D. G. J. Chromatogr. B 2002, 778, 157-164.
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[378] Gmeiner, G.; Gärtner, P.; Krassnig, C.; Tausch, H. J. Chromatogr. B 2002, 766, 209218. [379] Amorim, L. C. A.; Dimandja, J. –M.; Cardeal, Z. L. J. Chromatogr. A 2009, 1216, 2900-2904. [380] Waidyanatha, S.; Rothman, N.; Fustinoni, S.; Smith, M. T.; Hayes, R. B.; Bechtold, W.; Dösemeci, M.; Guilan, M.; Yin, S.; Rappaport, S. M. Carcinogenesis 2001, 22, 279286. [381] Toriba, A.; Kuramae, Y.; Chetiyanukornkul, T.; Kizu, R.; Makino, T.; Nakazawa, H.; Hayakawa, K. Biomed. Chromatogr. 2003, 17, 126-132. [382] Pigini, D.; Cialdella, A. M.; Faranda, P.; Tranfo, G. Rapid Commun. Mass Spectrom. 2006, 20, 1013-1018. [383] Pérez, C.; Velando, A.; Munilla, A.; López-Alonso, M.; Oro, D. Environ. Sci. Technol. 2008, 42, 707-713. [384] Fillmann, G.; Watson, G. M.; Howsam, M.; Francioni, E.; Depledge, M. H.; Readman, J. W. Environ. Sci. Technol. 2004, 38, 2649-2656. [385] Campo, L.; Addario, L.; Buratti, M.; Scibetta, L.; Longhi, O.; Valla, C.; Cirla, P. E.; Martinotti, I.; Foà, V.; Fustinoni, S. Toxicol. Lett. 2006, 162, 132-138.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 18
NEW TENDENCIES IN LIQUID CHROMATOGRAPHY FOR CONTROLLING PAHS POLLUTION R. Herráez-Hernández, P. Campíns-Falcó∗, J. Verdú-Andrés, A. Sevillano-Cabeza and L. Ferrando-Climent Department of Analytical Chemistry, University of Valencia, Valencia, Spain
ABSTRACT Different configurations were studied for the screening analysis of common polycyclic aromatic hydrocarbons (PAHs) in environmental waters by liquid chromatography (LC). Analyte enrichment on a traping microcolumn connected by column switching to the analytical column, and in-valve in-tube solid-phase microextraction (IT-SPME) were tested as alternatives to conventional solid-phase extraction (SPE) with disposable cartridges. For separation conventional LC, capillary LC and LC with a monolithic column were tested and compared, whereas UV-diode array detection (DAD) or fluorescence detection were used for signal monitoring. The compounds assayed were naphthalene, anthracene, fluoranthene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene and benzo[ghi]perylene. On the basis of the results obtained, new methods are presented for the on-line screening analysis of PAHs in several water matrices. The main advantages of the proposed procedures are: (i) the minimum off-line sample manipulation as the samples only need to be filtered; (ii) the rapidity, as the total analysis time can be reduced to less than 20 min, (iii) the improved detection limits, especially for the in-valve IT-SPME configurations, as they are compatible with the injection of very large sample volumes (up to 4.0 mL), and (iv) the total elimination of organic solvents in the sample treatment. Therefore, the proposed methods are environmentally friendly and costeffective alternatives for the routine monitoring of PAHs in waters. The occurrence of the tested PAHs in waters of The Comunidad Valenciana (Spain) was also tested.
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1. INTRODUCTION To date, many analytical procedures have been developed for the analysis of PAHs in water, soil, air and food matrices, most of them using LC or gas chromatography (GC). The conventional approach for the analysis of these pollutants in water samples involves the previous preconcentration of the analytes by liquid-liquid extraction (LLE) or SPE, often combined with solvent evaporation [2-8]. Recent research efforts are oriented towards the development of simplified and miniaturized sample treatment methods which reduce the time of analysis, the consumption of chemicals and the generation of wastes. In this respect, different alternatives have been proposed for PAHs based on the miniaturization of LLE [9, 10] or SPE [11, 12], but the most popular approach is solid-phase microextraction (SPME). SPME permits the detection and quantification of PAHs at low to sub ppb levels using for analyte enrichment either fibres [13-18] or stir bars [19, 20] coated with an extractive phase. The long adsorption times required to extract the analytes, up to several hours in some of the reported assays [19], is the most serious limitation of SPME for routine monitoring of PAHs. Ideally, an analytical method for monitoring of PAHs in waters should be simple, rapid, and sensitive. Sample conditioning should be integrated in the chromatographic system. However, owing to the low concentrations of PAHs expected, on-line sample conditioning would be only possible if the chromatographic system is compatible with the injection of large sample volumes. Another approach to improve the sensitivity in LC is the reduction of the column diameter dimensions. For example, Titato and Lancas demonstrated that the employment of capillary LC substantially increases the mass sensitivity in the analysis of PAHs with respect to conventional LC [21]. However, the sample volumes that can be injected in capillary chromatography are rather low and thus, some kind of analyte enrichment is still necessary. The assays reported for the analysis of PAHs by capillary LC generally involve conventional LLE or SPE [21, 22]. In this chapter we have evaluated different strategies for the sensitive analysis of PAHs in waters which combine the injection of large sample volumes and modern forms of LC. For analyte enrichment two approaches have been tested and compared: SPE into a trapping column connected to the analytical column by means of a switching arrangement and in-valve in-tube-SPME (IT-SPME). Analyte enrichment by column switching (CS) is a technique of routine use in conventional LC [7, 8, 23]. However, the employment of this technique in capillary LC is still rather limited [24]. As regards IT-SPME, different publications have demonstrated that this methodology is especially well-suited for the sensitive analysis of organic pollutants such as triazines, organophosphorous pesticides or phenols [25-27]. Recently, IT-SPME has been employed to preconcentrate and purify the extracts obtained after applying matrix solid-phase dispersion (MSPD) to extract PAHs from bivalve organisms [28]. However, the potential of IT-SPME for the sensitive analysis of PAHs in waters has not been fully exploited. For separation of the analytes capillary LC and LC with a monolithic column [29] have been tested and compared, whereas for signal monitoring UV-DAD and fluorescence detectors have been used. The reliability of the proposed methods has been tested by comparing the results obtained with those achieved by a classical approach based on
∗ Corresponding author: Tel.: + 34 963543002; Fax: + 34 963544436; e-mail address:
[email protected].
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the employment of SPE with disposable cartridges and conventional LC. Advantages, disadvantages and possible applications are discussed.
EXPERIMENTAL Reagents and Solutions All the reagents were of analytical grade. Naphthalene, anthracene, fluoranthene, benzo[b]fluoranthene, benzo[k]fluorantene, benzo[a]pyrene, dibenzo[a,h]anthracene and benzo[ghi]perylene (10.0 µg/mL in acetonitrile) were obtained from Dr. Ehrenstorfer (Augsburg, Germany). Working solutions of these compounds were prepared by dilution of the stock solutions with water. Acetonitrile of HPLC grade was obtained from J. T. Baker (Deventer, The Netherlands); tetrahydrofuran (THF) was purchased from Fluka (Buchs, Switzerland). Water was deionised and filtered through 0.45 μm nylon membranes (Teknokroma, Barcelona, Spain). All solutions were stored in the dark at 2ºC.
APPARATUS AND ANALYTICAL CONDITIONS SPE-Conventional LC-FLD The chromatographic system consisted of a quaternary pump, an automatic sample injector (Hewlett-Packard 1050 Series, Palo Alto, CA, USA) and a programmable fluorimetric detector (Hewlett-Packard, 1046 Series). The detector was linked to a data system (Hewlett-Packard, HPLC ChemStation) for data acquisition and calculation. According to the results of preliminary experiments obtained by injecting directly into the analytical column standard solutions of PAHs, the detector was programmed to measure the fluorescence intensity at the excitation/emission wavelengths pairs listed in Table 1. A LiChrospher 100 RP18, 125 mm × 4 mm i.d. (Merck, Darmstadt, Germany) column was used for separation. The mobile-phase was a mixture of acetonitrile-water in gradient elution mode (see also Table 1). The solvents were filtered through 0.45 μm nylon membranes (Teknokroma,) and degassed with helium before use. For SPE Envirolut PAH 1 g/6 mL cartridges (Variant, Harbor City, CA, USA) were used. Cartridges were conditioned by drawing successively 2 mL of acetonitrile and 5 mL of nanopure water. Then the samples (25 mL) were passed through the cartridges. All these operations were carried out by means of a vacuum station (IST VacMaster, Hengoed, UK). After sample loading the remaining water was manually eliminated from the cartridges by drawing the minimum volume of air with a plastic syringe. Finally, the analytes were desorbed from the cartridges with acetonitrile or THF, and 20 µL of the collected extracts were injected into the LC equipment. All the experiments were done in triplicate at ambient temperature.
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CS-Capillary LC-DAD A scheme of the system used for on-line enrichment by CS is shown in Figure 1a. The system consisted of a capillary pump (Agilent 1100 Series, Waldbronn, Germany) equipped with a Rheodyne model 7725 high-pressure six port injection valve (valve 1) with a internal loop (2 μL) or with an external loop (50, 100 or 200 µL). Analyte enrichment was effected into a trapping column containing a Zorbax SB C18 phase (35 mm x 0.5 mm i.d., 5 µm particle diameter) from Agilent. For separation of the analytes a Zorbax SB C18, 150 mm x 0.5 mm i.d., 5 μm particle diameter column (Agilent) was employed. Both columns were connected by means an automatic switching microvalve (valve 2) (Agilent 1100 Series). The UV-DAD detector (Hewlett-Packard, 1100 Series) was coupled to a data system (Agilent, HPLC ChemStation) for data acquisition and calculation. The UV chromatograms were registered from 200 to 450 nm. After filling the injection loop with the sample, valves 1 and 2 were rotated and the chromatographic run was started. In such a way, the sample was transferred from the injection loop to the trapping column; meanwhile, the effluent emerging from the trapping column was sent to waste. Next, the trapping and analytical columns were connected by rotating valve 2, and 0.5 min later valve 1 was turned to the load position. For separation of the tested PAHs a mobile-phase of acetonitrile-water in gradient elution was used (see Table 1). The solvents were filtered through 0.45 μm nylon membranes (Teknokroma) before use. All the experiments were done in triplicate at ambient temperature.
IT-SPME-Capillary LC-DAD A scheme of the system used for on-line enrichment by in-valve IT-SPME, separation and detection of PAHs is shown in Figure 1b. As observed, this system differs from that of Figure 1a in the injection device: the inner loop of the injection valve was replaced by a section of a capillary column such as those used in GC. This capillary column was a 70 cm x 0.32 mm i.d., 3-µm film thickness column coated with a polysiloxane polymer with 95 % methyl-5 % phenyl-substituted backbone (Teknokroma). The injection valve was connected directly to the analytical column (valve 2 was omitted). The other elements were those of the column switching-capillary LC-DAD system (Figure 1a). Conditions for separation and detection were those summarized in Table 1.The solvents were filtered through 0.45 μm nylon membranes (Teknokroma) before use. All the experiments were done in triplicate at ambient temperature.
IT-SPME-LC-FLD The chromatographic system was the same as Figure 1b, but the analytical column was a Onyx Monolithic C18, 100 mm × 3 mm i. d., (mesopores of ≈ 13 nm, macropores of ≈ 2 µm) column purchased from Phenomenex (Torrance, CA, USA); the signal was monitored with a programmable fluorimetric detector (Hewlett-Packard, 1046 Series). The detector was linked to a data system (Hewlett-Packard, HPLC ChemStation) for data acquisition and calculation.
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The mobile-phase was a mixture of acetonitrile-water in gradient elution mode, and flow rate was 1.0 mL/min. Conditions for separation and detection are also summarized in Table 1. The solvents were filtered through 0.45 μm nylon membranes (Teknokroma) and degassed with helium before use. All the experiments were done in triplicate at ambient temperature.
Analysis of Real Water Samples Different real water samples were analyzed by the proposed methods: river water, sea water, sewage water and harbour water. All samples were collected at different points of the coast of the Comunidad Valenciana (Spain). Samples were filtered through 0.45 μm nylon membranes (Teknokroma) after their arrival at the laboratory in order to remove any particulate matter. Each sample was analyzed in triplicate and all assays were carried out at ambient temperature.
RESULTS SPE-Conventional LC-FLD Method In this study SPE cartridges packed with a sorbent specially designed for PAHs as well as conventional LC with fluorimetric detection were used. In preliminary tests different elution and detection conditions were assayed for the separation of the selected PAHs. The elution program finally selected was that presented in Table 1. The FLD detector was programmed accordingly (see also Table 1). Cartridges were previously activated with 2 mL of acetonitrile, and then washed with 5 mL of nanopure water. After sample loading the cartridges were dried with air, and then the analytes were desorbed and chromatographed. Acetonitrile was the first option tested to desorb the analytes from the SPE cartridges. Aliquots of 1 mL of acetonitrile were successively passed through the cartridges, and the extracts were collected separately. Finally, 20 µL of each extract were injected into the chromatographic system. The obtained chromatograms revealed that most PAHs were desorbed in the second and third 1 mLfractions of acetonitrile. However, the most apolar PAHs benzo[a]pyrene, dibenzo[a,h]anthracene and benzo[ghi]perylene required up to 5 mL of solvent to be totally desorbed from the cartridges, and thus, the preconcentration achieved was considered unsuitable. In an attempt to avoid solvent evaporation THF instead of acetonitrile was tried as solvent for desorption. Nearly complete desorption of the analytes was observed with 1.5 mL of THF, which was then the option selected (Table 1). As an illustrative example, in Figure 2 are shown the chromatograms obtained for a blank and for an standard solution containing a mixture of the tested PAHs. As observed from this figure benzo[b]fluoranthene and benzo[k]fluoranthene partially overlapped. In an attempt to improve the resolution a variety of elution conditions were assayed but complete separation of these compounds could not be achieved in reasonable chromatographic times. Coelution between these PAHs seems to be a common problem in many of the procedures reported for PAHs using both LC and GC [6, 15, 17]. Nevertheless, the resolution achieved under conditions of Table 1 can be considered
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suitable for most purposes. The selectivity was also adequate as no matrix peaks were observed at the retention times of the analytes. This is illustrated in Figure 2c, which shows the chromatogram obtained for real sewage water in which none of the tested PAHs was found. The optimized conditions were applied to standard solutions of PAHs containing 5.0 ng/mL of each compound. The percentages of PAHs recovered were established by comparing the peak areas obtained for each PAH with those obtained from the direct injection of 20 µL of standard solutions containing an equivalent amount of the analytes. As listed in Table 2 the analyte recoveries ranged from 87 to 112 %. The intra- and inter-day reproducibilities were also tested; the RSD values encountered are listed in Table 3. The limits of detection (LODs), established as the concentration required to generate a signal-tonoise ratio of 3, were ≤ 1 ng/mL (see Table 4).
CS-Capillary LC-DAD Method The switching system used for on-line enrichment and chromatography of PAHs is shown in Figure 1a. Before each run the trapping and the analytical columns were equilibrated with the mobile phase. Then, the sample was placed in the injection loop by means of a syringe. At the beginning of the run valves 1 and 2 were rotated, so the sample was sent to the trapping column, and meanwhile, the effluent emerging from the trapping column was directly transferred to waste in order to protect the analytical column. After transfer the sample from the loop to the trapping column, both the extractive and the analytical columns were connected by changing the position of microvalve 2. At the same time or somewhat later the injection valve (valve 1) was also turned to its original position. In such a way, the mobile phase passed through the injection loop only to transfer the sample to the trapping column. In such a way, the time required for analyte desorption and separation could be significantly shortened, taking into account the the analytes were chromatographed under gradient elution. Initially, different acetonitrile-water gradient elution programs, as well as different time intervals for connecting the extractive and the analytical columns were assayed. In this study standard solutions containing 10.0 ng/mL of each PAHs were processed, and the volume of the injection loop was 100 µL. The mobile-phase flow rate was 20.0 µL/min. Under such conditions the time required to transfer the sample plug from the injection loop to the trapping column was c.a. 5 min. In order to ensure the complete transfer of the sample to the trapping column and to minimize the time required for chromatography the injection valve was turned to its original position at 5.5 min. Maximum analyte responses were achieved by rotating valve 2 at 5.0 min. On the other hand, all the tested analytes could be resolved under a variety of conditions with the exception of benzo[b]fluoranthene and benzo[k]fluoranthene. As in the SPE-conventional LC-FLD approach, these compounds eluted at similar retention times under a variety of conditions. However, they could be easily distinguished by their UV spectra. Thus, the elution conditions were adjusted to obtain satisfactory resolution of the other PAHs in the minimum chromatographic time (see Table 1).
Table 1. Conditions used in the analysis of PAHs by the tested procedures Conditions
SPE-conventional LC-FLD
CS-capillary LC-DAD
IT-SPME-capillary LC-DAD IT-SPME-LC(monolithic)-FLD
Sample treatment
Sample volume: 25 mL
Sample volume: 100 µL
Sample volume: 2 mL
Procedure:
Procedure:
Procedure:
- sample loading at 0 min - valve 1 rotation + valve 2 rotation at 5.0 min - valve 2 rotation at 5.5 min - valve 1 rotation
- sample loading - cleaning of the capillary with 100 µL of water - valve rotation
-
-
Separation
conditioning of the cartridges with 2 mL of acetonitrile + 5 mL of water sample loading drying of the cartridges desorption of the analytes with 1.5 mL of THF injection onto the LC equipment of 20 µL of the extracts
Elution program (acetonitrile:water, v/v):
Elution program (acetonitrile:water, v/v): -
at 0 min at 40 min at 42 min
-
60:40 80:20 100:0
at 0-7 min at 20 min at 22 min
Elution program (acetonitrile:water, v/v):
50:50 70:30 100:0
- at 0 min - at 12 min - at 13 min
Mobile-phase flow rate: 20.0 µL/min
Mobile-phase flow rate: 1.0 mL/min
Mobile-phase
Detection
Time (min)
λexcitation/emission (nm) 0-7.5 224 7.5-10.0 251 10.0- 20.0 235 20.0-26.0 301 28.0-30.0 300 30.0 302
λ (nm) 330 395 450 438 404 461
50:50 80:20 100:0
220 naphthalene fluoranthene, benzo[k]fluoranthene 250 anthracene 260 benzo[b]fluoranthene benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene 230
290
Compound: Time (min) 0-3.9 3.9-4.9 4.9-6.2 6.2-13.0
flow
rate: 1.0 mL/min
λexcitation/emission (nm) 224 251 235 301
330 395 450 438
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Table 2. Absolute recoveries obtained by the SPE with disposable cartridges, CS and ITSPME sample treatment procedures (values established at a concentration of 5.0 ng/mL in the SPE-LC-FLD and CS-capillary LC-DAD methods, and 1.0 ng/mL in the ITSPME-FLD method; n = 3) Compound
SPE
CS
IT-SPME
Naphthalene Anthracene Fluoranthene Benzo[a]anthracene Benzo[b]fluoranthe
104 ±11 98 ± 7 112 ± 10 99 ± 6
89 ± 3 95 ± 6 101 ± 4 99 ± 3
37 ± 4 45 ± 4 42 ± 2 32 ±2 26 ± 1
101 ± 7 94 ± 7 91 ± 9
105 ± 2 93 ± 2 95 ± 2
28 ± 1 33 ± 1 22 ± 1
87 ± 9
93 ± 2
26 ± 2
ne Benzo[k]fluorantene Benzo[a]pyrene Dibenzo[a,h]anthrac ene Benzo[ghi]perylene
Table 3. RSD obtained with the SPE-LC-FLD, CS-capillary LC-DAD and IT-SPMELC-FLD methods (values established at a concentration of 5.0 ng/mL in the SPE-LCFLD and CS-capillary LC-DAD methods, and 0.3 ng/mL in the IT-SPME-FLD method) Compound
Naphthalene Anthracene Fluoranthene Benzo[b]fluoranth ene Benzo[k]fluorante ne Benzo[a]pyrene Dibenzo[a,h]anthr acene Benzo[ghi]peryle ne
SPE-LC-FLD Intra-day Inter(n = 3) day (n = 6)
CS-
capillary
LCDAD
IT-SPME-LC- FLD
12 9 9 8
11 8 8 4
Intra-day (n = 3) 2 4 4 7
Inter-day (n = 6) 11 5 6 6
Intra-day (n = 3) 9 4 4 4
Inter-day (n = 6) 7 5 6 11
2
9
3
7
3
5
9 6
11 11
6 2
5 11
1 5
7 9
10
9
4
10
7
9
565
New Tendencies in Liquid Chromatography… Table 4. LODs (ng/mL) obtained with the four optimized procedures Compound
SPE-LCFLD
CS-capillary LC-DAD
Naphthalene Anthracene Fluoranthene Benzo[a]anthracene Benzo[b]fluoranthene Benzo[k]fluorantene Benzo[a]pyrene Dibenzo[a,h]anthracene Benzo[ghi]perylene
0.5 0.05 0.5 0.5 0.05 0.1 0.1 1
0.5 0.1 0.5 1 1 0.5 0.25 1
IT-SPMEcapillary LCDAD 0.1 0.02 0.05 0.1 0.1 0.1 0.2 0.025 0.025
IT-SPMELC-FLD 0.008 0.0015 0.008 0.006 0.006 0.006 0.007 0.005 0.0025
a to waste valve 2
valve 1
capillary column
pump
DAD
trapping column inner loop
b capillary or monolithic column
pump
DAD or FLD
IT-SPME capillary
Figure 1. (a) Scheme of the column switching capillary chromatographic system used for PAHs. System conditioning and sample loading: valve 1 (─) and valve 2 (─); sample injection and on-line enrichment: valve 1 (---) and valve 2 (---); transfer of the analytes to the analytical column: valve 1 (---) and valve 2 (─); analyte separation and detection: valve 1 (─) and valve 2 (─). (b) Scheme of the ITSPME device used for PAHs. For other experimental details, see text.
The effect of the sample volume was estimated by using external injection loops of 50, 100 and 200 µL. The times selected for rotating valve 2 were 2.5, 5.0 and 10.0 min, respectively, and in all instances the injection valve was returned to the load position 0.5 min later (see Figure 1a). The initial composition of the mobile-phase (50:50 acetonitrile-water, v/v) was kept constant in the 0-5, 0-7 and 0-12 min time intervals when injecting 50, 100 and 200 µL of the samples, respectively. Next, the gradient profile applied was equivalent to that shown in Table 1 for the method using 100 µL. Indeed, increasing the sample volume resulted in higher peak areas for the analytes, but also in longer retention times. When injecting 50 µL
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Dibenzo[a,h]anthracene
Benzo[ghi]perylene
Fluoranthene
200
Benzo[b]fluoranthene
300 Naphthalene
Fluorescence (A. U.)
400
Benzo[a]pyrene
500
Benzo[k]fluoranthene
Anthracene
of the samples, the signals obtained for most PAHs were close to their respective LODs, and some of the analytes were not detected. In contrast, all the PAHs tested were detected when using the 100 µL and 200 µL loops. The peak areas obtained with 200 µL of samples were only 1.4-1.8 times greater than those obtained for a sample volume of 100 µL (data not shown). In other words, increasing the sample volume increased the sensitivity but the improvement was lower than that expected. This is because peak broadening became more marked as the time of residence of the analytes in the chromatographic system increased. Although not tested, a change in the elution conditions would most probably improve the LODs by shortening the times of retention. As a compromise between sensitivity and analysis time, a sample volume of 100 µL was the option selected. The entire CS-capillary LC-DAD method is summarized in Table 1. In Figure 3 are depicted the chromatograms obtained under the selected conditions for a blank (water) and a standard solutions of the analytes at different wavelengths.
a 100
b c
0 0
10
20
30
40
Time (min) Figure 2. Chromatograms obtained by the optimized SPE-conventional LC-FLD method for: (a) a standard solution containing 20.0 ng/mL of each of the tested PAHs, (b) blank (nanopure water), and (c) a real waste water. For other details, see text.
The recoveries obtained under the proposed method were estimated by comparing the peak areas obtained for each PAH in the samples (5.0 ng/mL, each compound) with those obtained from the direct injection into the analytical column of 2 µL of standard solutions containing an equivalent amount of the analytes. The percentages obtained ranged from 89 to
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Dibenzo[a,h]anthracene
Benzo[ghi]perylene
60
Fluoranthene
Anthracene
Absorbance (mAU)
80
Benzo[b]fluorantene, benzo[k]fluoranthene
100
Benzo[a]pyrene
Naphthalene
105 % (Table 2). Data on the intra- and inter-day reproducibilities of the method are summarized in Table 3, whereas the LODs are listed in Table 4.
220 nm
40
20
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0 5
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Figure 3. Chromatograms obtained by the optimized column switching capillary LC-UV(DAD) method for a standard solution containing 20.0 ng/mL (up) and for blank (nanopure water) (down) at 220 nm and 290 nm. For other details, see text.
IT-SPME Based Methods IT-SPME is a well-established technique in which the analytes are extracted and concentrated into the stationary phase of a capillary column by aspirating and dispensing the samples from the capillary a number of times until equilibrium is reached or until sufficient analyte is extracted [30]. Alternatively, a volume as large as necessary of the sample is passed through the capillary until sufficient analyte is extracted to reach the desired sensitivity (one single extraction) [28]. For simplicity, the later approach was used in the present study. Obviously, in this form of IT-SMPE and for a given concentration of analyte, the responses depend on the sample volume passed through the capillary. On the other hand, according to previously described results the presence of small percentages of an organic solvent (such as acetonitrile) in the sample modifies the retention properties of the capillary coating [28]. Consequently, a systematic study was carried out in order evaluate the effect of the volume of sample to be processed, as well as the usefulness of adding acetonitrile to the
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samples. The concentrations of the PAHs in the samples were selected to produce peak areas of about the same order for all compounds, and ranged from 0.03 to 0.5 ng/mL. In this study, the extractive capillary column was coupled to a monolithic column, and fluorescence detection was used (see Figure 1b). Volumes ranging from 1.0 to 4.0 mL of standard solutions of the tested PAHs, prepared in water or in different acetonitrile:water mixtures, were passed through the capillary. Then, the solution remaining into the capillary was flushed with 100 µL purified water. Finally, the extractive capillary column and the separative monolithic column were connected by rotating the injection valve. As illustrative examples, in Figure 4 are depicted the results obtained for three PAHs of different polarity, fluoranthene (high-medium), benzo[a]pyrene (low-medium) and benzo[ghi]perilene (low). As expected, the increment of the sample volume had a positive effect on analyte responses, and nearly linear increment was observed for all the tested compounds within the tested interval. On the other hand, the addition of acetonitrile to the samples up to percentages of 10-20 % (v/v) increased the analytical signal for the most apolar compounds; this effect was not dependent on the sample volume. For example, a percentage of acetonitrile of 20 % (v/v) in the solutions loaded into the capillary column increased the peak areas of benzo[ghi]perilene by a factor of about 2.5. The percentage of acetonitrile that produced maximum analyte responses increased as the polarity of the analyte decreased. For example, maximum analyte responses for benzo[ghi]perilene were obtained when the solution loaded into the extractive capillary contained a percentage of acetonitrile of 20 % (v/v), whereas for benzo[a]pyrene a percentage 10 % produced the highest responses. For the most polar analytes such as fluoranthene, the presence of acetonitrile did not improve the signals. Percentages of acetonitrile of 30 % resulted in lower recoveries for all tested PAHs. On the other hand, good stability of the system was observed even when processing 4.0 mL of the samples, and the same capillary column was be repeatedly used without detecting losses in its extractive capabilities. The results obtained thought out this study demonstrated that with the IT-SPME-LC-FLD method, a sample volume of 2.0 mL would be suitable for the screening analysis of PAHs in waters according to the maximum levels fixed by the European regulations [1]. If required, the sensitivity can be increased by processing larger sample volumes; for the most apolar PAHs, the sensitivity can be further improved by adding acetonitrile to the samples in percentages of about 10-20 % (v/v). The percentages of PAHs recovered by the in-tube SPME procedure were established for a sample volume of 2.0 mL and without adding acetonitrile. The absolute recoveries were calculated by comparing the peak areas of the chromatograms obtained when injecting into the in-tube SPME device 2.0 mL of standard solutions of the PAHs (1 ng/mL, each compound) with those obtained by injecting directly 20 µL of a solution containing an equivalent amount of each PAH into the LC system. The results obtained are listed in Table 2. As observed, the absolute recoveries ranged from 22 to 45 % which are values typically encountered in this form of SPME [26, 28]. As an illustrative example, in Figure 5 are depicted the chromatograms obtained for a blank and for a standard solution of the PAHs under the optimized IT-SPME-LC-FLD approach. It is interesting to note that for the same mobile-phase flow rate, the employment of a monolithic column instead of a conventional column drastically reduced the time required for the separation. As it can be deduced by comparing Figures 2 and 5, the time required for the chromatographic run could be reduced from 35 min to 12 min, and with a minimum loss of resolution for the tested PAHs.
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20%
peak area (A.U.) % Acetonitrile
10%
1 mL
2 mL
3 mL
0% 4 mL
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1 mL
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Figure 4. Effect of the sample volume and the percentage of acetonitrile on analyte responses obtained by IT-SPME of: (a) fluoranthene (0.18 ng/mL), (b) benzo[a]pyrene (0.074 ng/mL), and (c) benzo[ghi]perylene (0.44 ng/mL). For other details, see text.
The IT-SPME device was also coupled to the capillary LC-DAD system. Figure 6 shows the chromatograms obtained under optimized extraction and separation conditions for a blank and for a standard solution of the tested PAHs (1.0 ng/mL). As observed in Figure 6, the chromatograms showed a profile similar to those obtained when by the CS-capillary LC-DAD approach (see Figure 3). However, analyte responses were much higher when applying the IT-SPME-capillary LC-DAD approach. The RSD and the LODs obtained by the IT-SPMEbased configurations are listed in Tables 3 and 4, respectively.
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Benzo[ghi]perylene
Dibenzo[a,h]anthracene
Benzo[k]fluoranthene Fluoranthene
Benzo[a]anthracene
Benzo[b]fluoranthene
Anthracene
2.0
Naphthalene
Fluorescence (A. U.)
2.4
Benzo[a]pyrene
It is interesting to note that peaks others than those corresponding to the tested PAHs were observed in the chromatograms of either a blank and a standard solution containing the tested PAHs. In additional experiments it was found that those peaks corresponded to different phtalates present in the purified water used either as a blank and to prepare the working solutions of PAHs. Those peaks were not detected neither when using the more selective FLD detection (see Figure 5), nor the less sensitive CS-capillary LC-DAD approach (see Figure 3).
b 1.6
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8
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Figure 5. Chromatograms obtained by the optimized IT-SPME-LC-FLD method for: (a) standard solution containing 0.3 ng/mL of each PAHs, and (b) for blank (nanopure water). For other details, see text.
Analysis of Real Samples Finally, the described methods were applied to the analysis of several samples of river water, sea water, sewage effluent and harbour water. All the analysed samples were collected at different points along the coast of the Comunidad Valenciana (Spain). None of the PAHs included in this study was found when applying the SPE-conventional LC-FLD and the CS-capillary LC-DAD methods, except naphthalene. This compound was
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New Tendencies in Liquid Chromatography…
Benzo[ghi]perylene
Benzo[a]anthracene
Benzo[b]fluoranthene, benzo[k]fluoranthene Benzo[a]pyrene Dibenzo[a,h]anthracene
Anthracene Naphthalene
Absorbance (mAU)
200
Fluoranthene
found in one of the harbour waters analyzed at a concentration close to its LOD, thus suggesting a high level of contamination due to fuel spills. In Figure 7a is shown the chromatogram obtained by the CS-capillary LC-DAD method for this sample.
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Time (min) Figure 6. Chromatograms obtained by the optimized IT-SPME-capillary LC-DAD method for a standard solution containing 1.0 ng/mL (up) and for blank (nanopure water) (down) at 220 nm and 290 nm. For other details, see text.
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Figure 7. Chromatograms obtained for real water samples: (a) harbour water sample by the CS-capillary LC-UV(DAD) method; (b) sewage water by the IT-SPME-capillary LC-DAD, and (c) sewage water (up) and standard mixture of the tested PAHs (down) by the IT-SPME-LC-FLD methods. Inset: comparison of the UV spectra obtained for the sample (---) and for a standard solution of naphthalene (─). For other details, see text.
New Tendencies in Liquid Chromatography…
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Naphtalene and in minor extension fluoranthene were found in some of the samples assayed when applying the IT-SPME-LC-FLD method, although the concentrations found were lower than the expected limits of quantification. In samples that contained the highest levels of naphthalene, this PAH was also detected when applying the IT-SPME-capillary LCDAD method. This is illustrated for one of the sewage samples analysed in Figures 7b and 7c. The peak corresponding to naphthalene could be easily detected in the IT-SPME-LC-FLD chromatogram not only because of its great intensity, but also because the chromatogram show less peaks corresponding to matrix compounds. The identification of this PAH in the sample was confirmed by the IT-SPME-capillary LC-DAD method by matching the UV spectrum of the peak of suspected naphthalene. It should be noted that not significant differences were found in the chromatogram profiles obtained for the different types of waters analysed.
Comparison of the Different Extraction/Separation/Detection Approaches Tested The four methodologies used in the present study are valid alternatives for the analysis of PAHs in waters. All of them are comparable in terms of reproducibility (see Table 3). As observed in Table 2, the absolute recoveries obtained for the tested PAHs were close to 100 % when using either off-line SPE into cartridges and on-line SPE into a trapping column packed with a C18 phase. When using IT-SPME for analyte isolation and preconcentration the alsolute recoveries obtained were significantly lower due to the modality of IT-SPME used. However, the total amounts of PAHs retained were higher as this form of IT-SPME is compatible with the injection of much larger sample volumes. As a consequence, the sensitivity ca be considerably improved (Table 4). The LODs obtained by using off-line SPE without solvent evaporation combined with conventional LC-FLD are comparable to those obtained by the CS-capillary LC-DAD method (see Table 4). In other words, although less sensitive detection is used the coupling of analyte enrichment by CS and capillary LC enables the detection of PAHs with sensitivity comparable to that obtained by the conventional method. The main advantage of the CScapillary LC-DAD approach is that off-line preconcentration operations are not required. According to the literature both approaches can be considered adequate for the analysis of PAHs in waste water and polluted waters [4, 5, 16, 17]. As regards IT-SPME, with the extractive capillary column used in the present study at least up to 4.0 mL of the samples can be processed resulting in maximum analyte responses. Although not tested, the sensitivity would be most probably increased by injecting larger sample volumes. The sensitivity can be further improved for the most apolar PAHs by adding acetonitrile to the samples in final percentages of about 20 % (v/v). When coupling IT-SPME to LC-FLD the direct processing of only 2.0 mL of the samples provided LODs clearly lower than the maximum concentrations fixed by the European regulations for natural waters [1]. Although less sensitive, the IT-SPME-capillary LC-DAD method offers improved confirmation of the analytes by matching the UV spectra. The application of this method to control and screening analysis of PAHs in natural waters would required the utilization of larger sample volumes, probably combined with the addition of an organic solvent such as acetonitrile to the samples. On the other hand, the employment of a monolithic column for the
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chromatographic separation results in a very simple and fast method, particularly well-suited for routine screening analysis. In Table 5 are also compared other relevant features of the four methodologies used in present study. When considering the off-line sample manipulation involved in the analysis, the three on-line methods proposed are clearly advantageous over the traditional approach, as samples only need to be filtered. Moreover, the employment of organic solvents for analyte enrichment and preconcentration is totally avoided. The consumption of extractive phases is also reduced to a minimum. For example, more than 500 real water samples have been processed in our lab by the IT-SMPE-LC-FLD method with the same extractive capillary column and without observing deterioration on its retention capabilities. Indeed, a modification of the chromatographic system is required if the on-line methods are going to be applied; in the conventional off-line method the chromatographic system does not need to be changed, but additional instrumentation for sample treatment such as a vacuum manifold was used. The CS-capillary LC-DAD approach requires a capillary trapping column and an additional switching microvalve over an standard capillary LC equipment. The ITSPME based methods only require the replacement of the inner loop of the injection valve by a capillary extractive column. Thus, the implementation of the IT-SPME-LC-FLD method only requires a minimum modification of conventional LC equipments. Thererefore, this is the most cost-effective approach. Table 5. Comparison of the tested methods. The higher the number of asterisks, the better is the method Features
Sensitivity Time for sample preparation Total time of analysis Off-line manipulation Consumption of solvents and extractive phases Instrumental requirements Cost of analysis
SPE-LC-FLD CS-capillary LCIT-SPMEDAD capillary LCDAD * * **
IT-SPME-LC*FLD ***
*
**
***
***
*
**
**
***
*
***
***
***
*
**
***
***
**
*
**
***
*
**
**
***
(*) LC with a monolithic column
CONCLUSION This chapter shows the reliability of different alternatives for the LC analysis of common PAHs in water samples that have been developed taking into account novel tendencies of Analytical Chemistry either for sample preparation or for analyte separation: CS-capillary
New Tendencies in Liquid Chromatography…
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LC-DAD, IT-SPME-capillary LC-DAD and IT-SPME-LC with a monolithic column-FLD. Such methods are clearly advantageous over a classical procedure based on SPE with disposable cartridges for a number of reasons: (i) the minimum off-line sample manipulation, as the samples only needed to be filtered; (ii) the rapidity, as on-line analyte enrichment is accomplished in a few min, (iii) the minimum consumption of extractive phases, as the extractive columns used (a C18 packed microcolumn or a segment of a capillary GC column) can be reutilized for several hundred samples; and (iv) the total elimination of organic solvents in sample preparation. The CS-capillary LC-DAD approach provides sensitivity adequate for the routine monitoring analysis of PAHs in sewage effluents, and identity confirmation by matching the UV spectra. Capillary LC-DAD is also compatible with IT-SPME, resulting in improved sensitivity. The best sensitivity is achieved when using FLD, even when combined with normal scale LC. The total analysis time can be further reduced by using a monolithic column for separation, thus resulting in a very rapid, sensitive, simple and cost- effective method. It can be concluded that the on-line methods described in this chapter are reliable alternatives to the classical methodologies for controlling PAHs pollution in waters, and more attractive in the context of a Green Chemistry.
ACKNOWLEDGMENTS The authors are grateful to the Spanish Ministerio de Educación y Ciencia (Project CTQ2008-01329/BQU) and to the Conselleria de Territorio y Vivienda de la Generalitat Valenciana (Application of EU Water Framework Directive 2000/60/EC on endocrine disruptors and other priority substances in coastal areas in the Comunidad Valenciana) for the financial support received.
REFERENCES [1] [2]
European Community Directive 98/80/EC, Brussels, November 1998. Miège, C.; Bouzige, M.; Nicol, S.; Dugay, J.; Pichon, V.; Hennion, M. C. J. Chromatogr. A 1999, 859, 29-39. [3] Martínez, E.; Gros, M.; Lacorte, S.; Barceló, D. J. Chromatogr. A 2004, 1047, 181-188. [4] Filipowska, A.; Lubecki, L.; Kowalewska, G. Anal. Chim. Acta 2005, 547, 243-254. [5] Bussetti, F.; Heitz, A.; Cuomo, M.; Badoer, S.; Traverso P. J. Chromatogr. A 2006, 1102, 104-115 [6] Zhou, Y.-Y.; Yan, X.-P.; Kim, K.-N.; Wang, S.-W.; Liu, M.-G. J. Chromatogr. A 2006, 1116, 172-178. [7] Gimeno, R. A.; Altelaar, A. F. M.; Marcé, R. M.; Borrull, F. J. Chromatogr. A 2002, 958, 141-148. [8] Oliferova, L.; Statkus, M.; Tsysin, G.; Shpigun, O.; Zolotov, Y. Anal. Chim. Acta 2005, 538, 35-40. [9] Wang, X.; Mitra, S. J. Chromatogr. A 2006, 1122, 1-6 . [10] Rezaee, M.; Assadi, Y.; Hosseini, M.-R. M.; Aghaee, E.; Ahmadi, F.; Berijani, S. J. Chromatogr. A 2006, 1116, 1-9.
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[11] Hsieh, Y.-N.; Huang, P.-C.; Sun, I.-W.; Whang, T.-J.; Hsu, C.-Y. ; Huang, H.-H.; Kuei, C.-H. Anal. Chim. Acta 2006, 557, 321-328. [12] El-Beqqali, A.; Kussak, A.; Abdel-Rehim, M. J. Chromatogr. A 2006, 1114, 234-238. [13] Negrão, M. R.; Alpendurada, M. F. J. Chromatogr. A 1998, 823, 211-218. [14] Djozan, D.; Assadi, Y. Microchem. J. 1999, 63, 276-284. [15] Doong, R.; Chang, S.; Sun, Y. J. Chromatogr. A 2000, 879, 177-188. [16] Popp, P.; Bauer, C.; Möder, M.; Paschke, A. J. Chromatogr. A 2000, 897, 153-159. [17] King, A. J.; Readman, J. W.; Zhou, J. L. Anal. Chim. Acta 2004, 523, 259-267. [18] Hu, Y.; Fu, Y.; Li, G.-K. Anal. Chim. Acta 2006, 567, 211-217. [19] León, V. M.; Álvarez, B.; Cobollo, M. A.; Muñoz, S.; Valor, I. J. Chromatogr. A 2003, 999, 91-101. [20] León, V. M.; Llorca-Pórcel, J.; Álvarez, B.; Cobollo, M. A.; Muñoz, S.; Valor, I. Anal. Chim. Acta 2006, 558, 261-266. [21] Titato, G. M.; Lancas, F. M. J. Liq. Chromatogr. Relat. Techno. 2005, 28, 3045-3056. [22] He, Y.; Lee, H. K. J. Chromatogr. A 1998, 808, 79-86. [23] Oliferova, L.; Ststkus, M.; Tsysin, G.; Zolotov, Y. Talanta 2007, 72, 1386-1391. [24] Lim, L. W.; Okouchi, Y.; Takeuchi, T. Talanta 2007, 7, 1600-1608. [25] Cháfer-Pericás, C.; Herráez-Hernández, R.; Campíns-Falcó, P. J. Chromatogr. A 2006, 1125, 159-171. [26] Cháfer-Pericás, C.; Herráez-Hernández, R.; Campíns-Falcó, P. J. Chromatogr. A 2007, 1141, 10-21. [27] Olejniczak, J.; Staniewski, J. Anal. Chim. Acta 2007, 588, 64-72. [28] Campíns-Falcó, P.; Verdú-Andrés, J.; Sevillano-Cabeza, A.; Molins-Legua, C.; Herráez-Hernández, R. J. Chromatogr. A 2008, 1211, 13-21. [29] Klodzińska, E.; Moravcova, D.; Jandera, P.; Buszewski, B. J. Chromatogr. A 2006, 1109, 51-59. [30] Kataoka, H. Anal. Bioanal. Chem 2002, 373, 31-45.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 19
PAHS IN SEDIMENTS ASSOCIATED WITH COAL AND COAL-DERIVED PARTICLES -- OCCURRENCE, MOBILITY AND RISK ASSESSMENT Yi Yang1 and Thilo Hofmann2 1
School of Resources and Environmental Sciences, East China Normal University 2 Department of Environmental Geosciences, Vienna University
ABSTRACT Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous hydrophobic organic contaminants in the environment. They tend to be associated with particles and are widely transported by flooding and atmospheric pathways, resulting in elevated concentrations in sediments/soils. Coal and coal-derived particles in natural sediments/soils can act not only as strong sinks for the PAHs, but also as very important sources of PAHs in sediments/soils. The understanding of the mobility of these contaminants from the sediments/soils, especially the sequestration of PAHs by coal and coal-derived particles is very important, because they can control the transportation, bioavailability, degradation and hence the potential risk of these contaminants in the environment.
1. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are of great concern, due to their persistence, toxicity and bioaccumulation. Once occurred in the aquatic system, these compounds tend to bound to particles because of their hydrophobic nature, and consequently accumulate in sediments. Thereby, sediments act as the important sink of these persistent organic pollutants (POPs), and pose the potential risk to the aquatic system. Coal and coal-derived particles with elevated PAHs concentrations produced by coal industry can be wild spread in the aquatic system through atmospheric transportation, runoff and flooding etc. Coal and coal-derived particles in natural sediments can act not only as strong sinks for the PAHs, but also as very
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important sources of PAHs in sediment. The occurence and mobility of these contaminants from the coal and coal-derived particles in sediments/soils is of significance, because sorption/desorption, especially sequestration of PAHs by these coal and coal-derived particles in sediments/soils can control their transportation, bioavailability, degradation and hence the potential risk in the environment.
2. OCCURRENCE OF COAL AND COAL-DERIVED PARTICLE ASSOCIATED PAHS IN SEDIMENTS 2.1. Identification of Coal and Coal-Derived Particles in Sediments Hard coal product has increased from less than 1 billion ton to almost 5 million ton from 1900 to 2005 [1]. Due to coal mining, coal industry, atmospheric transport, runoff, and flooding etc, unburnt coal and coal-derived particles can be wild spread in the aquatic system, and consequently settled in sediments. For example, unburnt coal particles can be released by open pit mining; coal stored at industrial sites for the production of coke, gas or steam, is subjected to erosion; moreover, coal naturally eroded into aquatic systems for sedimentary rock outcroppings containing coal seams. Occurrence of coal in sediments was reported from harbors such as Hamilton Harbour [2] and Roberts Bank coal terminal, in Canada [3]. Coal particles present in sediments made up 10.5 to 11.9 % dry weight of the soil mass in the vicinity of coal-loading terminals and was reported as non-hydrolysable solids. The classic method to identify coal and coal-derived particles in sediments is the coal petrography. Using optical microscopy enables the observation, identification, classification and quantification of coal and coal derived particles in natural sediments [4-8]. In a recent study, about 75 vol-% of the light fraction (i.e., the fraction with a density < 2 g/cm³, predominantly organic carbon) in a river floodplain soil were identified as hard coal particles [9]. Photomicrographs in Figure 1 show different coal and coal-derived particles identified by organic petrography techniques in sediment samples collected from Mosel River bank, where intensive coal mining activities happened in the former time.
Figure 1. Coal and coal-derived particles identified by organic petrography techniques.
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2.2. Elevated Concentration of Pahs in Sediments with Coal and CoalDerived Particles Coal is defined as a sedimentary rock containing more than 70% by volume of carbonaceous material with a three-dimensional macromolecular matrix ‘network’. In addition to the network structure, a multitude of small molecules, i.e. a ‘mobile phase’, is present within the network and is of particular environmental interest. These molecules can be released from the coal network more rapidly because they are less bound to the macromolecular matrix compared to the cross-linked molecules within the network. The number of conjugated aromatic rings per structural unit within the matrix increases with increasing coalification (maturity), ultimately resulting in graphite [10]. PAHs were often determined in hard coals with the concentration up to hundreds, and even few thousands of mg/kg. The characteristics of coal, including its PAH content and distribution pattern depend on (1) original biological raw materials (coal type) and (2) the degree to which the coal-bearing strata have been altered by the affects of pressure and temperature associated with burial over geologic time (coal rank) [11-15]. Due to the low density, coal particles are preferentially transported and can accumulate in remote areas, resulting in elevated PAH concentrations in sediments [4, 15]. For example, up to 80 mg/kg EPA-PAHs were found in bank sediment samples from the Saar and Mosel River (Figure 2). The light fraction, dominated by coal and coal-derived particles was collected by density separation. Although contributing 98%) and BaP (purity >97%) were purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. The substances were dissolved in dichloromethane (DCM) (Mallinckrodt Baker SA de CV EDOMEX, Mexico). Test substances (PAH) were dissolved in DCM. PAH diffusion in MS medium was previously determined (data not shown), therefore, test concentrations to evaluate removal of PAH varied as follows: 200 mg (kg of MS medium)-1 for PHE and PYR; 2 000 mg (kg of MS medium)-1 for BaP. In all cases MS medium was “as is” weight, i. e, not dry weight.
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2.4. Boiassays In vitro germination, root and stem length of plant species were evaluated with and without hydrocarbons (Reynoso-Cuevas et al., 2008). Pure DCM was used for the control samples; controls without solvent were also included. PAH solution (0.5 ml) was added to culture tubes just before hardening of the medium, shaken and maintained at room temperature for three days to evaporate the solvent; one seed was aseptically placed into each culture tube containing semi-solid MS medium supplemented with PAH test concentrations, the culture tube was closed with a plastic cap and sealed with parafilm. Sealed culture tubes were kept at 25 ºC, with 16 h photoperiod for 90 days for PAH removal studies.
2.5. Analytical Methods 2.5.1. Plants The two plant species were tested simultaneously, to achieve the best possible basis for comparing their individual outcome after 90 d culture. The plants were observed and monitored every 5 d to take note of changes in appearance and growth. The plants were sampled destructively five replicates per PAH concentration were analyzed and separated into roots and stems. 2.5.2. Hydrocarbons The liquid-liquid extraction of hydrocarbons of culture medium was made with DCM; three washings with 10 ml each, every 5 days during the 90 days of study. The reproducibility and recovery of the extraction method for the samples were satisfactory, with recovery averaged 96.7%±3.82 (standard deviation) for PHE; 98.2%±2.57 for PYR; 96.8%±2.74 for BaP. PAH identification and PHE and PYR quantification was carried out with a gas chromatograph, a Varian 3900 provided with FID detector and DB-PETRO (100 m, I.D. 0.25 mm) column was used; helium was the carrier gas (30 kPa). The initial oven temperature was 100ºC (holding time 1 min), increased to 180ºC at 20ºC min-1 and to 230ºC at 15ºC min-1 (holding time 5 min); then increased at 4ºC min-1 until 290ºC (holding time 23 min) and complete with 300ºC at 3ºC min-1 with a final holding time of 5 min. Injector and transfer lines were heated at 290ºC. Injection (1 μL) was performed with Varian CP8410 autoinjector. HPLC analysis for BaP was carried out on a Thermo separation products system consisting of a model ConstaMetric 3200 pump, an auto-sampler (model 545), and a UV detector (model 4100), λ 254 nm. BaP were separated on a Spherisorb ODS2 C18 column (15 cm x 2.1 mm, i.d.; 5 µm; film thickness), operated at 25 ºC, at a flow rate of 2 mL min-1 with acetonitrile:water (70:30) as the mobile phase. The sample injection volume was 20 µL. For calibration, standard solutions of BaP in hexane were used. The detection limit was 3 µg L-1. 2.5.3. Phytoremediation Factors The root concentration factor (RCF) describes the potential of a given xenobiotic to accumulate in the plant root, and makes no discrimination between surfaces accumulation and uptake into the root tissue.
In Vitro Phytoremediation Mechanisms…
RCF =
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Root final concentration (mg kg -1 ) MS medium concentration (mg kg -1 )
Stem concentration factor (SCF) indicates the potential of a given xenobiotic to accumulate in the plant stem.
Stem final concentration (mg kg -1 ) SCF = MS medium concentration (mg kg -1 ) Translocation factor (TF) points to the total of PAH that can be transported to the aerial parts of the plant.
Translocation (%) =
PAH content in stem (mg kg -1 ) × 100 PAH total content in whole plant (mg kg -1 )
2.6. Data Analyses All values presented for the chemical and biological analyses of plants are the means of five replicates. The mean and standard error were calculated for all data. PAH distributions in plants were one-way ANOVA tested. Statistical differences among individual treatments were assessed with Tukey-Kramer multiple comparison test at a significance level of α30% acetic acid was prerequisite for chitosan nanofiber formation. Concentrated acetic acid in water progressively decreased surface tension, and increased charge density without significant effect on solution viscosity Effect of several solvents and their mixtures with only strong acetic acid (80-90%) effective. Fiber diameter below 100nm. Several solvents investigated: formic acid, 0.2M acetic acid, and trifluoroacetic acid and their mixtures with methanol, ethanol, 1,4-dioxane, dichloromethane, N,Ndimethylformamide and dimethylsulfoxide. Only TFA and TFA/MC effective. Cross-linking with glutaraldehyde (GA) vapor. Insoluble in basic, acidic, and aqueous solutions. Effect of various solvents with the co-system of TFADCM most adequate due to low boiling point and surface tension. Antimicrobial test. Electro-wet-spinning using a coagulation bath containing ethyl alcohol in aqueous sodium hydroxide.
Ref [163]
[164]
[155]
[158] [157]
[193]
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Lingyan Kong, Gregory R Ziegler and Rajesh Bhosale Table 3. (Continued)
Material Chitosan
Solvent TFA-MC
Chitosan TFA Chitosan derivatives Hexanoyl chloroform chitosan Chitosan/PEO 50%- 90% Chitosan/ Acetic acid PEO/ surfactant Chitosan/ 2% acetic PEO acid Chitosan/ PEO
2% acetic acid
Chitosan/ UHMWPE O Chitosan/ PEO/HAp
3% acetic acid
Chitosan/ PEO Chitosan/ PEO
0.5 M acetic acid 90% acetic acid
Chitosan/ acetic acid PEO Chitosan/ PEO Chitosan/PVA Chitosan/ formic acid PVA Chitosan/ PVA
acetic acid
Carboxyme thyl chitosan
water
Technical highlights Effects of mechanical strength and permeability of fiber mats on nerve regeneration. High adsorption capacity for Cu(II) and Pb (II).
Ref [194] [160]
Pyridinium formate added to improve electrospinnability. Non-toxicity and cell proliferation support.
[189]
Surfactants can serve as carriers of lypophilic functional ingredients.
[165]
Crosslinked with GA vapor to reduce dissolution. Increased stiffness and decreased ductility of the electrospun mats. Pure chitosan fiber not achieved from aqueous acetic acid. Phase separation possibly occurring with results in chitosan and PEO rich fibers, respectively. Enhanced fiber formation with lowering UHMWPEO content below 5%.
[170]
Bone forming ability by the help of incorporate HAP. Potential interest for bone tissue engineering applications. Cell viability test. Potential interest for tissue engineering applications. Core-sheath geometry, with the PEO sheath serving as a template for the chitosan core. Matching rheological properties. Pure chitosan fiber obtained by removing the PEO sheath. Improved spinnability by hot air heating. Reduced beadlike defects and lowered PEO content (5%) Effect of blended ratio of PEO/chitosan. No biotoxicity and cell culture study.
[173]
Incorporated antimicrobial agents with AgNO3 more effective than TiO2. Antimicrobial activity also affected by average diameter and chitosan concentration. Nanoporous fibers obtained by removing the PVA. Fiber formation and uniformity improved by alkaline hydrolysis of chitosan. Water insolubility by heat-induced cross-linking.
[168]
[172]
[171] [174]
[166] [169]
[178]
[153]
[192]
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Table 3. (Continued) Material Chitosan 10/PVA Chitosan 100/PVA Carboxyme thyl chitosan/ PVA Chitosan/ PVA/HAp Carboxyme thyl chitosan/ PVA/HAp Chitosan/ PVA
Solvent FA & water FA & water water
acetic acid
acetic acid
Chitosan/ 90% acetic PVA acid Chitosan/other component Chitosan/ HFIP/TFA collagen Chitosan/ HFIP/TFA collagen 0.5 M Chitosan/ acetic acid collagen/ PEO Chitosan/ 90% acetic PLLA acid Chitosan/ TFA PLLA Cellulose/ chitosan HFIP/TFA Chitosan/ P(LLACL) Formic Chitosan/ acid PAA and chitosan/ PAMPS Chitosan/ TFA/DCM PLA
Technical highlights
Ref [155] [155]
No cytotoxicity. Potential application in wound dressing.
[191]
Cell proliferation test. Potential application in tissue regeneration. Cell culture test. Potential application in bone tissue engineering
[176]
Photocrosslinked by poly(ethylene glycol)-600dimethacrylate (PEGDMA) and photoinitiator 2hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1propanone (HEPK). Improved water resistance. No cytotoxicity. Lipase immobilization with pH and thermal stabilities of lipase improved.
[195]
[177]
[175]
[196] Collagen and chitosan miscible at molecular level.
[197]
Crosslinked with GA vapor resulting in increased Young’s modulus and reduced water absorption. Potential application in wound dressing. Cell culture study. Potential interest for biomedical applications. Potential application in tissue engineering.
[198]
Obtained by electrospinning and alkaline hydrolysis of cellulose acetate and dibutyryl chitin. Cell viability and morphology studied. Potential application as tissue engineering scaffolds.
[180]
Effect of pH on integrity.
[182]
Crosslinked with GA vapor. Inhibition of bacteria.
[199]
[183] [184]
[187]
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Lingyan Kong, Gregory R Ziegler and Rajesh Bhosale Table 3. (Continued)
Material Chitosan/ PAA Poly(chitos an-g-DLlactic acid) Poly(chitos an-g-DLlactic acid) Poly(chitos an-g-DLlactic acid)
Solvent Acetic acid
Technical highlights Effect of electrospinning temperature and blend ratio.
Ref [181]
Electro-wet-spinning using a coagulation bath containing ethyl alcohol in aqueous sodium hydroxide.
[200]
Different side chain lengths controlled by molar ratio of substrates. Potential application as tissue engineering scaffolds. Improved tensile strength and modulus.
[201]
O-
[202]
OH
O O O OH
O OH O
O NH
OH O
Figure 6. Chemical structure of hyaluronic acid.
Other Polysaccharides Hyaluronic Acid Hyaluronic acid (HA) is the acid form of hyaluronan, a naturally occurring linear polysaccharide consisting of repeating disaccharide units of D-glucuronic acid and N-acetyl-Dglucosamine (Figure 6). HA differs from other glycosaminoglycans in that it does not have sulfate groups. HA was first isolated by Karl Meyer from the vitreous body of the eye in 1934 [203]. HA can also be found in connective tissues, umbilical cord, and joint fluid. Because of its unique rheological properties, biocompatibility and ability of relieving inflammation, promoting cell proliferation and reducing scar formation, HA has been used in medical applications, for example in wound dressing, surgery, drug delivery, implants and arthritis treatment [204]. The electrospinning of HA nanofibers is challenging. The HA chains take the form of expanded, random coils entangling with each other at very low concentration. The resultant high viscosity and surface tension at relatively low concentration hinder electrospinning. Additionally, the insufficient evaporation of water is likely to refuse the nanofibers on the collector [205].
Fibers Spun from Polysaccharides
717
Table 4. Summary of reported electrospinning of HA or HA blend fibers. Polymer(s)
HA HA/Gelatin
Concentration (w/v) Gelatin fixed at 4.5 % 1.5 % 1.5 %
HA
2.5 %
H2O
74 nm
Zein/HA/PVP HA-DTPH/PEOa
15 % - 25 % 2.0 %
Ethanol/H2O DMEM
HA HA/Collagen
Up to 10% 10 %
1.8 µm – 3.7 µm 50 – 300 nm (85 %) 198 ± 45 nm 200 - 400 nm
Gelatin/HA
Blend ratio (w/w) 100/10 – 100/30 100/20 – 100/100
100:100
Solvent
Resultant fibers
Ref
H2O
200 – 240 nm
[206]
DMF/H2O DMF/H2O
120 – 250 nm 190 – 500 nm
[205] [205] [204, 207] [208] [209]
NaOH/DMF [210] 95/5 NaOH/DMF [210] 80/20 a HA-DTPH stands for 3,3’-dithiobis(propanoic dihydrazide modified HA. Fibers were cross-linked with 9.0% (w/v) poly(ethylene glycol)-diacrylate (PEGDA)
By modifying the electrospinning process to include an air-blowing system, Um et al. [204] were able to obtain hyaluronic acid nanofibers from very viscous HA solutions. They termed this apparatus “electro-blowing,” a combination of electrospinning and air blowing. The air flow provided additional pulling force to the polymer droplet. Meanwhile, the hot air also helped reduce solution viscosity and promoted solvent evaporation. In their studies, probably the first HA nanofibers ever spun, they determined the optimal process parameters including HA concentration, solution feeding rate, electric field, collector geometry and airblowing rate to get HA nanofibers of high quality. They aimed at fabricating water resistant HA nanofiber membranes through either the exposure of HA membranes to hydrochloric acid (HCl) vapor, followed by a freezing treatment at -20°C for 20-40 days, or the immersion of HA membranes in an acidic mixture of ethanol/HCl/H2O at 4°C for 1-2 days. Although both methods could produce hydrophilic, substantially water-resistant HA nanofibrous membranes (the treated membranes could keep their shape intact in neutral water at 25°C for about 1 week), the immersion method was shown to be more versatile and effective. Later on, the same group was able to use typical electrospinning setup to fabricate pure HA nanofiber from DMF/water solutions. DMF is a poor solvent for HA, but when mixed with water the solutions exhibited reduced surface tension and conductivity.
Dextran Dextran is a bacterial polysaccharide that consists of α-(1→6)-linked D-glucopyranose residues with some α-(1→2)-, α-(1→3)-, or α-(1→4)-linked side chains. Due to its biodegradability and biocompatibility, dextran has found various applications such as drug delivery [211] and scaffolds for cell proliferation [212]. One of the most intriguing properties of dextran for fiber spinning is its solubility; dextran is soluble in both water and organic solvents. Jiang et al. [213] was the first to report the successful electrospinning of dextran using water, DMSO/water or DMSO/DMF as
Lingyan Kong, Gregory R Ziegler and Rajesh Bhosale
718
solvents. Using water as a solvent, up to 10% of bovine serum albumin or lysozyme could be incorporated into dextran fiber with an average diameter of 2.5 µm, but decreasing BSA concentration to 5% could strikingly decrease the diameter to 500 nm. Since electrospun dextran fibers are soluble in water, a further cross-linking is necessary for the dextran membranes to be useful in medical applications. Thus methacrylated dextran was electrospun and photo-cross-linked by UV irradiation in the presence of 1% 2,2-dimethoxy-2phenylacetophenone (DMPA) as a photoinitiator. Another cross-linking method was utilized by Ritcharoen et al. [214] on their electrospun dextran membranes. GA was used as a cross-linking agent with MgCl2 as catalyst. Increasing curing temperature, curing time and catalyst usage were found to decrease swelling and weight loss of the dextran membranes in water without influencing the membrane morphology. In order to take advantage of its affinity for water soluble agents (e.g. protein) and compensate for the poor mechanical properties of dextran fibers, dextran was co-electrospun with poly(ε-caprolactone) (PCL) and PEG. By this technique, Jiang et al. [215] obtained coreshell structured fibers with PCL and PEG as shell and BSA-incorporated dextran as core. The study indicated that proteins can be electrospun into fiber membranes while retaining their bioactivity and released in a manner that is controllable through formulating electrospinning parameters such as feeding rate and polymer compositions. By electrospinning mixtures of poly(D,L-lactide-co-glycolide) (PLGA) and dextran in DMSO/DMF new porous hydrophobic/hydrophilic composite membranes were obtained [213]. The highly porous PLGA/dextran membranes were further studied for cell attachment, viability, proliferation and migration [216]. This study suggested use not only as a carrier for cells to accelerate wounds healing, but also as a potentially versatile scaffold for bioactive agents, for instance, drugs, genes or proteins to be delivered and released to the target sites.
OH
O OH OH
OH
O OH
O OH O OH
O OH OH
Figure 7. Chemical structure of pullulan.
O
Fibers Spun from Polysaccharides
719
Pullulan Pullulan is a linear polysaccharide produced extracellularly by a fungus Aureobasidium pullulans. The basic repeating unit of pullulan is a maltotriose trimer α-(1→4)Glup-α(1→4)Glup-α-(1→6)Glup- (Figure 7), although other structures may exist as maltotetraose, α-(1→4)Glup-α-(1→4)Glup- α-(1→4)Glup-α-(1→6)Glup-. The regular alternation of (1→4) and (1→6) bonds are believed to be responsible for several distinctive properties of pullulan, for instance, structural flexibility, enhanced solubility in water, adhesiveness, oxygen impermeability and fiber/film forming capacities [217, 218]. Hence pullulan has many potential biomedical, food, paper and electronic applications. Pullulan fibers have been disclosed in patents [219-222]. However, application of pullulan in fiber spinning has been limited, possibly due to its high cost. The only case of electrospinning pullulan containing fiber appears only very recently. Karim et al. [223] successfully electrospun pullulan/montmorillonite (MMT) blend nanofibers with diameters in the range of 50 to 500 nm from aqueous solutions. The introduction of MMT improved the tensile strength and thermal stability of the pullulan matrix. Starch Starch is among the most abundant biopolymers found on earth. Starch is found in plant tissues, such as leaves, stems, seeds, roots and tubers. It is also found in certain algae and bacteria. Starch exists in semi-crystalline granules of different size, shape and morphology depending on its botanical source. Nevertheless, most starches are composed of two structurally distinct molecules: amylose, a linear or lightly branched (1→4)-linked αglucopyranose, and amylopectin, a highly branched molecule of (1→4)-linked αglucopyranose with α-(1→6) branch linkages. Attempts to spin starch fibers have long existed in patents. Initially some tried producing amylose fibers, because the amylose component of starch is largely linear and thus was assumed to readily associate side-by-side under favorable conditions. For example, Hiemstra and Muetgeert [224] invented a process for extrusion of purified amylose in an aqueous sodium hydroxide solution into a concentrated aqueous ammonium sulphate/ammonium hydroxide solution at 30oC. Another process utilized a standard viscose spinning machine to spin amylose fibers from a 5% NaOH aqueous starch solution into a coagulating bath containing sulfuric acid and sodium sulfate at 50oC [225]. These production procedures either suffered from undesirable by-products (ammonium), or resulted in insufficient fiber strength. The high cost of purifying amylose was a further disadvantage as was the amylopectin component waste, which is more than amylose in common starches. Attempts to use native starch or modified starch for fiber spinning can be generally divided into two categories: spinning of thermoplastic starch and spinning of nonthermoplastic starch. A thermoplastic material behaves like a plastic that can be melted to a liquid by heating above a melting temperature and solidified to a glassy state by cooling. Thermoplastic starch (TPS) is based on starch blended with plasticizers, such as sorbitol and glycerol, so that it can be heated to a molten state without decomposition and processed on existing plastics processing machines. Attempts to spin TPS date back to 1951 by Horsak [226]. In this invention, starch fibers were obtained by adding glycerol to aqueous starch dispersions and extruding the compositions into a methanol/glycerol coagulating bath. Most other attempts have utilized melt spinning methods. For instance, Noda et al. [227] disclosed spinning water resistant starch fibers from melt mixtures of starch, fat/oil and plasticizers.
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Lingyan Kong, Gregory R Ziegler and Rajesh Bhosale
Others have tried adding another polymer, such as PVA which is also used in spinning cellulose and chitosan, to aid fiber formation [228]. Native and modified starches were blended with plasticizers and a small amount of PAM to make a melt processable composition, which then can be spun into fibers [229, 230]. Spinning TPS and copolyamide blends has also been described [231]. Spinning TPS fibers generally requires a significant amount of plasticizer that compromises fiber strength and raises cost of production. Non-thermoplastic starch (NTS) is generally made by blending starch with water and possibly other plasticizers. This mixture is suitable for wet or dry spinning, but not melt spinning since it decomposes before it can be melted. In such cases, cross-linking agents are employed to give water stability to the starch fibers. Eden and Trksak [232] described spinning of high amylose starch in saturated ammonium salt solutions into a coagulation bath containing ammonium salts. Hernandez et al. [233] also used ammonium salt in the coagulation bath to cross-link the resultant starch and starch/PVA fibers. Patents [234, 235] provide a refined process for spinning NTS. Cross-linking agents were added to the extruded starch mixtures, which were then forced through a die with multiple nozzles. Air flow was heated and used to dry and draw the starch fibers. In addition to conventional spinning of starch fibers, an electrospinning process for producing starch fibers was recently disclosed [236]. It provides a process for electrospinning of a TPS composition containing plasticizers and other polymers such as PAM. The resultant fibers were filled in molds to make flexible articles. Starch acetate was blended with cellulose acetate and spun via dry spinning [237, 238]. Bastioli et al. [239] invented a new spinning method where starch fibers are formed and coagulated when materials travel through the holes in a chamber. The resultant starch fibers could be used compatibly with cellulose fibers in paper making. Being both renewable and relatively inexpensive, starch is a popular biopolymer for replacing petroleum based materials. Starch based bioplastics, for instance, have received much attention, and there is much room to improve the properties and enlarge the uses of starch fibers.
CONCLUSION This chapter provides a general picture of fibers of either micro- or nano-scale spun from a variety of polysaccharides. It is unquestionable that in the field of biofibers, especially polysaccharide fibers, great progress has been made during the last few decades. However, despite their versatility in function and abundance in nature, the fiber forming capabilities of many polysaccharides have not been thoroughly exploited. As to the existing spun polysaccharide fibers, more progress will be needed to optimize their processing (e.g. environmentally harmless solvents, solvent and coagulant recycling, production scale up and production efficiency), enhance their properties (e.g. mechanical properties, average diameter, porosity, stability/integrity, reagent affinity and etc.) and broaden their functionalities (e.g. composite fibers, drug loading, nutrient incorporation and so forth). For the large variety of polysaccharides that have not been successfully spun, more research will be required. The increasing demand for bio-based materials will promote the development of polysaccharides fiber spinning technology.
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[213] Jiang, H., et al. (2004). Optimization and characterization of dextran membranes prepared by electrospinning. Biomacromolecules, 5(2), 326-333. [214] Ritcharoen, W., et al. (2008). Electrospun dextran fibrous membranes. Cellulose, 15(3), 435-444. [215] Jiang, H., et al. (2006). Modulation of protein release from biodegradable core-shell structured fibers prepared by coaxial electrospinning. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 79B(1), 50-57. [216] Pan, H., Jiang, H. & Chen, W. (2006). Interaction of dermal fibroblasts with electrospun composite polymer scaffolds prepared from dextran and poly lactide-coglycolide. Biomaterials, 27(17), 3209-3220. [217] Singh, R. S., Saini, G. K. & Kennedy, J. F. (2008). Pullulan: microbial sources, production and applications. Carbohydrate Polymers, 73(4), 515-531. [218] Leathers, T. D. (2003). Biotechnological production and applications of pullulan. Applied Microbiology and Biotechnology, 62(5), 468-473. [219] Nomura, T. (1976). Paper composed mainly of pullulan fibers and method for producing the same, U.S. Patent 3,936,347, Feb. 3. [220] Domoto, M. & Tsuji, K. (1978). Novel nonwoven fabric and method for manufacturing same, U.S. Patent 4,092,454, May. 30. [221] Ozaki, Y., Nomura, T. & Miyake, T. (1996). High pullulan content product, and its preparation and uses, U.S. Patent 5,518,902, May. 21. [222] Fujii, S., Mori, S. & Tabuchi, J. (1991). Nonwoven fabric of water-soluble resin fibers U.S. Patent 5,041,252, Aug. 20. [223] Karim, M. R., et al. Preparation and characterization of electrospun pullulan/montmorillonite nanofiber mats in aqueous solution. Carbohydrate Polymers. In Press, Accepted Manuscript. [224] Hiemstra, P. & Muetgeert, J. (1959). Process for the production of amylose articles by extrusion of aqueous sodium hydroxide solution thereof into concentrated aqueous ammonium sulphate solution, U.S. Patent 2,902,336, Sept. 1. [225] Kunz, W. B. (1962). Method of preparing amylose film, tubing, and the like, U.S. Patent 3,030,667, Apr. 24. [226] Horsak, D. (1951). Method of production of synthetic material from starch or starch containing substances, U.S. Patent 2,570,449, Oct. 9. [227] Noda, I., et al. (2007). Water stable fibers and articles comprising starch, and methods of making the same, U.S.P. Application 20070082573, Apr. 12. [228] Evers Smith, L., et al. (2006). Fibrous structures comprising a polymer structure, U.S.P. Application 20060275347, Dec. 7. [229] Bailey, V. A., Mackey, L. N. & Trokhan, P. D. (2004). Melt processable starch compositions, U.S. Patent 6,709,526 B1, Mar. 23. [230] Mackey, L. N., et al. (2006). Melt processable starch composition, U.S. Patent 7,041,369 B1, May. 9. [231] Buehler, F., et al. (1996). Starch-containing fibers, process for their production and products made therefrom, U.S. Patent 5,516,815, May. 14. [232] Eden, J. & Trksak, R. M. (1989). Process for spinning starch fibers, U.S. Patent 4,853,168, Aug. 1. [233] Hernandez, H. R., et al. (1979). Water insensitive starch fibers and a process for the production thereof, U.S. Patent 4,139,699, Feb. 13.
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[234] James, M. D., et al. (2004). Process for making non-thermoplastic starch fibers, U.S. Patent 6,811,740 B2, Nov. 2. [235] James, M. D., et al. (2007). Process for making non-thermoplastic starch fibers, U.S. Patent 7,276,201 B2, Oct. 2. [236] Gordon, G. C., et al. (2006). Electro-spinning process for making starch filaments for flexible structure, U.S. Patent 7,029,620 B2, Apr. 18. [237] Feuer, B. I. & Bronk, J. M. (1997). Starch acetate and blends thereof with metal chelates, U.S. Patent 5,693,279, Dec. 2. [238] Maheras, J. C., John, J., Hopkins, B. & Tatzlaff, H. (1995). Polymer blend composed of cellulose acetate and starch acetate and starch used to form fibers, films and plastic materials and a process to prepare said blend, U.S. Patent 5,446,140, Aug. 29. [239] Bastioli, C., Casale, B. & Zanardi, G. (1999). Device and process for the production of fibrious starch materials, U.S. Patent 5,866,251, Feb. 2.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 27
DEVELOPMENT AND CHARACTERIZATION OF STARCH BASED NANO-COMPOSITES Xiaozhi Tang and Sajid Alavi Kansas State University, Manhattan, Kansas, U.S.A.
ABSTRACT Starch-based biodegradable materials are considered one of the most promising candidates to replace certain types of conventional plastics. Starch is relatively cheap and available from a broad range of plants. However, the use of starch for packaging materials is restricted because of its sensitivity to water and inferior mechanical properties. The unique properties of nano-structured substances have opened windows of opportunity for the creation of high performance materials with a critical impact on starch-based materials. This chapter presents a review of ongoing research and development activities in our lab and in the wider scientific community related to starchclay nanocomposites. As a valuable way to further enhance properties of these nanocomposites, blends involving biodegradable polymers such as poly (vinyl alcohol) are also discussed. Melt intercalation, using extrusion processing, has proved to be an efficient way for preparation of starch-based nanocomposites with improved thermal, mechanical, and barrier properties. The chapter also elaborates the mechanisms of clay exfoliation and dispersion in the starch matrix, which are based on both chemistry and processing effects. Improved interfacial compatibility of the multiphase system (starch, biodegradable polymer, plasticizers, nanofiller, etc.), as well optimized processing parameters, can lead to the design of nanocomposites with enhanced properties. A special emphasis is also given to an overview of mathematic models for some critical nanocomposite properties, such as barrier and mechanical properties. The establishment of a mathematic model provides guidelines for tailoring clay modification, clay dispersion and processing parameters for fabricating starch-based nanocomposites with unique properties.
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1. INTRODUCTION Starch is the major form of stored carbohydrate in plants such as corn, cereal grain, rice, and potatoes. Starch is composed of a mixture of two polymers of α-glucose -- linear amylose and a highly branched amylopectin. Amylose molecules consist of 200-20,000 glucose units which form a helix as a result of the bond angles between the glucose units. Amylopectin is a highly branched polymer containing short side chains of 30 glucose units attached to every 20-30 glucose units along the chain. Amylopectin molecules may contain up to two million glucose units (Ray and Bousmina, 2005). Starch is one of the natural biopolymers most widely investigated for developing environmentally-friendly packaging materials to substitute for petrochemical-based nonbiodegradable plastic materials. Being an inherently biodegradable, renewable, and low-cost material, starch has high potential in food or non-food packaging applications. Starch in granular state has been used as filling agent for polyolefin and as a component in synthetic polymer blends. In such cases, a small amount of starch (6-30%) is blended with synthetic polymers like polyethylene (PE) or polypropylene (PP), increasing the products’ biodegradability (Evangelista et al., 1991; Bagheri, 1999). Starch can also be directly used to form edible or biodegradable films. Different sources of starch, high amylose starch, and modified starch (Wolff et al., 1951; Roth and Mehltretter, 1967; Mali and Grossmann, 2003; Mali et al. 2006) have been used to form self-supporting films by casting from aqueous solution. These films appear to have moderate oxygen barrier properties but poor moisture barrier and mechanical properties, which limit their commercial applications. Thermoplastic starch (TPS) is a relatively new concept and today, it is a primary researchy focus for the manufacture of starch-based biodegradable materials (Averous, 2004). Native starch is not thermoplastic, but in the presence of a plasticizer (water, glycerol, sorbitol, etc.), high temperature, and pressure, it melts and fluidizes, enabling its use in injection, extrusion and blowing equipment, just like synthetic plastic materials. Thermoplastic starches find use mainly in water soluble compostable foams, expanded trays, shape molded parts, and expanded layers as a replacement for polystyrene. TPS products with different viscosity, water solubility, and water absorption have been prepared by altering the moisture/plasticizer content, amylose/amylopectin ratio of raw material, and the temperature and pressure in the extruder (Mohanty et al., 2000). Several studies have focused on the plasticization of TPS using glycerol (Forssell et al., 1997), sorbitol (Gaudin et al., 1999), urea, formamide (Ma et al., 2004), dimethyl sulfoxide (Nakamura and Tobolsky, 1967), and low molecular weight sugars (Kalichevsky et al., 1993). Plasticizers can increase the flexibility and processibility of TPS. However, neither starch films nor thermoplastic starches can be used in many applications owing to their sensitivity to humidity and inferior mechanical properties, such as film brittleness caused by high intermolecular hydrogen bond forces. Therefore, there is a significant need for exploring new techniques to meet the challenges of developing high quality starch-based biodegradable materials.
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2. STARCH BASED NANOCOMPOSITES 2.1. Introduction to Nanocomposites It has been established recently that inorganic nanoparticles can be used as additives to enhance the performance of polymers in the context of packaging materials. Various nanoreinforcements currently being developed are nanoclay (layered silicates) (Pinnavaia and Beall, 2001), cellulose nanowhiskers (Neus Angles and Dufresne, 2000 and 2001), ultra fine layered titanate (Hiroi et al., 2004), and carbon nanotubes (Kumar, 2004). Among these, up to now only the layered inorganic silicates have attracted great attention by the packaging industry. This is not only due to their environmental friendliness, natural abundance and low cost but also due to their enhancements of base polymer properties and relatively simple processibility. Polymer layered silicate (PLS) nanocomposite technology was first developed for automotive applications by Toyota researchers in the late 1980s (Fukushima and Inagaki, 1987; Okada et al., 1987) and Toyota was the first company to commercialize these nanocomposites (nylon 6-clay nanocomposites) (Ray et al., 2006). In the following years, PLS nanocomposites have attracted increasing interest as a means for improving properties of synthetic polymers. These studies include nanocomposites of nylon 6-clay (Kojima et al., 1993a, 1993b), polyurethane-clay (Wang and Pinnavaia, 1998), polypropylene-clay (Kurokawa et al., 1996; Usuki et al., 1997; Kawasumi et al., 1997), polyimide-clay (Yano et al., 1993; Yano et al., 1997), polystyrene-clay (Vaia et al., 1995; Vaia and Giannelis, 1997) and polysiloxane-clay (Mark, 1996) etc. With only a small percentage of silicate content (≤5%), the nanocomposites exhibit remarkable improvement in materials properties when compared with pristine polymer or conventional micro- and macro-composites. These improvements can include increased strength and heat resistance, decreased moisture and gas permeability and lower flammability. Nanocomposites also offer extra benefits like keeping original material’s transparency, low density, better surface properties and recyclability (Giannelis, 1996). Potential applications of these nanocomposites include automobiles (gasoline tanks, bumpers, interior and exterior panels etc.), construction (building sections, structural panels), aerospace (flame retardant panels, high performance components), electronics (printed circuit boards, electric components) and pigments (Ray et al, 2006). In order to take advantage of their substantially enhanced properties, polymer nanocompsites have also been studied for food packaging applications including injection molded bottles for beverage or beer, coatings for paperboard juice cartons, and cast and blown films (Ray et al, 2006). Use of these nanocomposites for packaging with oxygen scavenging, reduced flavor scalping, increased heat resistance and high gas barrier properties has resulted in shelf-life of 3-5 years for packaged food (Sajilata et al., 2007).
2.2. Structure and Properties of Layered Silicates The most commonly used silicates in PLS nanocomposites include montmorillonite (MMT), hectorite and saponite, and their various modifications. Like talc and mica, which are
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better known minerals, these layered silicates belong to the general family of 2:1 layered silicates (or phyllosilicates) (Giannelis, 1996). Their crystal lattice consists of twodimensional, 1nm thick layers, which are made up of two tetrahedral sheets of silica fused to an edge-shaped octahedral sheet of either aluminum or magnesium hydroxide (Figure 1). The lateral dimensions of these layers vary from 30nm to several microns or larger, depending on the particular layered silicate. Stacking of the layers leads to a regular van der Waals gap between the layers called the interlayer or gallery. Isomorphic substitution within the layers (for example, Al3+ replaced by Mg2+ or Fe2+, or Mg2+ replaced Li+) generates negative charges that are counterbalanced by alkali and alkaline earth cations situated inside the galleries.
Tetrahedral (Si)
Octahedral (Al) ~ 1 nm d-spacing
Tetrahedral (Si)
Exchangeable cations
Gallery/ Interlayer
Figure 1. Structure of 2:1 layered silicates
In pristine layered silicates, the interlayer cations are usually hydrated Na+ or K+, showing hydrophilic surface properties. Obvious, in this pristine state, layered silicates are only miscible with hydrophilic polymers like starches. To render layered silicates miscible with hydrophobic polymers like polyethylene and polystyrene, one must convert the normally hydrophilic silicates surface to an organophilic one. Generally, this can be done by ionexchange reations with various organic cations (e.g. alkylammonium cations, cationic surfactant etc.). The organic cations lower the surface energy of the silicate surface and result in a larger interlayer spacing. Additionally, the organic cations may contain various
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functional groups that react with the polymer to improve adhesion between the inorganic phase and the matrix. Therefore, there are two particular characteristics of layered silicates exploited in PLS nanocomposites. The first is the ability of the silicate particles to disperse into individual layers. As a result, an aspect ratio as high as 1000 for fully dispersed individual layers can be obtained. The second characteristic is the ability to fine-tune their surface chemistry through ion exchange reactions with organic and inorganic cations.
2.3. Nanocomposite Synthesis and Characterization For nanocomposite synthesis, polymer chains must diffuse into the galleries between silicate layers to produce structures ranging from intercalated to exfoliated (Figure 2). Intercalation occurs when a small amount of polymer penetrates into the galleries, resulting in finite expansion of the silicate layers. This leads to a well-ordered multilayered structure with a repeat distance of a few nanometers, and is observed in systems with limited miscibility. Extensive polymer penetration leads to exfoliation or delamination of silicate layers. An exfoliated nanocomposite consists of nanometer thick platelets distributed homogeneously throughout the polymer matrix. In contrast, when the polymer and silicate are immiscible, the layers do not separate and exist as agglomerates or tactoids.
+ Layered silicate
Polymer
(Nanoclay)
Phase separated (Microcomposite)
Intercalated (Nanocomposite)
Intercalated disordered (Nanocomposite)
Exfoliated (Nanocomposite)
Figure 2. Possible structures of nanocomposites
The complete dispersion of clay platelets in a polymer optimizes the number of available reinforcing elements for carrying an applied load and deflecting cracks. The coupling
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between the tremendous surface area of the clay and the polymer matrix facilitates stress transfer to the reinforcement phase, allowing for such mechanical improvements. In addition, the impermeable clay layers mandate a tortuous pathway for a permeant to transverse the nanocomposites (Figure 3). The enhanced barrier characteristics, chemical resistance, reduced solvent uptake and flame retardance of polymer-clay nanocomposites all benefit from the hindered diffusion pathway through the nanocomposites.
Figure 3. Proposed model for the tortuous zigzag diffusion path in a polymer-clay nanocomposite when used as a gas barrier
Synthesis of PLS nanocomposites has initially been accomplished by one of two methods. The first involves intercalation of a suitable monomer followed by polymerization (in-situ polymerization) (Okada et al., 1987; Messersmith and Giannelis, 1993). Various mono and multifunctional monomers have already been used, yielding linear and cross-linked polymer matrices, respectively. The second method involves polymer intercalation from solution, (Ruiz-Hitzky and Aranda, 1990; Wu and Lerner, 1993) i.e., intercalation of dissolved polymer from a solution. However, for most commonly used polymers, both in-situ polymerization and intercalation from solution are limited because neither a suitable monomer nor a compatible polymer-silicate solvent system is always available. Vaia et al. (1993) discovered a more versatile and environmentally benign approach based on direct polymer melt intercalation. The process involves mixing the layered silicate with the polymer and heating the mixture above the softening point of the polymer. Accordingly some easy accessible processing techniques like hot-mixing (Vaia et al., 1993), melt-extrusion (Park et al., 2002; Tang et al., 2008a), ultrasonication (Lim et al., 2003) can be directly used for nanocomposite synthesis. Recently, a new alternative method for the preparation of nanocomposites was proposed, involveing a solid-state mixing at room temperature (ball milling) (Sorrentino et al., 2005; Mangiacapra et al., 2006). In this case, solid layered dispersion was promoted by the energy transfer between milling tools (generally balls) and polymer/inorganic particles were intimately mixed by mechanical grinding. Generally, the structure of nanocomposites can be characterized by two complementary analytical techniques, namely, X-ray diffraction (XRD) and transmission electron microscopy
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(TEM). XRD is used to identify intercalated structures by determination of the interlayer spacing (d-spacing). Intercalation of the polymer chains increases the interlayer spacing and according to Bragg’s law, it should cause a shift of the diffraction peak towards lower angles. As more polymers enter the interlayer spacing, the clay platelets become disordered, thus causing broader peaks and a wider distribution of such peaks, which implies exfoliation of the layered silicates in the polymer matrix (McGlashan and Halley, 2003). In addition, TEM images provide further evidence for the occurrence of intercalation or exfoliation. TEM allows a qualitative understanding of the internal structure, spatial distribution and dispersion of the nanoparticles within the polymer matrix through direct visualization. Both TEM and XRD are essential tools for evaluating nanocomposite structure. However, there are still some other techniques used to gain greater insight about nanocomposite structure, such as Differential Scanning Calorimetry or DSC (Giannelis, 1996), Nuclear Magnetic Resonance or NMR (VanderHart et al., 2001), Fourier Transform Infrared Spectroscopy or FTIR (Loo and Gleason, 2003).
2.4. Starch-Clay Nanocomposites Reinforcement of starch with nano-scale minerals like clay has been considered in order to improve the properties, including the resistance to water and mechanical properties of starch materials, without interfering in the biodegradability of the composites. De Carvalho et al. (2001) provided a first insight in the preparation and characterization of thermoplasticized starch-kaolin composites by melt intercalation techniques. After that, several studies have been performed on the preparation of starch-clay nanocomposites by melt intercalation involving hot mixing and extrusion in details (Park et al., 2002 and 2003; Wilhelm et al., 2003; Avella et al., 2005; Chen and Evans, 2005; Huang et al., 2005; Huang and Yu, 2006; Pandey and Singh, 2005; Chiou et al., 2007; Cyras et al., 2008; Chivrac et al., 2008). In our research group, melt extrusion processing method was used to prepare starchclay nanocomposites. The structures of the composites were characterized by XRD and TEM. Films properties (mechanical, barrier and thermal properties) were tested (Tang et al., 2008a,b,c). Preparation of starch-clay nanocomposites in our laboratory involved a two step procedure: native starches/water/plasticizer (e.g., glycerol)/silicate clays were premixed using a mechanical mixer, and were kept overnight in order to swell the granular starch in the presence of water and glycerol. Then the resulting swollen mixture was transferred into a corotating twin-screw extruder. The starches were gelatinized inside the extruder and the mechanical energy input helped the dispersion of silicate clays in the starch matrix. Figures 4 and 5 show the XRD patterns of composites with different types of nanoclaynatural montmorillonite and surface modified montmorillnite (Nanomer I30E) (Tang et al., 2008a). It is clear that the dispersion states of nanoclays in the starch matrix depended on the type of clay used. The natural MMT exhibited a single peak at 2θ = 7.21˚, whereas the starch/MMT hybrids showed prominent peaks at 2θ = 4.98˚ (Figure 4). It also can be seen from Figure 4 that starch blank exhibited a featureless curve in the range of 1-10˚ due to the amorphous character of gelatinized starch. The appearance of the new peak at 4.98˚ (dspacing = 1.77 nm) with disappearance of the original peak of the nanoclay at 2θ= 7.21˚ (d-
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spacing = 1.23 nm) and increase of d-spacing indicated the formation of nanocomposite structure with intercalation of starch in the gallery of the silicate layers of MMT.
Figure 4. XRD patterns of (1) natural montmorillonite (MMT), (2) corn starch blank (0% MMT), and (3 and 4) corn starch/nanoclay hybrids with 3 and 6 % MMT, respectively (Tang et al., 2008a).
Figure 5. XRD patterns of (1) original nanomer I30E, (2) corn starch blank (0% I30E), and (3 and 4) corn starch/nanoclay hybrids with 3 and 6% I30E, respectively (Tang et al., 2008a).
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From Figure 5, it can be seen that the organically modified nanoclay I30E alone exhibited an intensive peak at 2θ = 3.93 (d-spacing = 2.23 nm), whereas starch-I30E hybrids showed weak peaks just under the original peak of the I30E. This implied that little or no intercalation/exfoliation was achieved in the starch matrix. The above results clearly showed that compatibility and optimum interactions between starch matrix, organic modifiers (if any) and the silicate layer surface were crucial to the formation of intercalated or exfoliated starch-layered silicate nanocomposites. Nanomer I30E is an onium ion surface modified montmorillonite mineral. Compared with natural MMT, I30E is more surface hydrophobic, and therefore is not very miscible in the hydrophilic starch matrix. On the other hand, in the case of natural MMT, due to the strong interactions between small amounts of polar hydroxyl groups of starch and glycerol, and the silicate layers of the nanoclay (inorganic MMT Na+), the starch chains and glycerol molecules can intercalate into the interlayers of the nanoclay. TEM micrographs of typical starch-MMT and starch-I30E composites are presented in Figure 6. The TEM results corresponded well with the XRD patterns. Starch-MMT composites exhibited a multilayered nanostructure (Figure 6-a), whereas starch-I30E composites showed almost no intercalated morphology, but instead had particle agglomerates or tactoids (appearing as dark spots in Figure 6-b).
Figure 6. TEM images of (a) starch-6% MMT and (b) starch-6% I30E composites (Tang et al., 2008a).
Figure 7 shows the effects of clay concentration (up to 21 wt% MMT) on the structure of the nanocomposites. It can be seen that the only change was the intensity of peaks, which increased with higher clay concentration. There was no shift in any of the peaks with varying clay contents, indicating that the clay content did not have any significant effect on the occurrence of intercalation or exfoliation. Films based on above starch-clay composites/nanocomposites were made from ground extrudates and water vapor permeability (WVP) and tensile properties of the films were examined according to the ASTM standard methods E96-00 (ASTM, 2000) and D882-02 (ASTM, 2002).
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Figure 7. XRD patterns of (1) wheat starch blank (0% MMT), and (2, 3, 4, 5, and 6) wheat starch-clay nanocomposites with 3, 6, 9, 15, 21% MMT, respectively (Tang et al., 2008a).
Table 1 shows the effects of clay type on WVP of corn starch based composite films. At the same clay level, WVP of the starch-MMT composite films was significantly lower than that of films made from starch-I30E composites. There was no significant difference in WVP when the I30E content increased from 0 to 9%, while WVP decreased significantly with the addition of 3 to 9% MMT. It was obvious that the addition of I30E did not help in improving the barrier properties of the films, which indicated that improvement in film properties depended on the occurrence of the intercalation or exfoliation (formation of nanocomposites). Table 1. Effects of clay type on WVP of corn starch-based films (Tang et al., 2008a). WVP (g·mm/kPa·h·m2) Clay content Starch-MMT Starch-I30E 0% clay 1.61 ± 0.08a 1.61 ± 0.08ae 3% clay 1.42 ± 0.04b 1.63 ± 0.12e c 1.58 ± 0.08e 6% clay 1.06 ± 0.09 d 9% clay 0.77 ± 0.04 1.56 ± 0.14e Mean ± standard deviation of each analysis Means with the same letters are not significantly different (P 20 > 21. The adsorption efficiency is enhanced when the guest molecule is hydrophobic and has a good complementarity with the β-CD cavity. The adsorption of 20-22 was explained by the Freundlich isotherm. For experiments the branched amino acids L-valine, L-leucine and L-isoleucine 23-25 also were used, they were mixed with 20-22 in phosphate buffer solutions, and adsorption of mixed amino acids was studied. It was found that the adsorption efficiencies of 23-25 were lower than those of aromatic amino acids, this behavior being due to the fact that the benzene ring of 20-22 fits well to the CD cavity, while molecules of 23-25 are too small for CD cavity [70]. The adsorption efficiency of mixed aromatic amino acids decreased in the same order as for single aromatic amino acids, whereas the adsorption efficiency of 23-25 decreased in the order 24> 25> 23. The light-responsive aqueous solutions of macromolecules are intensively studied since they may be applied in sol-gel materials and in drug-delivery. Light is a rapid and clean trigger because no additives or stirring are necessary. Such materials may be used in design of functional viscosifiers, in control of the stability of emulsions and in solubilization of drugs or
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proteins, as well as in pharmaceutics and cosmetics. The noncovalent photobinding between polymers is a basis of light-responsiveness. The formation of responsive clusters of macromolecular chains, or gels of interconnected chains is achieved using hydrophobically modified polymers in presence of various additives, such as micelles of surfactants, CDs or proteins which bind to the hydrophobic side groups of the polymers. Hydrophobically modified polymers show the strong viscosification and high sensitivity to external stimuli. The use of mixtures of azobenzene-modified polymers with CD polymers allows to achieve photoviscosity swings by ca a decade at submillimolar concentrations of azo dyes [71]. It was established that polyacrylate modified with azobenzene, i.e. polymer N associates with β-CD polymer in aqueous solutions. Polymer N is a random copolymer containing sodium acrylate as a charged hydrophilic unit and a low fraction of 6-[4alkylamido]phenylazobenzene acrylamide as a light-responsive hydrophobic moiety. The βCD polymer is randomly crosslinked with EP. H2N H2N
COOH
H 2N
COOH
COOH H2N
COOH H 2N
COOH H 2N
COOH
HN
phenylalanine 20
HO tyrosine 21
tryptophan 22
leucine 24
valine 23
z
z y O
O- + Na
isoleucine 25
y
O
UV
NH
O
O-
Vis
X
O Na+
NH X O
O HN
HN
N
N
N
N x = CnH2n; n = 5 or 11 cis
tr ans polymer N
The association of polymer N with β-CD polymer is influenced by the density of photochrome on polymer N and the length of the spacer between the polymer backbone and azobenzene. Azobenzene molecules, due to their hydrophobicity, bind to CD cavities.The viscosity of formed inclusion complexes of β-CD with polymer N was studied. It was observed that the density is enhanced with concentration of β-CD polymer; at higher concentrations the viscosity increase leads to formation of large clusters. The light-triggered trans conformation of azobenzene units of polymer N results in considerable photoswitch of viscosity. Reversible viscosity swings, up to 6-fold, occur upon alternate exposure to UV and visible lights. However, it is noteworthy that the system of βCD/ polymer N complexes does not respond to light in dilute solutions.
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The viscosity of β-CD/ polymer N mixtures was measured in order to find conditions for the formation of clusters of polymers; it was observed that the highest viscosity was achieved in dark adapted samples, and dropped down upon exposure to UV light (predominantly cisazobenzene is formed). The initial viscosity was recovered upon exposure to visible light (predominantly trans-azobenzene is formed) [72]. It was established that the association of polymer N with β-CD polymer is considerably stronger than with any CD monomers studied (α-CD, β-CD and HP-β-CD). This association is favored by increasing the hydrophobicity of azobenzene groups, and increasing the density of azobenzene in polymer N chains. In the study of hydrogels formed from CD polymers [25, 73], it was found that the mixing of aqueous solution of host β-CD polymers 26 and 27 with hydrophilic guest polymers 28-31 bearing adamantyl groups affords flexible, easy to modify physical hydrogels [74]. The host CD polymers serving for entrapment of guest polymers 28-31 are easily obtained by reacting native β-CD and EP in sodium hydroxide solution. It was found that the crosslinking of β-CD with EP upon addition of toluene affords the water soluble, linear polymers 26, while without toluene the globular CD polymers 27 are obtained. Mixing of 26 with 30 in aqueous solution results in the strong increase of the viscosity. The formed hydrogel is the most stable when the stoichiometry of 26/30 is 1:1. The viscosity of the hydrogels is adjustable by varying the concentration and the length of the spacer in the guest polymer. When no spacer exists, as in 28, the polymer backbone of 28 prevents the complexation with the host polymer 26. In the case of a 3C-atom spacer as in 29(x=2), the adamantyl groups are the best accessible for 26.
Cl
+
O
β
26
Cl
β
O
27
Use of Cyclodextrin Polymers in Separation of Organic Species
n
m
n
O NH
NH
NH
m
O
O
O
X
spacer length
2 5 11
3C 6C 12C
NH x
O NaO3S
NaO3S
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29 30 31
HN
28
With the increased spacer length to 6-C-atom spacer as in 30 (x=5) or to 12-C-atom spacer as in 31 (x=11) the flexibility of the guest polymer also increases, and the adamantyl groups may build in into the polymer coil; this behavior results in difficult complexation and the lower viscosity of the hydrogel. The hydrogel formation depends also on the number adamantyl groups present in the polymer backbone. Moreover it was observed that for the formation of hydrogels with high viscosities the higher concentration of EP is required. The viscosity of hydrogels is also affected by pH changes. Under alkaline conditions the guest polymer is totally deprotonized; this results in the expansion of the polymer coil, therefore more adamantyl groups of the guest copolymer can be complexed by the host CD polymer, i.e. the aggregation of the hydrogel network increases. The obtained hydrogels are stable at pH range 6-10, and do not decompose by enzymatic treatment, for example by treatment with starch hydrolyzing enzyme taka-diastase from Aspergillus oryzae. Photo-induced polymerization of hydroxypropyl-β-CD/methyl methacrylate complex, i.e. HP-β-CD/MMA was performed in aqueous solution, at room temperature with Irgacure 2959, i.e. 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone serving as a photoinitiator [75]. The atom transfer radical polymerization (ATRP) and reversible addition fragmentation chain transfer (RAFT) [76] methods are also used to perform polymerization of the CD/guest monomer complexes in aqueous solutions.
OH O HO
O OH O Irgacure 2959
OH
838
Cezary A. Kozlowski and Wanda Sliwa R
(OH)14
N O TEMPO
32
HN
N O
33
O H N
N O
S
O OR
34
N S
N O
O
The UV-induced photopolymerization has many advantages over the traditional thermal polymerization; it requires lower polymerization temperature than thermal polymerization, and may be controlled by selecting the irradiation wavelength and light intensity. Photopolymerizations are highly efficient, therefore they are widely applied in coating industry, in adhesives and paints, and in dental restoration materials. It should be pointed out that UV light can be focused on a special place, thus the photopolymerization may be controlled at desired sites; this fact enables the use of photopolymerization in microdevice production. Moreover the UV-induced polymerization is environmentally friendly in contrast to the traditional thermal polymerization performed in organic solvents, this fact deserving a special attention. The photopolymerization of HP-β-CD/MMA complex, i.e. the photopolymerization of MMA in the form of HP-β-CD/MMA complex, carried out in aqueous solution, in the presence of a water soluble photoinitiator, Irgacure 2959, proceeds smoothly under UV irradiation. It should be pointed out that this process can be performed at room temperature [75]. The molecular weight of the obtained polymer and polymerization conversion decrease with higher UV intensity and with concentration of initiator. The reusability of unthreaded HP-β-CD was investigated. It was observed that HP-β-CD unthreaded from the polymer chains can entrap the insoluble monomer molecule and form again a soluble complex during polymerization. It is noteworthy that HP-β-CD is environmentally friendly. It was established that CDs labelled with TEMPO derivatives, i.e. spin labelled CDs 3234 undergo interaction with PEG 600 (PEG = polyethylene glycol) and with PPG 425 ( PPG = polypropylene glycol) in concentrated aqueous solutions. The EPR spectra of 32-34 are changed when they are complexed with PEG or PPG. It was observed that the relationship between rotational correlation times (τ) and solvent viscosity cannot be described by the Debye-Stokes-Einstein equation, this fact being due to self-aggregation of alkylene glycols in concentrated solutions. However the use of the fractional Debye-Stokes-Einstein equation, i.e. the relationship between relative τ values and relative viscosity is in accordance with the experimental data [77]. In the study of the interaction of 32-34 with PEG and PPG it was established that the complexation of 32-34 causes the decrease of αN values with higher concentration of PEG; this behavior results from the reduced hydrophilicity of the concentrated PEG solutions ( αN = hyperfine splitting). The same behavior was observed for complexation with PPG.
Use of Cyclodextrin Polymers in Separation of Organic Species
839
It was found that the mobility of 32-34 is different. The longest rotational correlation time (τ) was shown by 34, this is because the linker between the CD and nitroxide moieties in 34 is rigid, and therefore the spin probe reports on the movement of the whole complex 34/PEG. In 32 and 33, however the linker is more flexible, and therefore the τ values are strongly influenced by the local motion and are lower than in the case of 34. It was established that 32-34 form in aqueous solutions the self-inclusion complexes, whereas in PEG/PPG solutions the self-inclusion equilibrium is perturbed by host-guest interaction with polymer molecules. Moreover, the aggregation of PEG/PPG chains occurs, therefore the environment around the spin probes is less viscous as compared to the average viscosity in the bulk solution. The interaction of 32-34 with adamantane-functionalized dendrimers was also investigated. It is known that EPR parameters of 32-34 do not change considerably upon interaction with small molecules, therefore, in order to increase the sensitivity of EPR parameters to the host-guest interaction of 32-34, their complexation with adamantanefunctionalized poly(propylene imine) dendrimers has been performed. The first, second and third generation dendrimers, i.e. D 8, 16, 64 functionalized with adamantane groups were studied. It was observed that the strength of binding increases with the dendrimer generation; for the third generation, i.e. D 64 dendrimer, at high concentrations the formation of supramolecular aggregates occurs. The dendrimer molecules are large, this fact results in the increase of the τ values of 3234 upon complexation. The EPR spectra of 32 in the presence of each dendrimer were measured. For all dendrimers the increase of the τ values was observed, the most significant changes occurring for D16. Dendrimers D8 and D64 cause smaller, similar with each other, changes in τ values, this fact being due to their considerable flexibility. The τ values can serve for calculation of binding constants for complexation of 32 with adamantane functionalized dendrimers.
7. IMPRINTED CD POLYMERS Molecular imprinting is today intensively studied [78-80]. Molecularly imprinted polymers are matrices which mimic biological receptor systems, able to bind target molecules with affinity similar to that of antibodies and enzymes. This property of molecularly imprinted polymers was introduced during synthesis stage, when functional monomer was copolymerized with a crosslinker in the presence of a target analyte, i.e. the imprint molecule. The polymer forms binding pockets around the template molecule. After polymerization, upon the removal of the template, the binding sites, complementary in size an shape to those of the analyte, are revealed. Therefore, the polymer possesses the introduced memory, and is capable to selectively rebind the analyte [81]. It is known that molecularly imprinted β-CD polymer may be used as optical receptor for detection of organic compounds. The molecularly imprinted β-CD polymer was prepared from β-CD, using TDI as a crosslinking agent; in this procedure N-phenyl-1-naphthylamine (35) served as a template. The molecularly inprinted polymer was fluorometrically characterized using a fibre optic cable attached to a flow-cell. The above sensor was investigated for analytical determination of 35 [82].
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Cezary A. Kozlowski and Wanda Sliwa
It was found that the molecular imprinting afforded a better sensing signal by increasing the binding affinity and substrate selectivity toward template molecule than the control polymer which was prepared in the absence of template. It should be mentioned that the sensing receptor can be regenerated with acetonitrile and can be reused without any decrease in its intensity. The multiple surface plasmon resonance (SPR) sensor was applied for detection and quantification of dextromethorphan (36) with the use of molecularly imprinted β-CD polymer [81]. The cough suppressant 36 is a marker drug used to identify the activity of the CYP2D6 class of p450 monooxygenases. It was found that the sensor may be used to measure the consumption of 36 by CYP enzymes. Thin film of molecularly imprinted polymer can be prepared on a SPR chip, and the specific binding activity can be monitored as a change in angle for light reflected from the chip. However the preparation of molecularly imprinted polymer on the chip requires often a careful control of polymer composition. The most common method involves the free radical polymerization with the use of a photo-initiator, and this process can lead to changes of template structure; in the case of proteins their denaturation occurs. Other preparation procedures of molecularly imprinted polymers for SPR are atom transfer radical polymerization and polymer grafting method; these methods provide a better control of the film physical characteristics, but require a suitable catalyst for polymerization.
HN
35
Me
N H O Me dextromethorphan 36
To overcome these inconveniences the β-CD polymers were used. The β-CD polymers are advantageous for preparation of molecularly imprinted polymers due to easy polymerization process using simple neutral molecules such as EP or TDI as crosslinkers. In this procedure, β-CD itself can serve as a monomer to interact with template molecules. The polymerization process is fast and easy, and no initiator is necessary. Detection and quantification of 36 was performed by measuring the refractive index changes of multiple surface plasmons which result from the binding to template pockets within the thin layer of 36-imprinted β-CD polymer. Changes of refractive indexes alter the
Use of Cyclodextrin Polymers in Separation of Organic Species
841
angles at which surface plasmons are generated. The shift in SPR angles upon 36 binding to a molecularly imprinted polymer is measured by multiple surface plasmons. The preparation of 36-imprinted β-CD polymer begins with condensation of β-CD units with TDI in dimethylformamide. After initiation of polymerization, 36 is added; 36 forms inclusion complexes with β-CD units prior to completing the process. The obtained 36imprinted β-CD polymer is pipetted onto a rotating gold-coated glass slide to form a thin layer. The removal of template 36 from the film is achieved by washing with water. The same procedure, in the absence of 36, is used for preparation of the reference β-CD polymer devoid of 36. It was established that SPR technique provides the enhancement of sensitivity and the lower detection limit than conventional reflectance measurements. Molecular imprinting technique allows interaction of 36 with β-CD units during the polymerization stage; this behavior creates a specific arrangement of the β-CD units which can accomodate 36 molecules. In the reference polymer, however, 36 is absent during the synthesis, therefore the arrangement of β-CD molecules such as that in the imprinted β-CD polymer does not exist; the β-CD molecules are distributed randomly within the matrix, allowing the cavity blockage by each other which hinders the binding of 36 after the formation of polymer matrix. It was observed that the coupling of the specifity of a thin layer of 36-imprinted β-CD polymer film with the sensitivity of SPR method allows the rapid detection of 36. It should be pointed out that the above method is promising for monitoring drug–drug interactions. Preparation of artificial receptors which bind peptides in water is intensively studied in view of their potential use in separation and purification of bioproducts and in biosensing. Many works concern this theme [83, 84], however only few of them deal with recognition of structures of peptides in solution, and it should be pointed out that these solution structures govern biological functions and supramolecular assembling of peptides. It was found that the molecularly imprinted CD polymers may be used for the strict recognition of solution structures of oligopeptides. For this purpose the vinyl monomer of βCD was polymerized in water in the presence of oligopeptide template, and several β-CD molecules were immobilized complementarily to apolar and bulky groups in the template. Angiotensin II (AII) and its precursor angiotensin I (AI) were used as oligopeptide templates. Angiotensin II is an octapeptide hormone for blood pressure regulation, its precursor angiotensin I is decapeptide, which is converted to AII by enzymatic removal of its C-terminal His-Leu fragment. AI: AII: [Val5]-AI: [Val5]-AII:
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu Asp-Arg-Val-Tyr-Ile-His-Pro-Phe Asp-Arg-Val-Tyr-Val-His-Pro-Phe-His-Leu Asp-Arg-Val-Tyr-Val-His-Pro-Phe
Although AI and AII differ only by His-Leu fragment, the imprinted β-CD polymers are able to distinguish them in water and separate them from each other. The imprinted polymers can also recognize AI from [Val5]-AI differing from AI only by one amino acid (isoleucine of AI was replaced in [Val5]-AI by valine), and can recognize AII from [Val5]-AII where the same replacement (leucine into valine) was made [85].
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The molecular imprinting of β-CD was performed on the surface of silica gel. Acrylamido groups were introduced to the surface of silica gel, and in the presence of this modified silica gel mono-6-(N-acrylamido)-6-deoxy-β-CD and N,N’-methylenebis (acrylamide) were polymerized using radical initiator. It may be concluded that imprinted CD polymers successfully memorize solution structures of oligopeptides in water. Imprinted CD polymers are of use e.g. for separation and purification of target peptide hormone from complex mixtures, since the difference in only one amino acid can be recognized. The attachment of proteins to surfaces is of a great importance in biotechnological processes. In these applications the control of the adsorption strength, the protein orientation and retention of biological function is required. Such control can be made only when the binding of protein to the surface is specific. There are different methods to prevent the nonspecific adsorption of proteins onto surfaces, for example by adding surfactants or bovine serum albumin (BSA) to protein solutions, or by the widely applied method using self-assembled monolayers (SAMs) that are „protein-resistant”, such as SAMs with oligo(ethylene glycol), i.e. OEG chains. These SAMs prevent nonspecific interactions due to their loose packing and the well-hydrated nature. Such molecularly imprinted β-CD polymers, i.e. the β-CD molecular printboards were used as a platform for the immobilization of proteins through small multivalent orthogonal linker molecules. The modification of β-CD with seven thioether chains affording 37 was made to obtain ordered and densely packed SAMs on gold. The existence of multivalent host-guest interactions enables the formation of kinetically stable assemblies, and the complex formation by patterning, therefore such surfaces may be considered as „molecular printboards”. The monovalent blocking agent 38 was used to form a dynamic supramolecularly controlled OEG layer on β-CD molecular printboards. This process prevents nonspecific protein adsorption. Moreover it allows replacement by multivalent linker molecules, since multivalent interactions are stronger than monovalent ones. The above method was used for two proteins, namely for the typical test protein streptavidin 39a and for the bioengineered histidine-tagged, maltose binding protein (39b), i.e. His6MBP [86]. O R= HN
OR 7 37 O
O
OH
S 11
Ad 38
5
39a 39b
Me 11
Use of Cyclodextrin Polymers in Separation of Organic Species S
H N
NH HN
O O
O
O COOH
O O
843
O
3
O
N H
3 O Ad Ad
N
COOH Ad
HOOC 41
40
In order to achieve specific binding, the blocking agent 38 was used; it bears a single adamantyl group for specific and reversible interaction with β-CD SAMs and bears a hexa(ethylene glycol) unit for preventing nonspecific protein adsorption. The attachment to β-CD SAMs of 39a proceeds through the divalent linker 40, while the attachment of 39b proceeds through the Ni2+ complexed nitrilotriacetic acid–adamantyl linker 41. The analysis of the SPR (surface plasmon resonance) sensograms for the binding of 39a,b and BSA in the absence and in the presence of 38 indicates that in the absence of 38 the considerable nonspecific adsorption occurs, whereas the presence of only low concentration of 38 is sufficient to suppress the nonspecific interactions; in this case more than 80% of all β-CD sites are occupied by 38. The nonspecific adsorption is inhibited due to the binding of 38 to the surface; in this way the temporarily blocking the β-CD cavities and exposing the hexa(ethylene glycol) tails to the solution occurs. In order to prove whether the application of 38 allows the specific attachment of proteins to β-CD SAMs through orthogonal linkers, the binding of 39a through the linker 40 and the binding of 39b through the Ni2+ complexed linker 41 was studied. Since 39a is a homotetrameric protein with four identical biotin-binding sites, it can bind four equivalents of 40. It was observed that only two of the linkers, i.e. four adamantyl moieties bind four neighboring β-CD cavities of the β-CD SAM. Therefore it may be expected that the multivalency effect will make the binding of the complex 39a•(40)4 to the β-CD SAM much stronger than the binding of 38. In the case of 39b, the protein was premixed with a solution of Ni2+ ions, 41 and 38, and this solution was flowed over the β-CD SAMs already covered with 38. It was found that the specific binding of 39b and 41 occurs in the presence of the monovalent blocking agent 38. Thus, the new supramolecular blocking agent 38 inhibits nonspecific protein adsorption on β-CD molecular printboards. The linker 38 has similar binding constants to β-CD in solution and at the surface, and only a 0.1mM concentration of 38 is sufficient to inhibit nonspecific protein adsorption.
844
Cezary A. Kozlowski and Wanda Sliwa Ad
37
Ad
39a
Ad
-
38
Ad Ad
Ad
Ad Ad
Ad
Ad
40 Ad
38 Ad
Ad
Ad
Ad
Ad
Ad
39b
-
38
41
Ni2+ Ad
Ad
Ad
Ad
Ad
Moreover, the possibility to exchange 38 onto proteins was found; in this way the proteins can be attached to the surface by specific binding. This was shown for two proteins 39a and 39b, which are bound through differently functionalized linkers to β-CD SAMs; in this way the versatility of this method was shown. The above inhibition of nonspecific protein adsorption demonstrates that the use of molecular printboards as a general platform for the immobilization of proteins is very promising. An attention should be paid also to immobilization of Candida rugosa lipase 42 by CD polymers serving as a support. It was established that the immobilized lipase showed advantageous properties as compared to free enzyme. The immobilization of enzymes on solid supports increases their thermal stability and recoverability [87]. The covalent immobilization of enzymes usually begins with a surface modification or activation step. The immobilization of enzymes carried out by bifunctional species proceeds by formation of inter- and intramolecular crosslinkages between the enzyme molecules. Isocyanate derivatives and glutaraldehyde (GA) are used as crosslinkers for the immobilization of enzymes; in this reaction the amino groups of a protein form Schiff bases with glutaraldehyde [88, 89]. In experiments HMDI and GA served as activating agents for immobilization of lipase 42. To prepare β-CD polymers, the solution of β-CD in DMF was treated with HMDI in DMF and the resulting precipitate was filtered off; the obtained activated polymer is the support 43. The solution of 43 in acetonitrile was reacted with ethylenediamine in acetonitrile to give 44, which upon treatment with GA afforded the activated polymer which is the support 45 . For immobilization, the support 43 or support 45 was suspended by dissolving lipase powder in buffer solution, and incubated at 30 0C. The immobilized enzyme was lyophilized. The high thermal stability of β-CD polymer is due to its crosslinking; it is known that crosslinking results in increased thermal stability.
Use of Cyclodextrin Polymers in Separation of Organic Species
845
H 2N
42 O
method A
H N
O
N H
O
46 β
O C N
H N
N C O
HMDI O N O
OH 7
N H
C
O
support 43
H2N NH2
method B O
H N
O
N H
NH 2
O
GA
O
O
44
O
H N
O
H N
H N
N H
N
O
O
support 45 H2N
42 O O
H N N H
H N N
N
O
47
According to the method A, the support 43 reacted with lipase to give the immobilized lipase 46, while according to the method B, the support 45 reacted with lipase to give the immobilized lipase 47 [90]. The specific activity of 47, i.e. lipase immobilized by the support 45 is higher than that of 46, i.e. lipase immobilized by the support 43. This result is in accordance with literature data stating that GA is a convenient activating agent for immobilization of enzymes. It is known that pH is an important parameter, altering enzymatic activity in aqueous medium. Immobilization of enzyme may cause its conformational changes, resulting in variation of optimum pH. However, it was observed that the maximum activity is shown at pH 7 so by free as by immobilized by support 43 lipase, i.e. 46, indicating that no conformational changes occured during covalent binding. The free lipase is stable in the pH range from 6 to 7 while the immobilized lipase are stable in the pH range from 7 to 9, this fact indicates that the immobilization improves the stability of lipase in the alkaline region. The optimum temperature of the activity for the free enzyme is ca 350C, while for immobilized lipase so by support 43 or by support 45, it was 400C and 450C, respectively. Thus, the immobilized enzymes showed their catalytic activities at higher temperature. It should be pointed out that at higher temperature the free lipase can undergo denaturation, whereas the immobilized lipase can retain its catalytic activity. The above investigation has shown that the immobilization of lipase by covalent binding on β-CD-based, chemically modified polymer afforded immobilized lipases showing different properties than free enzyme. It is noteworthy that the immobilization procedure is simple and easy to carry out.
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Cezary A. Kozlowski and Wanda Sliwa
8. MEMBRANES BASED ON CD POLYMERS Phenols and their derivatives, as well as hydroquinone are used in industry for the synthesis of dyes, plastics, insecticides and pesticides; most of phenols are toxic, they are pollutants of water, and are harmful for various biological processes. Among a great number of methods for removal of phenols, adsorption is the most convenient one; many adsorbents, e.g. activated carbon [91] and modified bentonites [92] are known. Today the use of natural and chlorinated adsorbents for wastewater treatment is increasing due to their abundance and low cost. Adsorption of hydroquinone with the use of crosslinked chitosan / β-CD membranes was studied. Chitosan (CS), a derivative of the natural polymer chitin, is a nontoxic, biodegradable copolymer of glucosamine and N-acetylglucosamine. CS is used in treatment of wastewater, and as an ion exchanger [93]. CS, in the form of a soluble polymer dissolved in organic acid, is useful in construction of membranes, applied in food and pharmaceutic industries, as well as in biochemistry, medicine and agriculture [94,95]; one should point out its desirable membrane-forming ability, good hydrophilicity and biocompatibility [96]. The immobilization of CDs in crosslinked chitosan is promising, since the obtained polymers have physical properties along with inclusion and adsorption behaviors different so from CDs as from chitosan itself [97, 98]. Numerous crosslinking agents have been used to graft CDs to chitosan, e.g. EP, HMDI and 3-(3-dimethylaminopropyl)carbodiimide. In order to obtain β-CD grafted CS, at first β-CD polymer 48 was prepared from β-CD and EP, and then CS was reacted with trimethoxysilane 49 serving as a crosslinking agent. The obtained crosslinked CS 50a upon hydrolysis afforded 50b, which by treatment with βCD polymer 48 yielded β-CD-grafted chitosan 51. (OH)14
Cl O
2
O
O OH
OH
β−CD polymer 48
7
OH
NH2 HO O
O
Ac NH
O
O HO
HO
OH
4
O
O OH
NH 2
chitosan
NH2
O
O
Si(OMe)3
49
NH2
NH 4
R a OMe 50 b OH
OH
NH 4 Si
OH 50b
OH OH
SiR3
OH H2O/H+
48
O
NH β-CD grafted chitosan 51
4 Si
OH
O O
Use of Cyclodextrin Polymers in Separation of Organic Species
847
Chitosan/β-CD porous membranes were prepared using the following solvent casting procedure. The chitosan powder was dissolved in 2.0% (v/v) citric acid. The obtained solution of chitosan was treated with 49, and upon stirring the CD polymer 48 was added. The solution was cast into a membrane on a plastic dish and dried. The citric acid was removed by treatment with NaOH solution, and the NaOH excess was removed by soaking in water. Then the membrane was dried. It was established that the molar ratio of the epoxy groups of 49 to the free amino groups of chitosan, along with the evaporation time of solvent strongly influence the pore size and the water content of membranes. When the molar ratio of epoxy groups of 49 to the free amino groups of chitosan increases, the pore size and the water content of the membrane are lower. A longer time of evaporation of solvent results in larger pore size and lower water content in the membrane. Adsorption of hydroquinone was investigated using batch procedure, by shaking the hydroquinone solution with the membrane in a flask. It was found that the high initial concentration of hydroquinone enhances the adsorption capacity while the high temperature causes the increase of the initial adsorption rate along with the increase of the adsorption capacity.The adsorption is endothermic and has a character of chemisorption. The results of kinetic studies indicate that the adsorption is a pseudo-second order process [99]. The enantiomer separation with the use of membranes is a promising technique, more convenient than traditional methods, due to its high processing capacity, continuous operation mode and low energy consumption allowing its use in a large scale enantiomer separation processes. Supported liquid membranes show high chiral selectivity, however they are not stable; on the contrary, solid membranes with immobilized chiral carrier polymer are stable and therefore able to a durable enantiomer separation [100]. The enantiomer separation by solid membranes may be classified into diffusion selective and sorption selective types. In the former type, the membrane without a specific chiral selector, but consisiting of chiral derived polymer is used, whereas in the latter one the membrane has the immobilized chiral selectors. With the use of diffusion selective membranes, the enantiomer separation is achieved through chiral environment within membrane, while with the sorption selective membranes the enantiomer separation proceeds by interaction of enantiomer with chiral selectors immobilized on the membrane. It was established that chitosan is diffusion selective, and CD is sorption selective in membrane-mediated resolution processes, therefore the combination of these two chiral separation types is promising. Having this in mind, the chitosan/β-CD composite membranes of a semi-interpenetrating network were studied in the enantiomer separation of tryptophan (Trp) racemate, and the influence of CD content on the permselectivity and permeate flux of the CS/CD composite membrane was discussed [98]. The β-CD polymer was prepared by crosslinking β-CD with EP; for this purpose β-CD dissolved in NaOH solution was treated with EP. Then the EP excess was removed by acetone, and NaOH was removed by rinsing with water. In order to prepare CS/CD composite membranes, the solution of CS in aqueous acetic acid was treated with β-CD polymer, and glutaraldehyde (GA) was added as a crosslinker of CS. The Nylon-6 membrane substrate was immersed into the obtained suspension, then the membrane was taken out and spread on a glass plate. The membrane was dried to achieve the formation of CS/CD semi-
848
Cezary A. Kozlowski and Wanda Sliwa
interpenetrating network layer on the Nylon-6 substrate, washed with NaOH solution and water, and dried. Membranes were immersed in aqueous Trp racemate solutions. It was found that both βCD polymer and CS show selectivity toward L-Trp, the amount of adsorbed L-Trp was always larger than that of D-Trp. The CS shows higher selectivity than β-CD polymer since CS has the chiral environment effective in distinguishing between Trp enantiomers, whereas β-CD polymer is less selective due to larger cavity of β-CD compared with the size of Trp molecule. It was observed that the sorption ability of membranes increases with the β-CD content, due to higher affinity of β-CD polymer toward Trp, compared with the affinity of CS toward Trp. It was found that the increase of β-CD polymer content in the CS/CD composite membrane results in a significant decrease of its permselectivity, however, the permeate flux is enhanced. The above studied solution-diffusion process consists of three steps. In the first step the Trp enantiomers are selectively adsorbed on the membrane interface in the feed cell; in the second step both enantiomers diffuse through membrane with different diffusion rates; and in the third step the desorption of Trp enantiomers occurs on the membrane interface in receiving cell. The desorption rate is very high compared with diffusion rate, therefore the first two steps are decisive. It was found that in the separation of Trp racemate the diffusion selectivity is predominant over the sorption selectivity.
9. SELECTED SUPRAMOLECULAR CD POLYMERS In the study of CDs containing PEG, i.e. poly(ethylene glycol) chain, terminated by azobenzene unit, the thermal and photochemical conformational changes of trans-52 were analyzed in aqueous solution [101]. The results of the thermal study show that at low concentration trans-52 exists at 800C as a dethreading form 53. At 60oC trans-52 exists as a self-inclusion complex 54 where the CD cavity entraps the azobenzene moiety; and at 1oC the self-inclusion complex 55 where CD cavity encircles the carboxylic acid unit is present. It was observed that at high concentration trans-52 forms intermolecular complexes 56. The results of the photochemical study indicate that the irradiation of trans-52 or of its intermolecular complexes 56 with UV light leads to cis-52 in the form of a self-inclusion complex 57 in which the CD cavity encloses the azobenzene moiety, regardless to concentration. In the search for polymers for construction of intelligent materials [102, 103], it was observed that linear poly(ethyleneimine) PEI forms inclusion complexes with α-CD and with γ-CD in two ways: α-CD includes single strands of PEI, whereas γ-CD can include double strands of PEI; these latter inclusion complexes of γ-CD have been investigated [104]. In experiments the double strands of the PEG-PEI diblock-copolymer chain, grafted on the water soluble polymer dextran, i.e. PEG-PEI-dex were encircled by γ-CD to give supramolecular network, named double-axle intrusion complex. This network can modulate rheological properties in response to pH changes. The linear bifunctional PEG-PEI diblock copolymer was obtained via a cationic ring opening polymerization of 2-oxazoline, using PEG monotosylate as a macroinitiator.
Use of Cyclodextrin Polymers in Separation of Organic Species O O N
H N
O
N H
N
849
n
O
O
trans - 52
β
O
N
80 0C
N
dethreading form 53
high concentration 60 0C
intermolecular complexes 56
N N
low concentration
self-inclusion complex 54
Vis
UV
N
1 0C
UV
N N
N
Vis cis - 52 self-inclusion complex 57
self-inclusion complex 55
O
O O H
O OH HO
O
HO
m
HO
HO
N H
O
N H
H N
y
NH
n PEG-PEI-dex H N
H N
O
x H N
O
OH
H N
N H N H
H N H N
PEI-α-CD PEI-γ-CD
The double strands of the PEG-PEI chains grafted on dextran backbones intrude into the cavity of γ-CD in the same or in the opposite directions forming supramolecular networks. It was observed that the addition of γ-CD in only a low concentration (3% wt) to a PEG-PEIdex solution at pH 10 causes a considerable increase in the viscoelastic properties as compared to solution without γ-CD. The viscoelastic properties of PEG-PEI-dex-γ-CD change with pH, namely at pH 10 the full double-axle intrusion complex along the grafted chains exists, whereas at pH 4 the solution-like behavior is stronger, i.e. the network has a
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Cezary A. Kozlowski and Wanda Sliwa
looser structure or is partially dissociated due to the dethreading of γ-CDs. The γ-CD molecules slide out of the PEI chains since the protonation of secondary amine groups occurs. The double-axle intrusion complex of PEG-PEI chains with γ-CD is responsive to pH changes because the PEI chain is neutral at pH 10 and is totally protonated at pH 4. The following mechanism which leads to structure existing at pH 4 was proposed. Upon lowering solution pH from 10 to 4, the PEI segment repels the threaded γ-CDs, which move off the protonated PEI segment toward the neutral PEG segment, and other γ-CD molecules take the position on double strands of the PEG segments. Therefore, the double-axle intrusion complex is transformed into a longer structure, resulting in the formation of a looser network. It may be concluded that the rheological changes are due to molecular mechanical actuations of the double-axle intrusion system in supramolecular networks. Possible applications of these systems, such as muscle structures and stimuli-sensitive hydrogels may be modulated by several factors, e.g. by block length of the grafted chains or by degree of grafting [104]. One should point out that investigation concerning polyrotaxanes and polycatenanes containing CDs, especially former ones, is developing rapidly due to their interesting properties [105, 106]; among a great number of such species the following examples are shown below. In order to obtain polyrotaxanes with many α-CD moieties threaded on a PEG chain, the reaction of pseudorotaxane (58) with 2,4-dinitrofluorobenzene units serving as stoppers was performed to give polyrotaxane (59).
H 2N
NH2 n F
58
NO 2
NO2 NO2
O2N H N
H N
O2N
= α-CD
NO2
n
59
= PEG poly(ethylene glycol)
O H N
N H
60
n
hν (Vis) O
H N
N H
n H N
= β-CD = PPG poly(propylene glycol)
61
O
H N
n
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It was established that irradiation with visible light of the complex of β-CD with PPG (60) results in the photodimerization of 2-anthryl groups, affording β-CD polyrotaxane (61). Applications of supramolecular complexes of CDs with various polymers attract today a growing attention. These species are interesting for study of hydrogel formation as well as sol-sol and sol-gel transitions; they are also promising materials for construction of sensors and biological artificial systems.
CONCLUSION The above presented works concerning CD polymers provide some information on their synthetic procedures and applications. It is known that the understanding of biological processes is a challenge for researchers; in this field an increasing attention is paid today to investigation of CD polymers [107-111]. One should point out the important role of imprinted CD polymers which may serve for immobilization of proteins [112] and for recognition of structures of oligopeptides [85]. In recent years a growing amount of works concerning membranes based on CD polymers appears as a reflection of their usefulness in various separation processes [113-116], in the transport of organic species [113, 114] and metal ions [14-17, 115]. Today a rapid development of works dealing with supramolecular systems based on CD polymers [117-120] giving rise to fascinating structures is observed; they may serve as models for molecular recognition and are able to form hydrogels promising in the field of biotechnology. It should be pointed out that supramolecular structures, especially polyrotaxanes are promising for design of biological artificial systems and molecular machines [106].
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 30
PROPERTIES, APPLICATIONS AND CURRENT DEVELOPMENT OF EDIBLE POLYSACCHARIDE FILMS AND COATINGS Pau Talens1*, María José Fabra2 and Amparo Chiralt2 1
Grupo de Investigación e Innovación Alimentaria. Departamento de Tecnología de Alimentos. Universidad Politécnica de Valencia. Camino de Vera, s/n 46022. Valencia. Spain. 2 Instituto de Ingeniería de Alimentos para el Desarrollo. Universidad Politécnica de Valencia. Camino de Vera, s/n 46022. Valencia. Spain.
ABSTRACT Moisture, oxygen, carbon dioxide, lipid, flavor and/or aroma transfer between food components or between foods and their surrounding environment can provoke deterioration of food texture, flavour, color, aroma or nutritional values which results in food quality loss. Regulating the mass transfer in food systems by edible films and coatings can increase food-product shelf life and food quality. Besides their barrier properties, edible films and coatings can act as carriers for functional food additives, antioxidants, antimicrobial agents and nutrients; and due to their biodegradability nature, could have an impact on overall packaging requirements. Edible films and coatings are produced from edible biopolymers and food-grade additives. Film-forming biopolymers can be proteins, polysaccharides (carbohydrates and gums) or lipids. Plasticizers and other additives are combined with the film-forming biopolymers to modify the physical properties or functionality of films. The composition of the film must be chosen according to specific food applications, the type of food products and the major mechanisms of quality deterioration. Polysaccharide films and coatings are used to extend the shelf life of fruits, vegetables, seafood, meats and confectionary products by preventing dehydration, oxidation rancidity, surface browning and oil diffusion; and in some specific cases can improve the physicochemical, nutritional and sensorial properties of the products. The *
Corresponding author: Tel.: +34 963879836, Fax: +34 963877369, E-mail address:
[email protected] 858
Pau Talens, María José Fabra and Amparo Chiralt common polysaccharides used for edible films are: starches and their derivatives; cellulose and its derivatives; seaweed extracts; gums; pectins and chitosan. The objectives of this chapter are to (a) review research on polysaccharide filmformation and characteristics, (b) analyze mechanical and barrier properties (water vapour permeability, gas permeabilities and volatile permeability) of polysaccharidebased films, (c) summarize applications of polysaccharide films in food products, and (d) make conclusions as to the status of polysaccharide films and their future developmental direction.
INTRODUCTION In the last recent years, impressive advances have been made in the production of synthetic polymer films designed to protect foods. Nowadays, food companies and researchers are looking to edible films and coatings to add value to food products, increase shelf-life and/or reduce packaging. Edible films can be used for: coating fresh whole and pre-cut fruits and vegetables to reduce moisture loss, respiration and color change; coating frozen foods to prevent oxidation, as well as prevent moisture, aroma or color migration; coating nuts to prevent oil migration into surrounding food components; coating fragile foods such as breakfast cereals and freezedried foods to improve integrity and reduce loss due to damage; or for coating candies, cookies and/or nuts in ice cream to provide a moisture barrier and keep inclusions crisper. Edible films can also stabilize water activity gradients and preserve different textural properties possessed by different food components. For example, an edible film could be used to separate the crisp component of a pizza from the moist semi-solid component. The protective function of edible films and coatings may be enhanced with addition of antioxidants or antimicrobials to the films or coating. Depending on the nature of the food, food additives, such as, flavors, nutrients or colors can be incorporated into edible films and used to control location or rate of release of these additives in a food. A specific film-coating composition is selected as a function of the desired application for the edible food film to be produced. For example, when the purpose is to provide an individual barrier protection in opposition to moisture and oxygen in fresh fish, cheese, meat products or intermediate moisture foods, a film with low water and oxygen permeabilities is required; whereas, when the purpose is to control the moisture balance within heterogeneous foods such as pizzas, sandwiches or cakes, hydrophobic materials are required to make a film with good water barrier properties. Materials available for forming edible films and coatings fall generally into the categories of lipids, proteins or polysaccharides. The functional properties of edible films and coatings are greatly influenced by the physical and chemical characteristics of the materials used (Sothornvit and Krochta, 2000). Biopolymers can be used independently or in combinations. Films prepared by polysaccharides or proteins generally have excellent oxygen, carbon dioxide, aroma and lipid barrier properties; particularly at low relative humidity levels. They have desirable mechanical properties, making them constructive for improving the structural integrity of fragile products; but inaptly, their predominantly hydrophilic character results in them having poor water barrier characteristics. Due to their hydrophobicity, lipid compounds have been used as protective barrier layers to prevent moisture exchange between the food product and the surrounding medium, or between adjacent components within heterogeneous
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foods. Proteins and polysaccharides can be combined with lipids, as emulsion particles or multi-layered coatings in order to increase the resistance to water penetration (Pérez-Gago & Krochta, 2001; Morillon et al., 2002, Karbowiak, Debeaufort & Voilley, 2007, Hambleton et al., 2008) The use of polysaccharides as coating materials for food protection has long been recognized and grown extensively in recent years (Cuq et al., 1994, Nisperos-Carriedo, 1994, Phan The et al., 2002, Lacroix & Le Tien, 2005, Phan et al., 2009a,b). Polysaccharides filmforming materials include starches and their derivatives, cellulose and its derivatives, seaweed extracts, gums, pectins and chitosan. The use of polysaccharides presents advantages due to their availability, low cost and biodegradability. Furthermore, polysaccharides can be easily modified in order to improve their physicochemical properties. The sequence of polysaccharides is simple compared to proteins, which have 20 common amino acids. However, the conformation of polysaccharide structures is more complicated and unpredictable, resulting in much longer molecular weights than proteins. Most carbohydrates are neutral, while some gums are mostly negatively charged. Although this electrostatic neutrality of carbohydrates may not significantly affect the properties of formed films and coatings, the occurrence of relatively large numbers of hydroxyl groups or other hydrophilic molecules in the structure indicate that hydrogen bonds may play significant roles in film formation and characteristics. Some negatively charged gums, such as alginate, pectin and carboxymethylcellulose, show significantly different rheological properties in acidic than in neutral or alkaline conditions (Han & Gennadios, 2005)
POLYSACCHARIDES USED FOR EDIBLE FILMS AND COATINGS Polysaccharides are obtained from a variety of sources. Common polysaccharides used for edible films and coatings (Table I.1) include starches and their derivatives, cellulose and its derivatives, seaweed extracts, gums, pectins and chitosan.
Starches and Derivatives Starch is one of the most abundant natural polysaccharide, principally derived from tubers or cereals, consisting of a large number of glucose monosaccharide units joined together by glycosidic bonds. It is constituted by two types of molecules: the linear amylase and the branched amylopectin. The content of amylase in starch varies from 0 to 100%, depending on the botanic origins. Most starches, such as those from wheat, corn and potato, contain 20 to 25 percent of amylose and 75 to 80 percent of amylopectin. However, for amylomaizes, the amylose content can be higher than 50 percent and for 'waxy' maize it can be less than 5 percent (Li & Yeh, 2001; Singh et al., 2003). In unmodified forms, starches have very limited use in the food industry, but modified starches by disruption of hydrogen bonding or by chemical substitution, have significantly been playing important roles in the food industry. They have been used to modify physical properties of food products such as soups, sauces, snacks, batters and meat products; contributing mainly to texture, viscosity, gel formation, adhesion, binding, moisture retention,
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product homogeneity and film formation (Liu, 2005). For applications where viscosity, stability and thickening strength are desired, starches with a high content of amylopectin are required; whereas, for film-forming purposes and for the preparation of strong gels, starches with a high content of amylase are required (Nisperos-Carriedo, 1994).
Cellulose and Derivatives Cellulose, a structural component of the primary cell wall of green plants, many forms of algar and the oomycetes, is a polysaccharide consisting of a linear chain of several hundred to over ten thousand β(1→4) linked D-glucose units. Chemically, cellulose can be broken down into smaller polysaccharides called cellodextrins, or completely into glucose units, by treating it with concentrated acids at high temperature; however, hydrolysis of cellulose is relatively difficult compared to the breakdown of other polysaccharides. Many properties of cellulose depend on its degree of polymerization, or chain length; the numbers of glucose units that make up one polymer molecule (i.e.; cellodextrins), in contrast to long-chain cellulose, are typically soluble in water and organic solvents. Due to the high level of intramolecular hydrogen bonding, cellulose is insoluble in water and most organic solvents, but is odorless, biodegradable and has no taste. Compared to starch, cellulose is also much more crystalline. Whereas, starch undergoes a crystalline to amorphous transition when heated beyond 60-70 °C in water (as in cooking), cellulose requires a temperature of 320 °C and pressure of 25 MPa to become amorphous in water. Cellulose is the most abundantly occurring natural polymer on earth and is a cheap raw material. For film production, cellulose is dissolved in an aggressively toxic mixture of sodium hydroxide and carbon disulfide and then recast into sulfuric acid to produce cellophane films (Petersen et al., 1999). However, the usefulness of cellulose as a starting material for edible films and coatings can be extended by chemical modification to cellulose derivatives. The hydroxyl groups of cellulose can be partially or fully reacted with various reagents to afford derivatives with useful properties. Cellulose esters and cellulose ethers are the most important commercial materials. Generally, ether derivatives, like Methylcellulose (MC), Hydroxypropylcellulose (HPC), Hydroxypropylmethylcellulose (HPMC), Carboxymethy-lcellulose (CMC) or microcrystalline cellulose (MCC) have excellent filmforming properties and are used as raw material for edible films and coatings. Under controlled temperatures and pressures, alkali cellulose is allowed to react with methyl chloride to form MC; with propylene oxide to form HPC; with methyl chloride and propylene oxide to form HPMC and with sodium monochloroacetate to form CMC. MCC is formed by controlled acid hydrolysis of native cellulose. The level quantity of methoxyl, hydroxypropyl or carboxymethyl substitution affects the physical and chemical properties of the material. The number of substituted hydroxyl groups per monomeric units is known as the degree of substitution (DS). MC, HPC and HPMC, nonionic cellulose ethers, are commercially available in powder or granular form, and in varying molecular weights and DS. They are insoluble in hot water but are soluble in cold water and organic solvents (solubilitacion of MC in organic solvents depends of the degree of substitution, under 2.6 DS is partially soluble and upper 2.6 DS is complete soluble).
Table I.1. Common polysaccharides used for edible films and coatings. Structure, formula, properties and films characteristics. Polysaccharide
Structure & Formula
Polysaccharide Properties
(C6H10O5)n
Odorless, no taste. Modify physical properties of food products, contributing mainly to texture, viscosity, gel formation, adhesion, binding, moisture retention, product homogeneity and film formation
Starches
Cellulose (C6H1202)n Methyl cellulose
Hydroxypropyl cellulose
hydroxypropyl methylcellulose
Film characteristics Odorless, tasteless, and colorless. Low permeability to oxygen. Low cost of production. Physical characteristics, chemical resistance and mechanical properties similar to those of plastic films.
Insoluble in water and most organic solvents, odourless, no taste, biodegradable.
Transparent, flexible, odorless, tasteless, water-soluble, and resistant to oils and fats
Insoluble in hot water and soluble in cold water and organic solvents. The solubilization of MC in organic solvents depends of the degree of substitution.
No good moisture barriers. Excellent barrier against migration of fats and oils. It can be used to reduce oil absorption in fried products. Thermoplastic and capable of injection molding and extrusion. It can retard spoilage and moisture absorption in coated nuts and candies.
Insoluble in hot water and soluble in cold water and organic solvents.
Insoluble in hot water and soluble in cold water and organic solvents.
Effective film that can reduce oil absorption in certain reformed products.
Table I.1. (Continued) Polysaccharide
Structure & Formula
Soluble in hot and cold water but insoluble in organic solvents. It basic function is to bind water or impart viscosity to the aqueous phase thereby stabilizing the other ingredients or preventing synerersis.
Carboxymethyl cellulose
Alginate
Polysaccharide Properties
Sodium alginate (C6H7NaO6)n
Thickening, stabilizing, suspending, film forming, gel producing and emulsion stabilizes properties.
Uniform, transparent and watersoluble films. Poor moisture barriers Good oxygen barriers. Can retard lipid oxidation in foods, and can improve flavor, texture and batter adhesion. The treatment of alginate films with divalent cation (i.e. calcium) solutions converts these into insoluble films.
Water soluble galactose polymers. Extensively used as gelling and stabilizing agents in food industries.
Uniform, transparent and water soluble films with good mechanical properties.
Calcium alginate (C6H7Ca1/2O6)n Carrageenan
Film characteristics Forms a complex in the presence of casein, increasing the coatings formulation viscosity. It retains the firmeness of fruits and vegetables, preserves important flavor components of some fresh commodities, reduces oxygen uptake without causing carbon dioxide increase in internal fruits and vegetables, and improves the puncture strength of films based on caseinate
Table I.1. (Continued) Polysaccharide
KapaCarregeenan
IotaCarregeenan
lamdaCarrageenan
Structure & Formula
Polysaccharide Properties
Soluble in hot water. Form strong, rigid gels, some syneresis. Insoluble in most organic solvents Slightly opaque gel. Become clear with sugar.
Soluble in hot water. The addition of calcium ions ill induces the formation of a durable, elastic gel and increase gelling and melting temperature. Insoluble in most organic solvents. Partially soluble in cold water, fully soluble in hot water. No gel, random distribution of polymer chains. Range from low to high viscosity. Insoluble in most organic solvents. Compatible with water organic solvents.
Film characteristics Form excellent gel and film forming properties. Exhibits the highest tensile strength when compared with that of λ- and ι-carrageenan films. κ-carrageenan film containing potassium sorbate had great potential for antimicrobial food packaging, valued properties for extending shelf-life or increasing the safety of foods. Good mechanical characteristics. Are emulsion stabilizers, and decrease oxygen transfer.
Transparent and water soluble films with good mechanical properties.
Table I.1. (Continued) Polysaccharide
Structure & Formula
Agar produces perceptible gelation at concentrations as low as 0.04%. It forms strong gels characterized by melting points far above the initial gelation temperature.
Agar
Pulullan gum
[α-D-Glcp-(1_4)-α-D-Glcp-(1→4)-α-D-Glcp(1→6)]n (C6H10O5)n
Gellan gum
Polysaccharide Properties
Water soluble, insoluble in organic solvents and non-hygroscopic in nature. Its aqueous solutions are stable and show a relatively low viscosity as compared to other polysaccharides. It decomposes at 250–280 ºC. Gels are formed by dispersing the gellan gum in water, heating, adding cations, and then cooling to set. Gel texture can be modified by blending with other gums. It has good stability over a wide pH range (3.5-8.0)
Film characteristics Clear, transparent, strong and flexible films even at low moisture content levels. Similar water vapor permeability to starch films, arabinoxylan films or cellulose derivative films. Clear, odorless and tasteless. Good oxygen barriers. Generally, pullulan films are commonly combined with other polysaccharides or proteins to improve functional properties of edible films
Transparent, stronger and more brittle films than alginate or carrageenan films.
Table I.1. (Continued) Polysaccharide
Structure & Formula
Polysaccharide Properties
Pectin
Food additive which is mainly used for its gelling and stabilizing abilities. Very complex structure which depends on both its source and the extraction process. It forms gels in aqueous media containing sugar and acid.
Chitosan
Purified qualities of chitosans are available for biomedical applications. It is nontoxic, biodegradable, and biocompatible. Antimicrobial and antifungal activities
Film characteristics Can control water activity, preventing moisture loss from food by acting as a sacrificial agent. It can potentially limit fat migration, provide a barrier to gas, help to trap flavor and aroma, and carry and present antioxidants or antimicrobials. High-methoxy pectin forms excellent films Low-methoxyl pectin forms gels in the presence of calcium ions and can also be used for developing edible films. Highly permeable to water vapor. Excellent oxygen barrier properties. Good mechanical properties. Good antimicrobial activity.
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CMC, anionic cellulose ether, is available in a variety of types based on particle size, DS, viscosity and hydration characteristics for different food applications. It is soluble in hot and cold water but insoluble in organic solvents. It basic function is to bind water or impart viscosity to the aqueous phase thereby stabilizing the other ingredients or preventing syneresis. MCC, is used to gel a variety of sugar-based products, to stabilized a number of lowcalorie foods, to replace oil in emulsions, to control ice crystal growth, and for suspension of particulates such as chocolate in sterilized chocolate drinks (Dziezak, 1991)
Seaweed Extracts There are many different types of seaweed extracts used in the food industry. Generally, alginate, carrageenan, agar and furcellan are used for edible films and coatings. Alginates are known as potential biopolymer films or coating components, due to their unique and well-studied colloidal properties; which include, thickening, stabilizing, suspending, film forming, gel producing and emulsion stabilizes properties (Moe et al., 1995; Rhim, 2004). The component with these properties in consideration is a hydrophilic colloidal carbohydrate extracted with dilute alkali from various species of brown seaweeds (Phaeophyceae). Alginates are composed of 1–4 β-D-mannuronic acid (M) and α-Lguluronicacid (G). In the polymer chain, the monomers are arranged alternately in GG and MM blocks, together with MG blocks. The chemical composition and sequence of the M and G blocks are conditioned by the biological source and growth, and by the seasonal environment (Smidsrød, 1974). While the M-block segments develop in linear and flexible structures, the G-block residues give rise to fold and rigid structures and are responsible for the pronounced stiffness in the molecular chains. The ability of alginates to react with di- and trivalent cations is being exploited in the formation of alginate films. Carrageenans are water soluble galactose polymers extracted from red seaweed (Rhodophyceae), which are extensively used, as gelling and stabilizing agents, in food and pharmaceutical industries. The three main carrageenans, kappa, iota and lambda, differ only in the number of sulphate groups, 20, 33 and 41% (w/w) respectively. They are widely used in the food industry to improve thickening and texture qualities and to stabilize food products. Moreover, they are a renewable resource and commercially available at a reasonable cost. λcarrageenan is the most sulphated of the three main carrageenans mentioned, and adopts a coil conformation under all ionic and temperature conditions. While kappa and iota carrageenans form gels, lambda is unable to do it and is used as a pure thickener (Langendorff et al., 2000; Lizarraga et al., 2006). Iota-carrageenan is composed of altering α(1,3)-Dgalactose-4-sulphated and β(1,4)-3,6-anhydro-D-galactose-2-sulphate. In aqueous solutions, ι-carrageenans produce thermoreversible gels once cooling below the critical temperature; from that point on, the conformation changes from a random coil of single chains, to the formation of double helices of carrageenan chains, and consequently to gels (Yuguchi et al., 2002). Agar is a gum that is derived from a variety of red seaweeds, and, like carrageenan, it is a galactose polymer (Sanderson, 1981). Agar produces perceptible gelation at concentrations as low as 0.04%; and it forms strong gels characterized by melting points far above the initial
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gelation temperature (Whistler & Daniel, 1985). It is best known as a culture medium and is not used to great extent in foods. Agar based films have recently been tested for edible films (Phan et al., 2008, Phan et al., 2009). Furcellaran is extracted from seaweed Furcellaria fastigiata which is found in the waters surrounding Denmark. It is mainly used in Europe in producing jams and jellies, fruit juices, confectionery, milk puddings, chocolate milk and beer.
Gums Different varieties of exudates gums, seed gums and microbial fermentation gums are used for edible films and coatings. As described Nispero-Carriedo, 1994, exudate gums are structurally complex heteropolysaccharides obtained from natural exudates of different tree species. Gum Arabic; the dried gummy exudate from the stems or branches of Acacia Senegal and related species of Acacia; gum tragacanth; the dried gum exuded by the steams of Astragalus gummifer and other Asiatic species of Astragalus; gum ghatti; exudate of the Anogeissus latifolia tree and gum karaya; and the dried gummy exudate of the Sterculia tree are all examples of exudate gums that can be used for edible films and coatings. The uses of gum Arabic are based upon its action as a protective colloid or stabilizer and the adhesiveness of its water solutions. It has been used in confections and as a foam stabilizer and agent to promote adhesion of foams to glass. As coating has been used as a protective film on oily foods for eliminate moist, oily appearance and provide a low-calorie product. The gum tragacanth is mainly used as a thickener and stabilizer in salad dressings, sauces, bakery emulsion, toppings, ice cream, and confectionery. Its film-forming properties are useful in nonfood systems such as hair lotions, hand lotions and creams. Gum ghatti has been used effectively in food systems as an emulsifier and stabilizers, but films formed form ghatti dispersions are relatively soluble, brittle, and are not considered very useful. Gum karaya has been used as an emulsifier, stabilizer, or binder in the frozen desserts, dairy products, salad dressings, or meat products, but today it has been replaced by better stabilizers. Examples of seed gums that can be used for edible films and coatings are locust bean gum and guar gum. Both are soluble in water (locust bean gum must be heated to be dissolved) and have high viscosity. Though they are insoluble in organic solutions, they are compatible with other polysaccharides and proteins (Nispero-Carriedo, 1994). Locust bean gum generally is used as a thickener or viscosity modifier, binder of free water, suspending agent or stabilizer in chesses, frozen confections, bakery products, pie fillings, meats, sauces and salad dressings. Guar gum is used for controlling the mobility of dispersed or solubilized materials in water. It is used in dairy, bakery and meat products; as well as in beverages and salad dressings. Pullulan, xanthan and gellam gums are microbial polysaccharides that are edible and biodegradable. Pullulan is one of those commercially emerging biopolymers, synthesized by a yeast-like fungus known as Aureobasidium pullulans. It is a water soluble, random coil glucan gum that serves as a paradigm for the behavioral aspects of aqueous polysaccharides (Yalpani, 1988, Morris, 1995; Tsujisaka & Mitsuhashi, 1993, Singh, Saini & Kennedy, 2008). It is a regularly repeating copolymer, with the chemical structure {Æ 6)-a-D-glucopyranosyl(1 Æ 4)-a-D-glucopyranosyl-(1 Æ 4)-a-D-glucopyranosyl- (1Æ}n. Thus, the polysaccharide is viewed as a succession of a-(1 Æ 6)-linked (1Æ 4)-a-D-triglucosides i.e. maltotriose (G3).
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Pullulan’s solubility can be controlled, or provided with reactive groups, by chemical derivatization. Consequently, pullulan (and its derivatives) has wide potential for food, pharmaceutical and other industrial applications. Pullulan is water soluble, insoluble in organic solvents and non-hygroscopic in nature. Its aqueous solutions are stable and show a relatively low viscosity as compared to other polysaccharides. It decomposes at 250–280 ºC. It is moldable and spinnable, being a good adhesive and binder. It is also non-toxic, edible, and biodegradable (Singh, Saini & Kennedy, 2008). Pullulan membranes/films are being used as coating and packaging materials for foods such as instant food seasonings, powdered tea and coffee. Pullulan-coated paper also decomposes easily and does not contaminate the environment (Doman - Pytka & Bardowski, 2004). Xanthan gum is produced by fermentation from the organism Xanthomonas campestris. It contains five sugar residues: two β-D-glucopyranosyl, two β-D-mannopyranosyl, and one βglucopyranosyluronic acid residue (Jansson et al., 1975; Melton et al., 1976). Xanthan gum is soluble in both cold and hot water and has a high viscosity. It is used for its thickening, suspending and stabilizing effects in salad dressings, dry mix products, icings and frostings, confectionery, dairy products, fruit gels, sauces, syrups and baked goods. It can be used to provide uniform coating, good clinging qualities, improved adhesion in wet batters, and to prevent moisture migration during frying (Nisperos-Carriedo, 1994). Gellan gum is produced by the fermentation of a pure culture of Pseudomonas elodea. The gum has a linear tetrasaccharide as a repeating unit, consisting of (1Æ3)-β-Dglucopyranosyl, (1Æ4)- β-D- glucopyranosyluronic, (1Æ4) )-β-D-glucopyranosyl, and (1Æ4)-α-L-rhamnopyranosyl units. Gels are formed by dispersing the gellan gum in water, heating, adding cations, and then cooling to set. Gel texture can be modified by blending with other gums, especially the gelling gums. It has good stability over a wide pH range (3.5-8.0) (Nisperos-Carriedo, 1994).
Pectins Pectin is a family of heterogeneous branched polysaccharides consisting mostly of variably methylated galacturonan segments separated by rhamnose residues, some of which may be linked to short neutral sugar side chains. The rhamnose residues redirect the orientation of galacturonan segments to produce kinks, which upon aggregation, ensure open structures favorable for gel formation. Isolation of pectin from plant cell walls is achieved by breaking up the gel structure, usually stabilized by calcium cations, to solubilize large aggregates of pectin. Various grades of pectin are commercially available in different degrees of methyl esterification and in different ranges of molecular weights, or more accurately, different degrees of disaggregation (Hoagland & Parris, 1996).
Chitosan Chitosan is derived from chitin by deacetylation in the presence of alkali. Therefore, chitosan is a copolymer consisting of β-(1-4)-2-acetamido-δ-glucose and β-(1-4)-2-amino-δglucose units with the latter usually exceeding 80%. Chitosans are described in terms of the degree of deacetylation and average molecular weight; and, their importance resides in their
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antimicrobial properties in conjunction with their cationicity and their film-forming properties (Muzzarelli, 1996). This is the second most abundant polysaccharide on Earth, after cellulose, (Lezica & Quesada-Allue, 1990) and is commercially available from a stable, renewable source; that is, waste from the shellfish industry (Andrady & Xu, 1997).
POLYSACCHARIDE FILM FORMATION AND FILM CHARACTERISTICS Polysaccharide film formation and film characteristics depend of the type of polysaccharides used. Table I.1 summarize structure, formula and polysaccharide properties and Film characteristics For edible starch films, it is important to prepare a clear starch solution with proper solid concentration, in order to insure both the continuity of films and the ease of casting. A solid concentration of 10-15% is suggested for casting conventional starch films (Liu, 2005). With high concentrations, the solution is too viscous to be cast; and with low concentrations, the solution of completely solubilized starch polymers has low level gelling results, causing problems for the process of forming a continuous film of sufficient thickness (Protzman et al., 1967). To prevent the hydrolysis or oxidation of starch, the formation process of edible starch films must ensure that the starch polymers are completely gelatinized, disintegrated and solubilized (Lourdin et al., 1997). Complete solubilization of starches in water required high temperatures and can be facilitated using amylase-complexing agents like butanol. Aqueous starch solutions are normally unstable, and in this sense, it is necessary to keep the starch solution at a temperature above their gelation temperature prior casting. When the solid concentration is 10-15%, the gelation temperature is 60-74 ºC (Muetgeert et al., 1962). Generally, films produced from edible starches are odorless, tasteless, and colorless and exhibit physical characteristics, chemical resistance and mechanical properties similar to those of plastic films (Wolff et al., 1951). They have been utilized in the packaging and coating of food products because of their edibility, low permeability to oxygen and their low cost of production. The overall performance of starch films and coatings is highly likely to be customizable, because of the availability of a wide variety of starches and their capacity for physical and/or chemical modifications (Liu, 2002). They are commonly used in bakery, confectionery, batters and meat products (Thomas & Atwell, 1997). MC, HPC and HPMC are water soluble ethers with good film-forming properties. In order to avoid the formation of agglomerates the dissolution of these nonionic cellulose ethers must be done in two steps: dispersion and hydration. Wherever possible, they should be put into solution before other soluble ingredients are added or should be dispersed in water miscible nonsolvent such as glycerol, ethanol or propylene glycol and then add the slurry to water. The solutions of these cellulose ethers are stable at pH 2-11 and are compatible with surfactants, other water-soluble polysaccharides, and with salts. The procedure for the preparation of clear CMC solutions follows that of the nonionic cellulose ethers, except for pH conditions. CMC solutions are only stable at pH 7-9. CMC is compatible with a wide range of other food ingredients including protein, sugar, starches and others hydrocolloids. Edible coatings including MC, HPC, HPMC or CMC have been applied to a variety of foods to provide moisture, oxygen or oil barriers, and to improve batter adhesion. These
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cellulose ether films are generally transparent, flexible, odorless, tasteless, water-soluble, and resistant to oils and fats (Nispero-Carriedo, 1994; Lacroix & Le Tien, 2005). MC films do not have good moisture barriers, but do provide an excellent barrier against migration of fats and oils and it can be used to reduce oil absorption in fried products. HPC is thermoplastic and capable of injection molding and extrusion. It can retard spoilage and moisture absorption in coated nuts and candies. The film-forming characteristics of HPMC upon heating provide an effective film that can reduce oil absorption in certain reformed products. CMC forms a complex in the presence of casein, increasing the coatings formulation viscosity. It retains the firmeness of fruits and vegetables, preserves important flavor components of some fresh commodities, reduces oxygen uptake without causing carbon dioxide increase in internal fruits and vegetables, and improves the puncture strength of films based on caseinate (Lacroix & Le Tien, 2005). Alginates produce uniform, transparent and water-soluble films. Divalent cations are used as gelling agents (to induce ionic interactions, followed by hydrogen bonding) in the formation of alginate films (Kester & Fennema, 1986). Films and coatings can be made from a sodium alginate solution; these films and coatings can be produced by means of a rapid reaction with a cold application of calcium, forming intermolecular associations involving the G-blocks regions (Nisperos-Carriedo, 1994). The treatment of alginate films with divalent cation (i.e. calcium) solutions converts these into insoluble films (Pavlath et al., 1999). Alginates films are quite brittle; nonetheless, they may be further plasticized with glycerol (Nussinovitch & Hershko, 1996, Cha et al., 2002, Rhim, 2004, Immirzi et al., 2009). Alginate coatings are good oxygen barriers, can retard lipid oxidation in foods, and can improve flavor, texture and batter adhesion. Due to it high hydrophilic nature, alginate based films are poor moisture barriers; although alginate gel coatings can significantly reduce moisture loss from foods, because moisture is lost from the coating before the food dehydrates (Conca & Yang, 1993). Fabra et al., 2008 evaluated the effect of adding alginates to sodium caseinate-lipid films. In lipid free films, alginates improved the tensile properties of films, although water vapor permeability values increased. However, in sodium caseinate-lipid films, the addition of alginates to protein matrices produced less flexible, less stretchable and more permeable films. Carrageenan-based coatings have been applied to a variety of foods for a long time, they have been applied to incorporate antimicrobials or antioxidants, and to reduce moisture loss, oxidation, or disintegration (Lacroix & Le Tien, 2005). -carrageenan has one negative charge per disaccharide, with a tendency to form excellent gel and film forming properties, and exhibits the highest tensile strength when compared with that of λ- and ι-carrageenan films (Park, 1996). Choi et al., 2005 reported that the studied κ-carrageenan film containing potassium sorbate had great potential for antimicrobial food packaging, valued properties for extending shelf-life or increasing the safety of foods, when it is used as packaging or coating material. ι-carrageenanbased edible films have good mechanical characteristics, are emulsion stabilizers, and decrease oxygen transfer. The addition of lipids to form emulsified films decreases the water vapor transfer and could be used to encapsulate active molecules or aroma compounds (Hambleton et al., 2008, Fabra et al., 2009). Fabra et al., 2008 reported that λ-carrageenan improves tensile and water vapor permeability of sodium caseinate-oleic acid-beeswax films, though barrier properties of lipid-free films decreased with λ-carrageenan. The films made of agar are clear, transparent, strong and flexible even at low moisture content levels. Their water vapor permeability quality did not appreciably differ in comparison
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to starch films, arabinoxylan films or cellulose derivative films. Moreover, agar based films were found to be heat-sealable. Like with other polysaccaharides; antioxidants, antimicrobials, bacteriocins or antibiotics can be incorporated in agar based films to improve shelf life of foods. Phan et al., 2009b studied edible films based on the binary combination of agar, cassava starch and arabinoxylan; the study was conducted with investigative emphasis on their microstructure, moisture barrier and mechanical properties. They observed that mechanical properties of agar based films are degraded when cassava or arabinoxylan were added. The results suggest that agar can potentially provide a very good cohesive matrix, which contributes towards enhancing the mechanical properties of other polysaccharide based films. Pullulan films cast from aqueous solutions are clear, odorless and tasteless and have good oxygen barriers (Yuen, 1974, Conca & Yang, 1993). Pullulan films are commonly combined with other polysaccharides (alginate, carboxymethylcellulose) or proteins (whey protein, sodium caseinate) to improve functional properties of edible films (Tong, Xiao, & Lim, 2008; Gounga, Xu, & Wang, 2007; Kristo & Biliaderis, 2006; Kristo, Biliaderis & Zampraka, 2007). Tong, Xiao, & Lim, (2008) reported that pullulan films had lower water vapor permeability than alginate and carboxymethylcellulose (CMC) films (4.4 × 10−7, 9.7 × 10−7, and 1.3 × 10−6 g m/Pa h m2, respectively), but dissolved in water quicker than alginate and CMC films. By incorporating alginate and CMC into pullulan, water barrier and mechanical properties were weakened significantly. Blending pullulan with alginate or CMC up to about 17–33% (w/w total polymer) reduced film solubilization time in water. FTIR results indicated that blending pullulan with alginate and CMC resulted in weaker hydrogen bonds acting on –OH groups, compared to those of pure pullulan. The addition of pullulan (at low concentrations) to whey protein isolate films exhibited acceptable results that significantly modified oxygen permeability, water vapor permeability, moisture content and film solubility (FS); hence improving the potential characteristics of WPI-based films for food applications (Gounga, Xu, & Wang, 2007) Gellan gum films are transparent, stronger and more brittle than alginate or carrageenan (Nussinovitch & Hershko, 1996). Gellan films can also be used as a carrier of antimicrobials or antioxidants. In this sense, León & Rojas, 2007 evaluated edible gellan films as carriers for stabilizing l-(+)-ascorbic acid (AA) for nutritional purposes and its antioxidant effect on foods. Moreover, antimicrobial films incorporating nisin were found to have antimicrobial activity against Staphylococcus aureus, particularly those with higher contents of gellan gum. Studies showed that as the gellan gum content increased, so did the enhancements of the antimicrobial effects. These results suggest that films with a 70% blend of konjac glucomannan could be applied as a potential food package material for releasing active agents such as nisin (Xu et al., 2007). Pectin coatings have been investigated for their ability to retard moisture loss and lipid migration, and improve handling and appearance of foods. Generally, high-methoxy pectin forms excellent films and low-methoxyl pectin, derived by controlling esterification, forms gels in the presence of calcium ions and can also be used for developing edible films. Despite their hydrophilic character, pectinate coatings can control water activity, preventing moisture loss from food by acting as a sacrificial agent. It can potentially limit fat migration, provide a barrier to gas, help to trap flavor and aroma, and carry and present antioxidants or antimicrobials (Maftoonazad, Ramaswamy, & Marcotte, 2007). Addition of lipids may increase their resistance to water vapor transmission. Plasticized blends of citrus pectin give strong, flexible films; which are thermally stable up to 180ºC (Tharanthan & Kittur, 2003). Maftoonazad, Ramaswamy, & Marcotte, 2007 evaluated the moisture sorption behavior of pectin films formulated with different sorbitol contents. Based on changes observed in
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moisture sorption isotherms, they concluded that sorbitol strongly interacts with pectin polymers. Incorporation of sorbitol in pectin films resulted in lower equilibrium moisture contents at low to intermediate water activities (aw), but much higher moisture contents at aw > 0.53. Increasing moisture or addition of sorbitol to pectin films increased the elongation at break, but decreased the tensile strength, modulus of elasticity and Tg; even so, increasing the water vapor permeability of the films. Chitosan is of interest as a potential edible film component because of its excellent oxygen barrier properties (Hosokawa et al., 1990, Conca & Yang, 1993; Nisperos-Carriedo, 1994; Anker, 1996) and its good mechanical properties. However, the fact that it is highly permeable to water vapor limits its use (Butler, Vergano, Testin, Bunn, & Wiles 1996; Caner, Vergano, & Wiles, 1998), which is an important drawback since an effective control of moisture transfer is a desirable property for most foods. Acetic acid has often been the solvent for the production of chitosan films. Inherent antibacterial properties and the film-forming ability of chitosan make it an ideal choice for use as a biodegradable antimicrobial packaging material that can be used to improve the storability of perishable foods. It has been confirmed that chitosan films exhibit good antimicrobial activity, which can help extend the food shelf life (Kendra et al., 1989; Muzzarelli et al., 1990; El Ghaouth et al., 1991, 1994; Fang et al., 1994; Chen et al., 1996; Tsai et al., 2000, Coma et al., 2002, Dutta, Triphatti, Mehrotra & Dutta, 2009). In this sense, chitosan has exhibited high antimicrobial activity against a wide variety of pathogenic and spoilage microorganisms, including fungi, and Gram-positive and Gram-negative bacteria, especially in limiting the development of bacteria such as Listeria monocytogenes, which are unacceptable in foodstuffs because of their pathogenicity (Dutta et al., 2009). Due to the antimicrobial properties of these films, some research is based on the use of chitosan in developing “active” packaging systems (Brody, 2001). Active packaging is a type of packaging that changes the condition of the packaging to extend shelf-life or improve safety or sensory properties while maintaining the quality of the food. Thus, it is the packaging system possessing attributes beyond basic barrier properties, special attributes that are achieved by adding active ingredients in the packaging system and/or using functionally active polymers. The binding of antimicrobial to polymeric surfaces such as polyethylene (PE), polyvinyl chloride (PVC), polylactic acid (PLA), nylon and others has been achieved by different means; means ranging from simply spreading antimicrobial solutions onto the polymer surface, or by more sophisticated means such as combining the antimicrobials with binders. These binders can be of a cellulosic, or an acrylic co-polymer nature. Sometimes the antimicrobials have been covalently attached, with natural and synthetic cross-linkers like genipine, glutaraldehyde, formaldehyde etc. (Sebastien et al., 2006; Dutta et al., 2009). For instance, Sebastien et al. 2006 proposed a chitosan-loaded PLA film, consisting of high inhibitory properties, for usage against mycotoxinogen fungal strains.
POLYSACCHARIDE FILM PROPERTIES The main properties of edible films and coatings are their mechanical and barrier properties. Both properties are generally related to the physical and chemical nature of the polymers. Generally, polysaccharide-based films are very sensitive to humidity changes. At low relative humidities these films tend to crack. At high relative humidities these films swell and their barrier characteristics are markedly degraded.
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Mechanical properties reflect the durability of films and the ability of a coating to enhance the mechanical integrity of foods. Polysaccharide films are relatively stiff, and therefore plasticizers are needed to facilitate handling to achieve the desirable mechanical properties. As polysaccharide-based films are generally water-based, the most effective plasticizers are similar to the polysaccharide structure; therefore, hydrophilic plasticizers containing hydroxyl groups are best suited to this use. The plasticizers commonly used for polysaccharide-based films are glycerol, sorbitol, xylitol, mannitol, propylene glycol, polyethylene glycol and ethylene glycol (Sothornvit & Krochta, 2005). The effect of various plasticizers have been explored for films made from MC, HPC (Ayranci & Tunc, 2001), HPMC (Ayranci et al., 1997), locust bean gum (Aydinli & Tutas, 2000), gellan (Yang & Paulson, 2000), starches (Kim et al., 2002; Ryu et al., 2002) or pullulan (Kim et al., 2002). Barrier properties are important to separate food components or foods from the environment, which causes food deterioration. The most common barriers of interest include water, oxygen, carbon dioxide, aroma and oil barriers. At low to intermediate relative humidity levels polysaccharide-based films and coatings are good barriers against oxygen, due to their highly packed and ordered hydrogen-bonded networked structure (Yang & Paulson 2000), and other non-polar substances, such as aromas and oils. Nevertheless, due to their hydrophilic nature, they are indeed wettable by water and generally exhibit limited water vapor barrier ability, especially at high relative humidity levels. However, certain polysaccharides, applied in the form of high moisture gelatinous coatings, can retard moisture loss from coated foods by functioning as sacrificing agents rather than moisture barriers. Table I.2 shows the water vapor permeability (WVP) and tensile parameters (TS: Tensile strength, E: Elongation at break) of different polysaccharide based films. Comparisons are difficult because data were obtained in different studies using different film compositions, different test conditions (temperature and relative humidity) and with different methods of measurements. Polysaccharide films appear to have similar tensile strength and lower elongation at break values than synthetic polymers films. While agar, cassava and alginate films have the highest TS values, chitosan films have the lowest TS values. Because of the hydrophilic nature of polysaccharide films, they provide quite high water vapor permeability compared to synthetic materials such as low-density polyethylene (LPDE). Among polysaccharide films, starch films have the highest water barrier properties. The utilization of edible polysaccharide-based films and coatings as moisture barriers requires the formation of composite films. These multicomponent edible films and coatings are blends of polysaccharides and hydrophobic lipid materials such as edible fatty acids and waxes. In these film systems, the barrier properties may be improved by taking advantage of each component. Polysaccharides impart cohesion and serve as a structural matrix, while lipids are added due to their hydrophobic character. Composite films can be formed as a bilayer, when the lipid material is cast onto a dried polysaccharide-based film, or as an emulsion, when the lipid is added to the film-forming solution prior to film casting. Emulsion composite films have not achieved the excellent moisture barrier properties of bi-layer films. However, bi-layer films have the disadvantages of requiring two steps to be formed, plus they needed manipulation of either a molten lipid or a solvent. Furthermore, the films formed can suffer peeling which minimizes the water barriers. Achieving low water vapour permeabilities with emulsion composite films is favored by loading high hydrophobic lipidic materials, homogeneous distribution of lipids of small particle size, and the formation of continuous networks of interconnected lipids within the polysaccharide matrix (Krochta & The Mulder-
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Johnston, 1997; Wu et al., 2002 development and application of multicomponent edible coatings and films: a review). Properties of polysaccharide-lipid films have been studied extensively by different leading researchers in the field (Pérez-Gago & Krochta, 2001; Morillon et al., 2002, Karbowiak, Debeaufort & Voilley, 2007, Hambleton et al., 2008) Generally, protein-based films have more interesting mechanical and barrier properties than polysaccharides (Ou, Kwok, & Kang, 2004; Cao, Fu, & He, 2007). While proteins have a specific structure (based on 20 different monomers) which confers a wider range of functional properties, especially high intermolecular binding potential and can be modified easily, polysaccharide-based films are more readily disintegrated by absorbing water and in general have poorer barrier properties than protein-based films (Cuq et al., 1995). As a group, protein films appear to have lower oxygen permeabilities than non-ionic polysaccharide films. This may be related to their more polar nature and more linear (non-ring) structure, leading to higher cohesive energy density and lower free volume (Miller & Krochta, 1997). Biopolymer films made form mixtures of both, protein and polysaccharide, ingredients may advantageously use the distinct functional characteristics of each film-forming ingredient. Some works indicate that incorporation of polysaccharides into globular protein matrices may extend the functional properties of these ingredients (Zaleska, Ring & Tomasik, 2000; Turgeon & Beaulieu, 2001). Some edible films based on the blends of polysaccharides and proteins such as soluble starch–gelatin (Arvanitoyannis et al., 1997), hydroxypropyl starch–gelatin (Arvanitoyannis et al., 1998), soluble starch–caseinate (Arvanitoyannis et al., 1998), alginate or λ-carrageenan –sodium caseinate (Fabra et al., 2008) or some films based on the mixtures of polysaccharides such as starch–methylcellulose (Arvanitoyannis & Biliaderis, 1999), pulluan–starch (Biliaderis et al., 1999), chitosan– starch; chitosan–pulluan (Lazaridou & Biliaderis, 2002), agar-cassava starch and arabinoxylan (Phan et al., 2009b) were investigated. These publications demonstrated that depending on the interactions between components, these formulas can improve the mechanical and barrier properties of these films. For example, pectin and starch blends can be used to make a range of films with very good properties (Coffin & Fishman, 1993, Fishman & Coffin, 2005). Blends of pectin and chitosan can also be formed to improve properties of films (Hoagland & Parris, 1996). Xu, Kim, Hanna, & Nag, 2004, observed a decrease in water vapor transmission rates (WVTR) by combining chitosan with two thermally gelatinized corn starches. Composite films based on blends of pectin and κ-carrageenan increased mechanical properties, lowered glass transition temperatures, increased water permeability and hydrophilic properties with increased carrageenan content. These films revealed to be more permeable to water vapor than to oxygen and carbon dioxide, and the films selectivity (CO2/O2) was enhanced by adding ascorbic acid to the polymer matrix (Alves et al., 2006). Pectin can also form crosslinks with proteins under certain conditions (Thakur et al., 1997). Autoclaving enhances pectin-protein interactions, resulting in a three-dimensional network with improved mechanical and barrier properties.
Table I.2. Water vapor permeability (WVP) and tensile parameters of different polysaccharide based films. Film composition
Test conditions
Agar+15% glycerol Cassava+15% glycerol Arabinoxylan+15% glycerol iota-carragenan+ glycerol (1:0.3) HPMC
25ºC/22-99% 25ºC/22-99% 25ºC/22-99%
WVP (g mm/m2 d kPa) 11.2 ± 0.9 9.7 ± 0.9 11.7 ± 0.8
25ºC/30-100%
8.4 ± 0.9
25ºC/22-84%
8.6 ± 0.8
HPMC +15 % Gly
25ºC/22-84%
17.3 ± 0.5
Alginate-glycerol (1:0.5) Chitosan Chitosan + 2% OA Chitosan + whey protein Cellulose+PEG (1:0.4)
25ºC / 50-100% 5ºC/58-100% 5ºC/58-100% 25ºC/0-84% 25ºC/0-84%
12.28 ± 0.1
MC (4%) MC (3%) +PEG HPC (3%) +PEG Corn starch Amylomaize starch PVC PET LPDE LPDE HDLE
25ºC/0-52% 21ºC/0-85% 21ºC/0-85% 20ºC/33-98% 20ºC/33-98% 28ºC/0-100% 28ºC/0-100% 28ºC/0-100% 25ºC/50% 25ºC/50%
0.00324 ± 0.0001 0.002983 ± 0.0021 4.27 ± 0.15 7.950 ± 0.003 9.504 ± 0.004 3.17 ± 2.24 2.26 ± 1.39 0.62 0.17 0.031
TS (MPa)
E (%)
42.11 ± 3.27 35.17 ± 4.60 22.30 ± 2.97
6.51 ± 0.96 2.64 ± 0.73 5.46 ± 1.89
33.6 ± 3.1 12 ± 8 7±2 9.5 ± 0.6
14.0 ± 2.9 17 ± 8 11 ± 7 14.1 ± 0.59
23 ± 3
11 ± 1
9-17 17-35
500 300
Reference Phan The, Debeaufort, Voilley & Luu, 2009 Phan The, Debeaufort, Voilley & Luu, 2009 Phan The, Debeaufort, Voilley & Luu, 2009 Fabra, Hambleton, Talens, Debeaufort, Chiralt & Voilley, 2009 Phan The, Peroval, Debeaufort, Despré, Courthaudon & Voilley, 2002 Phan The, Peroval, Debeaufort, Despré, Courthaudon & Voilley, 2002 Rhim 2004 Vargas, Albors, Chiralt & González-Martínez, 2009 Vargas, Albors, Chiralt & González-Martínez, 2009 Di Pierro et al., 2006 Ayranci & Tunc, 2001 Nazan & Sahbaz, 2004 Park & Chinnan, 1994 Park & Chinnan, 1994 García, Martino, Zaritzky, 2000 García, Martino, Zaritzky, 2000 Shellhammer & Krochta, 1997 Shellhammer & Krochta, 1997 Shellhammer & Krochta, 1997 Briston, 1986 Briston, 1986
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APPLICATIONS OF POLYSACCHARIDE-BASED FILMS IN FOOD PRODUCTS A number of edible polysaccharide-based films and coatings have been used to extend the shelf-life of fruits, vegetables, seafood, meats and confectionary products by preventing dehydration, oxidation rancidity, surface browning and oil diffusion. In some specific cases, polysaccharide-based films can improve the physicochemical, nutritional and sensorial properties of the products. During the last decade, leading researchers in the field have studied the benefits of the application of polysaccharide-based films for food products. Alginates, carrageenans, cellulose, pectin and starch derivates have all been used to improve stored meat quality. Alginate films retard development of oxidative off-flavors when applied as an edible coating to precooked pork patties and reduce weight loss as well as microflora counts in frozen shrimp, fish, sausage and stored lamb carcasses (NisperosCarrierdo, 1994). The use of carrageenans as edible films and coatings have been applied for a long time on a variety of foods; they are applied as a carry source for antimicrobials or antioxidants; and, to reduce moisture loss, oxidation or disintegration when applied on fresh and frozen meat, poultry and fish, or to prevent superficial dehydration on ham or sausagecasings (Macquarrie, 2002), granulation-coated powders, dry solids foods, oily foods, etc. Furthermore, they have also been used in the manufacturing process of soft capsules (Tanner et al., 2002), and especially with the manufacturing of non-gelatin capsules (Fonkwe, Archibald & Gennadios, 2003). The ability of some water-soluble polysaccharides to form thermally induced gelatinous coatings has found wide application for its ability of reducing oil absorption during frying. Research conducted by Albert et al., 2002, compared different hydrocolloid materials (including gellan gum, κ-carrageenan-konjac-blend, locust bean gum, methyl cellulose, microcrystalline cellulose and three types of pectin) for their water and fat transfer properties, their film forming abilities, and their suitability for usage with fried foods. Various selected formulation and preparation methods were tested for their effectiveness and for their heat stability when applied on the control substance, a product made from a pastry-mix. Among the polysaccharide-based films, methyl cellulose was the best coating material for reducing fat uptake during frying. Researchers, García et al., 2002, used edible coatings from methylcellulose and hydroxypropylmethylcellulose to reduce oil uptake in deep-fat frying potato strips and dough discs. Methylcellulose coatings were more effective than the hydroxypropylmethylcellulose coatings in reducing oil uptake. Non-significant differences in texture of coated and uncoated samples were observed. Interesting research by Castro-Freitas et al., 2009, used a pectin-based coating in the deep-fat frying of preformed products made of either cassava flour or cassava purée. The coating treatments were efficient for the cassavapurée preformed products but not for the cassava-flour preformed products; and thus indicating that different products can show different responses with the same type of coating material. Bravin et al., 2006, investigated the effect of the deposition process, used for filmforming dispersion (spreading and spraying), and the effectiveness of edible coatings (made of cornstarch, methylcellulose and soybean oil) in controlling moisture transfer in moisturesensitive products. This research was conducted by coating crackers, a low water activity-type cereal food. They observed that film which was spread gave better water vapor barrier and
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mechanical properties than film that was sprayed, and that coated crackers had longer shelflife and higher resistance to water vapor transmission than uncoated samples at three different storage conditions (65%, 75% and 85% relative humidity). Moreover, in order to examine potential effects on egg quality properties, Caner & Cansiz, 2007, coated eggshells with three chitosan-based coatings (produced with organic acids: acetic-[C-AA], lactic-[C-LA] and propionic [C-PA]). All chitosan-coated eggs showed greater inner food quality than the noncoated eggs. When compared with the controlled, non-coated egg specimens, the coated eggs significantly kept better from weight loss; and better maintained the nutritional value amounts of minerals (especially calcium, iron and magnesium concentrations). The application of water-soluble polysaccharides on fruits and vegetables has become popular and extensive due to their ability to reduce O2 and increase CO2 levels in internal atmospheres. This effect modifies the internal atmospheres and reduces respiration rates, thereby prolonging the shelf-life of fruits and vegetables, in a manner analogous to controlled atmospheres. The effects of edible coatings and plastic packaging on the quality aspects of refrigerated white asparagus spears were studied by Tzoumaki et al., 2009, using different formulations based on carboxymethyl-cellulose and sucrose fatty acid esters; and pullulan and sucrose fatty acid esters. Both edible films exhibited a beneficial impact on the quality of asparagus by retarding moisture loss, reducing hardening in their basal part and slowing down the purple-color attainment process. Viña et al., 2007, studied the effects of combinations of polyvinylchloride film and starch-based coatings on quality aspects of refrigerated Brussels sprouts. They stored different combinations of polyvinylchloride film and coatings and uncovered samples at 0 °C for 42 days. All samples were removed every 14 days to determine commercial acceptability, weight loss, surface color and texture. Sprouts in all treatments maintained optimum quality conditions over the first 14 days. At the end of the storage period, it was concluded that browning of cut zones and losses in weight and firmness are better minimized in PVC-packaged sprouts when using polyvinylchloride film and coatings. Maftoonazad & Ramaswamy, 2005, evaluated the effects of a methylcellulose-based coating on the respiration rate, color and texture of avocados stored at room temperature. Coated avocados demonstrated lower respiration rates, greener color and higher firmness when compared with the uncoated samples. Furthermore, the appearance of brown spots and mesocarp discoloration normally associated with fruit ripening were delayed in the coated fruits. Ayranci & Tunc, 2004, found that coatings of methylcellulose and polyethylene glycol with stearic acid tend to lower the water loss rate of fresh apricots and green peppers. The inclusion of ascorbic acid or citric acid in the coating formulation, as antioxidants, lowered the vitamin C loss. Yaman & Bayoindirli, 2002, studied the effects of an edible coating (composed of sodium carboxymethylcellulose; sucrose esters of fatty acids; and, monodiglycerides of fatty acids) on the shelf-life and quality of cherries. The coating was effective in the reduction of weight loss and tended to increase firmness, ascorbic acid content, titratable acidity and skin color of cherries during storage time, and increased the shelf-life of the cherries by 21% at 30±3 °C and by 26% at 0 °C without perceptible losses in quality. The effects of edible chitosan coating on the quality and shelf-life of sliced mango fruits were studied by Chien et al., 2007. The chitosan coating retarded the deterioration of sensory qualities and delayed water loss; while increasing the soluble solid content, titratable acidity and ascorbic acid content. It also inhibited the growth of microorganisms. The data gathered reveals that applying a chitosan coating effectively prolongs the quality attributes and extends
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the shelf-life of sliced mango fruit. Casariego et al., 2008, demonstrated that chitosan films inhibited fungi growth, reduced ethylene production, increased internal CO2 and decreased O2 levels, when applied to tomatoes. The effects of alginate, pectin and gellan-based edible coatings on the shelf-life of fresh-cut melons and fresh-cut pears were investigated by OmsOliu et al., 2008a,b. The use of polysaccharide-based edible coatings on the fresh-cut samples increased their water vapor resistance and reduced their ethylene production. Pectin or alginate was able to reduce wounding-stress induced on fresh-cut melons and thus generated the accumulation of total phenolic compounds and other compounds with antioxidant properties. The Pectin-based coating seems to have best maintained the sensory attributes of this fruit. For the pears, coating formulations were integrated with N-acetylcysteine and glutathione; this not only reduced microbial growth (when compared with those samples not containing antioxidants) but was also effective in preventing fresh-cut pears from browning, for 2 weeks, without affecting the firmness of the fruit wedges. The increased vitamin C and total phenolic content observed in coated pear wedges (coatings with alginate, gellan and pectin including antioxidants) contributed in maintaining their antioxidant potential. Coatings with alginate or pectin best maintained sensory attributes of pear wedges for 14 days. The effect of alginate and gellan-based edible coatings on the shelf-life of fresh-cut apples, packed in trays with a plastic film, was investigated by Rojas-Graü et al., 2008. Polymers were crosslinked with a calcium chloride solution, to which the anti-browning agent N-acetylcysteine was added, being incorporated to the coatings formulation and helping to maintain firmness and color of the apple wedges during the entire storage time. The application of the edible coatings also retarded the microbiological deterioration of fresh-cut apples. Both edible coatings effectively prolonged the shelf-life of the apple wedges by 2 weeks of storage time. The controlled non-coated apple slices demonstrated considerable cut-surface browning and tissue softening from the very first days of storage, having evident conditions that limited their shelf-life to less than 4 days; a considerable degeneration when compared with the coated apples. The ability of polysaccharide-based coatings to extend the shelf-life of strawberry fruit was studied by different authors (Diab et al., 2001; García et al., 2001; Han et al., 2004; Han et al., 2005; Vargas et al., 2006; Ribeiro et al., 2007; Hernández-Muñoz et al, 2008; Campaniello et al., 2008; Almenar et al.; 2009). The physiological responses of strawberries coated with pullulan-based edible films have been studied by Diab et al., 2001. Application of a pullulan-based coating on strawberries resulted in substantial changes within internal fruit atmosphere composition, changes that were beneficial in extending the shelf-life of this fruit; the coated fruit showed much higher levels of CO2, a large reduction in internal O2, better firmness and color retention and a reduced rate of weight loss. García et al., 2001, used starch-based coatings to extend the storage life of strawberries. These authors analyzed the effects of a coating formulation (starch type, plasticizer, lipid and antimicrobial agent) with respect to fruit quality. Plasticizer presence reduced weight losses and maintained the surface color of fruits. Amylomaize coatings showed lower water vapor and gas permeabilities; and decreased weight losses for longer periods of time than corn starch coatings. Coatings with antimicrobial agents decreased microbial counts, extending storage life of coated fruits by 10 to 14 days, in contrast to the storage life of the controlled non-coated fruit samples. The addition of lipids to the formulations decreased the water vapor permeability of starch-based films, maintained the surface color of coated fruits and effectively limited fruit weight losses during storage. Composite starch-based coatings showed selective gas permeability (CO2
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higher than O2) which helps to delay the senescence of fruits. Han et al., 2004 used three chitosan-based edible coatings (chitosan, chitosan containing 5% Gluconal® CAL, and chitosan containing 0.2% -α-tocopheryl acetate) to extend the shelf-life and enhance the nutritional value of strawberries. These coatings significantly decreased decay incidence and weight loss, and delayed the changes in color, pH and titratable acidity of strawberries during cold storage. Coatings also reduced drip loss and helped maintain textural quality of frozen strawberries after thawing. The incorporation of calcium or vitamin C into chitosan-based coatings did not alter their anti-fungal and moisture barrier functions but significantly increased the content of these nutrients in both fresh and frozen fruit. Han et al., 2005, developed three 1% chitosan-based solutions for coating strawberries (chitosan in 0.6% acetic acid solution, chitosan in 0.6% lactic acid solution, and chitosan in 0.6% lactic acid solution plus 0.2% vitamin E). Coated strawberries were packed in clam-shell boxes and stored at 2°C and approximately 88% to 89% RH for 1 week. The samples were evaluated for their acceptance attributes by consumers and evaluated descriptively for their appearance, texture, and flavor by a trained panel. Results from the consumer evaluation on the 1st day of testing, on the 1st week after coating, indicated that chitosan coatings increased the appearance acceptance of the strawberries, but according to these consumers, coatings containing vitamin E decreased the acceptable appearance of strawberries. Chitosan coatings did not change consumer acceptability of flavor, sweetness, or firmness of the samples. Trained panel results after the 1st week of storage showed that chitosan-coated strawberries have similar sensory descriptors as those of fresh berries, whereas coatings containing vitamin E developed the waxy-and-white surface of the samples. The trained panel did not detect any astringency difference among all samples, indicating that 1% chitosan coating did not change the astringency of strawberries. Edible coatings based on high molecular weight chitosan combined with an oleic acid were used by Vargas et al., 2006, to preserve quality of coldstored strawberries. Coatings had no significant effects on the acidity, the pH and the soluble solids content of the sample strawberries throughout storage but they did slow down changes in their mechanical properties and did slightly modify their respiration rates. The addition of oleic acid enhanced chitosan antimicrobial activity and improved the water vapor resistance of chitosan-coated samples. Sensory analysis showed that coating application led to a significant decrease in strawberry aroma and flavor, especially when the ratio oleic acid:chitosan was high in the film. Ribeiro et al., 2007, studied the effects of the application of starch, carrageenan and chitosan based coatings (with and without calcium chloride) on the color, firmness, weight loss, soluble solids and microbiological growth of fresh strawberries. A minimum change of color and firmness loss was obtained for strawberries coated with carrageenan and calcium chloride. The minimum loss of mass was obtained for fruit with chitosan and carrageenan coatings, both with calcium chloride. In all cases, the addition of calcium chloride to the coatings reduced the microbial growth rate on the fruit. The minimum rate of microbial growth was obtained for strawberries coated with chitosan and calcium chloride. Hernández-Muñoz et al., 2008, studied the effect of chitosan coating combined with a postharvest calcium treatment on strawberry quality during refrigerated storage. The effectiveness of the combined treatment in extending fruit shelf-life was evaluated by determining fungal decay aspects, respiration rate, quality attributes and overall visual appearance. Chitosan coatings better delayed changes in weight loss, firmness and external color when compared to the untreated samples, and reduced respiration activity, thus delaying ripening and the progress of fruit decay due to senescence. Depending on the content level of
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chitosan, different antimicrobial activity levels were observed for the coatings. Also, the addition of calcium gluconate to the chitosan coating formulation increased the nutritional value by incrementing the calcium content of the fruit. Campaniello et al., 2008, coated strawberries with chitosan films. This research showed that chitosan coatings have the potential to prolong storage life of fresh strawberries; and, chitosan coatings on strawberries tend to inhibit fungi growth, reduce ethylene production, increase internal CO2 and decrease O2 levels. Almenar et al., 2009, reported the potential use of chitosan coatings on strawberries during the storage of the fruits to maintain desired flavor attributes. Different research has been conducted (Durango et al., 2006; Casariego et al., 2008; Adriano et al., 2009; Villalobos-Carvajal et al., 2009; Vargas et al., 2009) in order to analyze the ability of polysaccharide-based coatings to extend the shelf-life of carrots. Durango et al., 2006, applied a starch-chitosan edible coating on minimally processed carrots in order to evaluate a possible antimicrobial effect. The results of this study showed that the combined use of a chitosan coating and an edible antimicrobial yam starch promises to be a viable alternative for controlling microbiological growth in minimally processed carrots. Casariego et al., 2008, applied a chitosan coating on carrots and determined its effect on gas permeability, internal gas composition, and shelf-life. The coating inhibited fungi growth, reduced ethylene production, increased internal CO2 and decreased O2 levels. Adriano et al., 2009, propose a combined application of a chitosan-based coating and the use of modified atmosphere packaging as a postharvest treatment process to maintain quality and prolong shelf-life of carrots. The study showed that the use of a chitosan-containing edible coating preserved the overall visual quality of carrots and reduced surface whiteness during storage. The combined application of an edible coating containing chitosan, along with moderate O2 and CO2 levels, maintained quality and enhanced phenolic content in carrot sticks. Villalobos-Carvajal et al., 2009, developed hydroxypropyl methylcellulose-based coatings containing surfactant mixtures of sorbitan monostearate and sucrose palmitate in aqueous and hydroalcoholic media and analyzed the effect of the hydrocolloid/surfactant ratio; the hydrophilic–lipophilic balance of the surfactant mixtures; the solvent type used as a dispersing media on the water vapor barrier properties of the films; and, their optical effect when applied to carrot slices. The results showed that the solvent type, the hydrocolloid/surfactant ratio and the hydrophilic–lipophilic balance significantly affected viscosity, surface tension, and stability of the film-forming dispersions, which in turn had a great influence on the extensibility and final film structure of the coating on the carrot surface. All these aspects affected the water vapor barrier properties and color of coated carrot slices. Edible coatings based on high molecular weight chitosan, pure or combined with methylcellulose or oleic acid, were applied to fresh-cut carrots by simple immersion and with the application of a vacuum pulse by Vargas et al., 2009. Coatings improved sample appearance, since they diminished the occurrence of the white blush during storage. When applied by simple immersion, they neither conferred significant barrier properties nor the preservation of the mechanical properties of fresh-cut carrot samples. In contrast, the coating application with a vacuum pulse enhanced all the positive effects, since the water vapor transmission resistance of the test samples was significantly improved; additionally, a better preservation of color and mechanical response during cold storage was obtained with the test samples. Differences in film composition did not significantly affect the coating behavior; this is probably due to the variability dynamic induced by different factors when coatings were applied to the carrot surface.
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CONCLUSIONS Polysaccharide-based films and coatings have the potential to be used in a variety of food applications. They have been used to extend the shelf-life of fruits, vegetables, seafood, meats and confectionary products by preventing dehydration, oxidation rancidity, surface browning and oil diffusion. Due to their unique features, polysaccharide-based films and coatings, provide a promising technology to food companies for enhancing the quality and extend the shelf life of their products. However, commercial applications are still very limited. For industrial use, it is necessary to conduct scientific research to indentify the film-forming mechanisms of biopolymers in order to optimize their properties. This may be done by adjusting the formulation of film-forming solutions, incorporating functional ingredients and modifying film-forming conditions. More efforts are required to develop new materials and understand their functionality and interactions among the components used in the edible films and coatings. Considerable research is needed to improve the efficiencies of forming polysaccharide films and coatings on foods and to quantify the effects of the coatings on food quality using both instrumental and sensory evaluation. The development of new technologies to improve the film properties of active packaging and coatings is the major focus for future research.
REFERENCES Adriano, D. N., Simões, Tudela, J. A., Allende, A., Puschmann, R. & Gil, M. I. (2009). Edible coatings containing chitosan and moderate modified atmospheres maintain quality and enhance phytochemicals of carrot sticks. Postharvest Biology and Technology, 51, 3, 364-370. Albert, S. & Mittal, G. S. (2002). Comparative evaluation of edible coatings to reduce fat uptake in a deep-fried cereal product. Food Research International, 35, 5, 445-458. Almenar, E., Hernández-Muñoz, P. & Gavara, R. (2009).Evolution of Selected Volatiles in Chitosan-Coated Strawberries (Fragaria x ananassa) during Refrigerated Storage. Journal of Agricultural and Food Chemistry, 57, 3, 974-980. Alves, V., Costa, N., Hilliou, L., Larotonda, F., Gonçalves, M., Sereno, A, & Coelhoso, I. (2006). Design of biodegradable composite films for food packaging. Desalination, 199, 331-333. Andrady, A. L. & Xu, P. (1997). Elastic behaviour of chitosan films. Journal of Polymer Science: Part B: Polymer Phys., 35, 517-521. Anker, M. (1996). Edible and biodegradable films and coatings for food packaging literature review. Ski-Report. No: 623: Goteberg, SWEDEN. Arvanitoyannis, I., Nakayama, A. & Aiba, S. (1998). Edible films made from hydroxypropyl starch and gelatin and plasticized by polyols and water. Carbohydrate Polymers, 36(2), 105-119. Arvanitoyannis, I., Psomiadou, E., Nakayama, A., Aiba, S. & Yamamoto, N. (1997). Edible films made from gelatin, soluble starch and polyols, Part 3. Food Chemistry, 60(4), 593604.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 31
CARBOHYDRATE BINDING MODULES: FUNCTIONS AND APPLICATIONS Susana Moreira and Miguel Gama IBB, Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
1. ABSTRACT Carbohydrate-active enzymes (CAZymes) are associated to the synthesis and breakdown of complex carbohydrates and glycoconjugates. CAZymes, beside the catalytic domain (CD), usually present a substrate-binding module named carbohydratebinding module (CBM), which has independent fold and function. Nearly 7% of the CAZymes contain at least one CBM module. Presently, 58 CBMs families are described in the CAZy database (http://www.cazy.org/), presenting considerable heterogeneity in binding specificity, towards crystalline, amorphous and soluble polysaccharides, both between and within the families. CBMs are known to potentiate the activity of many enzymes, by targeting and promoting a prolonged interaction with the substrate. Since CBMs are functional and structurally independent of the other protein modules, several applications have been described using CBMs obtained by enzyme proteolysis or by DNA recombinant technology. The present revision focus on recent developments on CBMs applications in the biomedical, biological and biotechnological fields.
2. INTRODUCTION Carbohydrates play an important role in many biological and biochemical processes, including fertilization [1-3], cell differentiation and maturation [4, 5], protein folding and degradation [6]. Carbohydrates are also involved in a variety of recognition events, such as cell-cell and cell-matrix interactions [7, 8]. Many of these are immunologically relevant processes, namely inflammation [9], host-pathogen interactions [10], immune response [11] and health disorders, such as arthritis, Alzheimer’s disease [8], among others.
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Carbohydrate-active enzymes (CAZymes) play key roles in glycobiology. They are involved in the synthesis and degradation of complex carbohydrates and glyco-conjugates. CAZymes are classified in families according to the amino-acid sequence, correlating enzymes mechanism and folding rather than substrate specificity (http://www.cazy.org/). CAZymes may exhibit a modular structure, a module being defined as a structural and functional unit. These enzymes present, besides a catalytic domain (CD), a substrate-binding module named carbohydrate-binding module (CBM), the two modules being connected by a linker region [12, 13]. CBMs fold autonomously and, although not having enzymatic activity per se, are known to potentiate the activity of many enzymes, by targeting to and promoting a prolonged interaction with the substrate. The present revision does not include a description of CBM structures. Boraston et al published, recently, an excellent and comprehensive review on this subject [12]. Instead, we focus on recent developments on CBMs applications in the biomedical, biological and biotechnological fields.
3. THE CBM STORY The first CBM was described in 1986 by Tilbeurgh and coworkers [14-17]. These authors obtained two peptides by treating a cellulase from Trichoderma reesei with a protease; the higher molecular weight peptide retained the cellulolytic activity, while the smaller one exhibited cellulose affinity, therefore being designated a cellulose-binding domain. Later on, other CBM with different specificities were described. “Cellulose-binding domain” are thus a kind of a more general class of proteins, the “carbohydrate binding modules” [18, 19]. CBMs are present in a large variety of enzymes, with different functions and substrate affinities, crossing a wide range of species, from archea, bacteria and virus to eukaryotic organisms, including fungi, plant and mammalian. The CBM specificities include crystalline cellulose, non-crystalline cellulose, chitin, β-1,3-glucans and β-1,3-1,4-mixed linkage glucans, xylan, mannan, galactan and starch [12]. Furthermore, some CBMs display ‘lectin-like’ specificity, binding to a variety of cell-surface glycans [20]. The number of CBM families is still growing and since the last review on the subject [12] more than 10 new families were described. Recent findings establish a connection between CBMs and host-pathogen interactions [21], N-gycosylation in eukaryotic organisms [22], cell energy balance [23], among other functions. The knowledge of the CBM structures, elucidating their function and role in nature, may give rise to new biotechnological applications.
4. CBM CLASSIFICATION 4.1. CBM Families More than 300 proteins are currently classified in the CAZy database, including glycoside hydrolases (GHs), glicosyltransferases (GTs), polysaccharide lyases (PLs) and carbohydrate esterases (CEs). Usually, CAZymes present a modular structure, nearly 7% of the enzymes having at least one carbohydrate-binding module [13]. CBMs are also classified
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in families based on sequence similarities. Presently, 58 CBMs families are described in the CAZy database (http://www.cazy.org/). There is considerable heterogeneity in binding specificity, towards crystalline, amorphous and soluble polysaccharides, both between and within the families [12, 18, 24, 25]. A CBM is defined as a contiguous amino acid sequence within a carbohydrate-active enzyme, with a discreet fold having carbohydrate-binding activity [19]. CBMs contain from 30 to about 200 amino acids and exist as a single, double, or triple domain in proteins. The location within the parental protein can be either C- or Nterminal; occasionally, the CBM is centrally positioned within the polypeptide chain [26, 27]. A few exceptions include 1) CBMs that integrate the cellulosomal scaffoldin proteins and 2) those not associated with catalytic domains (rare instances of independent putative CBMs have been described) [12, 28, 29]. Besides having modular architecture with independent structure and function, in the more general case integrating a protein with catalytic activity, CBMs are distinguishable from other non-catalytic sugar binding proteins (such as lectins and sugar transport proteins) by the scarcity of hydrogen bonds between CBMs and their target ligands; instead, binding is dominated by hydrophobic interactions [12]. The three-dimensional (3D) structures indicate that CBMs from different families share structural similarity. The carbohydrate binding capacity can be attributed, at least in part, to several aromatic amino acids that constitute the hydrophobic surface [19, 30, 31]. Other features are also important for CBMs or CAZymes activity, namely the electrostatic environment (pH, ionic strength) and the presence of a linker [32, 33]. For instance, the enzymatic activity of many different cellulolytic enzymes is affected by the deletion, shortening or lengthening of the linker region bridging the CBM and catalytic modules [3337]. Such findings suggest that the two domains act in concert on the cellulose surface during catalysis, and that a flexible linker is needed for full cellulolytic activity.
4.1.1. Cellulosomes Cellulosomes are extracellular multiprotein complexes first identified in early 1980s, on the thermophilic anaerobic bacteria Clostridium thermocellum. Since then, several other cellulolytic bacteria and fungi have been reported to produce cellulosomes [38]. In 1999, a cellulosome holding a glycosyl hydrolase in the scaffoldin subunity was described, and later on Xu and colleagues (2004) reported another scaffoldin protein from a Bacteroides cellulosolvens cellulosome that includes a cellulase [38-43]. In general, two major types of subunit compose cellulosomes: the noncatalytic scaffoldin(s) and the catalytically active components. Each of these structures may be quite complex. The assembly of the cellulosome is facilitated by the high-affinity recognition between the scaffoldin cohesin and the enzymes dockerin modules. The scaffoldin often contains multiple cohesin modules, thereby enabling numerous different enzymes to be assembled into the cellulosome complex. In addition, in some species, such as Acetivibrio cellulolyticus, the cellulosomes present multiple scaffoldins with different cohesins [41]. The interaction cohesin-dockerins is type and specie-specific.
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Figure 1. Schematic representation of a cellulosome attached to the cell membrane. CD- catalytic domain; D- dockerin; C- cohesin, A- anchoring protein.
Another important cellulosomal component is the cellulose-specific binding module, the major determinant of substrate recognition. Only a few enzymes in cellulosomes contain a CBM; this is normally present in the scaffoldin protein [30, 38, 40, 44]. As shown for the first time by Goldstein and colleagues, the cellulose-binding protein A (CbpA) from C. cellulovorans, is a functionally independent domain of the scaffoldin protein [45]. Later on, Fierobe and coworkers, using a recombinant engineered cellulosome, showed that the proximity of the cellulosomal enzymes and the presence of the CBM3 in the scaffoldin is responsible for the synergy among the components, resulting in the efficient degradation of the native substrate [46].
4.2. CBM Types The CBMs may also be classified according to the topology of the binding sites, reflecting the macromolecular structure of the target ligand [12, 18]. Despite the large variability of carbohydrate structures, three types of binding topologies have been identified. This classification is based on both structural and functional similarities. Although the threedimensional (3D) structure of a number of CBMs has been solved, most CBMs have not been functionally characterized as yet. Furthermore, the binding pattern of CBMs determined so far vary widely, even within each family. However, it was shown that the modules are composed almost exclusively of β-strands arranged in a “jelly roll” motif, whose topography reflects the macroscopic nature of the target substrate. CBMs with this fold recognize several polysaccharides: crystalline and non-crystalline cellulose, chitin, β-1-3-glucans and β-(1-3)(1-4)-mixed glucans, xylan, mannan, galactan, and starch. Some CBMs display ‘lectin-like’ specificity, binding to a variety of cell-surface glycans [12, 18, 47]. Other families of β-
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sandwich CBMs are beginning to emerge with more complex glycan-binding specificities [48-51]. Based on the macroscopic nature of the target ligand, a classification of CBMs in three types, A, B or C, has been proposed. Table 1 presents the CBM families and corresponding type, A B or C. It should be noted that CBMs with type A and B-topology are found in the same family, while others still remains to be classified.
4.2.1. Type A CBMs Type A CBMs, with affinity for crystalline cellulose and chitin, display aromatic amino acid residues forming a planar hydrophobic surface that interacts with the glucosyl-pyranose ring of the substrate. These CBMs recognize multiple cellulose chains and strongly prefer insoluble microfibrils, such as cellulose or chitin, to soluble polysaccharides [12, 18, 19, 52]. Table 1. CBM types and families. CBMs Types Families
A 1, 2, 3, 5, 10 49
B 4, 6, 11, 15, 17, 22, 27, 28, 29, 30, 35, 36, 44 (?) 2, 20, 25, 26, 34
C 9, 13, 14, 18, 32, 40, 42
Unknown 8, 12, 16, 19, 21, 23, 24, 31, 33, 37, 38, 39, 41, 43
4.2.2. Type B CBMs Type B, the commonest class of CBMs, bind less-ordered plant structural polysaccharides such as amorphous cellulose, mannan, or xylan [12]. Conversely to type A, type B CBMs have a cleft that accommodates a single chain of the poly/oligosaccharide ligand [53], comprising several sub-sites able to interact with the individual sugar units of the polymeric ligand. Although ligands are recognized by aromatic side chains, similarly to type A CBMs, the side chains of tryptophan and tyrosine – and less commonly phenylalanine form planar, twisted or sandwich platforms for ligand binding [50]. For instance, CBM4 from xylanase of Rhodothermis marinus, a type B CBM, has a binding groove that recognizes a single polysaccharide strand [18, 54, 55]. Several applications of the CBM fused to other peptides by recombinant DNA technology were described (see below under the topic CBM applications). 4.2.3. Type C CBMs Type C CBMs have a solvent-exposed binding pocket or blind canyon, small binding sites which interact with mono or disaccharides. Thus, these CBMs are lectin-like, lacking the extended binding site grooves present in type B CBMs [12]. Indeed, type C proteins (i.e. CBM13, 14 and 18 families) were initially identified as lectins. Indeed, both kinds of protein are thought to share similar evolutionary origins. They are involved in toxin delivery, oligosaccharide synthesis, and in host-microbe interaction processes [12, 56]. For example, the β-trefoil fold of CBM13 is classified into the ‘Ricin-B-like family’ along with a bona fide lectin (ricin toxin B-chain) [57]. CBM42 also has structural similarity to ricin toxin B-chain, binding to small sugar units and displaying multivalency [58]. CBMs from families 6, 32 and 36 are structurally very similar to fucose-specific lectin of Anguilla anguilla, especially regarding the location of their metal ion and carbohydrate-
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binding sites [59]. Recently, two members of family CBM32 functionally related to lectins were described: they are galacturonic acid-binding proteins, present in the bacterial periplasmic space, that increase the substrate accessibility for pectin-degrading enzymes; however, remarkably, their action is independent of any CAZyme [48, 51].
5. CBM AT WORK In general, CBMs are linked to glycoside hydrolases that degrade insoluble polysaccharides. Although many of these modules target components of the plant cell wall or insoluble storage polysaccharides (cellulose, starch, glycogen), CBMs also bind soluble oligosaccharides such as malto-oligosaccharide [12]. Indeed, the non-catalytic CBMs are recognized as an essential component of several CAZymes and are thought to have three primary functions: proximity effects, substrate targeting and microcrystallite disruption [18, 52, 60, 61]. More recently, multivalency was also described for tandem CBMs [62, 63]. These functions are important in several biological mechanisms, such as substrate binding, mediation of protein-protein interactions or cell surface anchoring. Recently, putative cellulose-binding modules that do not bind cellulose were described. Three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were over-expressed, and surprisingly none bound to cellulosic substrates [64]. These results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the status of the family-3 CBMs and of the family-9 glycoside hydrolases is much more intricate and diverse than hitherto considered [64].
5.1. The Proximity Effect CBMs promote the association of the enzyme with the substrate, insuring a prolonged contact, and thereby increasing their effective concentration (proximity effect – Figure 2) [52, 65]. In fact, several studies show that enzymes fail to effectively perform when the CBM is removed by proteolysis or by recombinant DNA technology [12, 19, 66-69]. This effect is observed mostly in enzymes that act on insoluble substrates and in cellulosomes. This is the case of CBM3 from C. thermocellum, responsible for the cellulosome binding to the insoluble cellulose substrate [12, 70, 71].
5-2. The Targeting Effect CBMs have been shown to have selective substrate affinity, distinguishing different crystalline, amorphous, soluble and non-soluble polysaccharides (targeting function – Figure 3) [26, 68, 72-74]. In 2004, Boraston et al reviewed the mechanisms of polysaccharide recognition [18]. Since then, other CBMs with novel specificities were described. For instance, Henshaw et al showed that family 6 CBMs, present in two different β-agarases, bind specifically to the non-reducing end of agarose chains, recognizing only the first repeat of the
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disaccharide [61]. Recently, the high-resolution crystal structures of two CBM51 members, from two enzymes of C. perfringens, showed that they have highly similar β-sandwich folds. However, in spite of the structural similarity, one of the CBMs bind galactose residues, whereas the other revealed specificity for the blood group A/B antigens through nonconserved interactions [48]. The data suggest that CBMs have fine specificity for polysaccharide substructures. Thus, CBMs may be highly specific, subtle structural differences leading to diverse ligand specificity. This makes them an attractive system for biotechnological applications, namely as tools for the elucidation of protein-carbohydrate interaction mechanisms and as probes to identify different polysaccharides in plant cell-walls [75].
Figure 2. Schematic representation of the CBM mediated proximity effect. CAZymes with CBMs are able to bind to the insoluble substrates (such as crystalline cellulose) increasing the effective concentration of enzyme on substrate.
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Figure 3. Schematic representation of the targeting effect of CBMs, showing the specificity of the CBM type A for insoluble subtract (such as crystalline cellulose) and CBM type B for soluble derivates of cellulose (such as cellooligosaccharides)
5.3. The Disruptive Effect The concept of the CBM disrupting function (Figure 4), rendering the substrate more susceptible to enzymatic hydrolysis, emerged several years ago [18, 70]. It was first demonstrated in 1991, by Din et al. The non-catalytic cellulose-binding domain, isolated by these authors from endoglucanase A (Cellulomonas fimi), was able to disrupt the cellulose fibers, releasing small particles. Further, it was showed that the isolated catalytic domain did not disrupt the fibril structure, rather polishing the fibers surface [76]. Other cellulaseassociated CBMs with similar effect on cellulose fibers have been described [77]. Recently, it was showed that the CBM from CBHI (cellobiohydrolase I from T. pseudokoningii S-38) not only addresses the enzyme to the cellulose fibrils, but it also is involved in the structural disruption of the cellulose fiber surface [78]. The disruption effect was also reported for starch-binding modules [79] and for expansins, which have significant sequence identity with microbial cellulases [71, 80]. Recently, Vaaje-Kolstad and colleagues demonstrated that also chitin-binding modules have similar disruption ability. They showed that crystalline chitin is disrupted by a noncatalytic protein, leading to an increase in substrate access for a range of chitinases [63]. The modification of cellulose fibers with CBMs may lead to improved properties of textile and paper pulps [81].
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5.4. The Avidity Effect CBMs can be present in single, tandem or multiples copies within the enzymes architecture [19, 82]. It has been shown that they can bind their specific glycan targets when isolated from the parent molecule, behaving in a cooperative manner when organized in tandem [62, 83].
Figure 4. Schematic representation of the disruptive effect of the CBMs on polysaccharide fibers.
Boraston and coworkers identified a family 6 CBM present as a triplet in C. stercorarium. The multiple modules act cooperatively in the binding process. It has been suggested that the duplication or triplication of CBMs may, evolutionary, balance the loss of binding affinity of thermophilic glycosyl hydrolases at higher temperatures [62]. The analysis of CAZymes showed that the same enzyme may be linked to several CBMs (CBM multimodularity), with similar or dissimilar binding specificity. The authors speculate that the homogenous multimodularity increases the avidity of the CAZyme for the substrate, while heterogeneous multimodularity allows the enzyme to bind heterogeneous substrates [48, 51]. Recently, a recombinant protein containing tandem repeats of the CBM40 from a V. cholerae sialidase was constructed. Identical copies of CBM40 can be fused and manipulated in order to enhance its affinity through avidity [84]. This approach may be used for the creation of high affinity, multivalent CBMs, that may have broad application in glycobiology.
6. CBMS AND PHYSIOLOGIC FUNCTION Protein-carbohydrate recognition plays a pivotal role in key biological processes. These macromolecular interactions are central in host-pathogen recognition events, cell–cell communication, cellular defense mechanisms, protein trafficking, and on carbon recycling through the degradation of the plant cell wall.
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6.1. CBMs in Protein Turnover The protein turnover is an important mechanism for the regulation of numerous cellular processes. A major proteolytic route in eukaryotes involves ubiquitin (a highly conserved protein ubiquitously expressed in eukaryotes) and the 26S proteosome [6]. In most cases, selective recognition of the target proteins relies on protein–protein interactions mediated by the C-terminal domain of the F-box proteins. In mammals, the occurrence of F-box proteins with a C-terminal SBD (sugar-binding domain) that specifically interacts with high-mannose N-glycans on target glycoproteins has been documented [5]. The identification and characterization of sugar-binding F-box proteins demonstrated that also protein–carbohydrate interactions trigger the ubiquitin/proteasome pathway [6]. Recently, a close structural similarity of the protein malectin to CBMs of bacterial glycoside hydrolases was proposed. Malectin is a highly conserved protein anchored on the ER membrane (endoplasmic reticulum), with remarkable selectivity for Glc2-N-glycan, playing a role in the pathway of N-glycosylation. It has been speculated that malectin may function as a chaperon, or recruiting chaperons to protect the nascent polypeptide against aggregation, during the sensitive early synthesis period [22].
6.2. CBMs in Energy Balance and Diseases CBMs are also probably involved in the degradation of complex glycans, in human hosts, by glycoside hydrolases from pathogenic bacteria [85-87]. Recently, new CBM families have been discovered in secreted or cell surface-associated glycoside hydrolases from bacteria. These enzymes are often key virulence factors in pathogenesis [48, 88]. CBMs also play a role in several cellular mechanisms related with energy balance and disease. Recently, McBride and coworkers showed that the AMPK (AMP-activated protein kinase involved in energy balance at single cell and whole-body levels) is inhibited by glycogen and other oligosaccharides with α-1-6 branches, via its glycogen-binding domain (GBD) present in β-subunity [23]. The glycogen-binding domain has been reclassified as a member of the CBM48 [89]. It binds preferably oligosaccharides with more than five glucose units, but also tri, di, and monosaccharides. The comprehensive characterization of the CBM48 ligand recognition and binding may reveal important clues for the regulation of AMPK and its role in the cell. Another CBM with glycogen affinity belongs to laforin phosphatase, which is implied in a human disorder [26]. Several mutations in the N-terminal CBM were described [90]. The laforin’s CBM plays a role in protein dimerization [91], subtract binding [26], and interaction with malin (a single subunit E3 ubiquitin ligase necessary and sufficient to mediate ubiquitination) [92, 93]. It was demonstrated that the formation of the laforin-malin complex is a regulated process, where AMPK also plays a critical role [94]. Since laforin is involved in the glycogen metabolism, it might confer cancer resistance to energy deprivation-induced apoptosis [95]. Moreover, Gentry et al showed that the Plantae kingdom, which lacks laforin, possesses a protein with laforin-like properties called starch excess 4 (SEX4). Laforin and SEX4 (from Arabidopsis thaliana) are functional equivalents, suggesting that phosphatase laforin crosses evolutionary boundaries [96].
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The 3D structure of CBMs may elucidate the binding mechanism and specificity of several enzymes. New potential applications may emerge from this information. For example, solving the 3D structure of CBM43 from CtD-Ole e 9 (an olive pollen allergen belonging to group 2 of pathogenesis-related proteins) may be a relevant contribution for the understanding of the underlying biochemical function and help determining possible structure–allergenicity relationships, enabling the design of hypoallergenic peptides. Also, they may help elucidating the molecular basis of allergenicity and explaining why highly homologous proteins are allergenic while others are not [97, 98].
6.3. CBMs Roles in Plant: Growth, Defense and Degradation Cellulose is the major structural component of terrestrial and marine plant cell walls, although it is also produced by some marine animals and bacteria. The dimensions of cellulose fibers and the proportion of the crystalline and amorphous regions vary depending on its origin; moreover, the cellulose fibers in the higher plant cell walls are "encapsulated" by hemicellulose and lignin [29, 40, 99]. Therefore, there are a large number of enzymes acting on cellulose, due to the large variety of cellulosic materials and its different properties [29]. There are three different types of cellulases: endoglucanases (EG), cellobiohydrolases (CBH) and β-glucosidases. Together, they hydrolyze insoluble cellulose, both amorphous and crystalline, in a synergistic way, which is particularly relevant in the case of crystalline cellulose [100]. Considering that cellulose is one of the most abundant polymers in nature, it is not surprising to find organisms that modify and degrade cellulose across kingdoms and environments.
6.3.1. CBMs acting on polysaccharides: Cellulose, Starch, Chitin As referred previously, two main types of cellulolytic systems are currently recognized. Those based on ‘free’, soluble enzymes, produced mostly by aerobic microbes that secrete individual cellulases, which act synergistically on native cellulose; and those based on complexes of cellulolytic enzymes, or ‘cellulosomes’, produced by some anaerobic bacteria and fungi, which are usually attached to the outer surface of the microorganism [40, 101]. CBMs play a role in the phase transfer of a soluble free enzyme onto the insoluble substrate [68, 69]. CBMs are present in several polysaccharide-degrading enzymes, namely in hemicellulases [56, 58, 102], endomannanases [12], xylanases [56, 103], acetylxylanesterases [104] and β-glucosidases [71]. For many years, plant cellulases were thought, contrarily to bacteria and fungi cellulases, not to have CBMs. Nevertheless, putative CBM sequences were found in a tomato cellulose, in 1998 [105]. Since then, putative CBMs were described in peach [106], pear [107], strawberry [108] and mango [109]. However, only recently the first CBM from plant was described (present in a tomato cellulase), and a new CBM family was born (CBM49) [110]. The tomato cellulase is highly sensitive to proteolysis in the linker region, when expressed in a heterologous system. Therefore, the respective sequence was fused with a catalytic domain of another well studied cellulase, from T. fusca. It was shown that the fusion enzyme was able to bind and hydrolyze crystalline cellulose. CBMs are thus essential for the effective degradation of crystalline cellulose, but they are also involved in plant cell wall relaxation, expansion and biosynthesis. During the growth
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processes, plants respond to many different internal (metabolites, hormones) and external (light, water) signals. In many cases, the loosening of the cell wall arises as an answer to these signals, in order to enable turgor-driven cell expansion [71]. To enable cell wall loosening, the polysaccharide matrix has to be metabolized. Several enzymes play a role in this controlled process [111]. In fact, some of the plant cellulolytic enzymes are structure and functionally similar to those find in microorganisms. Besides its role on the cell wall degradation, several studies showed also the effects of CBMs on plant growth, development, and defense. Valdez et al presented the first report characterizing the role and function of CBMs in the enzymatic machinery of anabolic processes. They showed the importance of the N-terminal SBDs (CBM53) in the binding of starch and in the regulation and catalysis of starch synthase III from Arabidopsis thaliana [112, 113]. CBMs are also found in starch degrading enzymes, namely α-amylases, β-amylases and glucoamylases, which are widely distributed throughout many species of animals, plants and microorganisms [114]. Binding of the starch-degrading enzymes to its substrate is a critical step in starch hydrolysis, since it involves the phase transfer of a soluble enzyme to the insoluble substrate. Today, it is accepted that, similarly to other binding modules, SBMs play a pivotal role in the phase transfer [79]. The expression of artificial tandem repeats of a family 20 SBM, in an amylose-free potato mutant, resulted in the differential accumulation of SBM in the transgenic starch granules and in the production of granules with a different size [115]. Genetically fused SBMs can also be used as tools to anchor proteins (that do not have affinity for starch granules) inside starch granules, during their biosynthesis [116]. Modification of starch biosynthesis holds an enormous potential for the production of granules or polymers with new functions [117]. The understanding of the CBM-ligand interaction mechanisms may lead to the development of useful tolls for applications aiming at modulating the architecture of individual cells and even of entire organisms.
6.3.2. CBMs in Biomass conversion A significant attention is focused on the quest to replace petroleum with plant biomass for chemical and fuel production. Polysaccharide-degrading enzymes are key tools for this purpose. However, it has long been recognized that crystalline cellulose is recalcitrant to enzymatic hydrolysis, a major limitation in the production of fermentable sugars from plant biomass; therefore, CBMs that target enzymes to crystalline cellulose, promoting the hydrolytic activity, are particularly relevant for biomass conversion [18]. It has been shown that enzymes assembled in a scaffoldin protein act synergistically on the substrate hydrolysis. In addition, the CBM present in many scaffoldin proteins bind the enzymatic complex to the cellulose [40]; therefore, enzymatic complexes may be engineered as to optimize the enzymatic activities and the biomass conversion. In vitro evolution strategies utilize genes encoding thermostable proteins as suitable scaffolds. When developing thermostable enzyme, the starting material is an already stable backbone, thus improving the odds for evolution to optimize function at selected conditions for activity. An example of the application of this strategy is the diversification of the binding specificity of xylanase’s CBM4-2, from the thermophilic bacterium Rhodothermus marinus. Using this CBM, which has both high thermostability and good productivity in E. coli expression systems, a single heat stable protein could be developed with specificity towards different carbohydrate polymers [118], as well as towards a glycoprotein [119],
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demonstrating the potential of molecular biology for the development of different selective specificities, starting from a single protein scaffold [120]. The CBMs found in plant proteins, for instance tomato [110], opens new prospects for the use of CBMs in biofuel production. Plants with high expression levels of these proteins may be engineered, allowing a more efficient ethanol production. For instance, the overexpression of CBM or cellulase-containing CBM in vivo may modulate the cellulose structure as to become more accessible to other hydrolytic enzymes. Furthermore, biofuel research may also help uncovering exciting new uses for these enzymes.
6.4. Plant Development The interaction between cellulose microfibrils and the hemicellulose-xyloglucan network is believed to represent the major load-bearing structure in the primary cell wall. In physical terms, cell shape and size are governed by the mechanics of the cell wall. Cell expansion occurs via strictly regulated reorientation of the wall components and several enzymes play a rule in this mechanism [121]. In a in vitro study, it was showed that the cellulose-binding domain from C. cellulovorans modulates the elongation of different plant cells [122]. Further, using a model system of Acetobacter xylinum, it was shown that CBM enhances cellulose synthesis, which is a limiting factor in plant cell elongation [122]. In addition, there are several studies in vivo, using transgenic plants, showing that CBM-expressing plants have their growth behavior altered [123, 124]. For instance, the transgenic potato plant (Solanum tuberosum cv. Desiree) expressing the bacterial CBM3 from the C. cellulovorans CBPA, present significantly more rapid elongation of the main stem, mass accumulation and faster growth rate at earlier stage [123]. These findings suggest that the CBM may significantly alter plant growth, both in tissue culture and in vivo, under field conditions. Besides, CBMs from cellulases and expansins also play a role in cell-wall development. Expansins are plant proteins, expressed in several tissues, which catalyze the disruption of hydrogen bonds between cellulose microfibrils and matrix polysaccharides, promoting the cell expansion. They are involved in the regulation of growth and development [71, 125]. It has been shown that the over-expression of the potato expansin CBM, in transgenic tobacco plants, alter the cell wall structure, namely, stems exhibit enlarged xylem cells and thinner cells walls. However, the plant growth was not affected by the CBM expression [124]. Mixed results have been obtained when expansin genes were over-expressed in transgenic plants. Growth of tomato plants over-expressing endogenous expansin was significantly reduced [126]. However, the height of the tomato plants was not affected by the over-expression of expansin, although leaf size was slightly reduced [127]. Over-expression of OsEXP4 (expansin gene) resulted in pleiotropic phenotypes in adult transgenic rice plants [128]. A subpopulation of these transgenic plants was characterized by enhanced internodal elongation, while growth rate was inhibited in most of the rice transformants. The authors suggested that differences in expression levels may provide a possible explanation for these phenomena. According to this explanation, lower expression levels promote stem elongation, while higher levels promote leaf development with shorter internodes.
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6.5. Plant Defense Among eukaryotic plant pathogens, the genus Phytophthora contains more than 60 species that are pathogenic on a wide array of plants, originating diseases economically important worldwide. The cellulose binding elicitor lectin (CBEL), from Phytophthora parasitica nicotianae, contains two cellulose binding modules belonging to family 1, found almost exclusively in fungi (not detected in higher plants). CBEL is able to bind crystalline cellulose and tobacco cell walls in vitro, in a dose-dependent manner, but in contrast with cellulases, it does not possess any detectable enzyme activity on various polysaccharides [129]. Site-directed mutagenesis of aromatic amino acid residues located within the CBMs, as well as leaf infiltration assays using mutated and truncated recombinant proteins, showed that CBMs are sufficient and necessary for the expression of genes associated to plant defense. Moreover, it has been shown that the CBM1 from swollenin (a protein first identified in the saprophytic fungus Trichoderma reesei), is essential for the protein’s activity and capable of stimulating local defense responses in cucumber roots and leaves, affording for local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBM may be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction [130]. However, further studies will be necessary to elucidate how CBMs act on cells-wall defense mechanisms. The understanding of responses to cell wall damages may contribute to the development of strategies for the improvement of the resistance of plants to biotic stresses [131].
7. CBM APPLICATIONS The production and process development using CBMs obtained by enzymatic hydrolysis of the enzyme-containing CBMs was described by several authors [132-134]. The recombinant DNA technology allows for several structural and functional studies, and also for different applications using recombinant proteins fused with CBMs. Different applications of CBMs have been described: improvement of fibers in textile and paper industry; tags for recombinant proteinpurification and immobilization; probes for protein-carbohydrate interaction and microarrays. CBMs may also find applications in the modification of physical and chemical properties of composite materials, allowing the creation of new materials with improved properties. The CBMs expression in vivo may be also a valuable tool to modify plant characteristics, as discussed above [71]. Several studies have shown that CBMs may be used to modify the characteristics of enzymes. The basic approach in CBM engineering consist in the addition or substitution of a CBM in order to improve the enzyme stability or hydrolytic activity [135].
7.1. CBMs in the Paper Industry The Kraft pulping is the predominant process used in papermaking. This process involves the high temperature cooking of wood fibers in alkali, followed by the extraction of the colored lignin, using oxidative chemicals [136]. The treatment of wood pulp with hemicellulases has been shown to be a feasible way to enhance the extraction of lignin, in the
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quest for environmentally benign methods of paper manufacture [55, 136]. This enzymatic pre-treatment leads either to higher final paper brightness or to a reduction in the consumption of bleaching chemicals. Enzymes used in pulping can be used to increase the yield of fibers, reduce the refining energy requirements and finally to provide specific modifications to the fiber. Although the effect of enzyme treatment depends on the fiber type [137], several works showed the enhancement of the kraft pulping of sycamore chips and other pulp sources using cellulase, hemicellulase, and pectinase in pulp fibers pretreatments [55, 136, 138, 139]. However, the utilization of hydrolytic enzymes in paper production often results in the loss of tensile strength along with the desired performance [140]. Since CBMs, although not having catalytic activity, can modify the cellulose and starch materials, they have been also tested in the papermaking process. Several strategies were developed using CBMs or CBMs-conjugates [139, 141, 142]. It is well known that the incorporation of secondary fiber in paper production affects the final product quality and the papermaking process. Basically, the recycled pulps have lower fiber quality (smaller fibres with lower flexibility) and higher drainage resistance. These modifications lead to reduce inter-fiber bonding and consequently lower paper strength. Secondly, the sheet formation becomes more difficult, decreasing the paper machine runnability and increasing the energy consumption in the dryers [143]. Mooney et al. reported evidence that selective digestion of the smaller fiber pulp fragments results in increased drainage [137]. However, this effect could also be attributed to removal of cellulose microfibrils from the paper fiber surfaces. In either case, care must be exercised to keep treatments at a low dose, because endoglucanases that attack amorphous cellulose also cause rapid loss of fiber strength [139]. In an attempt to avoid the strength loss, Pala et al. [143] examined the use of cellulose-binding modules isolated from cellulases , following proteolytic digestion. At low doses, CBMs increased both drainage rates and paper strength properties, but at higher dosage rates, the beneficial effect on the strength parameters was less pronounced. The authors hypothesized that the beneficial effect on strength was attributable to an increase in the microfibrilation of the fiber surface. Increased drainage, however, may also be ascribed to the residual hydrolytic activity present in the used CBM formulations. Recently, Machado et al [144] demonstrated that a recombinant CBM from C. thermocellum conjugated with PEG effectively improves the pulps drainability, without significant effects on the strength parameters. Furthermore, the authors showed that the CBM alone does not modify the pulp properties, suggesting that the improved pulp drainability, reported by several researchers, is indeed a strictly interfacial effect, surface hydration playing a key role. Taken together, the results suggest that CBM technology may have valuable applications in paper recycling. CBMs can also be used combined with other molecules, as CBM-conjugates. Recently, Kitaoka and Tanaka described a CBM-based additive that enhances the paper strength [141]. Functional design of paper materials is in general achieved by addition of papermaking chemicals to an aqueous fiber suspension, at the wet end of a paper machine. The effect of additives is often affected by electrostatic interactions with contaminants present in this wetend step [142]. This limitation may be circumvented through the development of CBM-based papermaking additives, for improved retention and performance. CBMs obtained by hydrolysis of cellulase with papain andchemically bound to anionic polyacrylamide, retained the ability to interact with pulp fibres, leading to a good retention and high tensile strength under wet-end conditions [142, 145].
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Recombinant CBMs were also used to improve paper properties. The dry strength of a three-dimensional cellulose fiber network depends on the strength of the individual fibers, of the inter fiber bonds, and on the number and distribution of interfiber bonds. Inter-fiber bonding, which improves the stress transfer between the fibers under tensile deformation, is one of the most important factors affecting the overall stress development in the fiber web [146]. Levy et al. constructed a bifunctional protein, containing two-fused cellulose-binding modules (CBM3 from C. cellulovorans), able to mimic the chemistry of cellulose crosslinking [66], thus increasing the dry strength of paper. Interestingly, applying a single CBM to the paper also improved its mechanical properties, although to a lower extent. In addition, paper sheets treated with the fusion protein became more hydrophobic and demonstrated water-repellent properties [66]. Later on, the same authors constructed another bifunctional protein containing a cellulose and a starch-binding module. The treatment of paper fibers with the recombinant protein, together with corn-starch, improved the paper dry strength [146]. The significant improvement in the mechanical and surface properties of paper by CBMscontaining molecules demonstrates great potential for the bioengineering of novel papermodification reagents [66].
7.2. CBMs in the Textile Industry The textile industry requires large amounts of water, energy, and auxiliary chemicals [147]. The search for environmental-friendly methods has lead to the utilization of enzymes. Several enzymes have been used in textile processes in order to achieve improved and fashionable fabric properties. Among other enzymes for textile processing, amylases (used for desizing), cellulases (denim finishing), laccase (decolourization of textile effluents and textile bleaching) are commercially available [148, 149]. Textile fabric may also be treated with isolated CBMs or CBMs fused with other molecules or enzymes. Banka et al. demonstrated that a fibril-forming protein from T. reesei causes non-hydrolytic disruption of cotton fibers [150]. Lee et al. obtained images, by atomic force microscopy, of holes left in cotton fibers treated with inactivated CBH I. The holes are attributed to the penetration of fibers by the binding domain [151]. It has been shown that the surface of ramie cotton is roughened by treatments with CBM2 from C. fimi. Gilkes et al proposed that the treatment of cellulosic fibers with CBMs could be used in order to alter the dyeing characteristics of cellulose fibers [152]. Indeed, it was showed that CBM treatment increased the dye affinity of cotton fibers, especially in the case of acid dyes [153]. Lemos et al. (2002) developed a simple method to purify CBMs from fungal cellulases, by ultrafiltration following digestion with a protease. Using this process, it is possible to obtain gram amounts of CBMs, although with some contaminating enzymatic activity. Those CBMs were used in textile deppiling assays. The CBMs preparation - with residual hydrolytic activity - achieved a superior finishing deppiling (Figure 5). Further, using CBM-FITC (Fluorescein isothiocyanate) conjugates, Ramos et al demonstrated that the surface concentration of CBMs adsorbed to cotton fibers is very high [154]. Considering these results, it seems obvious that interfacial properties should be also considered in the design of depilling treatments. Fukuda et al used a new approach for the enzymatic desizing of starched cotton cloth. Sizing is required to prevent abrasion, fluffiness, and cutting of the warp during the weaving
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process. Among the several desizing methods, the use of enzymes (e.g.amylase) is well known as an environmental-friendly technology [155]. Instead of using an enzyme for desizing, Fukuda et al constructed a yeast strain that codisplayed glucoamylase and CBMs on the cell surface. The yeast cell acquired specific binding ability to cotton cloth with glucoamylase activity. Furthermore, the codisplaying strain showed greater activity than a strain displaying only glucoamylase activity [156]. The development of biotechnological tools for the modification of cellulose fibers may be achieved by combining CBMs, specially cellulose binding modules, with catalytic domains of enzymes that do not normally act on insoluble substrates (e.g. laccase, pectinase or lipase), or with other functional proteins/polypeptides (e.g. hydrophobic or chemically reactive) suitable for the modification of the textile surfaces. Since several CBMs belong to enzymes that act in extreme conditions, the CBM fusion proteins may also improve enzyme stability [103, 135, 157, 158]. Further, CBMs can be fused with bioactive molecules in order to functionalize the fabric tissue.
7.3. CBMs in the Food Industry Enzymes have been used for more than 20 years in poultry feed, mainly to improve the digestibility of cereals with high soluble non-starch-polysaccharide (NSP) levels, such as wheat, barley, oats and rye [159, 160]. It is well established that the inclusion of cell wall hydrolases in wheat, barley and rye-based diets of single-stomach animals, improves the efficiency of feed utilization, enhances growth and contributes to a better use of low-cost feed ingredients [161, 162]. Several studies on the potential application of CBMs for animals feeding are available [163, 164]. CBMs anchor cellulases and hemicellulases to their target substrates, therefore eliciting efficient hydrolysis of recalcitrant polysaccharides. Recombinant derivatives of a xylanase from C. thermocellum, containing a CBM6, were used to supplement poultry cereal-based diets. The data obtained showed that birds fed on a wheat-based diet supplemented with CBM-xylanase display an increased final body weight, when compared with birds receiving the xylanase catalytic module only [163]. Recently, Ribeiro et al studied the effect of supplementing a barley-based diet with a family11 β-glucan-binding domain, fused to a recombinant cellulase from C. thermocellum. The results showed that birds fed on diets supplemented with the recombinant proteins, containing the CBM11 or the commercial enzyme mixture, have improved performance when compared to birds fed with diets without the enzyme supplement [162]. Tarahomjoo et al presented a new strategy to enhance the viability of probiotics (living microbial cells as food supplement) in simulated gastric conditions. This strategy consists on the use of starch-binding modules displayed on the cell surface. It is known that starch granules partially hydrolyzed (with hollow cores) may be used to encapsulate living microbes, so protecting them from environmental stress [165, 166]. For this purpose, a protein containing the C-terminal region of a peptidoglycan hydrolase (an efficient anchoring domain to display heterologous proteins on cells) fused to the linker region and the SBM of the α-amylase (from Streptococcus bovis 148) was constructed. The fusion protein was able to bridge the cell surface of Lactobacillus casei NRRL B-441 (a well known probiotic bacteria) and corn starch. The potential usefulness of the SBM cell-surface display technique
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for the encapsulation of microorganisms and its delivery to the intestinal tract was demonstrated. This strategy does not involve any genetic modification of the probiotic strains [166]. Recombinant enzymes containing CBM may also find applicability on the food industry, for instance for the production of soy sauce using soybeans and optionally other vegetable ingredients, such as wheat and rice. During the production process, starch and other carbohydrates are degraded into sugars, used for aroma development by fermentation. It has been found that amylolytic enzymes comprising a CBM leads to an increased rate of starch hydrolysis, as compared to amylolytic enzymes without CBM, under conditions relevant for soy sauce production [167].
Figure 5. Images of cotton fabrics before and after depilling treatment. Non-treated fabric (a, b), enzyme treated fabric (c, d) and CBM treated fabric (e, f) observed by electronic microscopy at 30x magnification [adapted from 154].
7.4. CBMs as a Microarray and Probing Tool Microarrays of proteins and peptides make the screening of thousands of binding events possible, in a parallel and high throughput fashion; therefore, they are emerging as a powerful tool for proteomics and clinical assays. Microarrays consist of immobilized biomolecules spatially organized on substrates such as planar surfaces (typically coated microscope glass slides), microwells or arrays of beads. Immobilized biomolecules (probes) usually include oligonucleotides, PCR products, proteins, peptides, carbohydrates and other small molecules
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[168]. Several strategies are described to immobilize the probe on the support, including adsorption, physical entrapment or covalent binding. The CBM based microarray technology described by some authors offer fundamental advantages over current non-DNA microarray technology, such as retention of protein functionality after immobilization, ease of fabrication, extended stability of the printed microarray, integrated test for quality control (QC) and the capacity to print test proteins without a purification step [55, 169, 170]. These features, together with the intrinsic specificity of CBMs for individual carbohydrates and the facile modification with peptides and fluorescent molecules, allow for efficient production of protein and peptide microarrays. These can be used in a variety of potential applications technically impractical via conventional microarray technologies [55]. Ofir and colleagues developed a microarray system using an affinity-based probe immobilization strategy. They fused the exceptionally stable family-3a CBM, from the cellulosome of C. thermocellum, with antibodies or peptides. The recombinant proteins were immobilized on cellulose surfaces by specific adsorption and used for serodiagnosis of human immunodeficiency virus patients [168, 170]. Recently, Haimovitz and colleagues described a microarray system to analyse the cohesin-dockerin specificity. These authors immobilized recombinant CBM-fused cohesins on cellulose-coated glass slides, to which xylanase-fused dockerin proteins were applied. The fusion of dockerins with a thermostable xylanase was performed, allowing enhanced expression and proper folding. Using this elegant approach, cross-species interactions among type-II cohesins and dockerins was shown for the first time [171]. The plant cell wall biology studies require more sensitive and specific probes to target individual wall components. Traditionally, antibodies have been the primary workhorses for the spatial localization of cell wall polysaccharides. Currently, nearly 30 monoclonal antibodies directed toward specific arabinan, galactan, xylan, galacturonan, fucosylated xyloglucan, and cell wall glycoprotein epitopes are available, from academic and commercial sources [172]. Nevertheless, CBMs may be used for this purpose, since they present intrinsic specificity for individual carbohydrates. A quantitative fluorimetric method for the analysis of crystalline cellulose on fiber surfaces was developed. This method quantitatively shows differences in crystalline cellulose binding sites of differently processed pulp fibers. The results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of non uniform substrate surfaces, for example, during wood or pulping processes and possibly also during fiber biosynthesis [169]. The CBM4-2 from xylanase of Rhodothermus marinus was synthesized and utilized in vivo as a xylan-specific protein, for the analysis of hemicelluloses in wood and fibrous materials. It is well known that the CBMs specificity may be altered by genetic engineering; in particular, the CBM4-2 was modified through direct mutagenesis. Variants with specificity for two other polysaccharides were identified using phage display technology [118].
7.5. CBMs as a Protein Solubilization, Purification and Immobilization Tool Several works describe the use of CBMs as a tag for recombinant protein purification [24, 173-178] and enzyme immobilization [179-181]. Depending on the binding reversibility,
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different applications may be envisioned; CBMs with ‘irreversible’ binding has limited usefulness as an affinity tag for protein purification, because desorption may require strongly denaturing conditions. In turn, such a CBM may be a very useful tag for enzyme immobilization [182]. An obvious extension of the CBM-fusion technology is to enable a single-step purification and immobilization of fusion proteins by generating active CBM–Protein. Moreover, the utilization of the carbohydrate affinity system, such as cellulose, is attractive because it does not require a derivatized matrix, and cellulose is available in a variety of inexpensive forms, such as preformed microporous beads, highly adsorbent sponges or cloth and microcrystalline powders [181]. In fact, several CBMs were already commercialized as protein expression systems [179]. A cellulose-binding module from C. cellulovorans scaffoldin CbpA protein has been well characterized and commercialized as a fusion domain for protein purification, using a cellulose matrix (Novagen). In such applications, the use of CBMs offers many industrially attractive advantages. Since CBMs adsorb spontaneously to cellulose, very little or no pretreatment of the samples is required prior to immobilization [67, 183]. In addition, some CBMs seem to enhance the solubility of recombinant protein [184, 185]. Craig and colleagues described the design and application of a recombinant fusion protein containing a cellulose-binding domain (from C. cellulovorans) and an antibodybinding domain (protein LG), for direct immobilization of antibodies and cells onto regenerated cellulose hollow fiber membranes. Hollow fiber affinity cell separation is a monoclonal antibody based cell separation process. Cells are bound directly or indirectly via surface epitopes by monoclonal antibody or secondary ligand immobilized on the lumen side of hollow fibers. Deposited cells are fractionated, on the basis of adhesion strength, using the uniform shear field generated by the culture medium flowing through the hollow fiber modules with well-defined header geometry [186]. With this strategy, several problems associated to covalent binding are avoided: low coupling yield, random orientation of antibody, possible alteration of the structural properties of the hollow fiber membrane resulting from chemical cross-linking or protein degradation.
7.6. CBM as Bioremediation Tool Another field for CBM application is bioremediation. Richins et al. produced a bifunctional fusion protein, consisting of an organophosphate hydrolase (OPH) linked to a Clostridium-derived cellulose-binding module. The recombinant hydrolase is highly effective in degrading organophosphate compounds. Furthermore, the CBM enable the purification and immobilization onto different cellulosic materials, in a single step [181]. In this manner, OPH-activated cellulose materials are generated for a variety of relatively low cost applications, such as reactors with immobilized enzyme for the detoxification of hazardous organophosphates [181]. In another study, Xu et al presented a strategy to remove heavy metals from contaminated waters. They reported the cloning and expression of a bifunctional fusion protein, consisting of a synthetic phytochelatin linked to a Clostridium-derived cellulose-binding domain. Once again, the CBM enabled purification and immobilization of the fusions onto different
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cellulose materials, in a single step. The immobilized sorbents were shown to be highly effective in removing cadmium present in parts per million levels [187].
7.7. CBM as Biomedical Tool Cellulose is a chemically inert matrix that has stable physical properties, as well as low affinity for non-specific protein binding. It is pharmaceutically safe and relatively inexpensive. The binding of biomolecules to cellulose through a cellulose-binding domain further enhances its potential as a scaffold or carrier material. Maurice et al fused an antigen protein (from Aeromonas salmonicida) with a CBM (from C. cellulovorans), in order to develop a vaccine suitable for fish immunization. Vaccines vary in their efficacy depending on the antigen composition and accompanying adjuvant. Studies have shown that soluble immunogens rarely induce high titers of antibodies, unless strong adjuvants are used [188]. Surprisingly, binding Orbicell cellulose beads to a recombinant protein, Maurice and colleagues obtained a significant adjuvant effect. In addition, Orbicell cellulose beads were well tolerated by the fish and no deleterious response reactions were detected [188]. Guerreiro et al recently described the expression of AMPs (antimicrobial peptides) fused with a CBM3 from C. thermocellum in a bacterial host. AMPs are cationic molecules with a wide range of antimicrobial activities. The authors suggested CBM3 as a good candidate to overcome difficulties related to the expression of these molecules, namely associated to the small size and potential toxicity for host [189]. Furthermore the authors suggested the possible use of the fusion CBM-AMP to confer antimicrobial properties to cellulosic materials. CBMs were also described as a tool to adsorb bioactive peptides to carbohydrate-based materials [190-192]. Bacterial cellulose is being studied as a biocompatible scaffold for the engineering of cartilage and blood vessels, wound dressing, guided tissue regeneration, among other applications [193]. Andrade et al cloned and expressed a recombinant protein containing a cellulose-binding module (CBM3 from C. thermocellum cellulosome) fused with a tripeptide of Arg-Gly-Asp (RGD sequence is a ligand for integrin-mediated cell adhesion), showing that the bifunctional protein improved the fibroblast adhesion and spreading on bacterial cellulose [190]. Using a CBM with starch affinity (SBM20 from Bacillus sp. amylase) fused with RGD, it was possible to observe similar results for fibroblast adhesion and spread on starch-based hydrogel (Figure 6) [192]. The utilization of a recombinant CBM (a domain from the Celk gene from C. thermocellum) to stabilize single-walled carbon nanotubes (SWNTs) in water was recently described [194]. After production of SWNTs, the strong non-covalent interactions give rise to aggregated material. Functional molecules including surfactants, polymers, carbohydrates, nucleic acids and peptides or proteins have been reported to debundle and suspend SWNTs via a non-covalent adsorption. A family 4 CBM, cloned and over-expressed in E. coli, was successfully used to stabilize SWNTs. However, the mechanism of SWNTs - protein interaction has not been explained. Moreover, another recombinant CBM belonging to family 3 (type A) was also tested, but it did not show binding affinity for SWNTs. The authors suggested that, beside aromatic residues, higher-order protein structure could also play a key role [194].
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Figure 8. Results from fibroblast adhesion on starch-based hydrogel. Top – microscopic images showing cell morphology and spread on hydrogel treated with recombinant proteins (SBM or RGDSBM) and control hydrogel without recombinant protein and polystyrene plate. Bottom – results from viability assay of cell attached to the hydrogels or polystyrene plate [adapted from 192].
8. FUTURE PERSPECTIVES Although the functions of CBMs were firstly related to the activity of cellulase and other enzymes, the current CBM research heads in different directions. CBMs are involved in anabolic processes (such as oligosaccharide synthesis), host-microbe interaction, toxin delivery, recognition of complex glycan present on eukaryotic cell surface and extracellular matrix. CBMs may thus be used as tools to elucidate several carbohydrate-protein interactions and targets for the modulation of those processes. The determination of the 3D structures and mechanism of action of protein modules, such as CBM from family 6 or 2a [195] is still ongoing. The finding of new 3D structures may help elucidating the evolution of CBMs. The combined effect of CBMs from glycoside hydrolases in the recognition of host glycans by bacteria for pathogenesis, colonization, as a nutritional source, and evading the host immune system, defines a new avenue of CBM research, apart from plant cell wall recognition. In recent years, besides the utilization in textile or paper industry, the CBMs are seen as tools for biomedical application. Future studies may also reveal new avenues for biotechnology applications, such as design of antibacterial or anti-carcinogenic drugs, functionaliztion of biomaterials or cloth.
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[139] Lenting, HBM; Warmoeskerken, MMCG. Guidelines to come to minimized tensile strength loss upon cellulase application. Journal of Biotechnology, 2001, 89(2-3), 227-32. [140] Kitaoka, T; Tanaka, H. Novel paper strength additive containing cellulose-binding domain of cellulose. Journal of Wood Science, 2001, 47(4). [141] Yokota, S; Matsuo, K; Kitaoka, T; Wariishi, H. Retention and paper-strength characteristics of anionic polyacrylamides conjugated with carbohydrate-binding modules. BioResources, 2009, 4(1), 234-44. [142] Pala, H; Lemos, MA; Mota, M; Gama, FM. Enzymatic upgrade of old paperboard containers. Enzyme and microbial technology, 2001, 29(4-5), 274-9. [143] Machado, J; Araújo, A; Pinto, R; Gama, FM. Studies on the interaction of the Carbohydrate Binding Module 3 from the Clostridium thermocellum CipA scaffolding protein with cellulose and paper fibres. Cellulose, 2009, accepted. [144] Yokota, S; Matsuo, K; Kitaoka, T; Wariishi, H. Specific interaction acting at a cellulose-binding domain/cellulose interface for papermaking application. BioResources., 2008, 3(4), 1030-41. [145] Levy, I; Paldi, T; Shoseyov, O. Engineering a bifunctional starch–cellulose cross-bridge protein. Biomaterials, 2004, 25(10), 1841-9. [146] Feitkenhauer, H; Meyer, U. Integration of biotechnological wastewater treatment units in textile finishing factories: from end of the pipe solutions to combined production and wastewater treatment units. J Biotechnol., 2001, 89, 185-92. [147] Araújo, R; Casal, M; Cavaco-Paulo, A. Application of enzymes for textile fibres processing Biocatalysis and Biotransformation, 2008 332-349, 26(5). [148] Saravanan, D; Vasanthi, NS; Ramachandran, T. A review on influential behaviour of biopolishing on dyeability and certain physico-mechanical properties of cotton fabrics. Carbohydrate Polymers, 76 (2009) 1-7, 2009, 76, 1-7. [149] Banka, RR; Mishra, S; Ghose, TK. Fibril formation from cellulose by a novel protein from Trichoderma reesei: A nonhydrolytic cellulolytic component? World J Microbiol Biotechnol., 1998, 14, 551-8. [150] Lee, I; Evans, BR; Woodward, J. The mechanism of cellulase action on cotton fibres: evidence from atomic force microscopy. Ultramicroscopy, 2000, 82, 213-21. [151] Gilkes, NR; Douglas, Q; Miller, RC Jr.; Warren, A. inventors; University of British Columbia (Vancouver, CA), assignee. Methods and compositions for modification of polysaccharide characteristics. United States1998. [152] Cavaco-Paulo, A; Morgado, J; Andreaus, J; Kilburn, DG. Interactions of cotton with CBD peptides. Enzyme Microb Technol., 1999, 25, 639-43. [153] Ramos, R; Pinto, R; Mota, M; Sampaio, L; Gama, FM. Textile depilling: Superior finishing using cellulose-binding domains with residual enzymatic activity. Biocatalysis and Biotransformation, 2007, 25(1), 35-42. [154] Murai, T; Ueda, M; Yamamura, M; Atomi, H; Shibasaki, Y; Kamasawa, N; et al. Construction of a starch-utilizing yeast by cell surface engineering. Appl Environ Microbiol., 1997(63), 1362-6. [155] Fukuda, T; Kato-Murai, M; Kuroda, K; Ueda, M; Suye, S. Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulosebinding domain. Appl Microbiol Biotechnol., 2008, 77, 1225-32.
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[172] Kavoosi, M; Meijer, J; Kwan, E; Creagh, AL; Kilburn, DG; Haynes, CA. Inexpensive one-step purification of polypeptides expressed in Escherichia coli as fusions with the family 9 carbohydrate-binding module of xylanase 10A from T. maritima. J Chromatogr B Analyt Technol Biomed Life Sci., 2004, 807(1), 87-94. [173] Kavoosi, M; Sanaie, N; Dismer, F; Hubbuch, J; Kilburn, DG; Haynes, CA. A novel two-zone protein uptake model for affinity chromatography and its application to the description of elution band profiles of proteins fused to a family 9 cellulose binding module affinity tag. Journal of Chromatography A., 2007, 1160(1-2), 137-49. [174] Rodriguez, B; Kavoosi, M; Koska, J; Creagh, AL; Kilburn, DG; Haynes, CA. Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag. Biotechnol Prog., 2004, 20(5), 1479-89. [175] Shpigel, E; Goldlust, A; Eshel, A; Ber, IK; Efroni, G; Singer, Y; et al. Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain. Biotechnol Appl Biochem., 2000, 31(3), 97-203. [176] Ito, S; Kuno, A; Suzuki, R; Kaneko, S; Kawabata, Y; Kusakabe, I; et al. Rational affinity purification of native Streptomyces family 10 xylanase. Journal of Biotechnology, 2004, 110(2), 137-42. [177] Kavoosi, M; Creagh, AL; Kilburn, DG; Haynes, CA. Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli. Biotechnology and Bioengineering, 2007, 98(3), 599-610. [178] Xu, Y; Foong, FC. Characterization of a cellulose binding domain from Clostridium cellulovorans endoglucanase-xylanase D and its use as a fusion partner for soluble protein expression in Escherichia coli. J Biotechnol., 2008, 135(4), 319-25. [179] Ong, E; Gilkes, NR; Warren, RAJ; Miller, RC; Kilburn, DG. Enzyme Immobilization Using the Cellulose-Binding Domain of a Cellulomonas Fimi Exoglucanase. Nature Biotechnology, 1989, 7, 604-7. [180] Richins, RD; Mulchandani, A; Chen, W. Expression, immobilization, and enzymatic characterization of cellulose-binding domain organophosphorus hydrolase fusion enzymes. Biotechnology and Bioengineering, 2000, 69(6), 591 - 6. [181] Kwan, EM; Boraston, AB; McLean, BW; Kilburn, DG. A.J. WR. N-Glycosidase– carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation. PEDS, 2005, 18(10), 497-501. [182] Morassutti, C; De Amicis, F; Skerlavaj, B; Zanetti, M; Marchetti, T. Production of a recombinant antimicrobial peptide in transgenic plants using a modified VMA intein expression system. FEBS Letters, 2002, 519(1-3), 141-6 [183] Murashima, K; Kosugi, A; Doi, RH. Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans. Proteins: Structure, Function, and Bioinformatics, 2003, 50(4), 620 - 8. [184] Yeh, M; Craig, SJ; Lum, M; Foong, FC. Effects of the PT region of EngD and HLD of CbpA on solubility, catalytic activity and purification characteristics of EngDCBDCbpA fusions from Clostridium cellulovorans. Journal of Biotechnology, 2005, 116 233-44. [185] Craig, SJ; Shu, A; Xu, Y; Foong, FC; Nordon, R. Chimeric protein for selective cell attachment onto cellulosic substrates. PEDS., 2007, 20(5), 235-41. [186] Xu, Z; Bae, W; Mulchandani, A; Mehra, RK; Chen, W. Heavy metal removal by novel CBD-EC20 sorbents immobilized on cellulose. Biomacromolecules, 2002, 3(3), 462-5.
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[187] Maurice, S; Dekel, M; Shoseyov, O; Gertler, A. Cellulose beads bound to cellulose binding domain-fused recombinant proteins; an adjuvant system for parenteral vaccination of fish. Vaccine, 2003, 21(23), 3200-7. [188] Guerreiro, CIPD; Fontes, CMGA; Gama, FM; Domingues, L. Escherichia coli expression and purification of four antimicrobial peptides fused to a family 3 carbohydrate-binding module (CBM) from Clostridium thermocellum. Protein Expression and Purification, 2008, 59, 161-8. [189] Andrade, FK; Moreira, SMG; Domingues, L; Gama, FMP. Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate-binding modules fused to RGD. J Biomed Mater Res A., 2008. [190] Carvalho, V; Domingues, L; Gama, M. The inhibitory effect of an RGD-human chitinbinding domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films. Mol Biotechnol., 2008, 40(3), 269-79. [191] Moreira, SM; Andrade, FK; Domingues, L; Gama, M. Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence. BMC Biotechnology, 2008, 8(78). [192] Svensson, A; Nicklasson, E; Harrah, T; Panilaitis, B; Kaplan, DL; Brittberg, M; et al. Bacterial cellulose as a potential scaffold for tissue engineering of cartilage. Biomaterials, 2005, 26(4), 419-31. [193] Xu, Q; Song, Q; Ai, X; McDonald, TJ; Long, H; Ding, S; et al. Engineered carbohydrate-binding module (CBM) protein-suspended single-walled carbon nanotubes in water. Chem Commun., 2009, 337-9. [194] Michel, G; Barbeyron, T; Kloareg, B; Czjzek, M. The family 6 carbohydrate binding modules have co-evolved with their appended catalytic modules towards similar substrate specificity. Glycobiology, 2009.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 32
BIODEGRADATION OF CELLULOSE FIBERS AND ITS INHIBITION BY CHEMICAL MODIFICATION 1
Barbara Simončič*, 1Brigita Tomšič, 2Boris Orel** and 2Ivan Jerman
1
University of Ljubljana, Faculty of Natural Sciences and Engineering, Department of Textiles, Aškerčeva 12, 1000 Ljubljana, Slovenia. 2 National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
ABSTRACT Natural cellulose fibers are high-molecular polymers that are strongly susceptible to microbial degradation. Fungal and bacterial attack on the fibers cause depolymerization of cellulose macromolecules, which is reflected in decreased molecular weight and strength, increased solubility and a changed crystallinity. Although biodegradable textile fibers are classified as environmentally-friendly materials, the biodegradation process that occurs when the textile product is still in use could cause serious functional, aesthetic and hygienic problems because of textile deterioration, staining, discoloration and odor. To protect the cellulose against biodegradation, chemical modification is of great importance. In this research, therefore, fibers were chemically modified by the use of an antimicrobial finish on the basis of AgCl (AG) in combination with a reactive organicinorganic binder (RB), water and oil repellent finishes on the basis of fluoroalkylfunctional siloxane (FAS), perfluorooctyltriethoxysilane (PFOTES) in combination with di-ureapropyltriethoxysilane [bis(aminopropyl)terminatedpolydimethylsiloxane (1000) (PDMSU) and aminopropyl-perfluoroisooctyl polyhedral oligomeric silsesquioxane (AP2PF2IO4 POSS), as well as with an easy-care and durable press finish on the basis of imidasolidinone (DMeDHEU). Moreover, it was expected that these finishes would provide the active or passive antimicrobial properties of the modified fibers. Biodegradation of the finished cellulose fibers was carried out by the soil *
Corresponding authors: E-mail:
[email protected], Phone: 00386-(0)1-2003-231; Fax: 00386(0)1-2003-270 ** E-mail:
[email protected]); Phone: 00386-(0)1-4760-276; Fax: 00386-(0)1-4760-300
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Barbara Simončič, Brigita Tomšič, Boris Orel et al. burial test according to SIST EN ISO 11721-2:2003. The chemical and functional properties of the fibers were determined by means of DP, SEM, AFM, FT-IR, XPS, ICPMS, appropriate microbiological tests and static contact angle measurements of different liquids on the fiber surface. It can be seen from the study that AgCl embedded in RB imparts active antibacterial and antifungal properties to the coated fibers resulting in excellent microbial reduction and, consequently, strong inhibition of biodegradation. The presence of FAS, PFOTES-PDMSU and AP2PF2IO4 POSS coatings, which cause an increase in fiber hydrophobicity, as well as a high drop in surface free energy, results in decreased adhesion of microorganisms and impaired conditions of their growth. In the modification process of cellulose fibers by DMeDHEU, the formation of covalent bonds between the finish and the cellulose macromolecules strengthens the less ordered amorphous regions, resulting in a decrease of fiber swelling. This inhibits the penetration of microorganisms into the fibers, where biodegradation takes place. By applying a combination of FAS and DMeDHEU finishes, a synergistic action of the components is obtained in the coating. In this case, the protective properties of the coating against biodegradation are much higher than those obtained by single component finishing.
1. INTRODUCTION The study of biodegradation of cellulose fibers and their antimicrobial protection is a contemporary interdisciplinary research topic, which includes investigations in the fields of textile chemistry, polymer chemistry, biochemistry, microbiology, medicine, pharmacy, botany and ecology. Biodegradation is obviously an important and desirable process in waste textile materials, leading to their decomposition. On the other hand, it is strongly undesirable for textile products that are still in use, since an unpleasant odour, colour stains and discoloration of fabrics occur during the biodegradation process, which significantly decreases the applicable value of textile products from hygienic as well as aesthetic points of view (Dring, 2003; Gao, 2008; Vigo, 1983). Moreover, in the case of textiles that are used in public places and transportation, there is a possibility of transmission of pathogenic microorganisms from one person to another and therefore a great chance of transmission of various infections and illnesses. Due to mould formation and rotting of textile fibers, biodegradation is also an urgent problem in conservation and restoration of textile products of great historical importance and in preservation of the cultural inheritance. Cellulose fibers are classified as biodegradable. They are mainly comprised of cellulose, a high-molecular, water insoluble polymer of glucose. Since glucose is an important source of carbon, which is used by the microorganisms for their growth and development (Clarke, 1997; Glazer et al., 2001; Szostak-Kotowa, 2004), cellulose fibers are susceptible to bacteria and fungi as well as to algae, which, in the presence of moisture, heat and dirt, form a biofilm on the surface of the fibers, where they quickly multiply (Montegut et al., 1991). Due to the complexity of the nature of and interactions between different polysaccharides present in the cell wall of cellulose fibers and microorganisms, the biodegradation process is very complicated. In spite of the fact that the first scientific records of biodegradation of cellulose fibers date back to 1912 (Blackburn, 2005), this research field has not been fully investigated and, therefore, still remains open for further study. The most active microorganisms that cause biodegradation of cellulose are generally found in water, soil and air. The most active fungi, therefore, belong to the genera Chaetomium sp., Fusarium sp., Myrothecium sp., Memnoniella sp., Stachybotrys sp.,
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Verticillium sp., Alternaria sp., Trichoderma sp., Penicillium sp. and Aspergillus sp. The last two genera are of particular importance, since they include species that require relatively low humidity for their growth in comparison to other fungi. During biodegradation of cellulose, bacteria are of lesser importance than fungi, since they generally require conditions at which the fabric is saturated. Bacteria that degrade cellulose belong mostly to the genera Cytohaga sp., Cellulomonas sp., Cellvibrio sp., Bacillus sp., Clostridium sp. and Sporocytophaga sp. (Dring, 2003; Fischer et al., 1980; Itävaara et al., 1999; Raschle, 1989; Szostak-Kotowa, 2004). Bacteria degrade cellulose in different manner than fungi. While bacteria degrade cellulose fiber from its surface towards the inner parts of the fiber, degradation by fungi is just the opposite and proceeds from the inner parts of the fiber towards its outer side. In this case, the process of biodegradation starts at cracks in the surface or at the places where the fiber is cut off. Spores of fungi thus reach the lumen, from where their progression from the inner parts of the fiber toward the outer layers is possible (Szostak-Kotowa, 2004). During microbiological attack of cellulose, fungi and bacteria produce cellulolytic enzymes, which catalyze the chemical reactions and therefore dramatically increase the rate and ratio of the cellulose biodegradation process. Enzymes are specialized proteins with different specificities and modes of action. Their activity is often referred to as "the lock and key" model (Figure 1), in which enzyme active sites that are of specific geometric shapes (key) fit exactly into the sites of substrate (lock). There are at least three distinct enzymes: endoglucanase, cellobiohydrolase and β-D-glukosidase (Clarke, 1997; Glazer, 2001; SzostakKotowa, 2004), which act synergistically to hydrolyse the cellulose to lower oligosaccharides, cellobiose and glucose. The first step of the cellulose biodegradation process is ascribed to the activity of endoglucanases, mostly carried out in the amorphous regions of cellulose fibers, where they randomly attack and hydrolyse the β-(1→4) glycosidic bonds of cellulose to produce cello-oligosaccharides. Cellobiohydrolases degrade amorphous as well as crystalline cellulose, whereby they release disaccharide residues, i.e., cellobiose, from the non-reducing ends of cellulose molecules. The final step in the biodegradation of cellulose is the hydrolysis of cellobioses and soluble cello-oligosaccharides to glucose, which is caused by β-Dglukosidases (Clarke, 1997; Evans et al., 1998; Fischer et al., 1980; Itävaara et al., 1999). The mechanism of enzyme action in the cellulose biodegradation process is presented in Figure 2. Enzymatic degradation is reflected in a reduction of the degree of polymerisation of long cellulose chains, and damage to the fibers as well as a loss of breaking strength occur (Cao, 2002; Cao, 2004; Cao, 2005; Goynes, 1995; Seventekin, 1993; Szostak-Kotowa, 2004).
Figure 1. Mechanism of enzyme activity.
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Figure 2. Mechanism of enzymatic hydrolysis of cellulose; → denotes site of the attack of endoglucanase (A), cellobiohydrolase (B) and ß-glucosidase (C).
There are different possibilities of how to protect cellulose fibers against biodegradation. The chemical modification of fibers with antimicrobial agents is certainly one of the most effective methods. Of antimicrobial agents, biodices and biostates can be used, which differ in their chemical structure, the mechanism of antimicrobial action and their effectiveness (Dring, 2003; Gao, 2008; Vigo, 1983). Antimicrobial agents can be applied at different stages of fiber production. While in the spinning process of regenerative cellulose fibers, biocides are widely used as additives in the polymer master batch, in the case of natural cellulose fibers, chemical finishing with antimicrobial agents is the only possibility for fiber protection (Bajaj, 2002; Vigo, 1983). Regardless of the method of application, an antimicrobial agent kills microorganisms in contact with the fibers, resulting in a high inhibition of their growth. On the other hand, the rate and degree of cellulose biodegradation can also be successfully inhibited by application of chemical finishes, which are not active as antimicrobial agents, but their presence on the fibers significantly impairs the conditions for microbial growth. It could be denoted as passive antimicrobial activity. In this fiber modification process, it is taken into consideration that the amorphous regions of cellulose are much more susceptible to attack by microorganisms than crystalline cellulose, since the degradation process occurs at sites where fibers are in direct contact with the microorganisms, and that the activity of microorganisms is largely influenced by the environmental conditions, in which the presence of sufficient moisture is of great importance (Buschle-Diller et al., 1994; El-Morsy, 2001; Montegut, 1991; Park, 2004). It can thus be expected that any kind of chemical modification of cellulose fibers that leads to a strengthening of their amorphous
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regions and an increase in fiber hydrophobicity will effectively decrease fiber biodegradation. The first condition can be obtained by applying easy care and durable press finishes and the second with the use of water and oil repellent finishes. It is very important to note that both types of finish can modify the fiber chemical properties to such an extent that it causes reduced compatibility between enzyme active sites and the sites of cellulose substrate. Moreover, water and oil repellent finishes form a low energy polymer network on the fiber surface, which greatly decreases the adhesion between microorganisms and cellulose macromolecules (Zhao, 2004), as well as enzyme-cellulose interactions (Figure 3). Since the presence of water and oil repellent finishes greatly increase the hydrophobicity and oleophobicity of fibers in comparison to the untreated ones, this also greatly decreases the moisture in the fibers, which is crucial for the growth of microorganisms. This work consisted of two parts. In the first, we focused on the biodegradation process of cotton fabric, using a soil burial test to determine the resistance of cellulose fibers to the action of soil microflora. The rate and degree of the biodegradation process were demonstrated by scanning electron microscopy, measurement of the degree of polymerisation and the fiber breaking strength, as well as by Fourier transform infrared spectroscopy. The second part of the work was devoted to an assessment of the active and passive antimicrobial activity of different finishes on cellulose fibers. A silver-based finish, including AgCl salt, embedded in the organic-inorganic matrix was used for preparation of the active antimicrobial properties of cellulose fibers. For passive antimicrobial activity we used a non-formaldehyde containing product based on imidazolidinone, a fluoroalkylfunctional water-born siloxane (FAS), 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (PFOTES) in combination with diureapropyltriethoxysilane [bis(aminopropyl)terminated-polydimethylsiloxane (1000) (PDMSU), as well as aminopropyl-perfluoroisooctyl polyhedral oligomeric silsesquioxane (AP2PF2IO4 POSS) in combination with a diisocyanatohexyl (DICH) cross-linker. The presence of finishes on the cellulose fibers was demonstrated by scanning electron microscopy and energy-dispersive X-ray spectroscopy, atomic force microscopy, inductively coupled plasma mass spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and liquid contact angle measurements. The antimicrobial activity of finishes was investigated with the use of a soil burial test, as well as appropriate microbiological tests in which fungicidal activity was estimated for the fungi Aspergillus niger and Chaetomium globosum according to the modified DIN 53931 Standard Method, and bactericidal activity was estimated for the Gram-negative bacterium Escherichia coli using three standard methods: ISO 20645:2004 (E), AATCC 100-1999 and the EN ISO 20743:2007 Transfer Method. By employing the EN ISO 20743:2007 Transfer Method, it was possible to confirm the beneficial effect of low surface energy FAS, PFOTES and AP2PF2IO4 POSS finishes on the reduction of bacterial growth on treated cotton fabrics.
2. MATERIALS FOR FINISHING AND APPLICATION METHODS Plain-weave 100 % cotton woven fabric with a mass of 164 g/m2 was used in the experiments. In a pre-treatment process the fabric was bleached in an H2O2 bath, mercerised in a NaOH solution and neutralised in a diluted CH3COOH solution.
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Figure 3. A drop of water, including cellulolytic enzymes, on the surface of cotton fabric coated with water and oil repellent finish.
As an antimicrobial agent, iSys AG was used in combination with iSys MTX (CHT, Germany). The former is a dispersion agent containing AgCl (Ag) and the latter is a reactive organic-inorganic binder (RB). As water and oil repellent agents, we used Dynasylan F 8815 (Degussa, Germany), which is a fluoroalkylfunctional water-born siloxane (FAS), 1H, 1H, 2H, 2Hperfluorooctyltriethoxysilane (PFOTES) (ABCR), in combination with di-ureapropyltriethoxysilane [bis(aminopropyl)terminated-polydimethylsiloxane (1000) (PDMSU), as well as aminopropyl-perfluoroisooctyl polyhedral oligomeric silsesquioxane (AP2PF2IO4 POSS), in combination with diisocyanatohexyl (DICH) cross-linker. Synthesis of PDMSU and AP2PF2IO4 POSS was reported previously (Jerman et al., 2008a, b; Lavrenčič-Štanger et al., 2005; Vince et al., 2006). As an easy-care and durable-press finish, a non-formaldehyde containing product based on imidazolidinone (CHT, Tübingen), 1,3-dimethyl-4,5-dihydroxyethylene urea (DMeDHEU) was used. All products can be mixed with water to any desired concentration. The Ag-RB finish was applied to the cotton fabric by the exhaustion method. The Ag-RB sol-gel solution was therefore prepared using 3.0 g/l AgCl (which is equivalent to 0.15 % o.w.f.) in combination with 15 g/l RB with a liquid ratio of 1:50. The sample was immersed in the solution and left at room temperature with occasional stirring until equilibrium was achieved. The sample was then wrung to a wet pick-up of 80 ± 1 %, dried at 120 oC and cured at 150 oC for 1 minute. The cotton fabric was finished by 10 % FAS and 80 g/l DMeDHEU, as well as by their mixture using the pad-dry-cure method, including full immersion at 20 °C, wet pick-up of 80 ± 1 % at 20 °C, drying at 100 °C and curing at 150 °C for 5 minutes. The cross-linking reaction of the DMeDHEU finish with the cellulose hydroxyl groups was catalysed with 15 g/l hydrated magnesium chloride (Aldrich). The application of the 4 % PFOTES-PDMSU and the 4 % AP2PF2IO4 POSS-DICH sols to the cotton fabric was carried out by the pad-dry-cure method, including full immersion, wet pick-up of 100 ± 2 % at 20 °C, drying at 100 °C and curing at 140 ° for 15 min. Using the dip-coating technique, FAS, PFOTES-PDMSU and AP2PF2IO4 POSS-DICH sols were applied to smooth and polished aluminum AA 2024 alloy substrates for contact angle measurements. Heat treatment was performed at 140 °C for 15 min. The precursor concentrations in sols were the same as those used for the application to the cotton fabric.
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3. ANALYTICAL METHODS FOR THE EVALUATION OF MATERIALS AND FINISHES 3.1. Soil Burial Test Determination of the resistance of finished and unfinished cotton fabrics to the action of soil microflora was carried out by the soil burial test, according to ISO 11721-1:2001 and ISO 11721:2003 standards. In this standard process, a container was filled with commercial grade compost. The water content of the soil was 60 ± 5 % of its maximum moisture retention capacity. It was held constant during the experiment by spraying with water. The pH of the soil was between 4.0 and 7.5. Cotton fabric samples were buried in the soil for periods of 3, 6, 9 and 12 days. After the defined incubation time, the samples were removed from the test soil, lightly rinsed with running tap water and immersed in 70 % ethanol for 30 minutes before air drying.
3.2. Scanning Electron Microscopy (SEM) and Energy-dispersive X-ray Spectroscopy (EDXS) The morphology and composition of the coatings on the cotton fabrics were investigated by a JEOL JSM 5800 scanning electron microscope (SEM) equipped with an Oxford-Link ISIS 300 EDXS system with an ultra-thin window Si(Li) detector. The samples for SEM and EDXS analysis were coated with a ≈ 30-nm-thick carbon layer to ensure sufficient electrical conductivity and to avoid charging effects. Analyses were performed using a 10-keV electron beam, 200 to 500 pA beam current and X-ray spectra acquisition under a 35 o take-off angle. SEM micrographs were recorded using both secondary electron (SE) and backscattered electron (BSE) imaging modes. BSE compositional (Z-contrast) imaging was applied to emphasize and expose the difference between the added particles and the cotton fiber-matrix.
3.3. Atomic Force Microscopy (AFM) The topography of untreated and coated cotton fibers was measured by atomic force microscopy (AFM) with the use of atomic force microscope Solver Pro (NT-MDT Co.). Scanning in oscillating mode was performed. All images were obtained at ambient condition. The roughness parameter (rms) was calculated from the AFM images taken over 5 x 5 mm. Before the roughness calculation, a second-order plane was subtracted from each AFM image to take into account the curvature of the fibers. Two places were analyzed on each sample.
3.4. Breaking Strength Breaking strength was measured with an Instron 5567 dynamometer in accordance with SIST ISO 5081:1996. The relative reduction in breaking strength, qred, of the buried cotton
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samples compared with the unburied ones was calculated from the mean value of the breaking strength of ten specimens, using the following relationship:
q red ,t =
Ft Ft 0
(1)
where q red ,t is the loss of breaking strength of the buried cotton sample after burial time t, Ft is the breaking strength of the buried cotton sample after burial time t, and Ft0 is the breaking strength of the unburied cotton sample. Before testing, the samples were conditioned at 65 ± 2 % relative humidity and 20 ± 1 oC for 24 hours.
3.5. Degree of Polymerisation (DP) The DP of the cellulose samples dissolved in Cuoxam, a solution of cupric hydroxide in aqueous ammonia [Cu(NH3)4](OH)2, was determined viscosimetrically using an Oswald shear dilution viscometer.
3.6. Fourier Transform Infrared (FT-IR) Spectroscopy FT-IR spectra were obtained on a Brucker IFS 66/S spectrophotometer equipped with an attenuated total reflection (ATR) cell (SpectraTech) with a Ge crystal (n = 4.0). The spectra were recorded over the range 4000–600 cm-1, with a resolution of 4 cm-1 and averaged over 128 spectra. Before measurement, the studied samples were dried for 5 hours at 100 oC.
3.7. X-ray Photoelectron Spectroscopy (XPS) X-ray photoelectron spectroscopy (XPS or ESCA) analyses were carried out on a PHITFA XPS spectrometer (Physical Electronics Inc). The analyzed area was 0.4 mm in diameter and about 3–5 nm in depth. This high surface sensitivity is a general characteristic of the XPS method. Sample surfaces were excited by X-ray radiation from a monochromatic Al source at a photon energy of 1486.6 eV. C 1s, F 1s, O 1s, N 1s and Si 2p spectra were acquired with an energy resolution of about 1.0 eV with an analyzer pass energy of 58 eV. Quantification of surface composition was performed from XPS peak intensities measured on three different spots of the sample, taking into account the relative sensitivity factors provided by the instrument manufacturer (Moulder, 1995).
3.8. Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) The concentration of Ag in the finished cotton bulk samples was determined by ICP-MS on a Perkin Elmer SCIED Elan DRC spectrophotometer. A sample of 0.5 g was prepared in a
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
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Milestone microwave system by acid decomposition using 65 % HNO3 and 30 % H2O2. Three measurements were made for each sample, and the Ag concentration was given as a mean value.
3.9. Microbiological Tests 3.9.1. Fungicidal activity The fungicidal activity of the Ag-RB-treated cotton samples was estimated for the fungi Aspergillus niger (ATCC 6275) and Chaetomium globosum (ATCC 6205) according to the modified DIN 53931 Standard Method, in which synthetic nutrient-poor agar (SNA) (Nirenberg 1976) consisting of 1 g of KH2PO4, 1 g of KNO3, 0.5 g of MgSO4 × 7 H2O, 0.5 g of KCl, 0.2 g of glucose, 0.2 g of saccharose and 15 g of technical agar per 1 l distilled water was used instead of the prescribed malt-extract agar (MEA). SNA is a less nutritious cultural medium, allowing a more delicate colony growth and easier evaluation of the antifungal activity of Ag. Thirty μl of spore-suspension (105/ml were spread on each SNA plate. The inoculated plates were incubated at 29 oC for 24 hours. Samples of cotton fibers 5 × 5 cm were then placed on the medium and incubated at 29 oC for 7 and 14 days. After incubation, fungicidal activity was determined in terms of mycelial growth on and below the surface of the cotton fibers and the intensity of sporulation. To determine fungal development below the finished samples, the cotton samples were removed from the agar plate and the medium was examined microscopically. A droplet of cotton blue dissolved in lactic acid was placed on the medium and covered with a glass slip, allowing the detection of the stained fungal mycelium at low magnification. The degree of fungal growth was ordered in 8 grades from 00 to 5, where 00 indicates no growth, 0 fungal growth outside an inhibition zone surrounding the cotton specimen, [0] fungal growth up to the specimen’s edge, 1 fungal growth only on and below the specimen’s edge, 2 fungal growth on and below less than 25 % of the specimen, 3 fungal growth on and below 25–75 % of the specimen, 4 fungal growth on and below more than 75 % of the specimen and 5 100 % overgrowth of the specimen. The intensity of sporulation was assessed using the following symbols: – means clear, without mycelium; + weak, only mycelium; ++ noticeable growth, partly spores; and +++ strong overgrowth, extensive spore formation. 3.9.2. Bactericidal activity The antibacterial activity of coated cotton samples was estimated for the Gram-negative bacterium Escherichia coli (ATCC 25922) using three different standard methods: ISO 20645:2004 (E), AATCC 100-1999 and the EN ISO 20743:2007 Transfer Method. For the ISO 20645:2004 (E) standard method, known as the Agar diffusion plate test, two-layered agar plates were prepared. The lower agar layer consisted of 10 ml of ordinary agar; the upper layer consisted of 5 ± 1 ml agar inoculated with bacteria, whereby one ml of bacteria working solution with a concentration of 1–5 × 108 CFU was added per 150 ml of agar. Circular pieces of cotton, 25 ± 5 mm in diameter, were uniformly pressed on the agar and incubated for 24 hours at 37 ± 1 oC. After incubation, assessment was based on the absence or presence of bacterial growth in the contact zone between the agar and the sample and on the possible appearance of an inhibition zone, which was calculated from:
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H=
D−d 2
(2)
where H is the inhibition zone in mm, D is the total diameter of the cotton specimen and inhibition zone in mm, and d is the diameter of the specimen in mm. For bacterial growth, the contact zone under the samples was also determined with a microscope at 20-times magnification. For the standard method, the inhibition zone was measured in mm and the degree of bacterial growth was estimated in the nutrient medium under the specimen. The antibacterial effect of the studied samples was described as “good”, “limited” or “insufficient”. All tests were performed in duplicate. According to the AATCC 100-1999 standard method, circular swatches of finished cotton samples, 4.8 cm in diameter, were put into a 250 ml Erlenmeyer flask and inoculated with 1.0 ml of a nutrient broth culture containing 1–2 x 105 CFU of bacteria. An unfinished cotton sample was used as a control. After incubation at 37 oC for 24 hours, the bacteria were eluted from the swatches by shaking them in 100 ml of neutralizing solution for 1 minute. After making serial dilutions with sterilized water, the suspensions were plated on nutrient agar and incubated at 37 oC for 24 hours. The number of bacteria forming units (CFU) was then counted, and the reduction of bacteria, R, was calculated from:
R=
( B − A) 100 B
(%)
(3)
where A is the CFU recovered from the inoculated cotton sample swatch in the jar incubated over the desired contact period (24 hours), and B is the CFU recovered from the inoculated cotton sample swatch in the jar immediately after inoculation (at “0” contact time). Three treatments were performed on two samples for each finished cotton fabric. In addition, the reduction of bacterial growth on the finished samples was also estimated using the EN ISO 20743:2007 Transfer Method. This method enables an assessment of the bacterial reduction that is caused not only by the presence of antibacterial active agents (i.e., nanosilver particles) in the finishes, but could stem from the low surface energy of the oleophobic finished cotton (caused by FAS), which prevents or at least hinders the adhesion of bacteria and their consequent growth and the formation of a biofilm on the finished fabrics. The latter effect is called ‘passive antimicrobial activity’ to distinguish it from the inherent antibacterial effect of various chemical agents embedded in finishes, such as the silver nonoparticles used in this study. According to the EN ISO 20743:2007 Transfer Method, the agar plates were inoculated with 1 ml of a nutrient broth culture containing 1–3 x 106 colony forming units of Gram-negative bacteria Escherichia coli (ATCC 25922). The swatch of the test sample (3.8 cm in diameter) was plated on the agar surface and pressed down with a 200 g cylindrical weight for 60 ± 5 s. The test sample was then removed from the agar surface, placed in a 100 ml container with the transferred surface face up and incubated at 37 oC for 24 h in a humidity chamber. After incubation, 20 ml of neutralizing solution was poured on the test sample and it was shaken vigorously for 1 min. After making serial dilutions with sterilized water, the suspensions were plated on nutrient agar and incubated at 37 oC for 24 hours. The reduction of bacteria growth, RA, on the finished sample in comparison to the unfinished one was calculated as follows:
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
RA =
(U − T ) 100 U
(%)
937 (4)
where U is the number of bacteria forming units on the untreated sample after 24 hours incubation and T the number of bacteria forming units on the treated samples under the same conditions. Three treatments were performed on two samples for each fabric sample.
3.10. Assessment of Wetting Properties The static (equilibrium) contact angles of water (W), formamide (FA) and diiodomethane (DIM) on the coatings prepared on Si wafers and finished cotton samples were made on a DSA 100 contact angle goniometer (Krüss, Germany), which works on the principle of the goniometer-sessile drop technique. From the image of the deposited liquid drop on the surface, the drop contour was analyzed and the contact angle was determined by using the Young-Laplace fitting, which is the theoretically most exact method. With this method, the complete drop contour was evaluated taking into account the drop deformation caused by liquid weight, which, in addition to interfacial effects, also contributed to the drop shape. Liquid drops of 8 μl were deposited on different spots of the coated substrates to avoid the influence of roughness and gravity on drop shape. The average values of contact angles in 30 – 60 seconds from the deposition of the drop were calculated from at least five measurements on glass plates and from at least ten measurements on the studied fabric, minimizing errors due to roughness and heterogeneity. Contact angle measurements were carried out at 20 oC and ambient humidity. Van Oss and co-workers approach (van Oss et al., 1988a, b) was used for determination of the total surface free energy of the coatings, resolved to the corresponding apolar Lifshitzvan der Waals component,
γ SLW , which includes London dispersion, induction (Debye) and
orientation (Keeson) interactions, and a polar component, and electron-acceptor, energy components
γ SAB , due to electron-donor, γ S− ,
γ S+ , interactions. According to this theory, the solid surface free
γ SLW , γ S− and γ S+ can be obtained from the following expression:
WA = (1 + cosθ )γ L = 2 γ SLW γ LLW + 2 γ S+γ L− + 2 γ S−γ L+ if contact angles of three different liquids with known
(5)
γ LLW , γ L− and γ L+ are measured on the
same solid surface. Practice suggests that when using Equation (5), one of the chosen liquids should be non-polar. To perform the van Oss calculations, DIM ( γ L
LW
0,0 mJ/m2, and W ( γ L
= 21.6 mJ/m2,
γ L− =
γ L+ = 0.0 mJ/m2), FA ( γ LLW = 39.0 mJ/m2, γ L− = 39.6 mJ/m2, γ L+ = 2.28 mJ/m2)
LW
= 21.8 mJ/m2,
γ L− = 25.5 mJ/m2, γ L+ = 25.5 mJ/m2) were used.
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0 day
3 days
6 days
9 days
12 days
(A)
(B)
Figure 4. Photographs (A) and SEM images (B) of untreated cotton samples after 0, 3, 6, 9 and 12 days of the burial test.
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3.11. Assessment of Washing Fastness The washing fastness of coatings was determined by repetitive washing in an AATCC Atlas Launder-O-Meter Standard Instrument, which is widely used for evaluating laundry results on a laboratory scale. One wash in a Launder-O-Meter (ISO 105-C01:1989(E) standard method) provides an accelerated washing treatment corresponding to five home washings. The finished fabric samples were washed repetitively up to 10 times; the duration of the washing cycles was 30 min and was carried out in a solution of SDC standard detergent of concentration 5 g/l, previously heated to 40 °C, to give a liquid ratio of 50:1. After washing, the samples were rinsed in cold distilled water and then held under cold tap water for 10 min, squeezed and dried at room temperature. After drying, samples were also heattreated by ironing at 190 °C for 10 s. The quality of the coatings was assessed after the first and tenth washing cycles.
4. BIODEGRADATION OF UNTREATED CELLULOSE FIBERS 4.1. Fibers Buried in Soil The biodegradation process of cellulose fibers was carried out by the soil burial test and the results are presented in Figure 4. Photos of samples removed from the test soil after different incubation times showed that the speed of the rotting process caused by microorganisms in the soil accelerated over the time of burial. After 12 days of burial, the cotton fabric was degraded to such an extent that it fell to pieces. SEM images thus revealed major morphological changes due to the decomposition action of the soil microflora. In the initial stage of the experiment (0 day), the surface of the cotton fabrics was very smooth. After 6 days of soil burial, the development of superficial cracks in the case of the fibers could be observed. The intensity of this morphological damage increased with increasing soil burial time, whereby serious disintegration and defibrillation of the untreated fibers could be observed after 12 days of soil burial to such an extent that individual macrofibrils could be seen. 2000
1,2 1,0
1900
0,8
DP
qred,t
1800 0,6 1700 0,4 1600
0,2 0,0 0
3
6
9
12
1500
t (day)
Figure 5: Plots of the loss of breaking strength, qred,t, and DP of cotton samples against the burial time, t. - qred,t,
- DP.
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The biodegradation process was additionally demonstrated by breaking strength and DP measurements. The results shown in Figure 5 reveal that the breaking strength of the cotton samples rapidly decreased with increasing time of burial and reached a value of 0.002 after 9 days of incubation. The results also show that the loss of breaking strength was directly related to rupture of the β-(1→4) glycosidic bonds of the cellulose macromolecules (see Figure 2), resulting in a marked decrease of DP from 1923 for an unburied sample to 1551 for a sample buried for 12 days.
4.2. FT-IR Spectra of Degraded Fibers Important information about the biodegradability of cellulose fibers can be provided by infrared spectral analysis (Figure 6). An inspection of the measured ATR spectra of cotton samples after 12 days of burial revealed that microbial biodegradation led to cellulose structural changes. To provide a clear view of the changes appearing during the biodegradation process, the difference ATR spectra, obtained by subtracting the spectrum of unburied cellulose from that of the the buried one, were examined. In Figure 3A, negative band absorptions occur at 3340 cm-1 due to OH stretching, at 2918 and 2853 cm-1 due to CH and CH2 stretching and at 1456, 1429, 1370, 1336, 1318, 1280, 1160, 1105, 1053 and 1029 cm-1 due to C-C and C-O stretching vibrations, skeletal vibrations and ring vibrations in the cellulose fingerprint region as a result of the decreased intensities of these bands after biodegradation. Hulleman et al. (1994) suggested that the decrease of band intensities in the region 1500–900 cm-1 may be attributed to a decrease in cellulose crystallinity. This finding is in agreement with that obtained by Nelson et al. (1964), whereby the intensity of bands at 1429, 1370, 1335, and 1315 cm-1 (which were assumed to be very sensitive to changes in cellulose crystallinity and lattice type) decreased with decreasing crystallinity. This additionally confirmed the characteristic decrease in the 900 cm-1 band and its broadening after 12 days of burial (Colthup et al., 1990; Nelson et al., 1964). Simultaneously, significantly increased broad bands appear at 1640 and 1548 cm-1. This indicates that very important structural changes appear in the spectral region from 1700–1500 cm-1, reflected in the change of intensity of bands at 1640 and 1548 cm-1. A detailed analysis of the spectra showed that the significant increase of the band at 1640 cm-1, obtained for cotton samples buried for 12 days, was not caused by an increase of adsorbed water onto the degraded cellulose (Tomšič et al., 2007). Further inspection of the bands at 1640 and 1548 cm-1 also shows that their position and shape are not representative of the spectral absorption of aldehyde or carboxylic functional groups, which are produced in the cellulose biodegradation process. They seem to be more related to Amides I and II (Socrates, 2001; Vince et al., 2006). Their appearance in the cellulose structure could be explained by the presence of secondary polyamides due to the proteins that are produced during the growth of microorganisms on the fibers, which can be adsorbed on the degradable cellulose macromolecules. Microbial growth thus results in an increase of the bands at 1640 and 1548 cm-1. These results are in agreement with the vibrational spectra of bacteria grown on a culture media (Socrates, 2001).
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
4
941
A
Absorbance
3
2
1640
1
1548
0 4000
3500
3000
1500
1000
-1
Wave number (cm )
900
1548 1456 1429 1370 1336 1318 1280 1105 1160 1053 1029
1640
0
0,2
Absorbance
2918
3340
2853
B
4000
3500
3000
1500
1000
-1
Wave number (cm ) Figure 6. Measured infrared ATR spectra (A) of cotton samples before (—) and after (---) 12 days of burial. Difference ATR spectrum (B) obtained by subtracting the spectrum of unburied cellulose from that of the buried one.
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5. INHIBITION OF CELLULOSE BIODEGRADATION BY CHEMICAL MODIFICATION 5.1. Active and Passive Antimicrobial Protection The influence of chemical modification of cellulose fibers with AG-RB, FAS, DMeDHEU and FAS-DMeDHEU coatings on the biodegradability of cellulose fibers is presented in Figure 7. It is clearly seen from the results that the chemical modification of cellulose fibers with all the used chemical finishes inhibits the rate and degree of cellulose biodegradation, resulting in lower morphological damage after 12 days of soil burial in comparison with the untreated sample. These results are in accordance with the results of the loss of breaking strength that is directly influenced by the degree of sample degradation. It can be seen from Figure 8 that the AG-RB coating of the cellulose caused the highest inhibition of fiber biodegradation, followed by FAS-DMeDHEU, DMeDHEU and FAS coatings. Excellent antimicrobial properties of the AG-BR coating were expected, since it included AgCl, a well-known biocide, which provided active antimicrobial activity of modified fibers. However, the very high inhibition of the biodegradation process of cellulose fibers modified by DMeDHEU and FAS was not expected, since neither of the finishes used are antimicrobial agents. Nevertheless, the results indicated that the presence of either of the finishes on the cellulose fibers strongly impaired the conditions for the growth of microorganisms. This property was designated passive antimicrobial activity. Active and passive antimicrobial activity were proved by the appropriate microbiological tests. The properties of modified cellulose fibers were determined on the basis of FT-IR and EDXS spectroscopy, as well as sample wettability using static contact angle measurements.
5.1.1. Active antimicrobial activity of AG-RB finish Mechanism of biocidal activity of AgCl in the oxide matrix An AG-RB coating with the added AgCl salt was used for imparting antimicrobial properties to the cellulose fibers. Silver-based finishes are an important group of antimicrobial agents (Kissa, 1984; Simončič, 2003; Schindler & Hauser, 2004; Gao & Cranston, 2008), which act as an effective bactericide and fungicide even at low concentrations. These compounds are not chemically bonded onto the textile fibers and their antimicrobial activity is attributed to their gradual and persistent release from the textile into surroundings, where they act as a poison to a wide spectrum of bacteria and fungi. The anti-microbial mechanism of AgCl is poorly known. However, it seems that Ag+ ions released from AgCl act toxically towards unicellular micro-organisms by their binding to negatively-charged carboxyl, thiol and phosphate side groups in microbial cells (Gupta et al., 1998; Matsumura et al., 2003; Kim et el., 2007; Lee et al., 2007) and, among other things, also by interacting with thiol groups of proteins. Due to the abundance of sulphur-containing proteins on the bacterial cell membrane, Ag+ cations can react with sulphur-containing proteins inside or outside the cell membrane, which in turn affects bacterial cell viability. It was also proposed that the released Ag+ cations can interact with phosphorus moieties in DNA, resulting in deactivation of DNA replication, as well as with sulphur-containing proteins, leading to the inhibition of enzyme functions. At high concentrations, silver atoms and silver ions are also toxic to fungi (Jung et al., 2007). It
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
943
is also believed that silver is biocompatible and non-toxic to human cells at concentrations effective against microorganisms (Kusnetsov et al., 2001; Lee & Jeong, 2005), when in the form of non-agglomerated and well dispersed particles.
I
II
III
IV
V
(A)
(B)
Figure 7. Photographs (A) and SEM images (B) of untreated cotton sample (I) and samples coated with AG-RB (II), FAS (III), DMeDHEU (IV) and combination of FAS-DMeDHEU (V) finishes after 12 days of burial test.
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qred
0,8 0,6 0,4 0,2 0,0 0
2
4
6
8
10
12
14
t (day) Figure 8. Plot of the loss of breaking strength, qred,t, of untreated and chemically modified cotton samples against burial time, t. { - untreated sample, z - coated with AG-RB, S - coated with FAS, - coated with DMeDHEU, T - coated with combination of FAS-DMeDHEU.
To obtain uniform dispersion, embedment and controlled release of Ag+ particles, RB was used in combination with AG. RB is a reactive organic-anorganic binder forming an oxide matrix on the fibers in which silver particles can be physically bonded (Mahltig, 2005). The oxide matrix is biologically inert, and it does not represent a food source for microorganisms. Previous studies have shown that the presence of an oxide matrix increases the concentration of bounded silver as well as its uniform distribution and prolongs the time of Ag+ release into the environment (Akkopru & Durucan, 2007; Mahltig et al., 2004; Tomšič et al., 2008; Wang et al., 2006; Xing et al., 2007).
Structure of cellulose fibers modified by AG-RB finish Chemical modification of cellulose by the AG-RB coating was demonstrated by SEM micrographs (Figure 9) as well as EDXS and ATR spectra (Figure 10). Morphological changes and the distribution of the silver particles on the AG-RB finished cotton fabric can be observed from the SEM micrographs of the cotton fabric before (Figure 9A) and after (Figure 9B) the application of the AG-RB finish. The silver particles were spherical in shape and from 100 to 500 nm in size. Their presence on the finished cotton fibers was confirmed by the EDXS spectrum (Figure 10A). The bulk concentration of Ag particles on the cotton fibers, which reached 130 mg/kg was determined on the basis of ICP-MS analysis (Tomšič et al., 2009). The ATR spectrum of the AG-RB coating (Figure 10B) revealed bands of Si-O-Si linkages at 1130, 1075 and 1025 cm-1 (shoulder), showing that a silica network was formed during the condensation process, capable of incorporating Ag particles. It should be noted that the AG-RB coating was studied on a Si wafer since Si-O-Si bands at 1075 and 1025 cm-1 coincided with the strong bands of the cellulose fingerprint (Vince et al., 2006; Fir et al., 2007; Tomšič et al., 2008, 2009).
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
(A)
945
(B)
Absorbance
Figure 9. SEM images of untreated cotton fibers (A) and fibers coated with AG-RB finish (B). 1060 1075 1130
1452 1529 1581
1025 1354
896
925
0,4
1800
1600
1400
1200
1000
800
-1
Wave number (cm )
(A)
(B)
Figure 10: Measured EDXS spectrum (A) of cotton fibers coated with AG-RB finish, and ATR spectrum of AG-RB coating (B) on a Si wafer in the 1800-650 cm-1 spectral region.
Antibacterial and antifungal activity of AG-RB coating The results of the antibacterial and antifungal activity of AG-RB coating on the cellulose fibers are presented in Figure 11 and in Table 1. A comparison of photos of the growth of E. coli on nutrient medium covered with an untreated cotton sample (Figure 11, IA) and a cotton sample coated with AG-RB finish (Figure 11, IB) revealed that the presence of AgCl in the coating strongly suppressed bacterial growth. The release of Ag+ particles from the coating into its surrounding resulted in the formation of an inhibition zone greater than 1 mm. It is clearly seen from the microscopic images of the E. coli growth on the nutrient medium that no bacterial colonies (small shadowed spots) were present in the inhibition zone near the edge of the removed sample (Figure 11, IC), nor in the nutrient medium under the sample itself. Contrary to the coated cotton sample, an insufficient antibacterial effect was expectedly obtained for the untreated cotton sample (Figure 11, IA), caused by a lack of an inhibition zone and a heavy bacterial growth on the medium under the sample. The excellent antimicrobial efficiency of the AG-RB coating was demonstrated by the reduction of bacteria, R, shown by the AATCC 100-1999 standard method, whereby 100 % of bacterial reduction was obtained for the coated cotton sample and no bacterial reduction for the untreated cotton sample.
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Table 1. Antifungal activity of AG-RB coating on cellulose fibers for C. globosum and A. niger after 7 days of incubation at 29 oC, according to the modified DIN 53931 Standard Method (Tomšič et al., 2009). Fungus C. globosum A. niger
Growth A* B* 4 (> 75) [0] (0) 5 (100) 1 (5)
Intensity of sporolation A B +++ / +++ +
* A – untreated sample, B – sample coated with AG-RB finish.
(I)
(II)
(III) Figure 11. Growth of bacterium E. coli (I) and fungi C. globosum (II) and A. niger (III) on the nutrient medium covered with untreated cotton sample (A) and cotton sample coated with AG-RB finish (B). C) Microscopic observation of bacterial growth (IC) and of submersed mycelial growth of C. globosum (IIC) and A. niger (IIIC) on the nutrient medium near the edge of the removed cotton sample (1 – under the sample, 2 – in the surrounding of the sample). The antibacterial test was carried out according to the ISO 20645:2004 (E) standard method, and the antifungal test was carried out according to the modified DIN 53931 standard method.
It can also be seen from Figure 11 (II and III) and Table 1 that the concentration of AgCl in the coating was high enough to obtain antifungal properties of the modified cellulose fibers. Both fungi C. globosum. and A. niger grew all over the untreated cotton sample
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
947
(Figure 8, IIA and IIIA), but their growth was strongly inhibited in the case of the coated cotton sample (Figure 11, IIB and IIIB). The microscopic observation of submersed mycellal growth on the nutrient medium near the edge of the removed cotton sample revealed that the antifungal activity of AG-RB coating was lower for A. niger than for C. globosum. While the sample was totally unaffected by C. globosum (Figure 11, IIC), an inspection of the sample in contact with the medium previously inoculated by A. niger revealed that, in spite of the fact that there was no fungal growth present on the sample, a restricted mycelium with chains of candidia was found on some spots of the medium under the sample (Figure 11, IIIC). Nevertheless, these results clearly indicated that fungal growth was strongly attacked by the modified cotton fibers but not completely inhibited. The results of the microbiological tests are in accordance with the results of the biodegradation process of cotton fabric coated with the AG-RB finish. The very low damage caused by microorganisms in the soil even after 12 days of burial is certainly influenced by the active and highly effective antimicrobial and antifungal activity of AgCl in the coating.
5.1.2. Passive antimicrobial activity of FAS and DMeDHEU finishes Structure of cellulose fibers modified by FAS and DMeDHEU finishes The formation of a FAS nanocomposite network on the fiber surface, and the crosslinking of cellulose macromolecules with DMeDHEU in the amorphous regions of fibers (Schindler & Hauser, 2004) are schematically presented in Figure 12. The presence of FAS and DMeDHEU on the cellulose fibers was confirmed by FT-IR spectra (Figures 13 and 14) and XPS measurements (Figure 15) (Tomšič et al., 2007, 2008). Comparison of the spectra of untreated cotton fabric (Figure 14a), cotton fabric coated with FAS finish (Figure 14b), as well as FAS coating on Al substrate (Figure 13) revealed well resolved bands attributable to the stretching modes of the C–F groups (Church & Evans, 1995; Hoffmann et al., 1997; Hoque et sl., 2006, 2007; Jiang et al., 2005; Lenk, 1994; Monde et al., 1999; Rabolt et al., 1984) showing beside the band at 1245 cm -1 and the band at 1237 cm-1, while the band at 1204 cm -1, which was well expressed in the FAS coating on Al substrate, was blurred by the cellulose –OH bending (1200 cm -1) in the case of the coated cotton fabric. Moreover, the bands attributed to the Si–O–Si modes of silsesquioxane species formed when condensation proceeds in the presence of a textile substrate, could also not be assessed on the coated cotton fabric since the very strong bands ascribed to the cotton blurred the detailed absorption in the region from 1150–900 cm-1 (Fir et al., 2007; Tomšič et al., 2008; Vince et al., 2006). These results were fully confirmed by XPS measurements (Figure 15), showing the appearance of a fluorine (F 1s) peak at 689 eV and silicon (Si 2p) peak at 102 eV in the cotton fabric coated with FAS finish, since the XPS spectra of untreated cotton fabric revealed only two characteristic peaks belonging to carbon (C 1s) at 289 eV and oxygen (O 1s) at 533 eV. According to the cross-linking mechanism presented in Figure 12B, covalent bonds are formed between hydroxyl groups of DMeDHEU and cellulose molecules, in the condensation reaction during the curing stage of the finishing process (Schindler & Hauser, 2004). Since the finish molecules are small enough to enter the fibers, it was assumed that the cross-linking reaction occurs in the amorphous regions of the fibers. The incorporation of molecules of DMeDHEU into the cellulose structure was confirmed from the infrared spectral analysis. The ATR spectra (Figure 14c) revealed spectral changes in the 1800–1500 cm-1 region. For the finished cellulose, rather weak but characteristic bands attributed to the C=O and C–N
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Barbara Simončič, Brigita Tomšič, Boris Orel et al.
stretching vibrations of DMeDHEU appeared at 1760, 1700 and 1260 cm-1 (Socrates, 2001). The broad absorption band at 1645 cm-1, which is characteristic of the HOH bending vibrations of adsorbed water molecules (Kondo 1997; Łojewska et al. 2005; Hofstetter et al. 2006), occurred in the spectrum of untreated cellulose, while it was much weaker in the spectra of finished cellulose. Furthermore, the IR spectra of untreated and the treated cellulose samples showed a significant difference in the intensity and the shape of the band at 900 cm-1, which is very sensitive to conformational changes of the interglucosidal bond (Mathlouthi and Koenig, 1986), proving the presence of cross-linking between DMeDHEU and cellulose macromolecules.
...
CF3
CF3
CF3
CF3
CF3
CF3
(CF2)n
(CF2)n
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CH3 O
N O
CH2
CH3
N
N O
C N
O CH3
N O
CH3 CH2
C
O CH3
CH2
CH2 cellulose
(B) Figure 12. Formation of FAS nanocomposite network on the fiber surface (A) and cross-linking of cellulose macromolecules with DMeDHEU in the amorphous regions of fibers (B).
The results presented in Figure 16 show that the application of FAS coating to cotton fabric highly increased the hydrophobicity of the untreated cotton fabric (Figure 16, Ic and Id). On the other hand, the incorporation of DMeDHEU into the cellulose structure did not essentially decrease the hydrophilicity of the cellulose fibers (Simončič et al., 2008), which remained wettable by water (Figure 16, Ib). It can also be seen from the results that water formed drops of high contact angles on the FAS coated cotton fabric samples even after 12 days of burial (Figure 16, IIc and IId) indicating that FAS polymer network was not attacked by microorganisms. The coating was still therefore present on the fibers, in spite of the fact,
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
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that the rotting process of coated cellulose macromolecules was in progress during the burial time, causing fiber damage with holes partially located in the fabric structure.
1240 1204 1147
1,2 1,0
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0,8 0,6 0,4 0,2 0,0 3500
3000
1750
1500
1250
1000
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Wave number (cm-1)
Absorbance
Figure 13. Measured ATR spectrum of FAS coating on Al substrate.
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a 1800
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1500
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Wave number (cm-1) Figure 14. Measured ATR spectra of untreated cotton fabric (a) and of cotton fabric samples coated with FAS (b) and DMeDHEU (c) finishes.
Barbara Simončič, Brigita Tomšič, Boris Orel et al. Atomic concentration (at. %)
950
70 59,9
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50 40,1
40
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30 20 13,3
10
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0,0
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0 C 1s
O 1s
Si 2p
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Chemical element Figure 15. Surface composition of untreated cotton fabric (■) and cotton fabric coated with FAS finish (■) obtained by XPS measurements.
1.0
(I)
(a)
(b)
(c)
(d) (II)
(c)
(d)
Figure 16. Photographs of water droplet placed on the untreated and finished cotton fabric samples before (I) and after (II) 12 days of burial. a) untreated sample, b) coated with DMeDHEU, c) coated with FAS, d) coated with FAS-DMeDHEU.
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
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0,05
Absorbance
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1640 1548
0,03
d
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c b a
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Wave number [cm-1]
d c b
0,05
a 4000
3500
3000
1750
1500
1250
1000
750
Wave number (cm-1) Figure 17. Measured infrared ATR spectra of modified cotton fabric samples after 12 days of burial. a) coated with FAS-DMeDHEU, b) coated with DMeDHEU, c) coated with FAS, d) untreated cotton fabric.
Biodegradation of cellulose fibers modified by FAS and DMeDHEU finishes Comparison of the loss of breaking strength of cotton fabric during the burial time (Figure 8) revealed higher inhibition of the fibers rooting process in the presence of DMeDHEU than of FAS. This enables the conclusion that the mode of fiber protection of both finishes differs to each other and that the chemical modification of cellulose fibers with DMeDHEU provided more effective protection against microorganisms in the soil in comparison to that obtained with FAS. These results were proved by the FT-IR spectra presented in Figure 17, where the band intensities at 1640 and 1548 cm-1, characteristic for the Amide I and II, ascribed to the proteins produced during the growth of microorganisms on the fibers, are higher in the case of more degraded fibers. In order to discuss these results, we should consider both the binding mechanism of FAS and DMeDHEU to cellulose fibers as well as the mode of the microorganism action during the biodegradation process. It is clear that the formation of a FAS network on the surface of cotton fabric (Figure 12A) caused an increase of the fiber hydrophobicity, as well as a high drop in their surface free energy, resulting in a decreased adhesion of microorganisms and impaired conditions of their growth. Namely, the microorganism growth is strongly influenced by different factors among which the presence of water is of great importance. A high decrease of fiber wettability thus significantly inhibited microbial degradation at initial burial time, but the protection was much less effective when the fibers were exposed to microorganism action for a longer time. In contrast to FAS, the application of DMeDHEU assured sufficient protection of fibers, in spite of the fact that they remained hydrophilic. The covalent bonds that formed between DMeDHEU and the cellulose macromolecules in the
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Barbara Simončič, Brigita Tomšič, Boris Orel et al.
modification process (Figure 12B), strengthened the less ordered amorphous regions and held the macromolecular chains together, resulting in a decrease in cellulose chain mobility and fiber swelling. The powerful effect of the strengthening of amorphous region on inhibiting fiber biodegradation can also be explained by the manner of the cellulose biodegradation process, in which the action of the fungi is of great importance. Since fungi start to degrade fibers from their lumen outwards, the cross-linking of cellulose macromolecules inhibited the penetration of microorganisms into the amorphous region of the fibers where the biodegradation takes place. It can also be clearly seen from the results (Figure 5) that the FAS and DMeDHEU finishes act synergistically in the coating resulting in higher inhibition of the cellulose degradation process in comparison with single component FAS and DMeDHEU coatings. At the same time, microbiological tests according to the modified DIN 53931 and AATCC100-1999 standard methods revealed that neither DMeDHEU nor FAS provide active antifungal and antibacterial activity on the cellulose fibers.
6. SOL-GEL FINISHES FOR PASSIVE ANTIBACTERIAL ACTIVITY 6.1. Sol-Gel Precursors as Material of Choice It is clear that the antibacterial effect, closely related to the adhesion and colonization of bacteria on the cotton fabrics, is indirectly influenced by the low surface energy of the cotton fabrics. Zhao et al. (2004) demonstrated that by using a graded nickel-perfluoroethylene composite coating technique, it is possible to tailor the surface energy of membrane diffusers to optimum values, resulting in stable membrane filtration achieved by the retention of membrane permeability, which usually decreases due to the growth of a biofilm. It has been shown that the low dispersive part of the surface free energy in particular has a tremendous effect on reducing the attachment of E. coli. Like membrane diffusers, cotton fabric also has a porous structure and both substrates suffer problems of poor durability, lack of resistance to cleaning agents and are prone to leaching (Hamza et al., 1997). Chemical modification of cotton fabric with sol-gel based organic-inorganic hybrids containing perfluoroalkyl groups enables the formation of a nanocomposite coating of an organic-inorganic structure with extremely low surface free energy, providing excellent hydrophobicity and oleophobicity of fibers (Mahltig & Böttcher, 2003; Qing et al., 2002; Shao et al., 2004a, b; Tomšič et al., 2008; Yu et al., 2007). Mild processing conditions are required and single-step processing to impregnate the fabric is usually enough to impart multifunctional properties such as high contact angles for oil and water. Importantly, sol-gel hybrids exhibit excellent adhesion on cotton, attained through condensation between the -OH groups of the hydrolyzed silanes and those present at the surface of cellulose (Abdelmouleh et al., 2002; Vince et al., 2006). Since these studies have also shown that the sol-gel procedure enables the formation of textiles with radically new chemical and physical properties, which cannot not be achieved by the application of conventional chemical finishing agents, in addition to the FAS coating, novel sol-gel coatings were prepared with 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (PFOTES) in c-ombination with di-ureapropyltriethoxysilane [bis(aminopropyl)terminatedpolydimethylsiloxane (1000) (PDMSU) (Vilčnik et al., 2009) and with aminopropylperfluoroisooctyl polyhedral oligomeric silsesquioxane (AP2PF2IO4 POSS) (Figure 18) in
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combination with a commercial diisocyanatohexyl (DICH) cross-linker (Jerman et al., 2008). The letter represents a novel synthesised tri-functional POSS based silane precursor RxR’yR’’z(SiO3/2)8, (x+y = 8) bearing di-aminopropyl, di-perfluorohexiletil and tetra-isooctyl groups. It has a well defined cage-like structure with a stable inorganic Si-O core of dimensions from 1 to 3 nm surrounded by organic (R) substituents, resembling in this respect organically functionalized nanosized particles of SiO2. Most of the POSS known so far are not suitable for achieving low surface energy cotton finishes, because they do not prefer to form compliant gels as for example, when more common bridged silsesquioxanes (b-PS), prepared from different bi-podal alkoxysilane precursors (Shea & Loy, 2001) are used for cotton finishes. Stiff and structurally well defined polyhedral core of POSS prefer to form less compliant and more porous, water and oxygen permeable coatings and since usually they do not contain alkoxysilane reactive groups, the coatings could be washed from cotton fabrics and from other substrates relatively easy. In order to increase the fastness of POSS coatings on cotton fabrics and to overcome the absence of alkoxysilane groups in AP2PF2IO4 POSS, well proven to enhance fastness of b-PS finishes on cotton fabrics (Vince et al., 2006), POSS polyhedra were functionalized with di-amiopropyl groups, known by their reactivity towards isocyanato groups. This enables the possibility to add an appropriate diisocyanato cross-linker to the corresponding POSS solution and afterwards applied on cotton fabric.
6.2. Structure of Cellulose Fibers Modified by PFOTES-PDMSU and AP2PF2IO4 POSS Finishes The structure of PFOTES-PDMSU and AP2PF2IO4 POSS coatings was investigated by analyzing the IR ATR spectra. A closer look at the ATR spectra of the PFOTES coating deposited on an Al substrate (Figure 19) revealed the bands attributed to νa(CF2) mixed with rocking (CF2) (1240 cm-1), νa(CF2) + νa(CF3) (1209 cm-1) and νs(CF2) (1145 cm-1) vibrations of the perfluoro chains (Lenk et al., 1994). The assignment of the C-F bands was reliable enough for performing subtractive spectral analysis, which we used for the identification of the C-F groups and how they changed with respect to the Si-CH3 groups of the PFOTESPDMSU finishes on cotton fabric. Inspection of the spectra in Figure 20A showed that the bands attributed to the PFOTES-PDMSU/cotton sample are dominated by bands belonging to the cotton itself. Subtraction of the IR spectrum of cotton from the IR spectra of the finished cotton (Figure 20B), however, showed bands attributed to PDMSU at 1256 cm-1 (δ(Si-CH3)), 798 cm-1 (r(Si-CH3)) and CF bands (νa(CF2) mixed with rocking (CF2) (1240 cm-1), νa(CF2) + νa(CF3) (1209 cm-1) and νs(CF2) (1145 cm-1)) modes. In addition, strong asymmetric and symmetric ν(Si-O-Si) stretching bands of PDMSU chains at 1087 and 1020 cm-1 (Figure 20B) appeared, which can only be identified from the slight intensity changes of the main bands attributed to the cotton fabric in the region of 1200–1000 cm-1 (Chung et al., 2004; Langkilde & Svantesson, 1995). The bands of the urea-urea linkage were very weak, in contrast to the ATR spectra of cotton treated with PDMSU (Vince et al., 2006).
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Barbara Simončič, Brigita Tomšič, Boris Orel et al. OEt EtO
Si
F
F
F
F
F
F F
OEt F
F
F
F
F
F
(a) PFOTES O EtO EtO
Si OEt
N H
N H
CH3 O Si CH3
CH3 CH3 O Si Si CH3
n
CH3
O OEt N H
N H
Si EtO
OEt
(b) PDMSU
(c) AP2PF2IO4 POSS Figure 18. Structure of 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (PFOTES) (a), diureapropyltriethoxysilane [bis(aminopropyl)terminated-polydimethylsiloxane (1000) (PDMSU) (b) and aminopropyl-perfluoroisooctyl polyhedral oligomeric silsesquioxane (AP2PF2IO4 POSS) (c).
The ATR IR transmission spectra in Figure 21 confirmed the interactions between AP2PF2IO4 POSS and DICH since when the condensation of POSS and DICH proceeded, the band at 2269 cm-1 belonging to the isocyanate groups (which was accompanied by the appearance of the bands of the urea groups (Amid I and Amide II bands) disappeared from the spectrum. Furthermore, the presence of the band at 1116 cm-1 confirmed the formation of cube-like structures of AP2PF2IO4 POSS. As expected, the subtractive spectra (Figure 22) clearly demonstrated the presence of POSS on the cotton fabrics, inferred from the C-F bands (1240, 1210 and 1145 cm-1) (Lenk et al., 1994) and the (-SiO3/2) stretching band of the POSS
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
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polyhedra at 1116 cm-1. When DICH was added to the POSS/EtOH solution and applied on the cotton fabrics, the corresponding IR ATR spectra revealed the formation of bands characteristic of the formation of urethane bonds (Amide I and II bands at 1680 and 1540 cm-1), confirming the reactions between POSS and DICH. The similarity with the spectra shown in Figure 21 is striking, despite the presence of cotton fibers, which blurred the appearance of bands in the corresponding IR ATR spectra.
6.3. Hydrophobicity and Oleophobicity of the Sol-Gel Modified Fibers The results presented in Figure 23 showed that the presence of perfuoroalkyl groups in all three sol-gel hybrids, i.e., FAS, PFOTES and AP2PF2IO4 POSS, provided the hydrophobicity and oleophobicity of the coatings deposited on Al substrates, resulting in high contact angles for water, formamide and diiodimethane. The highest contact angles were obtained for the FAS coating, followed by the PFOTES-PDMSU and AP2PF2IO4 POSS coatings. The surface free energy values (Table 2) derived from the Van Oss Equation (5) revealed that all three coatings represent highly apolar low energy surfaces with γ STOT equal to 10.3 mJ/m2 for FAS, 14.5 mJ/m2 for PFOTES-PDMSU and 20.58 mJ/m2 for AP2PF2IO4 POSS. Concurrently, the −
+
polar electron-donor ( γ S ) and electron-acceptor ( γ S ) interactions decreased, reaching values lower than 0.8 mJ/m2 and 0.09 mJ/m2, respectively. The polarity of the coatings obtained from the Van Oss relation was very low, equal from 2 to 5 %. ν AS CF2 + ν AS CF3 1209
-CF2 1240
ν S CF2 1144
Absorbance
1105 -OEt 1083
-OEt
820
Si-O-Si 1040
1295
-OEt
-OEt
778
962
-CF2 1324
SiOH
1378
910
a
0,4
b 1800
1600
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1000
800
600
-1
Wave number (cm ) Figure 19. ATR spectra of non-hydrolysed (a) and hydrolysed (b) PFOTES deposited on an Al substrate.
1059 1034 1020
Barbara Simončič, Brigita Tomšič, Boris Orel et al.
a
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Wave number (cm ) (B) Figure 20. ATR IR spectra of cotton and cotton treated with PFOTES-PDMSU (A) and difference IR ATR spectra of PFOTES-PDMSU coatings obtained by subtracting the spectrum of cotton from the spectra of PDMSU-PFOTES/cotton (B).
Table 2. The surface free energy components of the coatings on Al substrates obtained with the use of Equation (5) according to the Van Oss and co-workers approach. Coating FAS PFOTES-PDMSU AP2PF2IO4 POSS
γ SLW
γ S+
γ S−
(mJ/m2) 9.97 14.06 20.12
(mJ/m2) 0.08 0.04 0.08
(mJ/m2) 0.79 0.32 0.61
γ STOT (mJ/m2) 10.30 14.30 20.58
Polarity (%) 5 2 2
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
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C=O Si-O-Si POSS
Absorbance
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1116
N=C=O
a
C-F
b Amide II Amide I 0,2
c 4000
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3000
2500
2000
1500
1000
-1
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*
Figure 21. IR transmission spectrum of DICH (a), AP2PFIO4 POSS (b) and a mixture of DICH and AP2PFIO4 POSS (c) deposited on an Al substrate.
Absorbance
Si-O-Si POSS
C-F Amide II Amide I
a 0,1
b 4000
3500
3000
1750
1500
1250
1000
750
-1
Wave number (cm ) Figure 22. Difference IR transmission spectrum obtained by subtracting the spectrum of untreated cotton from the spectrum of AP2PF2IO4 POSS/cotton (a) and IR transmission spectrum of DICH + AP2PF2IO4 POSS deposited on an Al substrate (b).
The results presented in Figure 24 show that, due to the rough morphology of woven fabrics (Tuteja, 2007), the contact angles for water increased from 121, 118 and 113°, observed on Al substrates, to 149, 147 and 151° for FAS, PFOTES-PDMSU and AP2PF2IO4 POSS. On the basis of the high water contact angles, the work of adhesion values (Figure 25), which were calculated from the left side of Equation (5), were very low because of the
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Barbara Simončič, Brigita Tomšič, Boris Orel et al.
decrease of the solid surface free energy of the coated cotton fabric, indicating that there are very weak adhesion forces between water and the cotton fibers. The SEM micrographs (Figure 26) and AFM measurements (Figure 27) of uncoated and coated cotton fibers showed that both fibers had very similar rms values (~ 30–50 nm). This means that the texture of the cotton fabric, and not the roughness of the individual fibers, played the decisive role in the observed enhancement of hydrophobicity. This was attributed to air, which is trapped in the fiber assembly in the rough fabric surface. Namely, we believe that in addition to the chemical structure, the surface porosity, geometry and roughness can also influence the liquid contact angles (Černe et al., 2008; Yu et al., 2007). The observed apparent contact angles on unwashed cotton fabrics were similar to those observed for cotton treated with a perfluorooctylated quaternary ammonium silane coupling agent (Isquith et al., 1972) or hexadecyltrimethoxysilane (Mahltig et al., 2004), but higher than those obtained for PDMSU treated cotton fabrics (Vince et al., 2006) and tetrafluoroethylene copolymers (Phani, 2006), indicating that the cotton fabrics became superhydrophobic. A comparison of the results in Figures 20 and 21 shows that, despite the lowest liquid contact angles observed on Al substrates, the AP2PF2IO4 POSS coating provided an excellent repellency of cotton fabric with a water contact angle higher than 150°. The reason for this could be the unique cube-like structure of the AP2PF2IO4 POSS coating on the surface of the cotton fabric (Figure 28). The chemical modification of the cellulose fibers treated with the sol-gel hybrids was washing resistant since its water repellent properties (Figure 24) were retained after consecutive washing, as reflected in the water contact angles, which changed from 151° (unwashed) to 141° (10 x washed) for AP2PF2IO4 POSS and from 147° (unwashed) to 142° (10 x washed) for PFOTES-PDMSU. Only FAS provided worse washing fastness since the initially high contact angles (149°) changed to 127° after 10 washings. It is important to note that the washing fastness of the coating is of great importance for textile products when in use. 140 120 100
θ (o)
80 60 40 20 0 W
FA
DIM
Liquid Figure 23. Contact angles of water (W), formamide (FA) and diiodomethane (DIM) on the Al substrates coated with FAS (■), PFOTES-PDMSU (■) and AP2PF2IO4 POSS (■).
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification
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155 150
θ (o)
145 140 135 130 125 120 0
2
4
6
8
10
Number of washings Figure 24. Static contact angle, θ, of water on finished cotton samples before and after repetitive washing at 40 oC. Coating: -z- FAS, -- PFOTES-PDMSU, -S- AP2PF2IO4 POSS.
35 30
2
Wa (mJ/m )
25 20 15 10 5 0 0
10
Number of washings Figure 25. Work of adhesion, Wa of the cotton fabrics coated with FAS (■), PFOTES-PDMSU (■) and AP2PF2IO4 POSS (■) before and after 10 washings.
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Barbara Simončič, Brigita Tomšič, Boris Orel et al.
A
B
C
D
Figure 26. SEM micrographs of uncoated cotton fibers (A) and fibers coated with PFOTES-PDMSU (B), AP2PF2IO4 POSS (C) and FAS (D) finishes.
A Figure 27. AFM of uncoated (A) and PFOTES-PDMSU coated (B) cotton fibers.
B
Biodegradation of Cellulose Fibers and its Inhibition by Chemical Modification F F
F
F F
F
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i-Oc
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Si O i-Oc O
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Si
Si O
i-Oc
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i-Oc
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i-Oc O
fibre surface
F
F
F
F
F
F F
F
F
F
F
F
i-Oc
F
F F
F
961
CH3
CH3
Figure 28. Schematic presentation of the AP2PF2IO4 POSS coating formation and its orientation on the surface of cotton fabric.
Table 3. Reduction, R, of bacteria Escherichia coli (ATCC 25922) according to the EN ISO 20743:2007 Transfer Method determined on finished cotton samples before and after 1 and 10 cycles of washing. Coating 0a Untreated AG-RB FAS PFOTES-PDMSU
/b 100 99.3 ± 0.1 100
Ra (%) 1a
/b 88.9 ± 3.4 99.7 ± 0,1
10a /b /b 28.9 ± 9.9 60.6 ±10.8
a
0 – unwashed sample, 1 – sample after the first washing cycle, 10 – sample after the tenth washing cycle; b /: no reduction of bacteria.
6.4. The Influence of Low Surface Free Energy of the Modified Cotton Fibers on the Bacterial Reduction In order to evaluate the low surface energy effect on the antibacterial properties and to distinguish it from the inherent antibacterial effect of the Ag particles, an alternative procedure for the assessment of bacterial growth was applied. Namely, the main problem encountered with the AATCC 100-1999 standard method is the need for complete wetting of the cotton fabrics when the antibacterial properties have to be assessed. We avoided this problem by using the EN ISO 20743:2007 Transfer Method, which gives the possibility of establishing, at least qualitatively, the contribution of the hydrophobicity to the antibacterial
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properties achieved. Inspection of the results shown in Table 3 shows that the antibacterial properties could be obtained with the application of an antibacterial active agent such as AgCl in an AG-RB coating, but also by applying hydrophobic and oleophobic FAS and PFOTESPDMSU finishes to the fabrics. It seems that the presence of FAS and PFOTES-PDMSU prevents, or at least hinders, the adhesion of bacteria and their consequent growth and the formation of a biofilm on the finished fabrics. This effect, which we called “passive antibacterial activity”, exhibited bacteria reduction even after 10 consecutive washings, suggesting the effective prevention of bacterial adhesion on low surface energy cotton fabrics. While no reduction of the growth of bacteria was observed for cotton coated with AG-RB after 10 repetitive washings, because of the gradual leaching of Ag cations from the coating the beneficial and long-lasting low surface energy effect of FAS and PFOTES-PDMSU in the absence of Ag particles was established, with a reduction in bacteria of about 28.9 ± 9.9 % for FAS and 60.6 ± 10.8 % for PFOTES-PDMSU. The latter results are in accordance with the washing fastness of the coatings, which was much higher in the case of PFOTES-PDMSU than with FAS.
7. CONCLUSION In this work we have presented the results of the chemical modification of cellulose fibres aimed at protecting the textile material against biodegradation. Namely, cellulose fibres are highly susceptible to microbial attack, resulting in worsened technological and applicable properties of textile products. This is a particularly crucial problem for textiles that are in use. The rate and degree of cellulose biodegradation is affected by several factors, among which the most important are the genera of microorganisms and the environmental conditions needed for microbial growth. In view of this, sufficient antimicrobial protection of cellulose fibres can be obtained in two ways. The first involves chemical modification of fibres with an active antimicrobial agent, e.g. biocide or biostat, which are able to kill or inhibit the growth of bacteria and fungi. This active antimicrobial activity was demonstrated with the use of a finish on a base of AgCl in combination with an organic-inorganic binder. The other possibility involves chemical modification with finishes which are not antimicrobial agents but their presence on cellulose fibres significantly impedes conditions for microbial growth and therefore improves fibre resistance against microbial attack. This kind of antimicrobial protection, which was denoted ‘‘passive antibacterial activity’’, was demonstrated by the application of easy-care and water and oil repellent finishes to cellulose fibres. While the easy-care finish strengthened the less ordered amorphous regions and therefore inhibited the penetration of microorganisms into the fibres, where biodegradation takes place, the water and oil repellent finish strongly decreased the fibre surface free energy, resulting in a highly decreased adhesion of microorganisms on the hydrophobic fibres.
ACKNOWLEDGMENT This work was supported by the Slovenian Research Agency (Programmes P2-0213, P10030 and Project M2-0104) and the Slovenian Ministry of Defence (Project M2-0104).
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 33
PATHWAYS TO BIOACTIVE OLIGOSACCHARIDES: BIOLOGICAL FUNCTIONS AND POTENTIAL APPLICATIONS Ellen C. Giese 1, Aneli M. Barbosa 2 and Robert F. H. Dekker 3* 1
Departamento de Química Analítica y Tecnologia de Alimentos, Universidad de Castilla-La Mancha, 13071 Ciudad Real, España. 2 Departamento de Bioquímica e Biotecnologia, CCE, Universidade Estadual de Londrina, CP 6001, 86051-990 Londrina-PR, Brazil. 3 Biorefining Research Initiative, Lakehead University, Thunder Bay, ON, Canada P7B 5E1.
ABSTRACT Oligosaccharides present specific physicochemical and biological properties that can be exploited for specific applications in foods and pharmacology. They can be produced through a number of different physical, chemical and enzymatic catalysed reactions from their parent polysaccharides as well as through transglycosylation reactions. This chapter examines the pathways leading to the production of bioactive oligosaccharides that have biotechnological applications. These carbohydrate oligomers constitute a nutritional type of “fiber” that benefits the growth of bifidobacteria and lactobacilli in the colon promoting human health and well-being. The use of oligosaccharides to modify biological responses was recently reported, and this has included their effects as antiinflammatory and anti-cholesterolaemic stimulating compounds. An overview of nutraceutical and biological functions of these carbohydrate fragments mainly for human health is also reported.
*
Corresponding author: E-mail:
[email protected] 970
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INTRODUCTION It was the English poet John Milton II (1608-1674), who in 1645 in his poem L'Allegro, first alluded to what can today be described as “polysaccharides’, when he wrote “..many a winding bout Of lincked sweetnes long drawn out..” This first mention perfectly describes what we know today as constituting “polysaccharides” from knowledge based upon the chemical and physical structures of these complex molecules, although in Milton’s time “polysaccharides” were unknown and had not been described. On this preface, polysaccharides are described as naturally-occurring biological macromolecules (winding bout and long drawn out) composed of sugar building blocks (linked sweetness) that exert highly varied functions in all living organisms. These biopolymers are formed by condensation of various monosaccharides linked through specific glycosidic bonds producing compounds of high molecular weight (MW) with peculiar physicochemical and biological properties (Kapteyn et al., 1996; Sutherland, 1998). Functionally, polysaccharides serve roles as skeletal structures and reserve sources of metabolic energy in Phyla comprising microorganisms, plants and animals, and more recently are known to modify biological responses, e.g., immuno-modulation (Leung et al., 2006; Chen & Serviour, 2007). Physical changes of native polysaccharides through modification by chemicals or enzymes have been used in attempts to increase their functionality for wider applications of these biopolymers (Hasegawa et al., 2006). Depolymerization of these biomacromolecules results in smaller fragments (oligosaccharides) with degrees of polymerization (DP) that may vary from two to anywhere up to twenty monosaccharide units, and this can be accomplished through hydrolysis using specific enzymes or dilute acids. The smaller fragments possess the same chemical constitution as the parent carbohydrate polymer, and can display unique functions, for example, they can participate as dietary fiber constituents as they are not digested within the human gastro-intestinal tract, and are fermented by the natural bacterial flora (lactobacilli and Bifidobacterium species) colonizing the human colon (Collins & Rastall, 2008). Many oligosaccharides are found naturally in plant species including fruits and vegetables, and some also in milk and honey. They include the disaccharides sucrose, lactose and trehalose, and various oligosaccharides made up of sucrose known as the “raffinose family of oligosaccharides” that are ubiquitous in the plant kingdom being present in many different varieties of seeds. They are next in abundance to sucrose as soluble sugars, and include raffinose (trisaccharide), stachyose (tetrasaccharide), and verbascose (pentasaccharide). Human milk constitutes up to 130 different heterogenic oligosaccharides built up of five monosaccharides: glucose, galactose, N-acetylglucosamine, L-fucose, and sialic acid (N-acetylneuraminic acid). Lactose (chief milk sugar) can be elongated through various glycosidic linkages to sialic acid and fucose to produce a range of different oligosaccharides in milk whose structural diversity offers potential health benefits; as nutrients for the colonic bacteria, and immunomodulatory agents. (Bote, 2006). Oligosaccharides are not digested by the human gastro-intestinal enzymes, and are passed intact into the large intestine where they are fermented by the colonic bacteria. Some disaccharides such as maltose and cellobiose can be prepared through hydrolysis (chemical or enzymatic) from starch and cellulose, resp. (Hirayama, 2002; Collins & Rastall, 2008), while higher oligosaccharides, e.g., hexa-, hepta- and nona- saccharides, have been obtained
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through hydrolysis by the action of specific enzymes (hydrolases), or via organic synthesis mediated by specific enzymes such as the glycosyltransferases (Monsan & Paul, 1995; Zhang & Kong, 2003; Yang & Kong, 2005). Oligosaccharides consist of a class of biomolecules that function in biological processes of recognition, like viral or bacterial infections, cell adhesion, cellular signal transduction and intercellular communication (Wong, 1995; Davies et al., 2001). Potential applications of these sugars in the areas of food, animal feeds, pharmaceuticals, cosmetics as well as prebiotics and immunomodulator agents has promoted new research to foster their production and to elucidate their biological and functional properties (Remaud-Simeon et al., 2000; Bote, 2006). The most studied oligosaccharides are shown in Table 1.
Methods for Obtaining Oligosaccharides The synthesis of oligosaccharides by chemical or biosynthetic approaches is more difficult than that of nucleic acids or peptides, due to the vast number of permutations for the combination of monomeric units of sugars and the introduction of stereo-specific glycosidic linkages (Tolstoguzov, 2004; Barreteau et al., 2006). Bioactive oligosaccharides can be obtained through synthesis (chemical or enzymatic), or by depolymerization/fragmentation of polysaccharides (physical, chemical or enzymatic) as described below. Chemical synthesis of oligosaccharides on solid phase supports (Garegg, 2004) can be conveniently performed by attaching the saccharide through the anomeric centre, or attachment through one of the hydroxyl positions, and the arrangement for attachment is dependent upon the mode of linkages required to build the glycose chain. There still is, however, the necessity of having to protect group sequences and their subsequent removal after the chain has been built. The procedure has been automated to synthesize complex oligosaccharides (e.g., a branched dodecasaccharide from glycosyl phosphate building blocks was assembled on an octenediolfunctionalized resin support, (Plante et al., 2001).
PHYSICAL PROCESSES Physical procedures are less used in the production and extraction of functional oligosaccharides because these approaches do not present selectivity and are mostly unspecific. One of the methods described in the recent literature is the use of ionizing radiation, hitherto used as a cross-linking agent between artificial polymers (Al-Assaf et al., 2006). The most common physical methods involve drastic changes of pH and temperature, and use of microwave irradiation or ultrasonication (Bekers et al.; 2005). Zhang et al. (2005) obtained mixtures of oligosaccharides of different molecular weights that retained the chemical structure and conformation of the parent polysaccharide, schizophylan (a β(1→3;1→6)-glucan from Schizophyllum commune). Degradation was achieved by ultrasonography using high-energy sound frequencies. The use of ultrasonication and microwaves at pH 1.5 was reported to cause the hydrolysis of xylan extracted from tamarind seeds, resulting in a series of xylo-oligosaccharides (Vodenicarová et al., 2006). Treatment with microwave irradiation was also used to obtain malto-oligosaccharides from acid hydrolysates of starch by replacing the conventional stage of thermal treatment (Warrand &
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Janssen, 2007). Studies on obtaining cello-, malto- and xylo- oligosaccharides by physical processes involving collisions using cyclotrons and electric quadrupole approaches have also been investigated (Pasanen et al., 2007).
CHEMICAL PROCESSES Among the procedures used in the chemical synthesis of oligosaccharides, those that stand out are condensation reactions and acid hydrolysis of polysaccharides. The first method has some drawbacks and the steps required for the separation and purification of the products are expensive and usually low yielding (Ning et al., 2003). An example is the production of gentio-oligosaccharides by the "Königs-Knorr" condensation reactions employing acetobromo sugars and preformed oligosaccharides (Kiyoshi et al., 1974). Organic synthesis can also take place in the presence of catalysts such as trimethylsilyl-trifluoromethanesulfonate and aceptors like 4,6-O-benzylidene-glucopyranoside, which have been widely used in the formation of β-(1→3)-type glucosidic linkages to synthesize derivatives with chemical structures corresponding to that of exopolysaccharides such as schizophylan, epiglucan and lentinan (He et al., 2002). Hydrolysis reactions employing acids to obtain oligosaccharides from parent polysaccharides needs to be strictly controlled in terms of the nature of the acid employed, acid concentration, and times and temperatures of hydrolysis. One of the drawbacks can be sugar degradation leading to the formation of degradation products such as furfural (from pentoses) or hydroxymethylfurfural (from hexoses) from the constituent residues of polysaccharides. Partial acid hydrolysis is recognised as an important and useful tool in the structural analysis of polysaccharides, and for the production of oligosaccharides (Vessels & Radding, 1993). These molecules are fragmented randomly generating a mixture of oligosaccharides that, once isolated and chemically characterized, aided by overlay of the various fragments, are used to deduce the structure of the biopolymer, such as the sequence of the sugar residues in the polysaccharide chain and the nature of the glycosidic bonds linking the sugars together (Pazur, 1994). In recent years, many oligosaccharides, particularly those of six or more monosaccharides units, were found to retain or enhance the potential biological properties presented by their parent polymers. One possible explanation for this finding was that fragmentation increased the solubility of oligosaccharides reducing their molecular weight (MW) compared to the parent polysaccharides, and this resulted in increased availablity to manifest bioactivity (Bland et al., 2004). In most cases their conformation was retained. Unlike the regio specificity of enzymatic hydrolysis, partial acid hydrolysis, a nonselective process of degradation, generates oligosaccharides of different molar mass (Schmid et al., 2001). Nevertheless, the advantage of acid hydrolysis is the possibility of being performed at high temperatures, promoting greater fluidity of the polymer samples submitted to this kind of treatment. The rate of total or partial acid hydrolysis of a polysaccharide is not uniform, since it is affected by factors such as glycoside anomeric configuration, size of the hemiacetal ring, conformation of monosaccharides, nature of the glycosidic bonds, presence of functional groups, and the intensity of inter- and intra- molecular interactions. Polysaccharides and oligosaccharides made up of sugars in the furanoside form (α-(1→5)arabinofuranosides of L-arabinans; and sucrose (a disaccharide) where one of the linked
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sugars is a fructofuranoside) are more easily hydrolyzed under mild acid conditions than those existing in the pyranoside form (starch or cellulose) (Belitz et al., 2009). Additionally, glycosidic bonds linking pentapyranoside sugars (xylopyranosides as in xylans) hydrolyze faster than the corresponding linkages comprising hexapyranoside sugars (glucopyranosides as in starch or cellulose), and glycosidic linkages of the (1→6) types are more labile to acid hydrolysis than (1→4), which in turn are more resistant than (1→3) and these are more resistant than (1→2) linkages (Biermann, 1988; Corradi da Silva et al., 2006). Also, there are different rates of hydrolysis displayed by polysaccharides of α and β anomeric configurations. For example, starch, which is comprised of glucose residues linked by α(1→4)-bonds is more easily hydrolysed than cellulose, a polymer linked by β-(1→4)glucosidic bonds; the anomeric effect. These structural features of sugar macromolecules therefore make partial acid hydrolysis an extremely valuable analytical tool, because different hydrolysis conditions can generate polysaccharide fragments of different sizes that aid in determining the structure of their parent molecules (Corradi da Silva et al., 2005). Therefore, if the reaction is strictly controlled as displayed by appropriate hydrolysis curves, the selection of acid, time and temperature of the reaction can generate fragments of different molecular weights that can be separated by chromatographic procedure ssuch as gel filtration, ion-exchange, adsorption onto Coal-Celite (Moon & Cho, 1997; Bao et al., 2001; Lee et al., 2004; Corradi da Silva et al., 2005), and capillary electrophoresis (Gonçalves et al., 2005). Appropriate hydrolysis curves indicate the time at which the depolymerization reaction should be stopped, and conditions must be controlled to reduce to a minimum the destruction of sugars, and even prevent the repolymerization of the fragments generated through acid reversion with the consequent formation of undesirable products (Biermann, 1988). Table 1. The principal oligosaccharides presenting biotechnological applications. Oligosaccharide Fructo-oligosaccharides Xylo-oligosaccharides Malto-oligosaccharides Isomalto-oligosaccharides Cyclomaltodextrins (cyclic gluco-oligosaccharides) Galacto-oligosaccharides Transgalacto-oligosaccharides Laminari-oligosaccharides Gentio-oligosaccharides Lactosucrose Lactulose*
Molecular Formula β-Fruf-(2→[1)-β-Fruf-(2]n→1)-β-Fruf-(2→1)-α-Glcp β-Fruf-(2→[1)-β-Fruf-(2]n→1)-β-Frufβ-Xylp-(1→[4)-β-Xylp-(1]n→4)-β-Xylpα-Glcp-(1→[4)-α-Glcp-(1]n→4)-α-Glcpα-Glcp-(1→[6)-α-Glcp-(1]n→6)-α-Glcpα-Glcp-(1→6)-α-Glcp-(1→[4)-α-Glcp-(1]n→4)-α-Glcpα-Glcp-(1→[4)-α-Glcp-(1]n→4)-Glcp n = 4, 5 or 6 β-Galp-(1→[4)-β-Galp-(1]n→4)-β-Galp-(1→4)-Glcp β-Galp-(1→[4)-β-Galp-(1]n→4)-β-Galp-(1→4)-Glcp β-Galp-(1→[6)-β-Galp-(1]n→6)-β-Galp-(1→4)-Glcp β-Glcp-(1→[3)-β-Glcp-(1]n→3)-β-Glcpβ-Glcp-(1→[6)-β-Glcp-(1]n→6)-β-Glcpβ-Galp-(1→4)-β-Fruf-(2→1)-α-Glcp β-Galp-(1→4)-Fruf
*Semi-synthetic compound Legend: Glcp, glucopyranoside; Fruf, fructofuranoside; Xylp, xylopyranoside; Galp, galactopyranoside; β-Galp-(1→4)-Glcp, lactose; β-Fruf-(2→1)-α-Glcp, sucrose.
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The most common acids employed in the hydrolysis of carbohydrates are sulfuric, hydrochloric and trifluoroacetic acids (Johansson et al., 2006). Trifluoroacetic acid (TFA), an organic acid, has been reported (Bao et al., 2001) to be an excellent acid catalyst for the hydrolysis of carbohydrates including polysaccharides, as it has the advantage of being easily removed by evaporation under vacuum, thus avoiding the neutralization step with barium carbonate that is frequently employed with sulfuric acid as catalyst for hydrolysis (Ukai et al., 1982). Fragments of mixed-linked oligosaccharides containing β-(1→3) and β-(1→4) linkages have been obtained from β-(1→3;1→4)-glucans extracted from cereals (oats and barley) employing acid (HCl, H2SO4 and TFA) hydrolysis at elevated temperatures (Johansson et al., 2006). The extraction of xylo-oligosaccharides from cotton seed xylan was achieved using sulfuric acid (0.125 M) at 90 °C (Sun et al., 2002). Another example of the value of partial acid hydrolysis in revealing the complex substitution pattern on polysaccharides was the isolation of an oligosaccharide structurally characterized as gentiohexaose (a β-(1→6)-linked gluco-oligosaccharide) from botryosphaeran (Silva et al., 2008), an extracellular β-(1→3;1→6)-glucan from Botryosphaeria rhodina (Barbosa et al., 2003). The hydrolysis was conducted in the presence of TFA (0.2 M at 100° C over 4 h). The production of oligosaccharides from complex polysaccharides is also an essential tool in determining the complex pattern of substitution displayed by these biopolymers. Partial acid hydrolysis employing TFA in the presence of boran-4-methylmorpholine (a stable acidreducing agent), for example, has been used to efficiently obtain pyruvylated and sulfonated disaccharide alditols from alkali-treated galactans (agarans) isolated from the red alga, Acanthophora spicifera (Rhodophyta) (Gonçalves et al., 2002). Polysaccharides of this type (agarans) (Gonçalves et al., 2005) consist of alternating β-(1→3)-linked D-galactose residues (sulfonated on C-2 with some residues carrying 4,6-pyruvylate acetal groups) and α-(1→4)linked L-galactose units. The L-galactose units are sulfated at C-6, and during reductive alkali treatment of the polysaccharides, cyclization occurs to form 3,6-anhydrogalactose residues. This selective hydrolysis procedure allows the cleavage of polysaccharide chains containing 3,6anhydrogalactose residues yielding a series of disaccharide alditols terminating in 3,6-anhydroL-galactinol with some β-D-galactose residues pyruvated at the 4,6-position, and other β-Dgalactose units retained their sulfate groups on C-2 (pyruvyl agarobiitol 2-sulfate) (Gonçalves et al., 2002). Depolymerization of polysaccharides through the Fenton reaction can be an alternative to acid hydrolysis, but the production of oligosaccharides through this reaction cannot be controlled. The Fenton reaction relies upon hydroxyl (OH•) radicals (highly reactive oxygen species) to attack the polysaccharide by abstracting a carbon-bonded H atom forming a carboncentred radical that leads to the formation of a “glycosulone” within the glycan chain. Upon reduction with borohydride, the polysaccharide chain is fragmented producing oligosaccharides. The pathway leading to the formation of the OH• radical by the Fenton reaction is: Cu+ + H2O2 → OH• + OH−+ Cu2+ In the method employed by Fry et al. (2001), OH• was generated in-vitro by mixing polysaccharide (xyloglucan and galacturonan) with catalytic amounts of copper (I), H2O2, and ascorbic acid, and led to the non-enzymatic scission of the polysaccharide chains producing a series of oligosaccharide fragments. The reaction is thought to occur naturally in plant cell walls such as pear fruit.
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Ozonolysis is another method that has been employed to produce oligosaccharide fragments through depolymerization of polysaccharides (Wang et al., 1998). All hydroxyl groups on the bacterial capsular polysaccharides from Streptococci (Group B) were first protected by acetylation, and the peracetylated polysaccharides subjected to ozonolysis, which selectively oxidized the most reactive β-D-aldosidic linkages to produce ester linkages at C-1. These in turn were cleaved with a nucleophile, thus fragmenting the polymer while simultaneously removing the O-acetyl-protecting groups on the sugar rings. Selective cleavage of the most sensitive linkage occurred randomly throughout the polymer chain, and yielded oligosaccharides with the same repeating-unit structure as the parent polymer. A chemo-enzymatic approach has led to the production of a series of chromogenic congeners of aryl linked [α-Xylp-(1→6)-β-Glcp-(1→4)-] and [β-Galp-(1→2)-α-Xylp(1→6)-β-Glcp-(1→4)-] gluco-oligosaccharides from plant xyloglucans (Ibatullin et al., 2008). These oligosaccharides were prepared from tamarind (Tamarindus indica) galacto-xyloglucans through hydrolysis using a crude Trichoderma reesei cellulase preparation that liberated a series of substituted β-(1→4)-linked gluco-oligosaccharides (glucotetraoses) having the motifs XylXylXylGlc and XylGalGalGlc (Glcp is glucose, Xylp is xylose, Galp is galactose). Galactose residues were removed by β-galactosidase. The resultant xylogluco- and galactoxylogluco- oligosaccharides were then treated by a variation of the “Königs-Knorr” glycosylation method to introduce chromogenic groups to yield new phenyl glycosides that formed convenient substrates for the precise assay of xyloglucanase activity, as well as substrates to elucidate the mode of action of these enzymes. Agro-industrial lignocellulosic materials (sugarcane bagasse, wheaten straw, corn cobs, rice husks) are a rich source of hemicellulose (esp., xylan) that can serve as substrates for the production of xylo-oligosaccharides. Hydrothermal treatment (e.g., autohydrolysis and steam explosion) of lignocellulosic residues solubilises the hemicellulose component (xylans) causing their partial depolymerization depending upon the conditions used. Steam explosion fractionated the constituent polymers in sugarcane bagasse with the hemicelluloses appearing in the liquor fraction in a partially-degraded form (Dekker et al., 1983) that can be further processed enzymatically to produce oligosaccharides of defined sizes for applications. Xylooligosaccharides have also been produced through autohydrolysis by autoclaving wheat straw slurries in dilute sulfuric acid to produce a liquor fraction where the degree of degradation of xylan fragments was dependent upon the severity of the autohydrolysis conditions. For example, at 190 °C for 2 mins (slurry in 0.5 % sulfuric acid), a series of xylo-oligosaccharides were produced that upon High Pressure Size-Exclusion Chromatography analysis revealed DP’s ranging in sizes from 9 to 25 (Kabel et al., 2007).
ENZYMATIC PROCESSES The use of enzymes to modify polysaccharides and expand their potential industrial applications has received wide attention (Vandamme & Soetaert, 1995). This approach makes use of the high specificity of enzymes in relation to their substrates. Enzymes can be employed to produce oligosaccharides, and among the different enzymatic reactions highlighted are hydrolysis and transglycosylation that are specifically catalysed by the glycan hydrolases, glycosidases and glycotransferases (Table 2).
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Ellen C. Giese, Aneli M. Barbosa and Robert F. H. Dekker Table 2. Microbial enzymes used in processes to produce oligosaccharides.
Oligosaccharide Fructooligosaccharides
Enzyme β-fructofuranosidase
βfructosyltransferase
Microorganism Schwanniomyces occidentalis
Aspergillus japonicus Aureobasidium pullulans Aspergillus japonicus
Reference Álvaro-Benito et al., 2007 Cheng et al., 2005 Yoshikawa et al., 2006 Dorta et al., 2006 Ghazi et al., 2005 Park et al. 2005 Barbosa, 1992
Xylooligosaccharides
xylanase
Aspergillus aculeatus Penicillium citrinum Trichoderma harzianum Rifai
Maltooligosaccharides
glucansucrase
Thermotoga maritima Streptococcus sobrinus
Jiang et al. 2004 Lee et al., 1997 Alcade et al., 1999 Nagarajan et al., 2006 Nakajima et al., 2004 Pan & Lee, 2005
β-galactosidase
Leuconostoc mesenteroides Bacillus subtilis Dictyoglomus thermophilum Saccharomyces carlsbergensis; Saccharomyces cerevisiae Aspergillus oryzae
β-glucosidase
Sterigmatomyces elviae Bifidobacterium Aspergillus niger Phanerochaete chrysosporium
Onishi & Tanaka, 1998 Rabiu et al., 2001 Yamashita et al., 2005 Kawai et al., 2004
Botryosphaeria rhodina; Trichoderma harzianum Rifai Arthrobacter sp Penicillium multicolor
Giese et al., 2006 Giese et al., 2006; Giese et al., 2009 Pang et al., 2005 Fujimoto et al., 2009
endo-β-1,4glucanase α-mannosidase
Aspergillus niger
Akiba et al., 1999
Penicillium citrinum
Maitin & Rastall, 2004
exo-α-L-arabinanase
Penicillium chrysogenum
Sakamoto et al., 2004
β-galactosidase
Sulfolobus solfataricus
Kim et al., 2006
α-amylase Isomaltooligosaccharides Galactooligosaccharides
Laminarioligosaccharides
trans-α-glucosidase
β-glucanases
Gentiooligosaccharides Cellooligosaccharides Mannooligosaccharides Arabinooligosaccharides Lactulose
β-glucosidase
Gaur et al., 2006
ENZYMATIC HYDROLYSIS REACTIONS Among the hydrolytic enzymes producing oligosaccharides are the glycan hydrolases. They catalyze the hydrolysis of polysaccharides producing an array of oligosaccharides of different molecular sizes that can comprise the same linkage arrangement, or a mixture of glycosidic linkages depending upon the linkage arrangements present in the parent polysaccharides (Grandpierre et al., 2008). The other class of hydrolytic enzymes is the glycosidases (glycosyl hydrolases) (Sutherland, 1984) that are ubiquitous and catalyze the
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hydrolysis of glycosidic bonds generally of low MW carbohydrate molecules such as oligosaccharides and glycoconjugates containing aglycon groups. They can also attack the short-chained appendage groups on polysaccharides, attacking them from the non-reducing ends, and glycosidases are also known that attack the internal linkages of branched chains such as those found on glycoproteins (Endo et al., 2006). Both types of hydrolytic enzymes have wide applications in processing foods by modifying biopolymers, and as analytical tools in the elucidation of chemical structures. The glycosidases and glycan hydrolases (EC 3.1.2.-) are stereo-selective and catalyze the hydrolysis of either α- or β- glycosidic linkages, but never both. Despite their high specificity and selectivity, their applications to produce oligosaccharides are limited due to the low yields of products obtained. One way of overcoming this problem is through controlling the time of hydrolysis, and substrate recycling to further hydrolyse unused substrate (Maitin & Rastall, 2004). To retain the fragments liberated and prevent their further hydrolysis, it is necessary to remove the product and this can conveniently be conducted in a membrane reactor that allows passage of products but not substrate. The catalytic action of hydrolytic enzymes (glycan hydrolases) attacking polysaccharides is further classified as either exo-, when they act from the non-reducing end of the glycan chain, or endo- when they attack the glycan chain internally at random; in both cases, this catalytic action can produce a mixture of mono-, di- and/or oligo- saccharides of varying sizes (Vandamme & Soetaert, 1995; Ramesh & Tharanathan, 2003). Among the most widely used glycan hydrolases to produce oligosaccharides are the amylases, the non-cellulose degrading β-glucanases, xylanases and inulinases. The αamylases are endo-acting enzymes (α-(1→4)-glucan 4-glucanhydrolases) and those produced by Bacillus subtilis, for example, have been used to produce malto-oligosaccharides from soluble starch (Nagarajan et al., 2006). Inulinases, enzymes hydrolyzing inulins (also known as fructans or fructosans; polysaccharides consisting of linear chains of β-(2→1)fructofuranosyl residues containing terminal glucose units), are of two types: exo-inulinases (β-D-fructofuranosyl fructohydrolases) and endo-inulinases (β-D-fructan fructanohydrolases), and attack inulin producing a series of β-(2→1)-fructofuranosyl-oligosaccharides (Kulminskaya et al., 2003; Cheng et al., 2005). The production of gluco-oligosaccharides from β-(1→3;1→6)-glucans (e.g., botryosphaeran and laminarin) constitutes a series of 1→3 (laminari-) and 1→6 (gentio-), as well as (1→3;1→6) mixed linkages, and are produced through the action of fungal β-(1→3)-glucanases such as those from Trichoderma harzianum (Giese et al., 2006; Grandpierre et al., 2008; Giese et al., 2009). These are just some examples of enzymes used to obtain oligosaccharides through the enzymatic cleavage of polysaccharides. They can also be used as tools to assist in the structural characterization of polysaccharides, and in determining their rheological and pharmacological properties (Giese et al., 2003). Processing of agricultural crops (sugarcane, cereals, maize, cassava), fruits (citrus, apples, pears) and vegetables (corn cobs) generates enormous quantities of waste residues enriched in carbohydrate polymers (cellulose, various hemicelluloses, pectins), and presents opportunities to produce oligosaccharides through the actions of glycan hydrolases. Enzymatic hydrolysis products arising from cellulose are only soluble to DP 6, and those of higher DP remain water insoluble unless derivatized (e.g., by acetylation) (Pan et al., 2006). Xylanases have been employed to hydrolyse corncobs to produce β-(1→4)-linked xylooligosaccharides (Moura et al., 2007). Xylo-oligosaccharides containing β-(1→3)-linkages
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have also been produced. The action of a novel ß-(1→3)-xylanase from the marine bacterium Vibrio sp. degraded the β-(1→3)-xylan of the green alga, Caulerpa racemosa var. laetevirens, to produce an array of oligosaccharides comprising β-(1→3)-linked xylose disaccharides and trisaccharides, β-(1→3)-linked xylo-trisaccharides containing (1→3)linked glucose (Glcp-(1→3)-Xylp-(1→3)-Xylp), and β-(1→4)-linked xylose (Xylp-(1→4)Xylp-(1→3)-Xylp and Xylp-(1→3)-Xylp-(1→4)-Xylp), as well as a mixture of β-(1→3)linked xylo-tetrasaccharides containing (1→3)-substitued glucose. The pattern of hydrolysates indicated C. racemosa β-(1→3)-xylan was a linear heteropolysaccharide containing (1→3)-Glcp and (1→4)-Xylp residues within the β-(1→3)-xylan chain (Kiyohara et al 2006).
TRANSGLYCOSYLATION AND REVERSE HYDROLYSIS REACTIONS The synthesis of oligosaccharides catalyzed by enzymes presents an interesting alternative approach to traditional chemical methods and enzymatic defragmentation of polysaccharides, as enzymes specifically allow the control of regio-selectivity and the stereochemistry of the final products (Perugino et al., 2004). The synthesis process involves two classes of enzymes, the glycosidases (EC 3.2.1.- ) and the glycosyltransferases (EC 2.4.-.-), and has allowed the production of complex oligosaccharides not produced by conventional methods involving depolymerization through enzymatic hydrolysis (Monsan & Paul, 1995; Shoda et al., 1998). Glycosyltransferase-catalysed synthesis of oligosaccharides was the subject of two excellent reviews (Weijers et al. 2008; Bojarová & Kren, 2009) where the reaction mechanisms among other topics were discussed in detail. Glycosyltransferases participate in both intramolecular and intermolecular transfer reactions (Ajisaka & Yamamoto, 2002). These enzymes are produced across the whole spectrum of organisms (bacteria, fungi, yeasts, plant and animal), and have been employed in free or immobilised forms to synthesize complex oligosaccharides (Nishiguchi et al., 2001). Cell walls containing cell-bound glycosyltransferases from yeasts (Saccharomyces cerevisiae and Pichia pastoris) have also been used as sources of enzyme for synthesizing oligosaccharides (Abe et al., 2003; Watanabe et al., 2006). The use of glycosyltransferases, however, on a scaled-up basis may be limited somewhat by the nature of the glycosyl donors, and esp., where these are nucleotide sugars such as UDP-glucose. A method to overcome this drawback can be through sugar nucleotide recycling, and bacterial coupling has been shown to be successful in regenerating sugar nucleotides, and additionally, the scale of production could be increased (Woodyer et al., 2006). The procedure makes use of whole bacterial cells for sugar nucleotide regeneration and the biosynthesis reactions. A disadvantage of this method, however, is the need to permeabilize the bacterial cells and the slow transport of sugar intermediates out of the cell. Where nucleotide sugars are replaced by sugar-1-phosphates, the synthesis reactions are catalysed by phosphorylases; the most common type being the glucosyl-phosphorylases. In these catalysed-reactions, the glucose moiety of glucose-1-phosphate is transferred to an acceptor glycoside to form glycosyl-glucosides with the liberation of inorganic phosphate. Another group of glycosyltransferases (e.g., glucosyltransferases or fructosyltransferases) use
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sucrose as donor molecules specifically to transfer the constituent glucose or fructose moieties for chain elongation to form oligosaccharides of the glucosyl- or fructosyl- types (Weijers et al., 2008). Glycosidases generally catalyze the hydrolysis of naturally occurring low MW oligosaccharides and glycoconjugates, but can also participate in the further degradation of low MW products arising from the enzymatic digestion of polysaccharides (Bhatia et al., 2002). Glycosidases also hydrolyse the branched substituents on polysaccharides in a sequential manner, as well as the internal linkages of branched oligosaccharide chains on glycoproteins (Endo et al., 2006). Glycosidases are of two types and can have “retaining” and “inverting” activities. The former releases products that have the same anomeric carbon configuration as the parent glycosidic substrate, while the latter inverts the products and have opposite configuration. Both retaining and inverting types of glycosidases differ in their mechanism of action (Bojarová & Kren, 2009). Glycosidases, as well as some glycan hydrolase enzymes, however, can also participate in the in-vitro synthesis of glycosidic linkages by undergoing a reverse reaction, (Faijes & Planas, 2007; Weijers et al. (2008); Bojarová & Kren, 2009), i.e., they reverse the direction in which the reaction would normally take place by reducing the activation energy (Ea) to the extent that the reaction favours the reverse direction. This can proceed in 2 ways: (i) thermodynamically, by controlling the shift in the equilibrium using high substrate concentrations, and (ii) kinetically, by use of activated glycosyl donors with excellent leaving groups such as fluorides and sugar nucleotides. Besides requiring high concentrations of substrate in thermodynamically-controlled reactions, these reactions can also operate in solvent systems of low water activity to synthesize products (Lieshout et al., 2004; Gaur et al., 2006). In reactions under kinetic control, there is the formation of an enzyme-glucosyl donor intermediate, which is retained by the nucleophilic amino acid groups on the enzyme molecule to extend the chain of oligosaccharides formed (Bhatia et al., 2002). These condensation reactions are simple to implement and, if the enzyme is chosen correctly, can present absolute selectivity (Ajisaka & Yamamoto, 2002; Maitin & Rastall, 2004). The regioselectivity of these reactions can be controlled by the type of enzyme, the nature of solvent system used, and the structure of the glycosyl acceptors (Murata & Usui, 2000). The main limitation to using glycosidases for synthesis reactions is the low product yields because of the possibility of reversion of the reaction, which promotes not only glycosylation, but can also catalyze the hydrolysis of the products formed which can serve as substrates for the enzymes (Shoda et al., 1998). This has recently been overcome through a novel approach first introduced in 1998 in Withers laboratory (Canada) through biomolecularly redesigning a β-glucosidase from Agrobacterium sp. engineered to replace the active site carboxylate nucleophile with a non-nucleophilic amino acid that still resulted in the correct folding of the enzyme but altered the activity of the mutated enzyme (Mackenzie et al, 1998). A mutation at glutamic acid 358 with alanine resulted in altered enzymatic activity compared to the original wild-type enzyme; hydrolytic action was inactivated, while the “mutant enzyme” showed a high capacity to transfer an activated glycosyl donor (fluoride, p-nitrophenol) onto a suitable acceptor glycoside. These redesigned “mutant” enzymes, named “glycosynthases”, represent a new tool for the synthesis of novel and complex oligosaccharides, and efficiently catalyzed the condensation of monosaccharides, but have lost their hydrolysis capacity through biomolecular engineering (Jakeman & Withers, 2002; Bojarová & Kren, 2009). These mutant enzymes have high regio- and stereo- selectivity, can easily be obtained, and are already
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widely used in the production of oligosaccharides at scaled-up levels (Moracci et al., 2001; Koizumi, 2003; Kim et al., 2005). This approach represents an important improvement over kinetically-controlled transglycosylation by wild-type glycosidases (Weijers et al., 2008). Glycosynthase-catalysed synthesis of polysaccharides has also been demonstrated, e.g., a “mutated glucan hydrolase” [(1→3;1→4)-β-D-glucanase from Bacillus] synthesized in-vitro a (1→3,1→4)-β-D-glucan (Faijes et al., 2004). In this approach, in-vitro synthesis by enzymatic polymerization of simple glycosyl donors through repetitive condensation has been accomplished by transglycosylation using wild-type retaining endo-glycosidases (under kinetically controlled conditions), as well as glycosynthases devoid of hydrolase activity. This glycomimetics approach is a powerful tool to synthesize polysaccharides with defined structures and morphologies as novel biomaterials (Faijes & Planas, 2007). Transglycosylation reactions have been employed to synthesize a wide variety of oligosaccharides using various enzymes and glycoside donors, and this has been achieved in high yields. The reaction employs carbohydrate donors (such as sucrose, lactose) having a glycosidic leaving group (e.g., fructose, galactose, resp.) attached at its anomeric carbon centre. The method can be carried out in aqueous solutions without organic solvents to give the transglycosylation product in high yields and high regioselectivity. Various fructooligosaccharides have been synthesized through transfructosylation reactions using sucrose (Yun, 1996; Ghazi et al., 2005; Watanabe et al., 2006). β-Fructosylfuranosidases from Aspergillus niger have been employed commercially to produce mixtures of fructooligosaccharides with DP’s up to 5 (Rivero-Urgell & Santamaria-Orleans, 2001). 6-Kestose (β-Fruf-(2→6)-β-Fruf-(2→1)-α-Glcp) was produced by a β-fructosyl-furanosidase from the yeast, Scwhanniomyces occidentalis, by transferring fructosyl units from sucrose (ÁlvaroBenito et al., 2007). Galacto-oligosaccharides are obtained through the transgalactosidation of lactose by the action of β-galactosidases. The final products of the reaction presented a range of glycosidic linkages between monomers of galactose with glucose at the reducing terminus. They can include β-galactosides of (1→2), (1→3) and (1→4) linkages with branches along the chain. These structural features are important, because they present resistance to gastric acidity when these oligosaccharides are used as prebiotics (Barreteau et al., 2006). The βgalactosidases also catalyze transgalactosidation reactions that have galactose-constituents as final products when acting on lactose, and synthesized oligosaccharides from this substrate with yields ranging between 25 and 70 % (Lee et al., 1997; Tzortzis et al., 2005). Probiotic bacteria (several Bifidobacterium spp.) that produce β-galactosidases have been employed to synthesize novel oligosaccharides from lactose through galactosyl transfer reactions (Rabiu et al., 2001). The linkages of the oligosaccharides synthesized varied between the bacterial species studied, and showed a range of different glycosidic linkages comprising (1→3)- and (1→6)- linked galactosides, mixed linkages comprising (1→2)-galactosides and (1→6)glucosides, and various glucosides containing (1→2)- (1→3)- and (1→4)- linkages. The αgalacto-oligosaccharides as prebiotics are also of interest as they are non-digestible and reach the colon intact where they promote growth of the probiotic bacteria. Among the α-galactooligosaccharides are raffinose and stachyose. An α-galactosidase from Lactobacillus reuteri was used to synthesize α-galacto-oligosaccharides in transgalactosylation reactions from raffinose and melibiose, and produced stachyose, and 2 trisaccharides (α-Galp-(1→6)-αGalp-(1→6)-Glcp and α-Galp-(1→6)-α-Galp-(1→6)-Galp), resp. (Tzortzis et al., 2003).
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Production of isomalto-oligosaccharides such as panose (α-(1→6)-linked maltose) involves transglucosylation reactions catalyzed by α-glucosidases using maltose as substrate (Hang & Woodams, 1995). Panose could also be produced from maltose using intact cells of Aureobasidium pullulans to selectively catalyze transglucosylation (Yun et al., 1994). A novel α-glucosidase from the basidiomyceteous yeast, Xantophyllomyces dendrorhous, acting on maltose synthesized several isomalto-oligosaccharides with α-(1→2), α-(1→4) and α-(1→ 6) glucosidic bonds of which the main transglycosylation products were panose and isomaltosyl-maltose (α-Glcp-(1→4)-α-Glcp-(1→6)-α-Glcp-(1→4)-Glcp) (Fernández-Arrojo et al., 2007). Panose and malto-oligosaccharides of DP 3-9 bearing α-(1→6)- and α-(1→3)glucosidic linkages were obtained in a reaction catalysed by an alternansucrase from Leuconostoc mesenteroides from sucrose and maltose (Sanz et al., 2005). Synthesis of laminari-oligosaccharides (β-(1→3)-glucose linked) through transglucosylation with β-glucosidases have also been described (Smaali et al., 2004). Two fungal βglucosidases (Sclerotinia sclerotiorum and Aspergillus niger) were used to synthesize a wide range of gluco-oligosaccharides from cellobiose, and produced β-(1→6)-branched β-(1→3) laminario-oligosaccharides corresponding to the structural domain of the polysaccharide epiglucan. The synthesis products also included cellotriose, cellotetraose, gentiobiose, and several mixed-linked oligosaccharides such as β-Glcp-(1→3)-β-Glcp-(1→4)-Glcp, β-Glcp(1→6)-β-Glcp-(1→4)-Glcp and a (1→6)-linked glucosyl-laminaribiose (β-Glcp-(1→6)-βGlcp-(1→3)-Glcp) (Smaali et al., 2004). A glucosyl-laminaribiose was also produced by a (1→3)-specific β-glucosidase from Phanerochaete chrysosporium using laminaribiose as substrate. The mode of substrate binding at the active centre of the enzyme was found to play a key role in the transglucosylation-catalysed reaction (Kawai et al., 2004). Gentio-oligosaccharides (β-(1→6)-glucose linked) have also been obtained through transglucosylation reactions with fungal β-glucosidases, but in this case, in the presence of high concentrations of glucose (Unno et al., 2005). From the foregoing, it is apparent that each enzyme showed specificity for the formation of particular β-(1→3), β-(1→4) and β(1→6) glucosidic linkages, and this specificity appeared to be dependent upon the source of the enzyme, and the reaction conditions used. Through transglycosylation reactions, xylo-oligosaccharides bearing O-alkyl groups were prepared from xylan in the presence of alcohols with primary alcohols being better acceptors for this type of reaction. Jiang et al. (2004) using a recombinant xylanase B preparation from Thermotoga maritima obtained alkyl-β-xylosides and alkyl-β-xylobiosides as final endproducts of the reaction. An endo-β-(1→4)-glucanase produced by A. niger was demonstrated to participate in the transfer of cello-oligosaccharides to acceptor molecules such as 1-octanol in the presence of cellopentaose as the donor molecule to produce 1-octyl-cellotrioside (Akiba et al., 1999). An exo-α-(1→5)-L-arabinanase produced by Pycnoporus chrysogenum in the presence of acceptor groups such as aliphatic alcohols, glycerol and some sugars (arabinose, fructose, galactose, glucose, rhamnose and xylose) showed transarabinosylation activity resulting in the formation of alkyl-arabino-oligossacharides (Sakamoto et al., 2004). Transglycosylation reactions have also been used as a tool to obtain polysaccharides, as in the case of an α-amylase from Dictyoglomus thermophilum, which acted on maltooligosaccharides and cycloamyloses to produce an amylose chain of about 200 kDa (Nakajima et al., 2004). Another example of reactions catalyzed by glucosyl transferases are the synthesis of cyclomaltodextrins (also known as the cyclodextrins, CD); cyclic oligosaccharides consisting of α-(1→4)-linked 6-, 7-, or 8- glucose units. They are formed
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enzymatically from starch by the action of microbial cyclomaltodextrin glucanotransferases [CGTase; α-(1→4)-D-glucan 4-α-(1→4)-D-glucano-transferases (cyclizing)] (Pulley et al., 1961). A novel CGTase (an isocyclomalto-oligosaccharide glucanotransferase) from Bacillus circulans has been described that acted on malto-oligosaccharides of DP>3, amylose and soluble starch to produce an unusual cyclomaltopentaose; cyclized by an α-(1→6)-linkage through an intramolecular α-(1→6)-glucosyl transfer reaction (Watanabe et al., 2006).
ISOMERIZATION REACTIONS Lactulose (β-Galp-(1→4)-Fruf) is a semi-synthetic disaccharide composed of fructose and galactose resulting from the alkaline isomerization of lactose. This disaccharide is present in significant concentrations in some food products since the isomerization can occur naturally, as for example, during heat treatment of milk (Barreteau et al., 2006). However, the isomerization reaction has several drawbacks, and the steps required for the separation and purification of these products presents high costs and results in low yields of lactulose. Several studies have investigated viability of biological production of lactulose by enzymes (Kim et al., 2005).
FERMENTATIVE PROCESSES TO OLIGOSACCHARIDES The use of fermentation processes for the production of oligosaccharides in-vivo is an interesting and viable alternative as isolated enzyme preparations are not required. The fermentative production of galacto-oligosaccharides from lactose by lactic acid bacteria and some yeasts is the most widely used procedure (Onishi & Tanaka, 1998). Galactooligosaccharides participating in the composition of fermented products like yogurt result from the fermentation of sugars in milk (lactose and galactose) by the probiotic bacteria present (Houdijk et al., 2002). The yeast Aureobasidium pullulans produces five β-fructofuranosidase isoenzymes when grown on sucrose as sole carbon source (Yoshikawa et al., 2006). During microbial growth, these enzymes catalyze transfructosylation reactions of sucrose leading to the formation of fructo-oligosaccharides and glucose as final products. Immobilized microorganisms have also been employed for the production of neofructo-oligosaccharides, e.g., cells of Penicillium citrinum immobilized in calcium alginate beads (Park et al., 2005). The potential application of by-products and agricultural wastes as substrates for microbial conversion into products of commercial interest has also been investigated. Sugarcane molasses, rich in sucrose, has been evaluated as a substrate for the production of fructo-oligosaccharides by Aspergillus japonicus and A. niger (Dorta et al., 2006). Fermentation is also a step leading to the pre-purification of oligosaccharides formed by transglycosylation reactions. For example, isomalto-oligosaccharides have been prepared from starch using a fermentation process that employed yeasts (Saccharomyces cerevisiae and Saccharomyces carlsbergensis) to remove metabolizable sugars formed during the reaction (glucose, maltose, and maltotriose) leaving the oligosaccharides of interest intact (Pan & Lee, 2005).
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FUNCTIONS OF OLIGOSACCHARIDES Applications in Foods and Beverages Oligosaccharides based upon glucose and maltose finds various applications in foods (baked products) and beverages, and these products are formed in-situ through enzymatic hydrolysis of starch (Dekker, 1994). For example, to improve the quality of flour for bread making, fungal α-amylases (Aspergillus oryzae) are often added to the flour resulting in the hydrolysis of the starch into α-limit dextrins (dextrins are defined as partially-degraded polysaccharides, although they are really oligosaccharides of high DP), and glucose and maltose. The in-situ β-amylases (exo-acting enzymes) present in flour further hydrolyse these dextrins to maltose, which is subsequently fermented by baker's yeast in making bread. Starch is marketed industrially in various grades as unmodified, modified and derivatized starches, or as starch syrups of varying degrees of hydrolysis. When hydrolysed to maltodextrins or syrups containing high maltose levels, they find use in foods and beverages as sweeteners. In starch saccharification, the limit dextrins produced during starch liquefaction are further hydrolysed to glucose, maltose and oligosaccharides depending on the desired properties of the final syrup. Starch syrups of different grades for commercial use can be produced using different amylases, or combinations of amylases. Syrups containing high maltose levels are generally quite sweet and find use in canned fruits, beverages and bakery products. Starch syrups of low maltose levels, but high maltodextrin (DP 3-9) content, can be produced by limited enzymolysis, and find use as nutritive bulking agents because of their clean bland flavour, lack of sweetness, low hygroscopicity and ease of solubility in water. Syrups of high maltose but low glucose content can be produced using β-amylase, which converts the amylose component of starch almost completely to maltose, while the amylopectin component is degraded to a β-limit dextrin. Thermolabile α-amylases (fungal) can also be used for this purpose and produce syrups of maltose contents up to 60 %. Alternatively, high maltose syrups can be obtained by supplementing β-amylase with pullulanase or isoamylase; enzymes that attack the α-(1→6)-glucosidic linkages of branch points in starch.
As Dietary Fiber Oligosaccharides have been used commercially as ingredients in cosmetics, medicines and agricultural products, as well as in food products as discussed above (Cano et al., 2006). These saccharides act as substitutes for sugar sweeteners of low-calorie value in foods, and are also added to juices and soft drinks to improve their organoleptic properties (Nakakuki, 2002; Kawase et al., 2001; Oku & Nakamura, 2002). They also act as anti-hygroscopic agents protecting food from the loss of water (Moon & Cho, 1997). Recently, studies on the development of new food and functional food ingredients (nutraceuticals) leveraged on new biotechnological processes to obtain oligosaccharides because of their increasing demand due to their peculiar properties (Moure et al., 2006). “Dietary fiber”, a term used to name substances present in foods, are resistant to digestive enzymes of the human gastro-intestinal tract (Phillips et al., 2008). The presence of fiber in food products is important as it assists the proper functioning of the digestive system and
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prevents the emergence of diseases such as short-bowel syndrome; a malabsorption disorder caused by the complete dysfunction of a large segment of the large intestine (Lairon et al., 2005). The change in people’s dietary habits in today’s world comprising both developed and developing countries has been linked to the increasing occurrence of chronic diseases such as obesity, atherosclerosis, hypertension, osteoporosis and even some types of cancers. Recent research showed a relationship between diabetes mellitus and a low consumption of dietary fiber in diets by adults (Ballesteros et al., 2001). The quality of life promoted during childhood has served as a call to food industries to develop functional food products according to the current consumption of diets rich in fat and low in fiber among children and adolescents (Vitolo et al., 2007). Use of additives such as oligosaccharides in foods like cakes (Moscatto et al., 2006), fermented lactic beverages (Oliveira et al., 2002) and yogurts (Aryana et al., 2007) (preferred by this age group) is an alternative to meet these requirements in populations. Oligosaccharides as prebiotics are dietary ingredients that comprise complex carbohydrates such as those discussed above. They are considered not digestible and are resistant to gastric acidity, as well as towards the actions of the salivary, gastric and intestinal enzymes. Prebiotics constitute nutritional ingredients fermented by the intestinal microflora, and selectively stimulate the proliferation of the bifidobacteria and lactobacilli at the expense of less-desirable organisms (Escherichia coli, clostridia, and proteolytic bacteroides) creating a healthy balance in the human host. This selective fermentation is known as the prebiotic concept as defined by Gibson and Roberfroid (Gibson & Roberfroid, 1995; Gibson, 2004). Prebiotics are known to possess platelet anti-aggregate activity that prevents infectious enteric pathogens adhering to the epithelial cells of the intestinal tract (Holzapfel & Schillinger, 2002; Shoaf et al., 2006). Prebiotics such as short-chain fructo-oligosaccharides possessing anti-inflammatory activity have also been described, and were effective in exerting their effects on induced inflammation in the distal colon of rats (Lara-Villoslada et al., 2006). Viable microbes that beneficially affect the health of individuals and provide a balance of intestinal microbial flora are known as probiotics. The most commonly employed probiotics are the bifidobacteria, lactic acid bacteria and some yeasts, and prevent, or can be used to treat, various gastro-intestinal disorders, such as lactose intolerance, constipation, food hypersensitivity and gastroenteritis (Klaenhammer, 2000; Blum et al., 2002). Recent research has shown the beneficial effects of probiotics on the equilibrium of intestinal bacterial in controlling diarrhea. The probiotic bacteria act by producing vitamins that allow the absorption of minerals and trace elements, and produce important digestive enzymes such as β-galactosidase (Holzapfel & Schillinger, 2002; Ramesh & Tharanathan, 2003). Therefore, the presence of bifidobacteria and lactobacilli constitute a criterion as an indication of good health by being part of the colonic microflora, and the ingestion of prebiotics is important for their maintenance. They also help protect against infections and allergies during childhood (Bruzzese et al., 2006). The fructo-oligosaccharides are considered the chief carbohydrates possessing prebiotic action, and consequently are added to various food products (biscuits, drinks, yogurts, breakfast cereals, jams and sweets) to stimulate and promote good health (Yun, 1996; Roberfroid, 2002). Fructo-oligosaccharides selectively promote the growth of Acidophillus, Bifidus and Faecium spp.; bacteria beneficial to the gastro-intestinal tract (Campbell et al., 1997; Collins & Rastall, 2008). By stimulating the intestinal flora, the fructo-oligosaccharides contribute to
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the increased production of organic acids in the colon, and thus inhibit the proliferation of pathogens sensitive to these changed conditions (Ten Bruggencate et al., 2003). The equilibrium produced in the gastro-intestinal flora is also responsible for other benefits to human metabolism, such as normalizing blood pressure in hypertensive individuals, change in the metabolism of gastric acid, reduce the absorption of carbohydrates and lipids, and improve metabolism in diabetics (Zafar et al., 2004; Ducros et al., 2005; Barreteau et al., 2006). Fructo-oligosaccharides are more soluble than the parent inulins from which they are derived, and likewise resist enzymatic hydrolysis in the human gastro-intestinal tract, but are fermented by the bacterial flora in the colon. They hence have a lower caloric value, although they are sweet with sugar sweetness levels between 30 to 50 per cent of commercial syrups, they can be used as an alternative sweetener. They also contribute a dietary fiber component in foods when added to dairy products (yogurt). (Kaplan & Hutkins, 2000) The xylo-oligosaccharides likewise act as prebiotics, and decrease the levels of sugars in the blood, and regulate fat metabolism (Alonso et al., 2003). Other oligosaccharides, such as lacto-sucrose, isomalto-oligosaccharides, galacto-oligosaccharides, raffinose and stachyose have also received attention, because they can act as growth factors and are used by Bifidobacterium spp. when included in different foods (Onishi & Tanaka; 1998; Kihara & Sakata, 2002; Oku & Ura , 2003; Yamashita et al., 2005; Collins & Rastall, 2008). Although bitter in taste, the gentio-oligosaccharides too have bifidogenic activity (Rycroft et al., 2001; Unno et al., 2005). Recent studies have shown that gentio-oligosaccharides of DP 2-3, showed better responses as a factor for the growth of Bifidobacterium spp. than those of a higher DP (4 and 5) (Sanz et al., 2006). In turn, the laminari-oligosaccharides are likewise able to stimulate the proliferation of Bifidobacterium spp. of the caecum (Salyers et al., 1977). Ingestion of foods containing prebiotics and probiotics can result in the generation of abdominal gas (flatus) by the intestinal microflora, and this can sometimes cause malaise. Minimizing gas generation has been studied through the synergistic action of adding mixtures of oligosaccharides to foods and this appears to diminish these effects (Ghoddusi et al., 2007). The effectiveness of each kind of prebiotic oligosaccharide depends upon its chemical structure and constitution. For example, only neoagaro-oligosaccharides of high DP have been shown to produce a prebiotic effect similar to those of fructo-oligosaccharides (Gong et al., 2006). The number of Bifidobacterium spp. in the intestine of children breastfed is considerably higher than those found in infants fed formula foods intended for children. Human milk contains oligosaccharides as discussed above that promote the proliferation of certain species of Bifidobacterium. In recent years, the demand for an ideal formula of probiotics and prebiotics in infant foods at the stage of lactation has increased (Puccio et al., 2007).
BIOLOGICAL ACTIVITY OF OLIGOSACCHARIDES Some excellent reviews on the biological activity of polysaccharides have recently been published (Leung et al., 2006; Chen and Serviour, 2007), but until lately, few studies have concentrated on bioactive oligosaccharides derived from these polysaccharides (Vos et al., 2007; Vos, 2008).
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Ellen C. Giese, Aneli M. Barbosa and Robert F. H. Dekker Table 3. Oligosaccharides as functional food ingredients: biological properties.
Oligosaccharide Xylo-oligosaccharides Fructo-oligosaccharides
Lactosucrose Mannooligosaccharides Guar gum hydrolysates Isomaltooligosaccharides Galactooligosaccharides Transgalactooligosaccharides Lactulose
Function Promotes the formation of vitamin B Enhancement of organic acids production and inhibition of pathogen proliferation in the stomach Increases the absorption of calcium by the intestine Reduction of blood pressure in hypertensive individuals Acts on lipid metabolism and decreases atherogenesis Prevents dental caries and relieves constipation in the elderly population Increases the resistance to fractures Promotes absorption of calcium in the post menopausal period of women Acts in the synthesis of short chain fatty acids in humans, and decreases serum triacylglycerol levels
Reference Alonso et al. 2003 Ten Bruggencate et al., 2003 Kishino et al., 2006 Hoshino-Takao et al., 2008 Suzuki & Hara, 2004 Kaneko et al., 1995; Chen et al., 2001 Van den Heuvel et al. 2000 Gibson, 2004 Vogt et al., 2006
The concept of functional foods emerged in Japan, and was regulated in 1991 and received the name “Foods for Specified Health Use” (Nakakuki, 2003). Functional foods are those that produce metabolic or physiological effects through the action of a nutrient or nonnutrient in the growth, development, maintenance and other normal functions of the body. Defined as products, functional foods contain in its composition some biologically active substances, which when added to a normal diet promotes metabolic or physiological processes resulting in reducing the risk of disease and maintenance of good health (Nagarajan et al., 2006). The addition of fiber in these foods must be indicated so that when ingested, it is not the lure of dietary nutrients such as lipids, minerals, proteins and carbohydrates that are digestible (Moura et al., 2007). Products intended for children are specially controlled, since metabolites (steroid hormones) formed through cholesterol metabolism are essential for the needs of the growing child, and some oligosaccharides are used as food additives by presenting hypocholesterolaemic action (Alliet et al., 2007). Fructo-oligosaccharides are known to increase the absorption of copper, calcium, magnesium and iron (Zafar et al., 2004; Ducros et al., 2005; Barreteau et al., 2006; Ohta, 2006; Borycka & Stachowiak, 2008), and also exert a positive effect on the treatment of hyperplasia in the caecum (Nzeusseu et al., 2006). The main biological properties of oligosaccharides as functional food ingredients are summarised in Table 3. Functional properties of oligosaccharides are directly related to their 3-D structure, which is responsible for interactions with other biomolecules (Almond, 2005; Collins & Rastall, 2008). Oligosaccharides have been extensively used for their rheological properties as thickeners in emulsions of glucose, amino acids and lipids when administered intravenously in postsurgery patients (Stegink et al., 1987). They have also been studied for use as drug carriers through controlled-release of active ingredients. They do this by presenting resistance to the
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digestive action of glycosidases and other hydrolases in the human system (Yuasa & Hashimoto, 2001; Coviello et al., 2005). The administration of these drugs has advantages over conventional therapies, which can be toxic due to the high amount of the drug’s active principle, or they can even be ineffective (Langer, 1995). Oligosaccharides such as the cyclodextrins, or those presenting triple helical conformations, can be used as carriers of small molecules such as peptides, or proteins and nucleic acids. These nanoparticles can serve to protect, transport and deliver drugs, and are quickly cleared from the bloodstream after intravenous administration (Lemarchand et al., 2006). Bioactive gluco-oligosaccharides derived from β-(1→3)-glucans stimulate antiinflammatory responses mediated by cells of the immune system through the induction of pro- and anti- inflammatory mediators such as cytokines, tumor necrosis factor-alpha (TNFα) and also interleukin-1 among others. The mechanism of anti-tumor action has been linked to the induction of various immune responses of the host, mainly by activating the natural killer cells (Pang et al., 2005). Gluco-oligosaccharides have excellent activity as immunostimulatory agents (Pang et al., 2005). For example, laminaritetraose and laminaripentaose stimulate phagocytic activity of granulocyte macrophages responsible for phagocytosis of necrotic tissue, and can stimulate cells in the immune system (Jamois et al., 2005). Oligosaccharides derived from curdlan, a bacterial linear β-(1→3)-glucan, increased the production of TNF-α by macrophages (Kataoka et al., 2002). A mixture of β-1,3-glucooligosaccharides (DP ≥ 4), prepared from laminarin using a bacterial β-1,3-glucanase, when incubated with human peripheral blood monocytes showed strong inhibitory activity against the proliferation of human myeloid leukemia U937 cells (laminarin did not). Although the β1,3-gluco-oligosaccharides and laminarin had no direct cytotoxic effect on U937, the cytotoxicity of the former stemmed from cytokines produced when the monocytes were stimulated by the enzymatically-degraded laminarin by inducing production of TNF-α (Miyanishi et al.,2003). Further studies using gluco-oligosaccharides from different algal laminarins confirmed this observation (Pang et al., 2005). The DP of gluco-oligosaccharides also appears to be crucial for their pharmacological implementation. For example, laminariheptaose showed distinct biological activity over laminarihexaose because of its spatial conformation (Lowe et al., 2001; Bland et al., 2004). Oligosaccharides containing pyruvic acid acetal groups are components of several bacterial antigenic factors, and are considered potential targets for immunological and synthetic vaccine development (Gonçalves et al., 2006; Nabarlatz et al., 2007). Introduction of ionic groups in these biomolecules not only increased their solubility in water, but promoted changes in intra- and inter- molecular hydrogen bonding, and increased the repulsion between the chains that changed their conformation and hence their biological activity (Zhang & Kong, 2003). Recent research reported that maltodextrins were effective in reducing up to 52 % of colon cancer in mice (Verghese et al., 2005), and xylo-oligosaccharides have been described as being more effective in treating cancer of the colon that fructo-oligosaccharides (Hsu et al., 2004).
CONCLUSION The oligosaccharides are a class of bioactive carbohydrates of importance to human health. Advances in technology used in the procedures for obtaining these carbohydrate
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oligomers are necessary for the development and their inclusion as functional products in foods. The use of enzymes in preference to physical and chemical processes in obtaining oligosaccharides has become possible through the advent of genetic engineering technology that select new mutant enzymes for the production of these biocatalysts. The awareness and the adequacy of the diet are necessary to ensure that all the benefits of these carbohydrates are present.
ACKNOWLEDGMENTS Dr. EC Giese is grateful to CAPES (Brazil) for a post-doctoral fellowship, and Dr. AM Barbosa acknowledges Fundação Araucária do Paraná (Brazil).
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Sakamoto, T., Fujita, T. & Kawasaki, H. (2004). Transglycosylation catalyzed by a Penicillium chrysogenum exo-1,5-α-L-arabinanase. Biochimica et Biophysica Acta (BBA). - General Subjects, 1674, 85-90. Salyers, A. A., Palmer, J. K. & Wilkins, T. D. (1977). Laminarinase (β-glucanase) activity in bacteroides from the human colon. Applied and Environmental Microbiology, 33, 11181124. Sanz, M. L., Côté, G. L., Gibson, G. R. & Rastall, R. A. (2005). Prebiotic properties of alternansucrase maltose-acceptor oligosaccharides. Journal of Agricultural and Food Chemistry, 53, 5911 916. Sanz, M. L., Côté, G. L., Gibson, G. R. & Rastall, R. A. (2006). Selective fermentation of gentiobiose-derived oligosaccharides by human gut bacteria and influence of molecular weight. FEMS Microbiology Ecology, 56, 383-388. Schmid, F., Stone, B. A., Mcdougall, B. M., Bacic, A., Martin, K. L., Brownlee, R. T. C., Chai, E. & Seviour, R. J. (2001). Structure of epiglucan, a highly side-chain/branched (1,3; 1,6)-β-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Scglecht. Carbohydrate Research, 331, 163-171. Shoaf, K., Mulvey, G. L., Armstrong, G. D. & Hutkins, R. W. (2006). Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infection and Immunity, 74, 6920-6928. Shoda, S., Fujita, M. & Kobayashi, S. (1998). Glycanase-catalyzed synthesis of non-natural oligosaccharides. Trends in Glycoscience and Glycotechnology, 10, 279-289. Silva, I. R., Monteiro, N. K., Martinez, P. F., Izeli, N. L., Vasconcelos, A. F. D., Cardoso, M. S., Corradi Da Silva, M. L., Barbosa, A. M., Dekker, R. F. H., Silva, G. V. J. & Moraes, L. A. B. (2008). Purificação e caracterização de uma gentioexaose obtida de botriosferana por hidrólise ácida parcial. Química Nova, 31, 1015-1019. Smaali, M. I., Michaud, N., Marzouki, N., Legoy, M. D. & Maugard, T. (2004). Comparison of two β-glucosidases for the enzymatic synthesis of β-(1;6)-β-(1;3)-glucooligosaccharides. Biotechnology Letters, 26, 675-679. Stegink, L. D., Zike, W. L., Andersen, D. W. & Killion, D. (1987). Oligosaccharides as an intravenous energy source in post-surgical patients: utilization when infused with glucose, amino acids, and lipid emulsion. American Journal of Clinical Nutrition, 46, 461-466. Sun, H. J., Yoshida, S., Park, N. H. & Kusakabe, I. (2002). Preparation of (1→4)-β-Dxylooligosaccharides from an acid hydrolysate of cotton-seed xylan: suitability of cottonseed xylan as a starting material for the preparation of (1→4)-β-D-xylooligosaccharides. Carbohydrate Research, 337, 657-661. Sutherland, I. W. (1984). Enzymes in the assay of microbial polysaccharides. Process Biochemistry, 19, 19-24. Sutherland, I. W. (1998). Novel and established applications of microbial polysaccharides. Trends in Biotechnology, 16, 41-46. Suzuki, T. & Hara, H. (2004). Ingestion of guar gum hydrolysate, a soluble and fermentable nondigestible saccharide, improves glucose intolerance and prevents hypertriglyceridemia in rats fed fructose. The Journal of Nutrition, 134, 1942-1947. Ten Bruggencate, S. J. M., Bovee-Oudenhoven, I. M. J., Lettink-Wissink, M. L. G. & Van Der Meer, R. (2003). Dietary fructo-oligosaccharides dose-dependently increase translocation of salmonella in rats. The Journal of Nutrition, 133, 2313-2318.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 34
AMIDATION OF CELLULOSE MATERIALS Nadege Follain
*
Centre de recherche sur les Macromolécules Végétales (CNRS) and Joseph Fourier University, BP 53, 38041 Grenoble Cedex 9, France.
ABSTRACT Native cellulose is a structural material that is biosynthesized as microfibrils by a number of living organisms ranging from higher and lower plants, to some ameobae, sea animals, bacteria and fungi. Depending on their origin, individual cellulose microfibrils have diameters from 2 to 20 nm, while their length can reach several tens of microns. The chemical modification of cellulose microfibrils is investigated for preparing new biobased materials with end-use properties in the fields of adhesion, textile, detergent, paint, cosmetic, medicine, food, etc. Among the possible chemical modifications, the selective oxidation of the primary alcohol group of polysaccharides has been studied for more than a half-century. Recently, a method for selectively oxidizing primary alcohol groups of polysaccharides has been described in literature without degradation of products. The technique is based on a reaction catalyzed by 2,2,6,6-tetramethyl-1-piperidine oxoammonium radical (TEMPO) in presence of NaOBr, generated in situ by NaOCl and NaBr, the catalyst being regenerated during the reaction. The chemical modification is a way to modify and introduce specific functionalities leading to the development of new biopolymers in macromolecular prodrug carrier, bio-based composites, nanocomposites, for example. The polymer must be biodegradable and / or biocompatible and must contain appropriate functional sites for chemical conjugation. Despites on large interest on natural and synthetic biodegradable polymers investigated, important efforts are continuing to search for new systems, notably on cellulose. In this report, the amidation of cellulose materials previously modified by carboxylation reaction is realized from the selection of some amines (cyclic and linear structures). The carboxylation is resulted from the TEMPO mediated oxidation of *
Corresponding author: University of Rouen, Laboratory « Polymers, Biopolymers and Surfaces », FRE 3101 CNRS & FR3038, 76821 Mont-Saint-Aignan cedex, France, Telephone: 33-235 14 66 98; Fax: 33-235 14 67 04; E-mail address:
[email protected] 1002
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cellulose, leading to partially or totally oxidized cellulose, in presence of carbodiimide which is known to increase the reactivity of carboxyl groups toward amidation but used rarely for polysaccharides holding carboxyl moieties. Few reports were found in the literature on the use of carbodiimide in the preparation of cellulose conjugates with amines in order to develop new modified cellulose materials. The goals of this report are to develop ways of preparing the cellulose conjugates which can be water-soluble materials or water-insoluble materials, to identify linkage with carboxylated cellulose materials through amide bonds and to understand the obtained results following by FTIR, NMR spectroscopies (at liquid and solid states) and electron paramagnetic resonance spectroscopy. The carboxyl groups content of oxidized cellulose materials after carboxylation and after amidation reactions are equally determined by titration curves of conductimetry and elemental analysis.
INTRODUCTION Native cellulose, structural material biosynthesized, is produced in the form of microfibrils having diameters ranging from 2 to 20 nm depending on their origin, while their length can reach several tens of microns [1]. These microfibrils are made of perfectly aligned cellulose molecules organized into a nearly defect-free crystalline arrangement. Some imperfections, referred to as amorphous zones, along the microfibril length are hydrolyzed by acid treatment to produce nanocrystals called cellulose whiskers [2]. The shape and size of nanocrystals are more or less fixed by cellulose origin: different samples like tunicin, cotton, bacterial cellulose, parenchyma cell cellulose produced different sizes of nanocrystals even under similar hydrolysis conditions. Recently, a method was found to catalyze the selective oxidation of primary hydroxyl groups in aqueous media [3-4]. This technique is based on a reaction catalyzed by 2,2,6,6tetramethyl-1-piperidine oxoammonium radical (TEMPO) in presence of NaOBr, generated in situ by NaOCl and NaBr, the catalyst being regenerated during the reaction [3, 5-6]. This method was firstly proposed for water-soluble polysaccharides [5-9] such as starch, inulin, amylodextrin, pullulan, amylopectin, chitosan. The method has been later extended to waterinsoluble polysaccharides such as cellulose, amylose and chitin [7-12]. In the case of cellulose I or native cellulose, the oxidation takes place only at the crystal surfaces which became negatively charged; a phenomenon that can be interesting for subsequent grafting or derivatization purposes [10]. Nevertheless, the cellulose can be fully oxidized to yield pure polyglucuronic acid only if regenerated or mercerized cellulose samples are used [9]. In the literature, the procedure leading to cellulose III allomorph from cellulose I is described from to an ammonia treatment [13]. This procedure known to increase the accessibility of crystalline cellulose [14] is realized at a solid-state that allows to maintain the integrity of the cellulose microfibrils while a substantial decrystallization combined to a reorganization of the intracrystalline hydrogen bond network of cellulose are achieved. By way of this procedure, the conversion of cellulose I into IIII improves its reactivity with respect to the TEMPO mediated oxidation system. If the cellulose I is used without any treatment as starting material, a partially conversion is realized due to its higher crystallinity and therefore the poor accessibility of primary hydroxyl groups. This approach introduced carboxyl groups at the surface of the crystals providing surface carboxylated cellulose nanocrystals materials. A number of applications have been described for carboxylated cellulose (also named oxidized
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cellulose) in the fields of gelation, complexation, antifloculation activities, adhesion, biological activities and as well as for textile, paper, reinforced bio-based composites. Polymers having polar groups like carboxylated cellulose are easily modified by esterification or amidation among others, the latter given generally better chemical resistance against hydrolysis. The establishment of an amide bond has been firstly proposed by Danishefsky [15] applied to a mucopolysaccharide. However, some reports claimed successful formation of amide link [15-16] whereas others reported failure in amidation [1718]. Recently, the use of N-hydroxysuccinimide like an activating reagent was found to be effective for the EDAC mediated amidation [10, 18]. Water-soluble carbodiimides such as EDAC present the advantage of being soluble in aqueous media and can be eliminated by simple dialysis. The grafting conditions of amines on oxidized cellulose were extensively studied [10, 18-22] and the procedures are further and still based on the Bulpitt and Aeschlimann works [18]. This procedure is applied to water-soluble carboxylated cellulose and equally water-insoluble carboxylated cellulose. The selection of single terminally aminated molecules like grafted reagents corresponded to primary linear and cyclic amines. In this chapter, an attempt to modify the cellulose material from totally oxidized cellulose (water-soluble cellulose) and partially oxidized cellulose (water-insoluble cellulose) samples is proposed to confer new features to expand the potential applications. The goals of this work are to describe a new way to get cellulose derivatives, to introduce the grafting mechanism, to investigate it from to a large range of characterization and to correlate a theoretical knowledge with the experimental results derived from conductimetry, elemental analysis and EPR, FTIR, NMR measurements. Considerable and promising efforts are going ahead to develop new macromolecular carrier systems and reinforcing structure in bio-based composites based on oxidized cellulose that is biocompatible and biodegradable polymer [23].
EXPERIMENTAL SECTIONS 1) Materials Cellulose A batch of cotton linters from Tubize Plastics, Rhodia (Belgium), was used as received. Chemicals For the oxidation reaction, 2,2,6,6-tetramethylpiperidine-1-oxyl radical (called TEMPO), sodium bromide and sodium hypochlorite from Aldrich were directly used. For the amidation reaction, N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS) provided by Sigma Chemical Co (Table 1) were used as reagents. For grafting, the amines were purchased from Aldrich Chemical Co: 4-amino 2,2,6,6tetramethylpiperidine-1-oxyl radical (called 4-amino TEMPO), n-octylamine, n-butylamine and 2-methoxyethylamine (Table 2).
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191.70
NHS
115.09
Formula
Table 2. Amines used for grafting reaction. Amines
Molecular weight (g/mol)
4-amino TEMPO
171.26
n-octylamine
129.15
n-butylamine
73.14
2-methoxyethylamine
75
Formula
Notations 1 eq of X represents the molar quantity of X for 1 mol of glycosyl unit (M = 162 g/mol).
2) Preparation of Cellulose Substrates Preparation of cellulose III by ammonia treatment In order to improve the accessibility of crystalline cellulose [24], the swelling of cellulose in ammonia or in molecules such as amines is a simple and classical way. This procedure leads to cellulose IIII from cellulose I (native cellulose) and has been frequently used to improve the reactivity of crystalline cellulose for the preparation of derivatives in better yields [25-26]. Indeed, this conversion carried out essentially at a solid-state process that keeps the integrity of the cellulose microfibrils while achieving a substantial decrystallization and a reorganization of the intra-crystalline hydrogen bond pattern of cellulose [27-28].
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Ammonia or amines enter the cellulose crystals as guests, which not only distort the crystals but also modify the conformation of the hydroxymethyl group within the cellulose lattice itself [29-30]. The protocol to prepare cellulose III [13] involved the use of exploded gaseous ammonia (EG-NH3) and corresponded to samples processed by Rhodia Acetow, following their patented process, which consists of treating cellulose samples with gaseous ammonia under high pressure followed by a rapid decompression [31-32]. This particular cellulose preparation was obtained from Rhodia, company which developed the technology for its use. The scientific basis for the “ammonia explosion” process has been achieved by Dale and collaborators [33-34].
HCl hydrolysis 20 g of cotton linters were hydrolyzed with 1 L of 2.5 M HCl at 100°C for 20 min. The hydrolyzate was filtered and washed with water until neutral pH. The weight loss resulting from the hydrolysis step was around 10% for cotton linters. It is estimated that this product loss corresponds to the hydrolysis of the amorphous zones.
TEMPO-mediated Oxidation Oxidation experiments were carried out on cellulose IIII and HCl-hydrolyzed cotton linters cellulose as previously published [12-13] with minor modifications [22]. In a typical run, cellulose samples (1.95 g, 12 mmol glycosyl units) were dispersed in distilled water (180 mL) for 3 minutes with a high speed T25 basic Ultra-Turax homogenizer (Ika-Labortechnik, Staufen, Germany). 90 mL of water used to wash the homogenizer was then added to the suspension. TEMPO (30 mg, 0.19 mmol), NaBr (0.63 g, 6.1 mmol) and NaOCl (1.76 M solution, 1.5 mL, 2.64 mmol) was stirred in 20 mL of water until complete dissolution. This solution was then added to the cellulose suspension, which was mechanically stirred and maintained at 20°C. The NaOCl (1.76 M solution, 11.5 mL, 20.24 mmol) was added dropwise to maintain the pH at 10 during the addition. After the total addition of NaOCl, the pH was maintained constant at 10 by adding 0.5 M NaOH solution until no more variation was observed indicating the end of the reaction. Methanol (5 mL) was then added to destroy the residual NaOCl and the pH was adjusted to 7 with 0.5 M HCl. A white suspension was obtained resulting from the oxidation of cellulose. After centrifugation, the supernatant, corresponding to the water-soluble oxidized cellulose with glucopyranose units completely oxidized, was separated to the water-insoluble fraction which corresponds to partially oxidized cellulose. The supernatant, referred to as polyglucuronic acid, was precipitated by adding an excess of ethanol, centrifuged, re-dissolved in water, dialyzed against water, and finally freeze-dried. The final yield of this process was between 90 and 95%. The waterinsoluble fraction was further purified by successive centrifugation, re-dispersion in water and finally by dialysis against distilled water to obtain the nanocrystal suspensions. Its final yield was about 92-95%. In the text, the samples are named surface carboxylated cellulose nanocrystals.
Grafting Reaction The grafting reaction between oxidized cellulose substrates and amines was performed according to Bulpitt and Aeschlimann [18] with minor modifications [22]. The typical run
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was achieved in aqueous media under stirring, with a typical addition of 2.5 mmol amine / 1 mmol glycosyl unit (2,5 eq). The pH of the solution was adjusted to 7.5-8 with 0.5 M HCl. N(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS) with ratios of 1.5 relative to glycosyl unit [22] diluted in 2 mL of water were added (1,5 eq). The pH was adjusted and maintained to 8 by adding 0.5 M HCl and 0.5 M NaOH solutions. The suspension was stirred during 24 h at 50°C and finally precipitated by adding an excess of ethanol. After filtration on a 0.5 µm membrane and washing with ethanol (3 times), the precipitate was redissolved in water, evaporated again to remove any trace of ethanol, re-suspended in water and finally freeze-dried.
3) Characterization Conductometric titration The residual carboxyl groups content of oxidized cellulose and grafted oxidized cellulose samples named degree of oxidation Do was determined by conductometric titrations [13]. Each dried cellulose (30-50 mg) was dissolved into 15 mL of 0.01 M hydrochloric acid solution. After 10 min of stirring (time to get stable suspension), the suspension was titrated with 0.01 M NaOH. The titration curves, exemplified in Figure 1, showed the presence of a strong acid corresponding to the excess of HCl and a weak acid corresponding to the carboxylate content.
Strong acid
Base
Weak acid
Figure 1. Typical conductometric titration curve of oxidized cellulose nanocrystals sample.
The carboxyl groups content of oxidized cellulose samples or degree of oxidation Do is determined by:
Do =
162 × n m − 36 × n
(1)
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After the grafting reaction, the residual carboxyl groups content Do1 is determined by:
Do1 = with
(162 + ( Ma − 4) × Do) × n m + ( Ma − 40) × n
162 (g/mol) corresponds to the molar mass of an unreacted cellulose unit Ma the molar mass of the amines m the weight of oven-dried sample (g) n (mol) the carboxylate content determined from : n = (V2 − V1 ) × c
(2)
(3)
where V1 and V2 are the equivalent volumes of NaOH (in L) and c the exact concentration of NaOH solution (mol/L). The degree of conversion (DC) which represents the number of grafted anhydroglucose units is deduced by the following equation: DC=1-Do1.
Infrared Spectroscopy Infrared spectra were recorded on a FT-IR Perkin-Elmer 1720X spectrometer. Samples were studied as KBr pellets (1% in anhydrous KBr). Spectra were recorded using 3600 cm-1 spectral width (between 400 and 4000 cm-1), 2 cm-1 resolution, and 32 scans were accumulated. Samples were studied as acidic form to avoid the superposition of sodium carboxylate peak with hydrogen bonds. For this, few milligrams of sample were suspended in 1 mL of water, 1-2 drops of 1M HCl were added and after stirring during 3-5 min the suspension was centrifuged and the precipitate was washed several times with water until neutrality. Liquid-state NMR Spectroscopy 13 C and 1H spectra were recorded with a BRUKER Avance 400 spectrometer operating at a frequency of 100.618 MHz for the 13C and 400.13 MHz for the 1H. Samples were studied as their sodium salt solutions in D2O (6-10 mg in 500 µL of solvent) at 30°C in 5 mm o.d. tubes. 13 C spectra were recorded using 90° pulses, 20000 Hz spectral width, 65536 data points, 1.638 s acquisition time, 2 s relaxation delay. From 4096 up to 10240 scans were accumulated depending on the sample solubility. Proton spectra were recorded with 4006 Hz spectral width, 32768 data points, from 4.089 up to 7.497 s acquisition time, 0.1 s relaxation delay and up to 128 scans. The 2D 13C-1H experiments were performed with 4006 Hz spectral width, 2048 data points, 0.266 s acquisition time, 1 s relaxation delay and 128 scans. Solid-state NMR Spectroscopy The NMR experiments were performed with a Bruker Avance 400 WB spectrometer operating at a 13C frequency of 100.62 MHz using the combined technique of proton dipolar decoupling (DD), magic angle spinning (MAS) and cross-polarization (CP). 13C and 1H field strengths of 100 kHz were used for the matched spin-lock cross-polarization transfer. The spinning speed was set at 12,000 Hz for all the samples. The contact time was 2 ms, the acquisition time 30 ms and the recycle delay 4 s. The deconvolution of the spectra was achieved following earlier procedure [35]. The position and width of the lines were maintained constant throughout a series of samples. The area corresponding to the integration
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of the C1 signal was set to one. The evaluation of the oxidation and crystallinity degree was made from the integration of the corresponding deconvoluted lines.
Electron paramagnetic resonance (EPR) EPR measurements were made with a Bruker EMX X-band continuous wave spectrometer equipped with a Bruker ER 4116 DM rectangular cavity operating at 9.658 GHz. Experiments were performed at room temperature (300K) with a hyper frequency power of 1 mW and a modulation amplitude of 0.5 G. The amplitude of the magnetic field modulation and microwave power were adjusted so that no line-shape distortion was observable. The received gain was 63,200 and the sweep time was 42 s. Absolute quantification was obtained by comparison with a TEMPO sample of known concentration after double integration of EPR spectra. Samples (2mg), i.e. 4-amino TEMPO, polyglucuronic acid-4-amino TEMPO and surfaced carboxylated cellulose nanocrystals-4-amino TEMPO, were dissolved in H2O (1mL) and loaded into a closed capillary tube (o.d. 0.7 mm) which was introduced in a standard EPR tube (o.d. 3 mm). In order to determine the degree of conversion DC from EPR data, taking into account cellulose substrates, the average molecular weight M of a glycosyl unit from derivatives was calculated. The number of 4-amino TEMPO (n 4-amino TEMPO) grafted with cellulose samples, was determined. –
with polyglucuronic acid sample:
M = 329 × DC + (1 − DC ) × 198 = 131 × DC + 198 n 4-aminoTEMPO =
DC =
which gives
–
DC × m DC × m = 131 × DC + 198 M
− 198 × n 4-aminoTEMPO 131 × n 4-aminoTEMPO − m
(4) (5)
(6)
with surfaced carboxylated nanocrystals sample:
M = 198 × Do1 + (162 × (1 − Do)) + (158 + Ma)( Do − Do1 ) M = 36 × Do + 131 × DC + 162 n4 − amino TEMPO =
which gives
DC =
DC × m DC × m = M 36 × Do + 131 × DC + 162
(−36 × Do − 162) × n 4 −amino TEMPO 131 × n 4 −amino TEMPO − m
(7)
(8)
(9)
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with 329 (g/mol) corresponds to the molar mass of a glucuronic unit grafted to 4-amino TEMPO 162 (g/mol) to the molar mass of an unreacted cellulose unit 198 (g/mol) to the molar mass of oxidized cellulose sodium salt (-COONa) (158+Ma) to the molar mass of glycosyl unit coupled to 4-amino TEMPO (Ma = 171.26 g/mol) m to the fraction of the dissolved sample which was introduced into capillary tube
X-ray Diffraction X-ray measurements were made on dried pellets of cellulose products. The X-ray diagrams were recorded on a Warhus vacuum flat plate X-Ray camera mounted on a Philips PW 1720 X-ray generator operated with Cu Kα radiation at 20 mA and 30 kV. Diffraction images were converted into 2θ-intensity profiles using specific software. Elemental Analysis The nitrogen content of the grafted samples was determined by elemental analysis. Indeed, the degree of conversion (DC) can also be calculated from the nitrogen content by using the following equation:
DC =
36 × Do + 162 14 × 100 − M a + 40 %N
(10)
where %N corresponds to the nitrogen content of the coupled samples determined by elemental analysis 162 (g/mol) to the molar mass of cellulose unit Ma the molar mass of 4-amino TEMPO
RESULTS AND DISCUSSION Before to dig into the core of this chapter, it is worth considering the cellulose as the main substrate, recalling the knowledge about its structure and underlining some of its organization principles which are key points for all chemical modifications.
1) Organization and Chemical Structure of Cellulose Since cellulose is reproduced by nature with fixation of CO2 gas, cellulose is the most abundant organic polymer on the Earth. Cellulose is the structural component of the primary cell wall of green plants, many forms of algae and the oomycetes. Some species of bacteria secrete it to form biofilms and also certain sea animals like tunicates. Cellulose is the most common organic compound on Earth. For an example, about 33 percent of all plant matter is cellulose. Cellulose chains are organized into microfibrils which constituted the backbone of cell wall and presented a very high resistance to tension. The cellulose fiber is composed by
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macrofibrils themselves composed of bundles of microfibrils and each microfibril, in turn, is composed of bundles of cellulose chains (Figure 2). The cellulose microfibrils are linked via hemicellulosic tethers to form the cellulose-hemicellulose network (macrofibrils), which is embedded in the pectin matrix containing lignin and proteins. Primary cell walls characteristically extend (grow) by a mechanism called acid growth, which involves turgordriven movement of the strong cellulose microfibrils within the weaker hemicellulose/pectin matrix, catalyzed by expansion proteins. The outer part of the primary cell wall of the plant epidermis is usually impregnated with cutin and wax, forming a permeability barrier known as the plant cuticle. The cellulose microfibrils represent about 20 to 30% of the dry mass of parietal material and occupy about 15% by volume of the wall. For the differentiate cells with a secondary wall, the proportion of cellulose reaches 40 to 90% of parietal mass. The main sources of cellulose are issued to i) primitive organisms like bacteria (ex: Acetobacter xylinum), algae (ex: Valonia, Cladophora, Microdictyon); ii) plants (ex: wood, cotton, flax, ramie, jute, parenchyma of sugar beet pulp…); and iii) envelop of sea animals belonging to the Ascidians family (ex: tunicate). cellulose fibril
macrofibril microfibril
cellulose chains
Figure 2. Structure of cellulose fiber.
1.1) Chemical structure Cellulose was discovered in 1838 by the French chemist Anselme Payen, who isolated it from plant matter [36]. From to Payen’ works, the basic cellulose formula has been determined by Weillstatter and Zechmeister [37-39]. Cellulose, organic compound with (C6H10O5)n formula, is a linear homopolymer, consisting of several hundred to over ten thousand ß(1→4)-glycosidic linked D-glucopyranose units (Figure 3) with three hydroxyl groups on C2, C3 and C6 carbons [40]. The repetitive unit is named cellobiose which is constituted by two D-glucopyranose units placed at 180° giving the molecule its linear characteristic.
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Crystallographic investigations of D-glucose and cellobiose [41] established unambiguously that the D-glucose residues had the 4C1 chair conformation. The two chain ends are chemically different: one end has a D-glucopyranose unit in which the anomeric carbon atom is involved in a glycosidic linkage whereas the other end has a D-glucopyranose unit with free anomeric carbon atom (Figure 4). This cyclic hemiacetal function is in an equilibrium in which a small proportion is an aldehyde which gives rise to reducing properties at this end of the chain: the cellulose chain has a chemical polarity. Determination of the relative orientation of cellulose chains in the three-dimensional structure has been and remains one of the major problems in the study of cellulose. So, in the cellulose crystal, two arrangements are possible: either an organisation in parallel chains with reducing chain end placed in the same side or an organisation in antiparallel chains with alternate position.
6
3’
4 5 3
5’ 2’
2 1
4’
6’
1’
n
Figure 3. Schematic representation of a cellulose chain.
Figure 4. Chain ends of cellulose chain.
1.2) Crystallinity and polymorphism of cellulose The 4C1 chair conformation of D-glucopyranose units added to the ß(1→4)-glycosidic link induces to a straight chain polymer with no coiling or branching occurs, and the molecule adopts an extended and rather stiff rod-like conformation, aided by the equatorial conformation of the glucose residues. The free hydroxyl groups present in the cellulose are likely to be involved in a number of intra and inter molecular hydrogen bonds which may give rise to various ordered crystalline arrangements (Figure 5). The intra molecular hydrogen bonds are realized between i) hydrogen O3-H and heterocyclic oxygen O5 on a neighbor cycle and ii) hydrogen O2-H and oxygen O6-H on a neighbor cycle (Figure 6). The inter molecular hydrogen bonds are performed with oxygen O3-H and primary hydrogen O6-H on a neighbor chain (Figure 6).
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Figure 5. Schematic representation of cellulose chains showing the hydrogen bonds (dashed).
Figure 6. Representation of intra and inter molecular hydrogen bonds into cellulose.
The intra molecular hydrogen bonds confer the rigidity to cellulose chain whereas the inter molecular hydrogen bonds ensure the cohesion of crystalline structure formed by the all cellulose chains. The combination promotes the alignment in parallel beams leading to regular arrangement of rigid molecules. In the case of cellulose, these crystalline arrangements are usually imperfect, in terms of crystallinity, crystal dimensions, chain orientation and then the purity of the crystalline form must be taken into consideration. The crystal density can be gauged from the crystallographic data which leads to suggest the importance of the amorphous components generally present. By infrared spectroscopy, the degree of crystallinity can also be estimated as a function of the relative intensity of specific bands [42]. Four principal allomorphs have been identified for cellulose by its characteristic X-ray diffraction pattern: I, II, III and IV [43]. The relationships among the various allomorphs are
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shown schematically (Figure 7). The natural form of cellulose, called cellulose I or native cellulose, is the most abundant form. Its highly complex and not yet completely resolved three-dimensional structure results of co-existence of two distinct crystalline forms cellulose Iα and Iβ. This was a major discovery and led to a revival of interest in the study of cellulose structure [44-45]. Cellulose II, named regenerated cellulose, is generally obtained by regeneration of cellulose from solution or by mercerization (alkaline medium) [46]. The transition from cellulose I to cellulose II is not reversible and this implies that cellulose II is a stable form compared with the metastable cellulose I.
Figure 7. Relationship between various allomorphs of cellulose.
The preparation of cellulose III is realized with ammonia treatment and allows to obtain the form cellulose IIII or the form IIIII from to cellulose I and cellulose II, respectively. Cellulose III can be transformed into cellulose IV after treatment at high temperature in glycerol: cellulose IVI and IVII obtained from cellulose IIII and IIIII, respectively. After relative recent studies by electron diffraction [47], it is now accepted that cellulose IVI is a disordered form of cellulose I (native cellulose form). This allomorph, at native state, may be observed in some mushrooms but also in primary cell walls of cotton [48].
1.3) Crystalline structures The X-rays diffraction is the oldest technique used to characterize the crystallinity of cellulose. First work suggested that native celluloses with different origins seem to crystallize in different arrangements whereas from Valonia and bacterial sources, cellulose presents a same crystalline unit cell. The crystalline structure of cellulose is refined with new techniques based on solid-state 13C NMR spectroscopy which confirmed the existence of two families of native cellulose [44-45]. From a detailed analysis of the carbon atom couplings, Vanderhaart and Atalla [44-45] established that native cellulose corresponded to a composite of two distinct crystalline phases named a one-chain triclinic structure Iα and a two-chain monoclinic structure Iβ. The fractions of Iα and Iβ crystalline phases in any native cellulose samples depend on the origin of the cellulose. This model was supported by electron diffraction study of native cellulose from algal cell wall [49] and by computational prediction [50]. In general, the celluloses produced by primitive organisms (bacteria, algae etc.) are enriched in the Iα
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phase whereas the cellulose of higher plants (woody tissues, cotton, ramie…) consists mainly of the Iβ phase. Indeed, the crystallographic studies of envelop of sea animals like tunicate show crystalline form uniquely composed of the Iβ phase [51]. The discovery of the crystalline dimorphism of cellulose was the starting point for a number of research projects of which the aim was to evaluate the properties of each allomorph and procedures for their interconversion. Details of the crystalline structure of these two forms were reported by Kono [52] using 13C NMR technique and by Nishiyama [53-54] using synchrotron X-ray and neutron fiber diffraction. The estimation of phase’s composition of native cellulose is possible using different techniques such as FTIR [55], 13C NMR [56-57] and synchrotron-radiated X-ray diffraction [58]. The Iα phase is a metastable from which can be converted to the more stable Iβ form by annealing in different medium [59-60]. The Table 3 and Figure 8 report crystallographic characteristics of allomorphs of native cellulose. The crystalline forms of cellulose II, III and IV are organized in monoclinic unit cell as observed for cellulose Iß whereas the cellulose Iα is oriented in triclinic unit cell [53]. Each step of interconversion induces change in crystallographic features of cellulose and in chemical assignment of C1, C4 and C6 specific carbons (Table 4 and Table 5). Table 3. Crystallographic characteristics of allomorphs of cellulose. Allomorph Iα
Unit cell Triclinic
a (Å) 6.74
b (Å) 5.93
c (Å) 10.36
Iβ
Monoclinic
8.01
8.17
10.36
Angles (°) α= 117 β = 113 γ = 97.3 γ = 97.3
Figure 8. Orientation of unit cells of monoclinic cellulose Iß and triclinic cellulose Iα.
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Table 4. Chemical shifts of C1, C4 and C6 carbons characteristic of cellulose allomorphs. Allomorph Cellulose Iα Cellulose Iß Cellulose II Cellulose IIII Cellulose IIIII Cellulose IVI Cellulose IVII Amorphous
Chemical shifts (ppm) C1 C4 106.0-106.3 89.8-90.2 105.0-105.2 89.1-89.3 105.8-106.3 88.7-88.8 105.3-105.6 88.1-88.3 106.7-106.8 88.0 105.6 83.6-84.6 105.5 83.5-84.6 105 108
C6 66.2-66.5 65.5-66.1 63.5-64.1 62.5-62.7 62.1-62.8 63.3-63.8 63.7 63
Table 5. Characteristic diffraction angles of cellulose allomorphs. Allomorph Cellulose I Cellulose II Cellulose IIII Cellulose IIIII Cellulose IVI Cellulose IVII
2θ Diffraction angle (°) 110 110 200 012 14.8 16.3 22.6 12.1 19.8 22.0 11.7 20.7 20.7 12.1 20.6 20.6 15.6 15.6 22.2 15.6 15.6 22.2 20.2
The mercerization of native cellulose, consisting on swelling of cellulose in alkaline medium following by washing with water, induces that the conversion from cellulose I to cellulose II is as a function of crystallinity of cellulose, NaOH concentration, time and temperature of reaction. The cellulose II presents an antiparallel arrangement of cellulose chains inside the unit cell of crystal. This arrangement allows the establishment of more number of hydrogen bonds than in native cellulose explaining also a better stability of this allomorph. Concerning the cellulose III, numerous studies were focus on reversible conversion from cellulose I to cellulose III based on the use of electronic microscopy [61-62], solid-state NMR [29] or X-ray diffraction [63]. It is interesting to note that the conversion from cellulose I to cellulose IIII, in the case of Valonia cellulose, was accompanied by an important decrystallisation and fragmentation of the cellulose crystal. The reverse transition resulted in partial re-crystallization but this did not allow complete restoration of the damage done to the morphological surface. Characterization by electron diffraction revealed that the uniplanaruniaxial orientation of the crystalline cellulose microfibrils was lost completely during the stage of swelling and washing necessary for the conversion into cellulose IIII. Washing with methanol resulted in the formation of irregularities into which were inserted crystalline domains of small dimensions. The final material which crystallized in the cellulose I form was obtained by treatment with hot water and characteristically displayed an increase in the accessible surface and consequently reactivity.
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In spite of some minor differences the results agree sufficiently well to propose a model in which the cellulose chains has almost perfect two-fold symmetry and is compatible with occurrence of two intermolecular hydrogen bonds between consecutive residues.
1.4) Microfibrils of native cellulose The microfibrillar structure of cellulose has been established beyond doubt through the application of electron microscopy [64-65] and great variations in dimensions, depending on origin, have been reported [47-48, 66]. The application of transmission electron microscopy [67-72] has established with certainty that the microfibril is the basic crystalline element of native cellulose [1, 67, 68-70, 73]. Different levels of structural organisation of cellulose are now well characterized. An experimental protocol using exo-cellulases finally reached the proof of parallel arrangement in the family of native cellulose [74]. Recent investigations using complementary enzymatic and chemical staining of reducing ends have supported this model [75] and, at the same time, produced precise descriptions of the orientation of the chains relative to the crystal axes. Hence the crystalline microfibrils possess the same polarity as the chains of which they are composed. In addition to the crystalline phases, native cellulose contains disordered domains which can be considered like amorphous. Native cellulose may be assigned to a semi-crystalline fibrillar material. Presence of disordered phases was supported by experimental results from solid state 13C NMR characterization, tensile tests of cellulose fibers, wide-angle (WAXS) and small-angle X-ray scattering (SAXS). 1.5) Whiskers of cellulose Depending on their origin, the microfibril diameters range from about 2 nm to 20 nm for lengths that can reach several tens of microns (Figure 9). As they are devoid of chain folding and contain only a small number of defects, each microfibril can be considered as a string of cellulose crystals, linked along the microfibril by amorphous domains (Figure 10). High crystalline cellulose
20 nm
Tunicate
10-15 nm
Valonia
Ramie
5 nm
3.5 nm Wood
3 nm Primary cell wall Low crystalline cellulose
Figure 9. Schematic representation of range of microfibril size from different sources.
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Amorphous domains
Cellulose microfibril Figure 10. Schematic representation of amorphous and crystalline domains of cellulose microfibril.
Some imperfections arose from dislocations at the interface of microcrystalline domains along the microfibril length. These imperfections were used to advantage by treatment with acid to produce monocrystals called “whiskers” having the same diameter as the starting microfibrils but much shorter length. These monocrystals corresponded to nearly perfect crystalline arrangement. After hydrolysis, the monocrystals of cotton linters are shorter than those issued to long microfibrils like Valonia cellulose. These microcrystals reach lengths of 0.1 micron and widths from 10-50 Å and have a shape value of 20. At the other end of the spectrum, cellulose microfibrils from parenchyma (low crystalline cellulose) are produced by a mechanical treatment which, contrary to the hydrolysis, allows disruption of the microfibrils without affecting the original length. As a result, microfibrils several microns long and 20-30 Å wide are obtained. It follows that microfibrils with high section (∼ 25 nm) presented few per cent of amorphous domains whereas for cotton with 5 nm section and for primary cell wall like sugar beet pulp with 3 nm section, the amorphous domains corresponded to about 30% and 70%, respectively [1]. The smaller microfibrils, the more surface chains are and the more non-crystalline component is rising correspondingly.
1.6) Cellulose as starting materials for grafting purposes Currently, many search laboratories are involved in cellulose research and development for either industrial use or structural knowledge increase. To precise crystallographic description remains a fundamental target. In this chapter, we decided to realize grafting reaction on cotton cellulose that is a fiber issued to cotton plant for which the stem is covered with flowers to 5 lobes. The benefits arising are ovoid capsules that open to release seed wrapped a white duvet called Linter. In order to obtain cellulose monocrystals (“whiskers”), an acid treatment of cellulose microfibrils is advocated. The amorphous regions act as structural defects and are responsible of the transverse cleavage of the microfibrils into short monocrystals under acid hydrolysis. This procedure largely described is used to prepare highly crystalline cellulose particles. Under controlled conditions, this transformation consists of the disruption of amorphous regions surrounding and embedded within cellulose microfibrils while leaving the microcrystalline segments intact. Highly crystalline cellulose consists generally of a stiff rod-like particle called “whiskers” (or hydrolyzed nanocrystals in this chapter). Geometrical characteristic of cellulose nanocrystals depend on the origin of cellulose microfibrils and acid hydrolysis process conditions such as time, temperature and purity of materials. The most studied cellulose sources are: Valonia [76], cotton linters [77], wood pulp [78] and sugar beet pulp [35, 79].
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Two acid treatments can be possible [80] to form nanocrystals. The use of sulfuric acid hydrolysis leads to stable aqueous colloidal suspension which could form chiral nematic ordered phases when the suspension concentrations exceed a critical value, and the nanocrystals present a negatively charged surface (sulfate esters groups). The cellulose nanocrystals suspensions resulting from hydrochloric acid hydrolysis have minimal surface charges and are organized into small aggregates.
2) Oxidation Reaction of Cellulose Samples 2.1) Choice of oxidation reaction Oxidation of polysaccharides has been studied in detail by numerous investigators, but, because of the presence of several reactive groups, it is not easy to attain high selectivity, and only a limited number of reagents are available for this purpose [81]. Because it is insoluble in water and most common organic solvents, there are especially difficulties with cellulose. In view of the structure of an anhydroglucose unit of cellulose, one has to account for the presence of three reactive groups available for oxidation: one primary (C6) and two secondary (C2, C3) hydroxyl groups. Direct oxidation of hydroxyl groups provides interesting routes for the introduction of carbonyl and carboxyl groups into polysaccharides. Oxidation of secondary hydroxyl groups has been achieved by various oxidation reagents, e.g., periodate and hypochlorite, which results in oxidative scission of 1,2-diols and the formation of dialdehyde and dicarboxylic structures, respectively [82-83] (Figure 11).
Figure 11. 2, 3-dicarboxy derivative after sodium chlorite treatment.
Full oxidation of monosaccharides by nitric acid to aldaric acids has been an established technique for more than a century. Analogously, oxidation of polysaccharides such as cellulose or starch by nitrogen dioxide (N2O4) yields 6-carboxy starch and 6-carboxycellulose respectively [84]. Subsequent hydrolysis at rigorous conditions (0.5-2M HCl at 150°C) of these materials yields D-glucuronic acid. A drawback of the oxidation with nitrogen dioxide is that depolymerisation may be an important side reaction. An improvement of the process with respect to this aspect can be achieved by conducting the reaction when the polysaccharide is dissolved in 85% phosphoric acid and with sodium nitrite as the oxidant: better yield versus a more important depolymerisation [85-86]. In the recent decade, catalytic oxidation of carbohydrates transposed to polysaccharides using highly regio-selective and stable nitroxyde radical (2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)) has become one of the most promising procedures to convert polysaccharides into the corresponding polyuronic acids. The method is very suitable for
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selective oxidation of primary hydroxyl groups into aldehydes and / or carboxylic acid groups. Contrary to enzymatic or metal-catalyzed oxidation, the TEMPO mediated oxidation is highly effective in the conversion of high molecular weight polysaccharides. Other advantages to be mentioned in connection to the TEMPO-oxidation process are the high reaction rate and yield, the high selectivity, the catalytic process, just a modest degradation of polysaccharides throughout the process. This process was first utilized some 15 years ago, but to this day, to our knowledge, no commercial process is in operation. After the first publications on TEMPO mediated oxidation of polysaccharides and other types of alcohols, numerous papers have been published.
2.2) Mechanism of reaction The oxidation of primary alcohol groups in natural polysaccharides, catalyzed by 2,2,6,6tetramethylpiperidine-1-oxyl radical (TEMPO) has been recently proposed as a more selective, faster and better-controlled method [3, 5-6] as opposed to the traditional procedure using nitrite/nitrate in concentrated phosphoric acid [84, 87]. TEMPO and its analogues belong to a class of compounds which usually are referred to as nitroxide radicals. This compound is secondary amine nitrogen oxide with the general chemical structure given below (Figure 12).
Figure 12. General chemical structure of nitroxide radicals.
2,2,6,6-tetramethyl-1-piperidine oxoammonium radical (1) is known to be a stable radical because of the unpaired electron delocalized between the nitrogen and the oxygen atom. The NO-bond energy has been estimated to be 420 kJ/mol which approximately corresponds to the energy of one and a half bond. The most striking feature of nitroxyde radicals is their high or extreme stability toward dimerization or decomposition and inertness to typical organic molecules. However, the stability largely depends on the structure of the radical compound and especially on the nature of the substituents attached next to the nitroxyl group. For instance, hydrogens attached on the α and α’-carbons are a source of instability leading often to disproportionate the radical. About cyclic nitroxide radical such as TEMPO, the presence of electro-donor methyl groups on carbons indicated an exceptional stability and inertness. That induced high redox properties help characterize the radical TEMPO [4]. The cyclic nitroxide radical of the piperidine is interrelated by one-electron transfer oxidation or reduction reactions: either the radical can be oxidized by several reagents (oxidant) to give a nitrosonium ion (2) [88] or reduced to give the hydroxylamine (3) by reducing agents (Figure 13). In all studies, the nitrosonium ion is assumed to be the responsible oxidizing specie. The oxidation potential of TEMPO is not negligible, but in order to function as efficient oxidants of organic substrates, it converted into the more powerful nitrosonium ion. The nitrosonium ion shows selectivity towards primary hydroxyl functions over secondary ones.
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Figure 13. Nitroxyde radical TEMPO.
In most of works, TEMPO radical is utilized as oxidation catalysts: the nitrosonium ion is continuously regenerated in situ by a primary oxidant. The main advantage based on that the radical can be added in catalytic amounts. The co-oxidant must be inert towards organic substrates to oxidize. The procedure for the in situ generation of TEMPO was first described by Semmelhack [89]. Following the method developed by Davis and Flitsch [90] using a mixture of sodium hypochlorite, sodium bromide and 2,2,6,6-tetramethyl-1-piperidine oxoammonium radical, the TEMPO-NaBr-NaClO system was applied to a wealth of products including many polysaccharides. This method was first proposed for water-soluble polysaccharides [3, 5-8], namely starch, inulin, amylodextrin, pullulan, alternan, amylopectin, chitosan, galactomannan, and later extended to water-insoluble products [7-12], such as cellulose, amylose, and chitin. The principle of the catalytic system involved TEMPO in presence of two primary cooxidants: sodium hypochlorite and sodium hypobromite. The reactional mechanism is presented in Figure 14.
Figure 14. Reactional mechanism of TEMPO mediated oxidation.
The catalytic mechanism of TEMPO mediated oxidation is based on the regeneration in turn of nitrosonium ion (2) in presence of regenerating oxidant which is NaOBr (Figure 14). NaOBr is the product of oxidation of NaBr by NaOCl (primary co-oxidant) and then reacted on TEMPO radical to give the nitrosonium ion. At alkaline pH, the nitrosonium ion is
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continuously regenerated throughout the conversion of hydroxylamine (3), which is formed after substrate oxidation, into the radical (1). The nitrosonium ion reacted with primary hydroxyls of polysaccharides to get aldehyde groups releasing the hydroxylamine (3). In turn, the aldehyde was modified in carboxylic acid with the same principle. So, two equivalents are necessary during the reaction: the former to obtain aldehyde groups and the latter to converse the aldehyde groups to carboxylic acid groups. The products obtained by this procedure present lower molecular weight than those obtained via a two step method with for an example NaIO4 and NaClO2 (methods known for the glycolic oxidation of polysaccharides) inducing the perspectives for biodegradability are better [91]. Another advantage is that the product will be cheaper since it is prepared in a single, simple step with a relatively inexpensive reagent. For native cellulose (allomorph I of cellulose), the oxidation led to a two-phase solution: water-soluble and water-insoluble fractions. These two fractions corresponded to the totally oxidized polyglucuronic acid and the surface carboxylated cellulose nanocrystals, respectively. The oxidation runs were carried out according to the above-mentioned scheme. Sodium hypochlorite, used to regenerate the catalyst, is added dropwise during the reaction. It has been shown that a slow addition of the oxidant led to a higher yield and products that were less coloured (so less degraded!). The sodium bromide allowed to the regeneration of sodium hypochlorite. The oxidation must be realized at pH rigorously kept at 10. Indeed, with higher pH, a severe degradation by β–elimination of cellulose occurred. With an acid pH, the secondary hydroxyl groups could be oxidized [4].
2.3) Influence of oxidation parameters Oxidant. The reagent sodium hypochlorite should be added gradually to avoid undesired reactions such as glycol cleavage due to the presence of excess sodium hypobromite [92]. Indeed, the amount of primary oxidant determines the yield of the oxidation reaction. Solvent. Herrmann [93] reported on a water-based system (acetic acid – water) that only gave the aldehyde groups. These results suggest that the conversion of aldehydes to carboxyl groups predominantly takes place in the water phase. Since the presence of water is essential for the latter conversion [4], it is plausible that only the hydrated form of aldehyde intermediate can be oxidized further (Figure 15).
Cell
6
TEMPO mediated oxidation
Cell
6
H2O
Cell
6
TEMPO mediated oxidation
Cell
6
Figure 15. TEMPO mediated oxidation of primary alcohols via the hydrated aldehyde intermediate.
pH value. Significant for TEMPO mediated oxidation reactions of polysaccharides is the high chemoselectivity for the primary alcohol groups, which probably is due to the steric hindrance of the secondary hydroxyl groups. The researches on the oxidation of cellulose showed that both the rate of oxidation and the nature of the products are determined by the pH of the solution. It was established that in the TEMPO-NaBr-NaClO system, the pH optimum rate of TEMPO is found at pH 10 [13, 22, 95]. If the pH value is lower than 9.5, an
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undesired oxidation of secondary hydroxyls functions by hypobromite during the reaction is observed in competition with the oxidation of primary hydroxyls ones [5]. With higher pH than 10.5, a severe degradation by β–elimination of cellulose occurred [6]. Temperature: Low temperature during reaction is essential for minimizing the degradation of the polymer. In previous studies [94], the TEMPO-NaBr-NaClO system is usually carried out at 0-5°C (in an ice batch). To accelerate the reaction, the oxidation was to perform at increased but still moderate temperature (20°C) without loss of selectivity and presence of side reactions [22].
2.4) Characteristics of cellulose substrates In general, the evolution of the oxidation was followed by the sodium hydroxide consumed to neutralize the carboxylic acids generated in C6 carbons (conductometric titration). The increase of NaOCl concentration up to 2 eq involved an increase in the degree of oxidation (Do) of cellulose samples. 0,4
100
0,35 80 0,3
60 Yield (%)
Do
0,25 0,2
40
0,15 0,1
20 0,05 S Cotton linters nanocrystals 0
0 0
0,5
1
1,5 eq(NaOCl)
2
2,5
Figure 16. Evolution of degree Do (…) and yield of oxidation (−) versus the amount of added NaOCl for cellulose nanocrystal samples.
For partially oxidation leading to surface carboxylated cellulose nanocrystals, the TEMPO mediated oxidation led to approximately constant molar yields of oxidized nanocrystals more or less constant, 92–95% with cotton linters (and for soluble fractions: 4– 7%) (Figure 16). The global yields for hydrolyzed samples were always 100%. Montanari [95] indicated that the cellulose samples which are not hydrolyzed before oxidation presented lower global yields with increasing NaOCl concentrations. The values decreased to around
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90% for cotton linters. The degradation of cellulose samples explained this difference between hydrolyzed and nonhydrolyzed samples. As a result, in this chapter, hydrolyzed cellulose samples are privileged: few loss of material correlated with no degradation and no side reactions. A constancy in the Do value as of 1 equivalent of NaOCl is shown (Figure 16). This maximal value not corresponds to the theoretical value which would be 2 mmol of NaOCl/mmol of glycosyl unit (2 eq). This value depends on the crystal size and corresponds roughly to the amount of primary hydroxyl groups easily accessible to the reactants, i.e. surface hydroxyl groups. In addition, the Figure 16 showed efficient molar yields with the constancy in value indicating no degradation of cellulose samples with the NaOCl concentration. From these observations, the TEMPO mediated oxidation of cellulose nanocrystals is performed with 1 mol NaOCl / 1 mol glycosyl unit (1 eq). The carboxyl groups content of surface carboxylated cellulose nanocrystals was determined by conductometric titrations with very reproducible results and the value is 0.15 for cotton linters (Figure 16). For totally oxidized polyglucuronic acid (from cellulose IIII), a constant molar yield in the order of 100% is obtained. The conversion into cellulose IIII involved starting material which is rapidly and fully oxidized yielding polyglucuronan whereas for native cellulose, around 10% of water-soluble product is obtained. The accessibility improve is due to the increase in distance between chains in the crystal (confirmed by the density variation from 1.61 (cellulose I) to 1.54 (cellulose IIII)) and also the fact that hydroxyl groups are less involved in hydrogen bonds network. Similar results are shown in the literature [13, 28]. A constancy in the Do value as of 1 equivalent of NaOCl is shown (Figure 16). This maximal value not corresponds to the theoretical value which would be 2 mmol of NaOCl/mmol of glycosyl unit (2 eq). This value depends on the crystal size and corresponds roughly to the amount of primary hydroxyl groups easily accessible to the reactants, i.e. surface hydroxyl groups. In addition, the Figure 16 showed efficient molar yields with the constancy in value indicating no degradation of cellulose samples with the NaOCl concentration. From these observations, the TEMPO mediated oxidation of cellulose nanocrystals is performed with 1 mol NaOCl / 1 mol glycosyl unit (1 eq). The carboxyl content of surface carboxylated cellulose nanocrystals was determined by conductometric titrations with very reproducible results and the value is 0.15 for cotton linters (Figure 16). For totally oxidized polyglucuronic acid (from cellulose IIII), a constant molar yield in the order of 100% is obtained. The conversion into cellulose IIII involved starting material which is rapidly and fully oxidized yielding polyglucuronan whereas for native cellulose, around 10% of water-soluble product is obtained. The accessibility improve is due to the increase in distance between chains in the crystal (confirmed by the density variation from 1.61 (cellulose I) to 1.54 (cellulose IIII)) and also the fact that hydroxyl groups are less involved in hydrogen bonds network. Similar results are shown in the literature [13, 28]. The solid-state NMR characteristics of surface carboxylated cellulose nanocrystals and liquid-state chemical shift data of polyglucuronic acid are given in Table 6 with the main appearance of the peak near 175 ppm and the FTIR peak at 1730 cm-1 corresponding to carboxyl moiety. The FTIR characterization of these oxidized cellulose samples is shown in Table 7.
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Table 6. The 13C NMR characteristics of polyglucuronic acid (liquid-state) and surface carboxylated cellulose nanocrystals (solid-state). Liquid-state 13C NMR, δ (ppm)
Solid-state 13C NMR, δ (ppm)
C1 C2 C3 C4 C5 C6 oxidized
C1 C2-3-5 C4 amorphous C4 crystal C6 amorphous C6 crystal C6 oxidized to C=O
103.5 73.75 75.25 81.95 76.30 175
105 70-77 83.5 88.7 62.4 65 174.8
Table 7. FTIR characteristics of oxidized cellulose.
OH CH C=O (free COOH)
FTIR (in acid form) cm-1 Water adsorbed onto the 3340 surface of nanocrystals 2900
1640
1730
The oxidation of hydroxyl moieties allows the presence of carboxylic functions, which can be grafted easily.
3) Grafting Reaction 3.1) Mechanism of grafting reaction The aim of this chapter is to guide the chemical modification to the grafting reaction of amines into cellulose samples. Commonly, grafting techniques of carboxylic derivatives largely used carbodiimides as grafting reagents with in particular N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC). EDAC is a water-soluble condensing reagent generally used as carboxyl activating agent for amide bonds with primary amines. EDAC has been used in peptide synthesis [96], modification of polysaccharides [15], crosslinking proteins to nucleic acids [97-98] and preparation of immunoconjugates [99]. For polysaccharides, the typical amidation procedure was described by Danishefsky [15] applied to a mucopolysaccharide. In many studies [16-18], their reaction conditions were followed: mixing of all components at pH 4.75. But, some reports claimed successful formation of amide link [15-16] whereas others reported failure in amidation [17-18]. Recently, the use of N-hydroxysuccinimide like an activating reagent was found to be effective for the EDAC mediated amidation [10, 18]. The amide bond formation between carboxylic acid functions and amines is catalyzed by carbodiimide throughout activation of carboxylate functions (Figure 17). Carbodiimide reacts
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with carboxyl groups to form an unstable, intermediate O-acylurea which, in the absence of nucleophiles, rearranges to a stable N-acylurea by way of a cyclic electronic displacement. From this active O-acylurea, a more hydrolysis-resistant and nonrearrangeable active ester intermediate is formed to graft primary amines to oxidized cellulose samples (Figure 17). NHS, commonly found in organic chemistry, is further used as an activating reagent. Activated acid functions (NHS-ester intermediate) react with amines to form amide bonds whereas nonactivated carboxylic acid functions would just form a salt with an amine. NHS then reacts to form a less labile activated acid and EDAC is released subsequently like watersoluble derivative of urea.
Na+
n
under acidic conditions or without NHS n
N-acylurea
n
O-acylurea n
NHS-ester intermediate
n
Coupled cellulose samples
Figure 17. Grafting reaction onto oxidized cellulose samples.
3.2) Influence of grafting conditions At low pH, carbodiimides are considered as unstable [15, 17-18]. In fact, under acidic conditions, the unstable intermediate O-acylurea is readily formed and rearranges by the cyclic electronic displacement to the stable N-acylurea. Indeed, this ON migration occurred in presence of a primary amine [17] and any amide formation occurs. Furthermore, nucleophilic addition to the ester formed requires amine to be presented in an unprotonated form. Only a limited number of amines have pKa values in the suitable range of Danishefsky’ procedure and are consequently few reactive with active NHS-ester intermediate. In fact, in most cases, primary amines have typically pKa higher than 9.
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Therewith, the NHS-ester intermediate allows for the grafting reaction to be carried out at neutral or slightly basic pH (about 7-8.5) and consequently yields products by reaction with primary amines. The combination of these observations suggested amidation under alkaline conditions. Few authors have already presented this way better than Danishefsky’ procedure to form amide bonds onto cellulose in an alkaline medium [22, 100].
3.3) Grafting reaction onto polyglucuronic acid For having polyglucuronic acid as starting material, we decided to use cellulose III obtained from native cellulose by ammonia treatment. Therefore, cellulose III presented crystalline structures with primary hydroxyl groups relatively accessible and could be highly oxidized, leading to almost totally soluble polyglucuronic sample. The TEMPO mediated oxidation has been applied to cellulose IIII leading to complete solubilization of product. This procedure has been frequently used to improve the reactivity of crystalline cellulose for the preparation of derivatives. The grafting reaction was carried out on polyglucuronic acid in the presence of carbodiimide and NHS with linear and cyclic amines. The conditions were 2.5 mmol amine / 1 mmol glycosyl unit (2.5 eq). A mixture of EDAC and NHS (1.5 eq each) is diluted in 2 mL of water [22]. In this chapter, at stable pH 8, the mixture of EDAC and NHS was added. The solution became turbid and the pH value tending to increase is maintained to 8 by adding 0.5 M HCl and/or NaOH solutions. At the end of grafting reaction, the samples were collected. The products issued to grafting reaction were analyzed by conductimetry, elemental analysis, FTIR, NMR and EPR spectroscopies. Degree of conversion. The degrees of conversion calculated from conductimetry, elemental analysis and EPR results from equations 3,6,10 are summarized in Table 8 with a significant agreement [22]. We obtained degrees of conversion close to 13%, 33%, 39% and 56% with n-octyl amine, 4-amino TEMPO, n-butyl amine and 2-methoxy ethyl amine, respectively [22]. These results indicated a decreasing dependence with the increase of carbon chain length of amines due to the variable electronic distribution along the amine chain, the steric effects and more probably to a decrease in solubility. But, these informations can not fully explain the results. In fact, the amine structures are characterized by inductive and mesomer effects and so the pKa values have to be into account. In aqueous medium, the protonation of amines provides ammonium salts (RNH3+). As during the grafting reaction the pH was maintained at around 8, the amount of ammonium ions in solution was lower for amines with pKa values nearest to 8. Thus, for instance, 2-methoxy ethyl amine (pKa=9.2) in solution was presented less ammonium ions than for n-butyl amine (pKa=10.6). Table 8. Degrees of conversion from polyglucuronic acid. Amines n-butyl amine n-octyl amine 2-methoxy ethyl amine 4-amino TEMPO
Conductimetry 0.39 0.13 0.56 0.33
Elemental analysis 0.43 0.15 0.59 0.37
EPR analysis 0.36
Amidation of Cellulose Materials
1027
1550
1730 1650
Since the nucleophilic addition to the NHS-ester intermediate requires the amine to be presented in an unprotonated form and carried out at slightly basic pH (as above-mentioned), then amines which will be less protonated should present a better reactivity. That’s observed with 2-methoxy ethyl amine which presents the lowest pKa value and the higher degree of conversion in the range tested. From these results it appears that the grafting can be directly correlated with the amines reactivities. FTIR spectroscopy. By FTIR spectroscopy, the products issued to grafting reaction are presented in Figure 18. Characteristic carboxyl acid peak appears at 1730 cm-1 with two other distinct bands at 1650 and 1550 cm–1 located in the zone related to the (-CONH-), corresponding respectively to the (C=O) stretching band and to the (-NH) bending vibration band [101]. Indeed, as already reported by several authors [18, 102-103], the carboxylic acid band is well separated from the amide group (-CONH-) bands at 1650 and 1550 cm-1. The FTIR analysis clearly shows that an amide bond has been formed during the grafting reaction and a correlation between degrees of conversion and the intensity of characteristic peaks is indicated.
polyglucuronic acid with 4-amino TEMPO
polyglucuronic acid with 2-methoxy ethyl amine
1624
1724
polyglucuronic acid with n-butyl amine
1640
polyglucuronic acid in acidic form
cellulose
cm-1 4000
3600
3200
2800
2400
2000
1600
1200
800
400
Figure 18. FTIR spectra of cellulose, polyglucuronic acid, polyglucuronic acid-n-butyl amine, polyglucuronic acid-2-methoxy ethyl amine and polyglucuronic acid-4-amino TEMPO.
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Liquid-state NMR characterization. The chemical shift data of polyglucuronic acid-nbutyl amine, 2-methoxy ethyl amine and polyglucuronic acid-4-amino TEMPO are reported in Table 9 and Table 10. The characteristic signals related to carbons of polyglucuronic acid are observed, and among them, several new resonances are detected. The presence of an extra signal well separated from the (-COONa) C6 resonance is observed at 170.30 ppm with linear amines and 174.75 ppm with 4-amino TEMPO and assigned to (-CONH-) acetamide carbon according to 13C NMR data already published by several authors [21, 104-105]. The presence of this signal supported the amide bond formation [22]. The other new resonances in the spectra of polyglucuronic acid grafted to amines are equally attributed. In order to view typical NMR results, the 13C NMR spectrum of polyglucuronic acid-2-methoxy ethyl amine is reported in Figure 19. Table 9. Chemical shift data of polyglucuronic acid with n-butylamine and 2-methoxy ethyl amine. All the experimental data have been extrated from previous work [22]. Polyglucuronic acid with n-butyl amine 13 H δ (ppm) C δ (ppm) H1 4.69 C1 103.5 H2 3.56 C2 73.75 H3 3.81 C3 75.25 H4 3.83 C4 81.95 H5 4.05 C5 76.30 C6 carbonyl 175.65 C6 acetamide 170.30 H3C 1.08 CH3 13.90 H2C 1.51, 1.69 CH2 20.35, 31.20, -HN-C 40.25 1
Polyglucuronic acid with 2-methoxy ethyl amine 1 13 H δ (ppm) C δ (ppm) H1 4.44 C1 102.85 H2 3.36 C2 73.20 H3 3.51 C3 75.60 H4 3.60 C4 81.15 H5 3.81 C5 75.70 C6 carbonyl 175.25 C6 acetamide 170.25 O-CH3 3.28 O-CH3 58.65 HaHb-C1 3.28 O-C2 70.60 HcHd-C2
3.45
-HN-C1
39.60
Table 10. Chemical shift data of polyglucuronic acid-4-amino TEMPO. All the experimental data have been extrated from previous work [22]. 1
13
H δ (ppm)
H1 H2 H3 H4, H4* H5
4.69 3.55 3.82 3.85 4.04
H4’
2.60
C δ (ppm) C1 103.35 C2 73.75 C3 75.25 C4, C4* 81.95 C5 76.30 C6 carbonyl 175.65 C6 acetamide 174.75 15.35, 25.90 CH3
H2C3’, H2C5’
1.68
C4’
39.45
H3C-C2’, H3C-C6’
1.43
C3’, C5’
43.75
C2’, C6’
56.40
Amidation of Cellulose Materials
1029
Figure 19. 13C NMR spectra in D2O of polyglucuronic acid-2-methoxy ethyl amine. All the experimental data used have been extracted from previous work [22].
The 1H spectra of polyglucuronic acid grafted amines were analyzed and the different proton signals assigned by 2D-COSY (spectrum not presented) and 2D 13C-1H HMQC experiments. For illustration, the 2D 13C-1H HMQC spectrum of polyglucuronic acid-2methoxy ethyl amine was presented in Figure 20a. All the characteristic proton signals of oxidized cellulose (H1, H2, H3, H4, H5) were easily identified and are in agreement with assignment already published [12, 106]. The presence of characteristic signals both in the 1H and 13C NMR spectra confirmed the grafting onto C6 carbons of polyglucuronic acid. The 2D 13C-1H HMBC spectrum presented in Figure 20b reported strong cross-peaks between C6 acetamide (O=C-NH) and Ha and/or Hb protons of amine carbon C1. This signal confirmed and corroborated the formation of the chemical bond between amine and carboxyl groups of the glucuronic unit. An additional strong connectivity between C6 acetamide (O=CNH) and Hc and / or Hd protons of amine carbon C2 across 4 linkages suggesting a “w” conformation, confirmed the coupling between the carboxy group and 2-methoxy ethyl amine. For the grafting of 4-amino TEMPO, the 13C NMR data are reported in Table 10. The 13C signals detected are attributed to the nitroxide radical, as already published by Irwin [107] with resonances at 56.40, 43.75, 39.45, 25.90, 15.35 ppm assigned to quaternary, tertiary, secondary and primary (25.90 and 15.35 ppm) carbons, respectively and the signal at 174.75 ppm to (-CONH-) acetamide C6 carbon. The evidence of coupling between polyglucuronic acid and 4-amino TEMPO was supported and highlighted by the 2D 13C-1H HMBC NMR experiments (Figure 21) where long range coupling constant could be observed via cross-peak correlations. The presence of a strong cross-peak between C6 of polyglucuronic acid and H4’ of nitroxide radical is observed. This correlation corresponds to a connectivity across three bonds and clearly indicates the amide bond formation. Strong connectivities between C1 and
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H4* and H1 and C4* corresponding to inter residual three-bond correlations over the glycosidic linkages are equally observed.
Figure 20. 2D 13C-1H spectra in D2O: a) HMQC of polyglucuronic acid-2-methoxy ethyl amine; b) HMBC of polyglucuronic acid-2-methoxy ethyl amine. All the experimental data used have been extracted from previous work [22].
Amidation of Cellulose Materials
1031
Figure 21. 2D 13C-1H HMBC spectrum in D2O of polyglucuronic acid-4-amino TEMPO. All the experimental data used have been extracted from previous work [22].
With n-butyl amine
With 2-methoxy ethyl amine
With 4-amino TEMPO Figure 22. Chemical structure of amines grafted polyglucuronic acid.
Based on these results and observations, the chemical structure of the grafted polyglucuronic acid can be established and reported in Figure 22.
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We observed that the limitation of the grafting reaction is the result combined of amine reactivity connected to pKa value and pH solution, the presence of alkaline medium for carbodiimide mediated reaction and steric effects inherent in all molecules. The liquid-state NMR characterization probed the effectiveness of grafting reaction throughout the presence of characteristic strong cross-peaks reported in the 2D NMR experiments. This first encouraging part has led us to develop the amines-grafting onto water-insoluble substrates of cellulose. We headed towards cellulose nanocrystals that are partially oxidized in surface.
3.4) Grafting reaction onto surface carboxylated cellulose nanocrystals From native cellulose, a partial conversion into surface carboxylated cellulose nanocrystals has been realized owing to its high crystallinity and its poor accessibility of primary hydroxyl groups engaged in both intra and inter molecular hydrogen bonds in the native cellulose structure. The oxidation led to a two-phase solution: water-soluble and waterinsoluble fractions. These two fractions corresponded to the totally oxidized polyglucuronic acid and the surface carboxylated cellulose nanocrystals, respectively. The grafting reaction by carbodiimide mediated amidation process is carried out onto surface carboxylated cellulose nanocrystals with nitroxide radical (4-amino TEMPO). A suspension is obtained. Thereafter, the purification of products included precipitation, dialysis and freeze-drying steps. The reaction yields were of 98% with cotton linters. This slight loss of materials can been explained by partial degradation of grafting product. The importance of material loss depended on the crystal size of cellulose whiskers which is more important when the soluble fraction is high. The products issued to grafting reaction were analyzed by conductimetry, elemental analysis, FTIR, NMR and EPR spectroscopies. Degrees of conversion. The degrees of conversion of products are calculated. The results are summarized in Table 11. When using the conductimetry and elemental analysis and the aforementioned equations 3 and 10, there is a remarkable concordance in the DC values. The average value is 30% (Table 11). Table 11. Degrees of conversion after reaction with 4-amino TEMPO.
Degree of conversion (%) Substrate Surface carboxylated cotton linter cellulose nanocrystals
Conductimetry
Elemental analysis
EPR analysis
NMR measurement
31
32
28
30
FTIR spectroscopy. The Figure 23 reported the FTIR results for cotton linters cellulose. Characteristic absorption bands near 3340-3338 cm-1 assigned to the (OH) stretching band and near 1640 and 2900 cm-1 attributed to water molecules adsorbed onto the surface of carboxylated nanocrystals and (-CH) stretching vibration belonging to the anhydroglucose unit, with strong intensity, are observed. The carboxyl acid peak appears at 1726 cm-1 and shows significantly reduced intensity compared with that of the unreacted cellulose nanocrystals. Two other distinct bands at 1630 and 1550 cm–1 are also observed, and
Amidation of Cellulose Materials
1033
corresponded to the (C=O) stretching band (amide I) and to the (-NH) bending vibration band (amide II), respectively [101] as above-mentioned. Indeed, the carboxylic acid peak at 1726 cm-1 is well separated from the amide group (-CONH-) bands, as already reported by several authors [18, 102-103]. In agreement with present results, Araki and collaborators [10] have also observed two absorption bands at 1657 and 1544 cm-1 corresponding to amide I and amide II absorptions resulting to the effective binding of PEG-NH2 onto rodlike cellulose microcrystals. A significant reduction in the intensity of the free (COOH) stretching vibration band (1730 cm-1) is also noted in comparison with the spectrum of ungrafted oxidized nanocrystals confirming a reduction of the number of (-COOH) functions. These observations are similar to the polyglucuronic acid samples spectra. This result confirms the amidation occurring at the C6 carbon. A slighty higher intensity of the 800 cm-1 peak assigned to (-CH-) bending vibration band (δC-H) is also noted in the spectrum of the grafted sample. The presence of (-CH-) groups on the 4-amino TEMPO involved the amplitude. The FTIR results confirmed the conductometric and elemental analysis data. Similar observations have been made when polyglucuronic acid in solution was used as starting material [22] for grafting.
Figure 23. FTIR spectra of surface carboxylated cotton linters nanocrystals reacted with 4-amino TEMPO.
EPR Spectroscopy. Electron Paramagnetic Resonance (EPR) spectroscopy has been shown to be a reliable technique to characterize and quantify the microstructural and dynamic properties of various species.
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The EPR spectra of surface carboxylated cotton linter cellulose nanocrystals-4-amino TEMPO and an aqueous solution of 4-amino TEMPO as standard reference are presented in Figure 24. The three symmetrical well-resolved derivatives of Lorentzian lines are generally characteristic of the presence of free radical in solution. For conjugate sample, the lines broadening (particularly the lines at high field) clearly show a restrictive motion of the spin label linked to the cotton linters cellulose nanocrystals. It is therefore an indication that the nitroxide moiety has been successfully grafted on the nanocrystals. By double integration (area) of the labeled nanocrystals spectrum, the absolute number of coupled radicals is obtained (Table 11) and the degree of conversion calculated (equation 9) is close to those obtained with others characterizations.
6000
standard 4000
surface carboxylated cotton linter cellulose nanocrystals-4-amino TEMPO
2000
0
-2000
-4000
-6000 3400
3410
3420
3430
3440
3450
3460
3470
3480
Gauss
Figure 24. EPR spectra of 4-amino TEMPO (standard) and surface carboxylated cotton linter cellulose nanocrystals-4-amino TEMPO.
Solid-state NMR characterization. During the past decade, high resolution solid-state NMR spectroscopy has proven to be a powerful tool in the investigation of structural features of cellulose materials [35, 95, 108]. The 13C NMR spectra of cellulose raw materials presented characteristic signals in agreement with the literature: resonances at 62.4 and 65 ppm assigned to disordered and crystalline regions of C6 carbons of cellulose and between 70-77 ppm in the form of two very intense peaks the resonances assigned to C2, C3, and C5 carbons. The signal at 83.5 and 88.7 ppm are attributed to disordered and crystalline regions of C4 carbons, respectively, and C1 carbons located at 105 ppm [9, 35, 95, 109]. The resulting cellulose nanocrystals samples were characterized by solid-state NMR spectroscopy. The Figure 25 illustrated the CP-MAS 13C NMR spectrum after oxidation and amidation. After oxidation, the major change concerns the appearance of the carboxyl groups signal at 174.8 ppm. The Do is evaluated by the integration of the signal at 174.8 ppm which displays high amplitude. A crystallinity index [35] was estimated by comparing the surface area of the C4 signals at 83.5 and 88.7 ppm corresponding to the carbons in disordered and crystalline regions, respectively.
Amidation of Cellulose Materials
(c)
1035
surface carboxylated cotton linter cellulose nanocrystals-4-amino TEMPO whiskers 174.8 171.5 MPO
(b)
(a)
surface carboxylated cotton linter cellulose nanocrystals whiskers 174.8
HCl-hydrolyzed cotton linter cellulose zed cotton linter
160
120 (ppm)
80
40
0
Figure 25. CP-MAS 13C NMR spectra of (a) hydrolyzed cotton linter samples, (b) surface carboxylated cotton linter cellulose nanocrystals and (c) surface carboxylated cotton linter cellulose nanocrystals-4amino TEMPO.
After grafting by amidation, we observed the presence of characteristic carbons signals at 40 ppm corresponding to the C3’, C5’ and C4’of 4-amino TEMPO, the notable reduction of
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resonance due to the disordered regions of C6 carbons (62.4 ppm), the strong reduction in intensity of the signal at 83.5 ppm attributed to disordered regions of C4 carbons, and the decrease of the carboxyl signal of carboxylated nanocrystals at 174.8 ppm. Indeed, the C5 carbons resonance at 77 ppm presents a reduced intensity revealing a change in spatial environment due to the grafting to C6 carbons. In addition, a new resonance is detected at 171.5 ppm well separated from the (-COONa) C6 resonance (174.8 ppm). Based on the aforementioned results [19, 22, 104, 107], this new resonance corresponded to (-CONH-) acetamide carbon resulting from the grafting of 4-amino TEMPO on carboxylated nanocrystals. Irwin and collaborators [107] have also observed resonances at 40 ppm assigned to C3’, C5’ and C4’of 4-amino TEMPO during the preparation of the amidated poly(galacturonic acid). The Table 12 reveals that the carboxyl group content decreases with the grafting of 4amino TEMPO concommitant with the reduction of the disordered regions of C6 carbons resonance at 62.4 ppm, whereas the crystalline contribution at 65 ppm remains constant. This is a strong indication of the selectivity of reaction which occurs only at the surface of nanocrystals and / or disordered regions. The combination of this reduction and the presence of the acetamide resonance at 171.5 ppm confirm that the grafting occurred in the disordered regions of C6 carbons. The decrease of the C4 signal at 83.5 ppm (disordered regions) corroborates it. Furthermore, it is worth noting that the crystallinity index does not evolve during the grafting reaction (Table 12). This observation is a strong indication that the integrity of the crystallites is not altered by the amidation. In addition, the presence of acetamide carbon on the conjugate induce modifications in environment of C4 and C6 carbons in term of change in disorder, which are observed by a reduction in the intensity of corresponding resonances at 62.4 and 83.5 ppm. This phenomenon is attributed to the amine-grafting onto surface of nanocrystals. Table 12. Results of the quantitative analysis of CP-MAS 13C NMR spectra.
Cellulose samples cotton linter cellulose hydrolyzed Cotton linter cellulose surface carboxylated cotton linter cellulose nanocrystals surface carboxylated cotton linter cellulose nanocrystals-4-amino TEMPO a
NaClO (molar ratio)a
C=Ob (174.8 ppm)
C6crystc (68-63 ppm)
C6amorc (63-58 ppm)
C6totald (68-58 ppm)
0
0
0.54
0.37
0.92
C4crystc (CIe) (91-86 ppm) 0.69
0
0
0.68
0.22
0.90
0.75
0.25
1
0.10
0.68
0.13
0.81
0.72
0.28
1
0.07
0.68
0.09
0.77
0.72
0.19
Mol NaClO/mol glycosyl unit. Carboxyl groups (Do) obtained by integration of the signal at 174.8 ppm. c Crystalline and amorphous contents determined by deconvolution. d Obtained by integration of the signal at 62.4 ppm. e Crystallinity index. b
C4amorc (86-81 ppm) 0.33
Amidation of Cellulose Materials
1037
4) Discussion on the Reactivity of Grafted Cellulose Substrates Polyglucuronic acid. The main restriction of amine-grafting on totally oxidized cellulose is focused on the protonation state of amines during the carbodiimide mediated amidation accentuated by the decrease of solubility with carbon chain length increase. The pKa value must be taken into account. Higher the pKa value of amine is, smaller the grafting is. The use of amines presenting pKa values close to 9 would be highly relevant for grafting mediated by EDAC in aqueous conditions. As a result of this less protonated state of molecule, amines present a better reactivity. In addition, the length of the amine carbon chains involved steric effects that minimize the amide bond formation onto C6 carbons of oxidized cellulose. A similar response is observed with large amine like 4-amino TEMPO. To knowledge of authors, few reports presented conversion oxidized C6 carbons to acetamide carbons as presented in this chapter. The conversion is thus driven by the ability of amines to be protonated in alkaline conditions. Surface carboxylated cellulose nanocrystals. The average DC value for the cotton linter cellulose is near 30%. In fact, a maximum of about one third of carboxylated functions of cellulose are really grafted. Similar results are reported in the literature, for instance, with the grafting of single terminally aminated PEG (20%) or phenylpropanolamine (6%) onto waterinsoluble oxidized cellulose [10, 19]. For attempting to understand the results, the organization of cellulose in plant cell walls and the organization of cellulose nanocrystals resulting from oxidation must be considered. According to current models and recent works [49, 95, 110-112], the cellulose microfibrils present square section with exposed surfaces corresponding to the [ 110 ] and [ 110 ] crystal planes of the cellulose lattice (in reference to the cellulose unit cell defined by Sugiyama [49]). As opposed to the core of the microfibrils, which is considered to be in a crystalline arrangement, the microfibril surfaces are organized in a different manner. The difference between surface and core chains is clearly evidenced in CP/MAS 13C NMR spectra of cellulose: different resonances at C4 carbons are attributed either to the core or surface cellulose chains [35, 113]. One distinguishes surfaces accessible to oxidation located at the exterior of microfibrils aggregates and inaccessible surfaces located inside the microfibrils aggregates. This organization in aggregates is not modifying by HCl hydrolysis that only converts microfibrils aggregates into nanocrystals aggregates. In addition, regarding the description of the [ 110 ] and [ 110 ] crystal surfaces, the oxidation took place only one-half of the surface hydroxymethyls of cellulose and the other half being toward the core of the crystalline domains (Figure 26). Surface hydroxylmethyl groups accessible to oxidation
Core of cellulose nanocrystals with inaccessible primary hydroxyl groups to oxidation
Figure 26. Scheme of cellulose chain at the surface of cellulose crystal.
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Theoretically, if all surface hydroxylmethyl groups were transformed during carboxylation, the maximum grafting would be 50%. The experimental degrees of conversion opposed to the theory. It is worth interesting to make some comments on the experimental results of grafting. In first observation, even if the HCl hydrolysis broadly involved the production of nanocrystals exempt to amorphous domains, crystals aggregates are obtained. Without HCl hydrolysis, the crystals are more highly aggregated [95]. After carboxylation, the maximal Do of cellulose substrates corresponded to 15%. Even if the surface oxidation improved the individualization of the crystals, less one-half of the surface hydroxymethyls of cellulose is accessible. A part of these accessible surfaces is entangled inside cellulose aggregates throughout hydrogen bonds network between nanocrystals embedded in aggregate. The oxidized samples consist rather of crystallites made of two or three individual crystals. On the basis of these informations, the 50% of conversion is not reachable. Further, the partial individualization of the crystals achieved a modification of accessibility rate of carboxylated functions for grafting since the aggregates made of two or three individual crystals are organized in different and inhomogeneous manner. We can envisage a large scale of arrangements from a packing of two or three individual crystals (parallel until displacement across of nanocrystals). The least favorable cases for grafting reaction are presented in Table 13 in comparing with nanocrystals totally individualized with 50% conversion. We can consider that the limit of DC values corresponds to 33% up to 50%. By and large, the new calculated DC value resulting from the least favorable case with three nanocrystals by aggregates is very close to the experimental results exhibited in this chapter. Presumably, the steric effects resulting to the amine grafted adding to bulk organizations of aggregates could partly explain the experimental results. Table 13. Schematic representation of most limiting cellulose nanocrystals organizations for the amine-grafting reaction. Cellulose nanocrystals organization ocrystal totally individualized
ulose nanocrystals made of three individual crystals
Accessibility
DC calculated
100%
50%
A : 75%
A B
B: 50%
33,3%
C
C: 75%
h total overlapping ellulose nanocrystals made of two individual crystals
A : 75%
A B
h total overlapping
37,5% B: 75%
Amidation of Cellulose Materials
1039
CONCLUSION In this chapter, we described the grafting on polyglucuronic acid samples and surface carboxylated cellulose nanocrystals with single terminally aminated molecules. To achieved it, we used linear and cyclic amines presenting pKa values at around 9 – 11. Polyglucuronic acid was issued by TEMPO mediated carboxylation of cellulose IIII. Surface carboxylated cellulose nanocrystals were obtained by oxidation of HCl-hydrolyzed cellulose in order to realize a preferential surface oxidation leading to higher nanocrystals yields. The products resulting from grafting reaction were analyzed by conductimetry, elemental analysis, FTIR, NMR and EPR spectroscopies. After carboxylation of cellulose by TEMPO mediated oxidation, the main observations corresponded to the appearance of the carboxyl groups signals near 175 ppm with the carboxyl peak at 1730 cm-1. The NMR characterization of cellulose substrates indicated that oxidation is selectively realized on C6 carbons. Upon amidation, a covalently-linked cellulose with a 1:2,5:1,5 mol ratio of cellulose:amine:carbodiimide is formed. We demonstrate that, by the formation of a more hydrolysis-resistant and nonrearrangeble active ester intermediate from the active O-acylurea, the primary amine-grafting onto oxidized cellulose is possible. With polyglucuronic acid, the reaction is driven by protonation state and solubility of amines in the alkaline conditions used. With surface carboxylated cellulose nanocrystals, the results widely vary from the surface oxidation level and the morphology of carboxylated samples, i.e. nanocrystal aggregates made of two or three individual nanocrystals. By NMR characterization, the presence of acetamide signal is proven accentuated by the observation of two FTIR peaks at 1650 and 1550 cm-1 corresponding to the (C=O) stretching band and to the (-NH) bending vibration band of (-CONH-) functions. For the grafting of 4-amino TEMPO, EPR technique equally allowed to ensure the amide bond. The combination of NMR, EPR spectroscopies, conductimetry and elemental analysis providing the undisputed evidence that the amine-grafting reaction is realized with a good agreement between calculated results. Through this work, some parameters are identified as key drivers to success. In order to validate the experimental results, some possibilities are suggested in open discussion concerning the reactivity of cellulose substrates. With totally oxidized cellulose such as polyglucuronic acid, the grafting reaction is limited by the molecular weight of primary amines, their solubilities at reactional temperature and especially the unprotonated state of amine at the pH used. Indeed, on the basis of reactional mechanism, the active ester intermediate must obviously react with amine groups of molecule. With partially oxidized cellulose like nanocrystals, the reaction is more dependent on substrate and particularly on the accessibility of reactive functions that are carboxylated groups. The cellulose chains organization limited the number of reactive functions to one-half of the surface hydroxymethyls of cellulose preventing partly the efficiency of amidation to one-third grafting.
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ACKNOWLEDGMENT N. FOLLAIN is grateful to CNRS for financial support during post-doctoral position at CERMAV. The author thanks Mrs M-F MARAIS (CERMAV, CNRS) for preparing the cellulose samples. The author also acknowledges the help of Dr Serge GAMBARELLI (CEA-Grenoble, DRFMC/SCIB) for the EPR characterization, M. TRIEWEILER for NMR data (CERMAV, CNRS) and Dr. Henri CHANZY for valuable discussion during this work.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 35
D/L-HYBRID GALACTANS FROM RED SEAWEEDS: DO THEY REALLY EXIST? Marina Cianciaa,b and Alberto S. Cerezo*b a
Cátedra de Química de Biomoléculas, Departamento de Biología Aplicada y Alimentos, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, 1417 Buenos Aires, Argentina. b CIHIDECAR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina.
ABSTRACT Several points concerning the presence in red seaweeds of galactans with cooccurrence of carrageenan and agaran structures (DL-hybrid galactans) are addressed: a. b. c. d. e. f.
Analysis of D- and L-galactose enantiomers and their methylated and cyclic derivatives. Spectroscopic, enzymatic and chemical methodologies. Systems of galactans, in which the presence of D- and L-galactose has been determined (DL-galactan systems). The possible deviant structures of carrageenans and agarans in DL-hybrid galactans. The isolation of “pure” carrageenans and agarans from DL-galactan systems. The formation of molecular complexes between carrageenans, agarans and carrageenans/agarans. DL-hybrid galactans or molecular complexes: Evidences in favor of each hypothesis.
* Corresponding author, e-mail:
[email protected], phone/fax: 54 11 4576 3346.
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INTRODUCTION Galactose-containing polysaccharides are the main matrix polysaccharide components in the majority of the red seaweeds. These galactans have structures based on a linear chain of alternating 3-linked β-galactopyranosyl residues (A-units) and 4-linked α-galactopyranosyl residues (B-units). The A units always belong to the D-series, whereas the B units include residues of the D- or L-series, many times occurring as 3,6-anhydrogalactopyranosyl moieties (Stortz, Cases & Cerezo 1997a). The simplest classification is made according to whether the 4-linked residues belong to the D- or L-series: by this token, two different classes of galactans are defined, namely carrageenans and agarans, respectively. Classification of carrageenans takes into account the sulfation pattern of the A-unit; the κ-family comprises 4-sulfated polysaccharides, while the λ-family includes those with 2sulfation (Fig. 1). Less important are the β-family, in which the A-unit is not sulfated and the ω-family with sulfation on C-6 of this unit.
(a) -
O3SO
Α−unit CH 2OH
Β−unit -
O O
O
O
CH 2OH
O OR
O O
O3SO O
O 3SO
OH
CH 2OH
λ-carrageenan
HO
O O
C
OSO3-
C
O OSO 3-
-
-
μ-carrageenan R=H ν-carrageenan R=SO3-
(b) CH2OH
O OH
κ-carrageenan R=H ι-carrageenan R=SO3-
OH
OR
O
OH
O HO
O
O O3SO
OSO 3-
O
O OSO3-
cyclized λ-carrageenan (θ−carrageenan)
Figure 1. Idealized structures of carrageenans of the kappa-family (a) and of the lambda-family (b)
Classification of agarans is not so clear cut as that of carrageenans, but these products can be grouped between two extreme structures, one of them is agarose and the other is obtained replacing the 3,6-anhydro-derivative by L-galactose 6-sulfate (Fig. 2), giving rise to the so called “Yaphe´s third extreme” (Duckworth & Yaphe 1971). The latter is important in porphyrans. In addition, a third group, in which B-units with the D- and L-configuration are on the same molecule, is under discussion (Fig. 3). They are usually known as “hybrid” or intermediate galactans (agaroid-carrageenan hybrids, according to Takano, Hayashi & Hara 1997). Nevertheless, the denomination “hybrids” has to be used with due care. In the field of carrageenans, it was used to describe products whose structures contain different idealized repeating units (i.e. κ/ι-carrageenans). To avoid name uncertainties, we use for them the term
D/L-Hybrid Galactans from Red Seaweeds: Do they Really Exist?
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“carrageenan hybrids” and adopt the term “D/L-hybrid galactans” for galactans, in which B units with D- and L-configuration are assumed to be in the same molecule. We also define “DL-galactan system” as the system of galactans, in which the presence of α-D- and α-Lgalactose or their 3,6-anhydro-derivatives, and consequently of carrageenan and agaran structures, can be experimentally detected. OH CH2OH
OH
a O OH
O O
O ROH2C
O
OH
HO
b
O
CH2OH
O
OH O
HO
O
R=H+ or SO3- (porphyran)
Figure 2. Structure of the basic backbone of agarans (a) and of agarose (b)
a) With the L-galactose (B-units) interspersed in the chain: -G-DA-G-DA-G-LA-G-DA-G-D-G-DA-G-LA-G-DA-G-D-G-L-G-LA-G-DA-G-DAb) Forming a carrageenan-agaran block copolymer: -G-DA-G-DA-G-D-G-DA-G-D-G-DA-G-D-G-LA-G-LA-G-LA-G-L-G-LA-G-LA-G-Ljunction zone 1
Figure 3. Possible structures of DL-hybrid galactans
The division of red algal galactans in carrageenans and agarans led to call their parents seaweeds carrageenophytes and agarophytes, respectively. Carrageenophytes are usually members of some families of the order Gigartinales, while typical agarophytes usually belong to the orders Gelidiales and Gracilariales. This clear-cut separation has been upset because careful studies using enantiomeric analysis on the polysaccharide systems produced by seaweeds from the Gigartinales, known to yield carageenans, indicated that they also biosynthesize small amounts of L-galactose containing galactans (Ciancia, Matulewicz & Cerezo 1993, 1997, Estevez, Ciancia & Cerezo 2000, 2001; Storz, Cases & Cerezo 1997b). In time, it was also found that some agarophytes produce, not only agarans, but also small amounts of galactans with carrageenan structures (Errea & Matulewicz 2003). This means that if the division between carrageenophytes and agarophytes seaweeds is maintained, it should refer only to the major types of polysaccharides produced by the seaweeds. The “DL-hybrid” galactans that have a carrageenan predominant structure were called “DL-hybrid carrageenans” and those with a predominant agaran structures, “DL-hybrid agarans”. These are the limits between which the DL-hybrid galactans are defined with a
1
The letter codes refer to the nomenclature of Knutsen, Midlabodski, Larsen & Usov (1994).
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Marina Ciancia and Alberto S. Cerezo
molar ratio D-:L-galactose going from 1:0 in “pure” carrageenans to 1:1 in “pure” agarans. Intermediate values are interpreted as DL-hybrid galactans.
ENANTIOMERIC ANALYSIS In some cases, detection of the presence of carrageenans, agarans and DL-hybrid galactans was carried out, or at least aided, by the regular use of 13C NMR spectroscopy for screening of extracts or, in purified fractions, for structural determinations. This is possible due to the fact that carrageenan and agaran dissacharidic backbone units are diastereomeric (Figs. 1 and 2). The position of the signal of C-1 of the galactose residues in these products depends not only of the type of bond with the neighboring unit [α-(1→3) or β-(1→4)], but also of the relative configurations of both, the residue under consideration, and the neighboring residue (Usov, Yarotskii, & Shashkov 1980, Usov 1984). Besides, substitution produces second-order effects. For instance, an A unit (3-linked β-D-galactose) of a noncyclized agaran gives a signal at 103.7 ppm, while the same unit in a non-cyclized carrageenan appears around 104.8 ppm. For the B unit, C-1 of an α-L-galactose unit appears at 100.9-101.1 ppm, while that of α-D-galactose, at 96.1 ppm; 3,6-anhydro-α-L-galactose, at 98.0-98.5 ppm and 3,6-anhydro-α-D-galactose, at 95.1-95.7 ppm (Usov, Yarotskii, & Shashkov 1980, Usov 1984). However, the use of 13C NMR spectroscopy for the determination of enantiomeric forms of the monosaccharide units in these types of polysaccharides is difficult due to the complexity of these polymers and their high molecular weight. Several broad bands are usually found in the anomeric region and weak signals can be lost, even if more elaborated chemical analysis (see later) shows that the corresponding units are present. Besides, methoxyl and sulfate substitution on C-2 shifts greatly the anomeric signals. Given the low sensitivity inherent to 13C NMR spectroscopy and the complexity of the spectra in the anomeric zone, low percentages of minor enantiomers may not be detected. In fact, it is well documented that usually galactan structures present in 5-10 % together with other predominant structures, are poorly detected by 13C NMR spectroscopy. Thus, negative results in the spectra of carrageenans or agarans do not exclude the presence of small, but still significant, amounts of 4-linked α-L- or α-D-galactose moieties, respectively, or their 3,6anhydro-counterparts. The use of bidimensional techniques is beginning to overcome these difficulties. Several other methods have been developed to assign the D- or L-configuration of sugar units in galactans, as optical rotation determinations of the polysaccharide hydrolyzate by acid or enzymatic methods (Aspinall 1982). The first one may only be used with homopolymers, which should be devoid of other optically active matter. The second, gives only gross results, and should be used with care with sugars for which the specificity of the enzymes is unknown. A method combining the use of infrared spectroscopy, partial methanolysis and further analysis of the fragments by liquid chromatography was proposed (Whyte, Hosford, & Engar 1985). The authors noticed a signal at 940 cm-1 in the spectra of agars, which represented at least 70 % of the intensity of that at 905 cm-1, and was small or absent in spectra from carrageenans.
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More elaborated determinations, which override the above described problems, involve acid hydrolysis of the polysaccharide to obtain the mixture of free monosaccharides, which are then treated with a chiral reagent to give a mixture of diastereomeric compounds. These compounds are analyzed using GLC with regular columns (Gerwig, Kamerling, & Vliegenthart 1978, Leontein, Lindberg, & Lonnngren 1978, Gerwig, Kamerling, Vliegenthart, 1979, Little 1982, Cases, Cerezo, Stortz 1995, Lindqvist & Jansson 1997) or high-field 1H NMR spectroscopy (York, Hantus, Albersheim, & Darvill 1997). In another approach, monosaccharide enantiomers are separated as different sugar derivatives using chiral GLC columns (Lindqvist & Jansson 1997, Heinrich, Konig, Bretting, & Mischnick 1997). However, the use of glycosides as chiral derivatives produces multiple peaks in the chromatogram for each component sugar that correspond to the anomers of the pyranosic, furanosic and the open forms of the sugars, in a proportion determined by their equilibrium. Thus, overlapping of peaks makes their assignment difficult, even when using capillary GLC. This fact demands the use of routine GC/MS with ion monitoring for the analysis, as total ion chromatogramans are too complicated (Takano, Matsuo, Kamei-Hayashi, Hara, & Hirase 1993). Another drawback is that methylated products present in small but significant amounts may not be observed due to their partitioning in several peaks. This problem was overcome by transforming the anomeric carbon atoms of the sugars obtained during hydrolysis of the polysaccharide into another group, as amino (by reductive amination) or carboxylate (by oxidation). An HPLC method was developed, which involves a coupling reaction with (-)TBMB carboxylic acid and per-O-acetyl pyranosyl bromides to give diastereomeric 1-OTBMB carbonylated sugars, which could be separated by HPLC (Nishida, Bai, Ohrui, & Meguro 1994). Application of these methods to the analysis of carrageenans and agarans has still the drawback that during acid hydrolysis performed in the usual conditions, degradation of 3,6anhydrogalactose occurs. A qualitative analysis of the presence of both isomers of 3,6anhydrogalactose may be obtained by partial reductive hydrolysis of the polysaccharide sample and detection of the acetylated derivatives of agarobiitol and/or carrabiitol by GLCMS (Usov & Ivanova 1992). The absolute configuration of 3,6-anhydrogalactose units has also been assigned by partial methanolysis and determination of the presence of agarabiose and/or carrabiose derivatives (Takano, Matsuo, Kamei-Hayashi, Hara, & Hirase 1993). Later, this procedure was superseded by the development of a method that involves a partial oxidative hydrolysis to yield the 3,6-anhydrogalactonic acids, which are separated and quantified by GLC, after conversion to the acetylated diastereomeric sec-butyl esters (Errea, Ciancia, Matulewicz, & Cerezo 1998). Care should be taken when the 3,6anhydrogalactosidic linkages are hydrolyzed to carry out their oxidation or reduction, as their rates of hydrolysis may be markedly affected by the presence of substituents (methoxyl or sulfate groups) on C-2. Therefore, their quantitation may show large errors (Chiovitti, Bacic, Craik, Kraft, Liao, Falshaw, & Furneaux 1998). Many seaweed galactans contain not only galactose and 3,6-anhydrogalactose, but also their mono-O-methyl derivatives in one or more of the four available positions. Each of them may individually belong to the D- or L-series (Cases, Stortz, & Cerezo 1994). In this case, a reaction of the hydrolyzed mono-O-methyl-galactoses with chiral 1-amino-2-propanol by reductive amination followed by acetylation and GLC analysis was used successfully. The procedure resolves all enantiomeric pairs, but that of 2-O-methylgalactose, which can be separated if the amine is replaced by α-methylbenzylamine (Cases, Cerezo, & Stortz 1995).
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This method was then extended to determine the configuration of 3,6-anhydrogalactose and its 2-O-methyl-derivative, using also derivatization with one of the enantiomers of αmethylbenzylamine as chiral amine. Moreover, a one-pot technique was developed to determine the proportion of both enantiomers of 4-linked 6-sulfated α-galactose and their 3,6anhydro-derivative in a polysaccharide, and, as results of this analysis, the percentages of precursor structures of carrageenan- and agaran-types (Navarro & Stortz 2003). Obviously, this method can be used also in the methylation analysis of polysaccharides, improving the structural information (Errea, Kolender, & Matulewicz 2001). With that purpose, the absolute configuration of tetra-, tri-, di- and mono-O-methylgalactoses was also assigned with the aid of GC/MS of trimethylsilylated derivatives of 2-octyl-L-glycosides (Takano, Kamei-Hayashi, Hara, & Hirase 1992, Takano, Matsuo, Kamei-Hayashi, Hara, & Hirase 1993). Table 1 summarizes the methods available for enantiomeric analysis of red seaweed galactans by application of gas chromatographic techniques. In spite of some remaining difficulties, at present, full chiral recognition of the monosaccharides in the original polysaccharide, as well as the partially methylated sugars in a permethylated galactan is an absolute requisite for a fine structural study of red seaweed galactan structures. Table 1. Summary of the methods available for enantiomeric analysis of red seaweed galactans by application of gas chromatographic techniques1. Reaction involved2
1. Hydrolysis, 2. Chiral ROH/H+ 1. Hydrolysis, 2. NaCNBH3/(S)-MBA 1. Hydrolysis, 2. NaCNBH3/(S)-AP 1. Mild hydrolysis, 2. MMB 1. Mild hydrolysis, 2. Br2, 3. H+, 4. NaBH4, 5. SOCl2, 6. s-BuOH
1
Derivative
Comments Many peaks (anomeric forms) per sugar. Glycosides3,4 Severe overlapping if methylated Gals are present Only for 2-O-Me-Gal enantiomers. 5 Aminoalditols Enantioselective for other Gal units 2-O-Me-Gal enantiomers are not Aminoalditols5 Separated. Quantitation of acid-stable sugars is possible. Only for 3,6-AnGal in consecutive Disaccharide alditols6 carrabiose or agarobiose units.
Esters7
Only for 3,6-AnGal
1. Hydrolysis, 2. NaCNBH3/(S)-AP Repeat twice, mild then strong hydrolysis
Aminoalditols8
3,6-AnGal and 2-O-Me-Gal enantiomers not separated. Quantitation of acid-stable sugars and 3,6-AnGal is possible.
1. Hydrolysis, 2. NaCNBH3/(S)-MBA Repeat twice, mild then strong hydrolysis
Aminoalditols8
Only for 3,6-An-2-O-Gal, 3,6-AnGal and 2-O-Me-Gal
Adapted from Navarro & Stortz 2003. 2 MBA=methylbezylamine, AP=aminopropanol, MMB 4morpholine borane. 3Gerwig, Kamerling, & Vliegenthart 1978. 4Leontein, Lindberg, & Lonnngren 1978. 5 Cases, Cerezo, & Stortz 1995. 6 Usov & Ivanova 1992. 7 Errea, Ciancia, Matulewicz, & Cerezo 1998. 8 Navarro & Stortz 2003.
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DL- Galactan Systems in Red Seaweeds It has been found that carrageenophytes of related families of the order Gigartinales, as Gigartinaceae (i.e. Chondracanthus canaliculatus, Gigartina skottsbergii, G. canaliculata, G. leptorinchos, Sarcothalia crispata studied as Iridaea undulosa, Chondrus crispus), Phyllophoraceae (i.e. Mastocarpus stellatus, Gymnogongrus torulosus, G. griffithsiae) and Solieriaceae (i.e. Anatheca dentata, Kappaphycus alvarezii) produce agarans usually in small amounts, but, at least in one case, up to ∼ 30% of the total galactans (Craigie & Rivero Carro 1992, Ciancia, Matulewicz, & Cerezo 1993,1997, Stortz & Cerezo 1993, Estevez, Ciancia, & Cerezo 2000, 2001, 2004, 2008, Chopin, Kerin, Mazerolle 1999, Talarico, Zibetti, Faria, Scolaro, Duarte, Noseda, Pujol, & Damonte, 2004, Nunn, Parolis, & Russell, 1971, 1981). On the other hand, agarophytes, like those of the Ceramiales (i.e. Rhodomela larix, Digenea simplex, both from the Rhodomelaceae), but also in some cases of the Bangiales (i.e. Porphyra columbina) and Gelidiales (i.e. Pterocladiella capillacea), were found to produce not only agarans, but also carrageenans as minor components (Takano, Yokoi, Kamei, Hara, & Hirase 1999, Takano, Shiomoto, Kamei, Hara, & Hirase, 2003, Navarro & Stortz 2003, Errea & Matulewicz 2003), while other groups of red algae of the orders Bonnemaisoniales, Halymeniales, Plocamiales, Rhodymeniales, biosynthesize both families of galactans in variable quantities (Usov 1992, Stortz and Cerezo 2000, Takano, Shiomoto, Kamei, Hara, & Hirase 2003). However, not only the biosynthetic role of these mixed systems, but also their molecular structures are still unclear. No evidence for neat separation of these DL-galactan systems into carrageenans and agarans has been achieved and variations in composition and substitution levels shown in different fractionations might be ascribed to high, but normal, compositional and structural dispersion. However, no proof of D/L-hybrid galactan structures has been found either. Whether these carrageenan- and agaran-structures are forming blocks in copolymers, or if they are different molecules forming molecular aggregates, is still not known. There is a strong need for verification of the existence of these types of molecules, however, until now they have only been made evident by the exhaustive usage of fractionation techniques and homogeneity determinations, before carrying out structural determinations. Considering the fact that partial hydrolysis has up-to-date produced only agaran or carrageenan fragments, it is possible that the agaran-carrageenan domain, if it exists, should correspond to a junction zone of a block copolymer (Fig 3). Thus, the yield of DL-hybrid oligosacharides (those which contain both α-D- and α-L-galactose units) in a random partial hydrolysis should be very low. Moreover, considering that agaran and carrageenan may self-complex in solution, the irregular junction zones might be slightly more susceptible to hydrolysis, and the yields of those oligosaccharides might be even lower than those expected in a random hydrolysis process. Taking into account that agarophyte taxa of the order Bangiophycidae are considered as ancestral pool from which the evolutionary higher taxa of the Florideophycidae have arisen (Oliveira & Bhattacharya 2000), the biosynthetic capacity to produce agarans may represent a primitive condition among the Florideophyceae, as suggested by the fact that seaweeds of the order Ahnfeltiales are agarophytes. These organisms would be interpreted as ancestral to taxa that contain carrageenans as major product in their cell walls (Fredericq, Hommersand, & Freshwater 1996).
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The most important types of galactans (i.e. agarans and carrageenans) are not always biosynthesized by monophyletic groups of red algae, but they are also present in paraphyletic or polyphyletic groupings of these seaweeds. This fact could be a consequence of the multiple origin of each type of polysaccharides along the red algal evolution. However, the chemical structure of these products within the red seaweeds seems to be a stable chemotaxonomic character, useful for the characterization at genera or family level, but it is highly variable at ordinal or higher groupings (Estevez, Ciancia, & Cerezo 2008). Some DL-galactan systems from “classical” and “non-classical” carrageenophytes and agarophytes are discussed below, most of the examples were chosen to ilustrate fractionation methodology and enantiomeric analysis.
1. DL-GALACTAN SYSTEMS FROM “CLASSICAL” CARRAGEENOPHYTES Seaweeds from three families of the order Gigartinales: Gigartinaceae, Solieriaceae and Phyllophoraceae produce the most valuable carrageenans and have been known as typical carrageenophytes. Craigie and Rivero-Carro (1992) were the first to show the presence of DL-galactan hybrids in a screening of galactans from gametophytes of the Gigartinaceae (Gigartina canaliculata, Chondrus crispus, G. leptorhynchos) and Phyllophoraceae (Mastocarpus stellata, previously included in the Petrocelidaceae 2 ) and reported it in the XIVth International Seaweed Symposium, but these results were never published in detail. Several groups were working in the field at that time, and in 1993 two papers were published: One reported the presence of L-galactose containing galactans in the polysaccharide system biosynthesized by gametophytes of Gigartina skottsbergii (Ciancia, Matulewicz, & Cerezo 1993) and the other one, in the system from tetrasporophytes of Iridaea undulosa (Stortz & Cerezo 1993) and, in 1994, two Ph.D. Thesis appeared (Noseda 1994, Ciancia 1994), starting the detailed, chemical, enantiomeric and spectroscopic studies of these systems.
a. DL-Galactan Systems from Cystocarpic Plants of the Family Gigartinaceae. Matrix galactans from cystocarpic plants of the Gigartinaceae are completely extracted at room temperature and they are made up of similar amounts of polysaccharides gelling at low concentrations of KCl (κ/ι- carrageenans, together with small amounts of L-galactose- and 3,6-anhydro-L-galactose-rich galactans) and non-gelling galactans (partially cyclized μ/νcarrageenans, together with small amounts of L-galactose- and 3,6-anhydro-L-galactose-rich galactans) (Ciancia, Matulewicz, & Cerezo 1993, 1997, Flores, Cerezo, & Stortz 2002). Water-soluble polysaccharides from gametophytes of Gigartina canaliculata, Chondrus crispus, G. leptorhynchos were fractionated by precipitation with potassium chloride, and the 2
Names are given according to the currently accepted classification, as informed by Guiry & Guiry 2009.
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KCl-soluble products (partially cyclized μ/ν-carrageenans, together with small amounts of Lgalactose- and 3,6-anhydro-L-galactose-rich galactans) were submitted to an alkaline treatment (cyclization of the μ/ν- to κ/ι-carrageenans, Fig. 1). The cyclized derivatives were, again, precipitated with KCl separating new soluble fractions. Compositional and enantiomeric analysis of these fractions showed the presence of D- and L-galactose in molar ratios near unity, suggesting that these products were agarans (Craigie & Rivero-Carro 1992). The mixture of galactans from cystocarpic samples of I. undulosa (Cc) were fractionated (Table 2) and treated as depicted in Fig. 4 (Flores, Cerezo, & Stortz 2002). Bulk precipitation of Cc with 2M KCl yielded 62 % of an insoluble product (Ci, κ/ι-carrageenans) and 31% of a soluble one (Cs, μ/ν-carrageenans). Alkaline treatment of Ci and Cs proceeded with excellent yields (93-95%), giving CiT and CsT in which the precursor units (4-linked α-galactose 6sulfate and 2,6-disulfate) were cyclized. Both products were fractionated by precipitation with increasing concentrations of potassium chloride, yielding three fractions insoluble at 0.1M, 1M, and 2M KCl and one soluble at the latter concentration (Table 3). The main fraction obtained from CiT (CiTi-0.1), which precipitated at 0.1M KCl, represents 92% of the original weight and has a composition very similar to that of the original fraction; it does not contain L-galactose. Minor fractions precipitating at higher concentration of KCl (Table 3) showed increasing amounts of L-galactose. Ce
soluble Cs
insoluble Ci
1 M NaOH/ NaBH4
1 M NaOH/ NaBH4
CsT
CiT
0.1 M KCl insoluble CsTi-0.1
soluble
0.1 M KCl soluble
insoluble CiTi-0.1
1 M KCl insoluble
soluble
2 M KCl
CsTi-2
insoluble CsTi-2
insoluble
insoluble CiTi-1
soluble CsTs
soluble CiTs
Figure 4. Fractionation of the gametophytic carrageenan from I. undulosa
insoluble CiTi-2
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Table 2. Yields and analyses of the native carrageenan (Cc) from Iridaea undulosa and from fractions obtained by precipitation with KCl1. Carrageenan Yield %
Gal:3,6-AnGal:sulfate Molar ratio Cc 1:0.57:1.14 Ci 62 1:0.59:1.14 Cs 31 1:0.55:1.23 1 From Flores, Cerezo, & Stortz, 2002
Ratio per 100 monosaccharide units 3,6-AnGal Sulfate 36 73 37 72 35 79
Table 3. Yields and analyses of the fractions obtained by alkaline treatment and fractionation of carrageenans Ci and Cs from Iridaea undulosa1. Range of Yield Monosaccharides4,5 (moles %) precipitation % Ara Xyl Man Glc Gal M, KCl DLCiT 932 1 1 95 3 CiTi-0.1 0.0-0.1 92 4 93 CiTi-1 0.1-1.0 5 tr tr 1 tr 94 4 CiTi-2 1.0-2.0 1 tr 1 96 3 CiTs 2.0 sol. 2 2 7 14 2 58 16 CsT 953 2 tr 5 84 7 CsTi-0.1 0.0-0.1 81 4 tr 4 88 4 CsTi-1 0.1-1.0 9 2 2 1 11 77 7 CsTi-2 1.0-2.0 90 %), while the fraction soluble in 2 M KCl (F3) had ∼ 60 % of agaran structure (Table 6). A part of F3 (F3i) retrograded and the remaining soluble moiety (F3s) was submitted to alkaline treatment and KCl fractionation, to give a new soluble product (T4, 40 % of the alkali treated product). Although a small amount of κ-structure was still detected, T4 is mostly an agaran ([α]D= - 40º), constituted by 3-linked non sulfated β-Dgalactose and β-D-galactose sulfated on C-2 or C-2 and C-4, and 4-linked α-L-galactose, partially sulfated on C-3 and partially methylated on C-2; also, a significant quantity of 3,6anhydro-α-L-galactose 2-sulfate was detected. Other agarans extracted from this seaweed have structures similar to T4, with quantitative variations in the proportion of these structural units, in spite of their solubility behavior (Estevez, Ciancia, & Cerezo 2004). A 13C NMR spectroscopic study of the hot-water extract of G. torulosus from New Zealand (as Anhfeltia torulosa) showed κ- , ι- and μ/ν-structures in a ratio 32:46:22 (Furneaux & Miller 1985). Later, chemical studies with extensive use of fractionation procedures showed that the red seaweed G. torulosus biosynthetizes a system of carrageenans similar to that of cystocarpic plants of the Gigartinaceae (i.e. KCl-insoluble carrageenans of the κ/ιcarrageenan hybrid type, in this case, with predominance of ι-structure, together with KClsoluble μ/ν-carrageenans). Nevertheless, enantiomeric analysis of the component sugars and structural units of the fractions showed in all the cases the presence of L-galactose containing galactans.
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Table 6. Analysis of the raw extract obtained from Kappaphycus alvarezii (C), of fractions obtained from it by KCl fractionation (F1-F3) and fractions obtained from treated F3s by KCl-fractionation (T1-T4). Data for a commercial κ-carrageenan from the same seaweed (K) are also shown1. Frac- Range of Yield Carbo- Sulfate tion precipi- % hydrates as SO3K tation % % M, KCl
Monosaccharide composition [α]D Mw KDa (moles %) º Gal 2-L- 6-D- AnGal D- DD- L- Gal Gal D- L- Xyl Glc 2 K n.d. 28.0 49.5 1.0 - 48.4 1.1 - - + 51.8 140 3 C 65.6 n.d. 23.9 50.2 3.1 tr. tr. 40.0 3.8 1.1 1.8 + 48.6 30 C´ 82.0 45.0 22.0 47.8 tr. tr. 1.2 44.6 3.1 1.3 2.0 n.d. 125 F1 0.1-0.2 67.0 47.1 25.4 49.9 1.3 tr. tr. 45.0 3.8 tr. - + 51.2 42 F2 0.2-0.5 7.0 49.1 26.9 48.8 1.6 tr. tr. 46.6 3.0 - tr. + 56.7 42 F34 2.05 15.0 n.d. 22.8 41.6 18.4 5.4 3.3 14.7 tr. 10.5 6.1 n.d. n.d. F3s 10.6 55.0 25.1 35.9 19.2 4.3 1.3 5.1 26.9 7.3 n.d. 24 T1 Insol.6 14.8 24.0 10.0 39.4 31.2 2.3 3.5 3.6 3.0 12.9 4.1 n.d. n.d. T2 0.1-0.2 18.2 51.6 31.8 43.9 4.5 - 1.0 45.7 1.7 1.4 1.8 + 20.0 n.d. 5 T4 2.0 40.0 48.0 19.3 29.0 14.2 6.1 2.8 5.3 4.6 18.9 16.2 - 40.0 n.d. F3i 4.4 27.1 11.6 33.3 24.1 5.5 2.3 2.5 3.8 14.7 13.8 n.d. n.d. 1 2 From Estevez, Ciancia, & Cerezo 2004. n.d.=not determined. 3tr.=traces. 4By dissolution of F3 in water a soluble (F3s) and an insoluble product (F3i) were obtained. 5Soluble in 2.0 M KCl. 6 Insoluble in water.
Table 7. Analysis of the hot water extracts C1-C4 from Gymnogongrus torulosus and of fractions obtained from C1 by KCl fractionation1. Ext- Range of Yield2 Sulfate Monosaccharide composition [α]D Mw Carra- Agaran5 KDa geenan5 % (moles %)4 racts precipi- % as º Gal 6- AnGal Xyl Glc % tation SO3K % Gal M, KCl D- L- D- D- L- D- DC1 31.1 29.9 54.2 6.1 tr.3 28.7 9.6 1.4 tr. + 25.0 44 67 33 F1 0.4-0.5 44.9 29.2 47.4 1.4 - 38.1 11.4 1.7 - + 40.0 56 74 26 F2 1.2-1.4 21.1 26.3 54.8 tr.2 - 36.7 6.0 2.5 - + 47.9 77 88 12 F33 2.04 16.1 15.8 36.5 25.4 - 16.8 5.8 8.6 4.3 - 15.5 18 25 75 C2 10.1 22.9 54.5 5.9 2.5 22.4 11.0 1.4 2.4 + 18.4 45 65 35 3 C3 7.6 20.2 54.7 6.8 3.3 14.2 15.7 1.0 4.3 + 13.8 23 52 48 C4 3.6 17.1 42.6 8.4 2.7 26.9 3.7 6.4 9.3 - 3.5 18 70 30 1 2 From Estevez, Ciancia, & Cerezo 2001. Yield of C1-C4 are given for 100 g of the residue of exhaustive extraction at room temperature. Yields of fractions F1-F3 are given for 100 g of C1. 3 tr.=traces. 42.6 % and traces of 3-Gal were found in F3 and C3, respectively. 5Calculated considering Xyl and Glc as contaminants.
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Sequential extractions with water at room temperature produced three fractions (A1-A3) in low yields. All of them contained significant amounts of L-galactose and 3,6-anhydro-Lgalactose, comprising 28-38 % of agaran structures. Further sequential hot water extractions also yielded products, in which both galactose and 3,6-anhydrogalactose were present in Dand L-configurations (Estevez, Ciancia, & Cerezo 2008). Fractionation of the first hot water extract (C1, Table 7) with KCl gave two insoluble fractions, F1 and F2 (Table 7), in which the 3,6-anhydrogalactose 2-sulfate units were in the D- and L-forms, while the 3,6anhydrogalactose was in the D-form (Estevez, Ciancia, & Cerezo 2004). The KCl-soluble fraction (F3) was treated with alkali and the cyclized derivative was further fractionated with KCl. All the fractions contained D- and L-galactose (Table 8). An insoluble subfraction (F3T1) with 8.6 % sulfate and [α]D= - 114.3º contained 91 % of agaran structures, while other (F3T4) with 19.0% sulfate and [α]D= + 46.3º contained about 50% of each carrageenan and agaran structures; the soluble fraction (F3T6) was mostly agaran (73 %). Considering that F3T2 and F3T4 precipitated with KCl and that a chain of a few carrabiose units is too short for gelation (Rees, Williamson, Frangou, & Morris 1982), the carrabiose units should not be interspersed regularly in the backbone, but grouped. This is in agreement with the block-structure theory (Takano, Hayashi, Hara 1997) and with the isolation of carrageenan and/or agaran fragments from the partial hydrolysis of the polysaccharides from Anatheca dentata (Nunn, Parolis, & Rusell 1971) and Grateloupia divaricata (Cryptonemiales) (Usov & Barbakadze 1978). Table 8. Analysis of the fractions obtained by alkaline treatment of F3 from Gymnogongrus torulosus and further fractionation with KCl1. Frac- Range of Yield3 Sulfate Monosaccharide composition (moles [α]D Mw Carra- Agar6 tion2 precipi- % %) as an6 º KDa geenan tation Gal D-3- L-3- AnGal DSO3K % % M, KCl % D- L- Gal Gal D- L- Xyl F3T1 Ins. 12.9 8.6 43.9 32.1 6.2 - 10.0 7.8 - 114.3 39.7 9 91 F3T2 0.1-0.2 16.7 27.3 41.2 10.3 tr.4 31.2 15.8 1.4 + 33.3 35.0 50 50 F3T4 0.6-1.0 12.5 19.0 55.1 4.6 tr. 19.2 19.8 1.3 + 46.3 45.0 50 50 34.6 16.1 45.9 29.3 4.2 - 8.3 4.2 8.1 - 56.9 22.5 27 73 F3T6 2.05 1 From Estevez, Ciancia, & Cerezo 2001. 2Fractions obtained in small yield are not shown. 3Yields of F3T1-F3T6 are given for 100 g of alkali-treated F3. 4tr.=traces. 5Soluble in 2.0 M KCl. 6 Considering Xyl as contaminant.
F3T1 and F3T6 are composed by similar structural units, although F3T1 is less sulfated, 3-linked non sulfated β-D-galactose and β-D-galactose substituted on C-4 or C-6 (possibly with sulfate on C-4 and with single stubs of β-D-xylose on C-6) or C-2 and C-4, and 4-linked α-L-galactose, partially sulfated on C-3 and, possibly, 3,6-anhydro-α-L-galactose 2-sulfate units. Carposporophytes of G. torulosus also biosynthesize small amounts of L-galactose containing galactans (about 4 % of agaran structures) (Estevez, Ciancia, & Cerezo 2002). Raw water extracts and their alkali-treated derivatives from six species of red algae of the genus Callophycus (Solieriaciae) were studied by Fourier Transform Infrared spectroscopy as well as 1H and 13C NMR spectroscopy without detecting any absorption that could be assigned to agaran structures (Chiovitti, Basic, Craik, Munro, Kraft, & Liao 1997). Also, hot
Marina Ciancia and Alberto S. Cerezo
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water extracts of two seaweeds of the genus Rhabdonia (R. coccinea and R. verticillata) (Solieriaceae) showed 13C NMR spectra without any absorption corresponding to agarans (Chiovitti, Liao, Kraft, Munro, Craik, & Basic 1996), suggesting the necessity of the chemical determination of the absolute configuration of the galactose and derivatives. These are two examples of chemical work carried out with a different aim, where different information is obtained.
c. DL-Galactan Systems from Tetrasporophytes of the Family Gigartinaceae Galactans from tetrasporic plants of the Gigartinaceae are also extracted at room temperature and they are mainly carrageenans of the λ-family. The galactans from tetrasporophytes of I. undulosa (Stortz & Cerezo 1993) and G. skottsbergii (Noseda 1994) gave, by fractionation with KCl, fractions which precipitated at high concentrations of this salt (λ-carrageenans), and a fraction soluble in 2 M KCl. These soluble fractions comprised structures of the λ-family, together with unusual units and L-galactose-containing galactans. By fractionation of the soluble product from I. undulosa, T3 ([α]D= +11.0º, 30% of galactose in the L-form, Table 2) by anion-exchange chromatography (Stortz, Cases, & Cerezo 1997) fractions containing the expected λ-carrageenans, together with mixtures of λ-structures and agarans (or DL-hybrids) and some unusual polysaccharides were isolated (Table 9). These results suggest that there could also be some polysaccharide structure (F10) leaving the usual unit alternancy. Table 9. Analysis of fractions obtained from the soluble tetrasporic carrageenan (T3) from Iridaea undulosa by alkaline treatment and KCl-fractionation1.
1
Fraction
Yield %
Sulfate as SO3Na %
[α]D º
F2 F5 F10 F14 F17 F20 F30A F30B
14.6 6.0 4.8 13.6 3.6 2.0 4.0 6.8
21.1 6.7 15.2 23.3 22.3 29.1 19.9 27.8
- 20.3 + 4.4 - 26.6 - 29.2 - 10.8 + 37.6 + 10.6 + 43.7
From Stortz, Cases, & Cerezo 1997b. 2F2 and F10 respectively.2tr.=traces.
Residues/100 sugar residues D-Gal L-Gal D-Glc SO3Na 49.8 43.0 4.92 85 13.4 41.1 45.5 36 58 36.0 45.1 13.82 53.0 47.0 108 64.8 28.7 6.5 109 93.1 6.9 146 76.2 21.5 2.3 124 100 tr. tr. 162 contain 2.3 and 5.1 % of L-Rham,
The crude tetrasporic carrageenans from G. skottsbergii yielded also a fraction soluble in 2M KCl (T7), its analysis, as well as that of the alkali-treated derivative (T7T1), indicated a D-:L-galactose ratio of 4:1. Carrageenans from the tetrasporic plants of Gigartina lanceata and G. chapmanii have also been studied. 13C NMR spectra of the extracts and of the alkali-treated products gave no evidence of the presence of L-galactose or of its 3,6-anhydro-derivative which, according to the above described results, should be present (Falshaw & Furneaux 1998). These results suggest that this methodology may not be adequate to detect small, but still significant,
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percentages of minor diasteromeric structures, due to its inherent low sensitivity, as well as the low solubility and viscosity of the solutions, and the usual complexity of the spectra of these raw materials (see above).
2. DL-GALACTAN SYSTEMS FROM OTHER RED SEAWEEDS OF THE ORDER GIGARTINALES Members of the family Kallymeniaceae produce complex, highly sulfated, non-gelling, D- and L-galactose containing galactans. Few reports of galactans from seaweeds of the genus Callophyllis informed the presence of carrageenans in C. rhynchocarpa (Usov & Klochkova 1992, Usov, Ivanova, & Shashkov 1983); carrageenans and agarans were found in C. rangiferina (Chopin, Kerin, & Mazerolle 1999) and in C. cristata (Usov & Klochkova 1992). Polysaccharides from C. hombroniana have mainly a θ-carrageenan structure (Fig. 2), minor quantities of λ-carrageenans, and a galactan structure corresponding to a θcarrageenan, desufated on C-2 of the 3,6-anhydrogalactose units. An agaran structure comprising α-L-galactose units was also found in significant amounts (Falshaw, Furneaux, & Stevenson 2005). Carrageenans of the λ/θ/α-type were informed for C. variegata, based on infrared spectroscopy (Chopin, Kerin, & Mazerolle 1999). Further studies on polysaccharides from gametophytic samples of this seaweed extracted with water at room temperature (Rodriguez, Merino, Pujol, Damonte, Cerezo, & Matulewicz 2005) showed only traces of L-galactose or its derivatives. However, fractionation by precipitation with KCl yielded fractions with low positive optical rotations consistent with the presence of carrageenan and agaran structures. Accordingly, low but significant quantities of L-galactose (3,6-anhydrogalactose was always in the D-configuration) were detected (Table 10). F3 (soluble in 2 M KCl) was submitted to alkaline treatment giving, after fractionation of the modified polysaccharides with KCl, a major soluble fraction, F3T3 (Table 10). The agaran moiety in these fractions was constituted by 3-linked β-D-galactose units, mainly sulfated on C-2 and non-sulfated α-L-galactose residues, similar to those found in C. hombroniana (Falshaw, Furneaux, & Stevenson 2005). Other seaweed of the family Kallimeniaceae, Cirrulicarpus gmelinii, was also found to produce DL-hybrid galactans (Usov & Klochkova 1992). Hence, it was reported that the ratio agaran/carrageenan, as well as the major type of polysaccharides, might prove to be a significant chemotaxonomic marker for the genus and others of the family Kallymeneaceae, as there is still some taxonomic controvercy about these seaweeds (Miller 1997, Chopin, Kerin, & Mazerolle 1999).
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Table 10. Analysis of fractions isolated from the raw extract obtained from Callophyllis variegata by KCl-fractionation and of the main fraction obtained from alkali-treated (F3T3) by further fractionation with KCl1. Frac- Range of Yield2 Sulfate tion precipi% as tation SO3K M, KCl %
5 Monosaccharide [α]D Mw Carra- Agaran 3 5 composition % º KDa geenan (moles %) % Gal 3DDD- L- Gal AnGal4 Xyl F1 1.20-1.25 9.7 33.6 64 7 3 15 8 + 10.8 185 84 16 2 F2 1.80-2.00 11.0 40.9 60 10 16 2 + 13.5 356 77 23 73.3 31.6 57 10 2 20 6 + 20.0 95 77 23 F3 2.006 F3T3 2.006 94.0 29.4 59 11 23 7 + 5.7 n.d. 76 24 1 From Rodríguez, Merino, Pujol, Damonte, Cerezo, & Matulewicz 2005. 2Yields are given as percentages of the total recovered for F1-F3 (65.1 %). 3Small quantities of Glc and Man were detected in F1-F3. 4Only 3,6-anhydro-D-galactose was detected by 13C NMR spectroscopy; for F3 and F3T3 this was confirmed by an analytical procedure. 5Considering Xyl, Glc, and Man as contaminants. 6Soluble in 2.0 M KCl.
It was found that Endocladia muricata (Endocladiaceae) produces the expected carrageenans, but also 6 % of agaran (White, Hosford, & Engard 1985). On the other hand, funorans, a mixture of galactans from Gloiopeltis spp. also belonging to the family Endocladiaceae, now included in the order Gigartinales, have been considered sulfated agarans (Lawson, Rees, Stancioff, & Stanley 1973, Penman & Rees 1973), mainly constituted by 6-sulfated agarose and its precursor structure. However, fractionation of the galactans of G. complanata by precipitation with cetylpyridinium and redissolution with 4 M KCl at 4º C, allowed to isolate a highly sulfated fraction in small yield, mostly constituted by D-galactose residues and with a highly positive optical rotation ([α]D = + 64º). By redissolution of the remaining precipitate in the same solution, but at 100º C, an agaran was isolated; this product was further fractionated to give a fraction that precipitated in 0.5 M KCl, mostly 6-sulfated agarose, and a soluble fraction, rich in precursor units (Takano, Hayashi, Hara, & Hirase 1995). Later, a DL-hybrid galactan fraction obtained from another species of the same genus, G. furcata, obtained in a similar way (PS3, [α]D = + 23º), was studied in depth (Takano, Iwane-Sakata, Hayashi, Hara, & Hirase, 1998). By partial acid hydrolysis and fractionation di- and tri-saccharides containing only D-galactose or alternating D- and L-galactose (or their derivatives) were isolated and characterized (Table 11). No evidences of alternating carrageenan and agaran disaccharide units were found. Structural determination by methylation analysis and 13C NMR spectroscopy indicated that the carrageenan moiety was constituted by 3-linked β-D-galactose (4)2,4-disulfated units and 4-linked 3,6-anhydro-α-Dgalactose residues, or their precursor (α-D-galactose 6-sulfate). Some seaweeds of the family Dumontiaceae, like Constantinea rosa-marina, C. subulifera, and Cryptosiphonia woodii, were also found to produce “DL-hybrid galactans” (Chopin, Kerin, & Mazerolle 1999). Sulfated galactans from Schyzimenia binderi are DL-hybrids, with predominance of carrageenan structures devoid of 3,6-anhydrogalactose. Sulfation was found on C-2, C-4, or both positions, on the 3-linked β-D-galactose units and on C-3 on the 4-linked α-galactose residues (Matsuhiro, Conte, Damonte, Kolender, Matulewicz, Mejías, Pujol, & Zúñiga 2005).
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Polysaccharides obtained from other species of this genus had previously been found to be either carrageenans (Whyte, Foreman, & De Wreede 1984) or agarans (Bourgougnon, Lahaye, Quemener, Chermann, Rimbert, Cormaci, Furnari, & Kornprobst 1996). Significant quantities of uronic acids were detected in some cases. Table 11. Disaccharides and trisaccharides obtained by partial acid hydrolysis of PS3 from Gloiopeltis furcata1. β-D-Gal(1→4)L-Gal β-D-Gal(1→4)D-Gal α-L-Gal(1→3)D-Gal α-D-Gal(1→3)D-Gal β-D-Gal(1→4)3,6-anhydro-L-Gal 6-O-Me-β-D-Gal(1→4)D-Gal α-L-Gal(1→3)β-D-Gal(1→4)L-Gal α-D-Gal(1→3)β-D-Gal(1→4)D-Gal β-D-Gal(1→4)α-L-Gal(1→3)D-Gal β-D-Gal(1→4)α-D-Gal(1→3)D-Gal 1 From Takano, Iwane-Sakata, Hayashi, Hara, & Hirase, 1998.
1 2 3 4 5 6 7 8 9 10
3. DL-GALACTAN SYSTEMS FROM RED SEAWEEDS OF THE ORDER HALYMENIALES Algae from the Halymeniales biosynthesize complex sulfated galactans. Most of them have sulfate at C-2 of the β-D-galactose units and they formally belong to carrageenans of the λ-family. The presence of D- and L-galactose residues has been reported in members of different genera of the order Halymeniales, as Grateloupia (Usov, Miroshnikova, & Barbakadze 1975, Usov & Barbakadze 1978, Usov, Yarotsky, & Shashkov 1980, Sen, Das, Sarkar, Suddhanta, Takano, Kamei, & Hara 2002, Wang, Bligh, Shi, Wang, Hu, Crowder, Branford-White, & Vella 2007) Halymenia (Fenoradosoa, T. A., Delattre, C., Laroche, C., Wadouachi, A., Dulong, V., Picton, L., Andriamadio, P., Michaud, P. 2009), Pachymenia, (Farrant, Nunn, & Parolis, 1971, 1972, Parolis, 1978, 1981, Miller, Falshaw, & Furneaux 1995), Phyllymenia (Nunn & Parolis 1969, Parolis 1981) and Aeodes (Allsobrook, Nunn, & Parolis 1971, 1974, 1975). Acetolysis of the polysaccharide of Aeodes ulvoidea (Grateloupiaceae) resulted in the isolation of ten oligosaccharides most of them arising from non-cyclized carrageenans, but one, obtained in small amount (about 0.1%) was 4-O-β-D-galactopyranosyl-2-O-methyl-Lgalactose and other, a tetrasaccharide, isolated in 0.03 % yield ( [α]D = - 60˚), was suggested to have alternating D- and L-galactose residues (Allsobrook, Nunn, & Parolis 1975). Other major characteristics of the galactans from the genus Aeodes were: methylation on C-2 of the α-D-galactose units, substitution of the same units by single stubs of 4-O-methyl-α-Lgalactose residues, and sulfation, mostly on the β-D-galactose units, possibly on C-2, and also on C-6 of some of the α-galactose residues (Nunn & Parolis 1968, Allsobrook, Nunn, & Parolis 1975).
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Cryptonemia crenulata (Halymeniaceae) has been reported to produce ι-carrageenans (Saito & Oliveira 1990). Nevertheless, later preliminary results suggested highly complex polysaccharides (Chopin, Kerin, & Mazerolle 1999). In a more detailed research (Zibetti, Noseda, Cerezo, & Duarte 2005) the seaweed was extracted with water at room temperature (C1) and with a hot phosphate buffer (pH 6.5) (C2), these extracts were purified by precipitation with 2M KCl in which 94% of C1 and 97% of C2 remained soluble (C1S and C2S, Table 12). C1S and C2S were fractionated by ion exchange chromatography to give subfractions C1S-1 – C1S-3 and C2S-1 – C2S-4, respectively. C2S-2 was refractionated on the same column giving C2S-2a – C2S-2d. In all the fractions, when enantiomeric analysis was carried out, the D- and L-forms of galactose, 3,6-anhydrogalactose and 2-Omethylgalactose, together with the L-form of the 3,6-anhydro-2-O-methylgalactose were found. The percentage of carrageenan structures was 28-38 %, while that of agaran structures was 62-72%. Table 12. Analysis of some of the galactans obtained from Cryptonemia crenulata1. Fraction Yield2 Sulfate Pyruvic Monosaccharide units3 [α]D % as (moles %) acid º SO3Na % G+D:L DA:LA LA2M D2M:L2M Xyl % C1S4 3.76 26.0 7.9 1:0.17 0.18:0.04 0.14 0.10:0.02 0.17 + 25.5 C1S-1 9.6 22.3 n.d.5 1 0.21 0.04 0.08 0.16 + 14.0 C1S-2 18.0 26.5 2.2 1 0.02 0.07 0.05 + 12.0 C1S-3 56.0 16.0 0.7 1 0.15 0.09 0.11 0.18 + 12.0 C2S4 6.63 27.7 3.7 1:0.17 0.04:0.14 0.11 0.07:0.02 0.06 + 27.0 C2S-1 3.2 20.1 n.d. 1 0.12:0.01 0.10 0.10 0.05 + 11.0 C2S-2 24.0 25.1 1.9 1 0.06:0.04 0.10 0.08 0.14 + 6.5 C2S-3 54.2 28.3 0.5 1:0.09 0.09:0.20 0.14 0.05:0.02 0.05 + 23.0 C2S-4 3.3 17.5 n.d. 1 0.09:0.06 0.13 0.03 + 4.0 C2S-2c 25.0 14.0 4.5 1 0.14 0.15 0.09 n.d. C2S-2d 55.0 20.4 0.4 1:0.11 0.01 0.09 0.05:0.03 n.d. 1 From Zibetti, Noseda, Cerezo, & Duarte 2005. 2Yields of C1S and C2S are given per 100 g of dry seaweed, yields of fractions are given per 100 g of the parent compound. 3Knutsen´s nomenclature. G+D=β-D- plus α-D-galactose and L=α-L-galactose. 4Soluble in 2.0 M KCl. n.d.=not determined.
4. DL-GALACTAN SYSTEMS FROM AGAROPHYTES OF THE ORDER CERAMIALES All the representatives of the Ceramiales studied contained agarans with a wide range of substituents. The first time that a carrageenan structure, even though in small quantity, was identified in the polysaccharide system from an algae belonging to this order was in a fraction of the extract from the red seaweed Rhodomela larix (Takano, Yakoi, Kamei, Hara, & Hirase 1999), previously considered as producing highly methylated agarans (Usov & Ivanova 1975). However, partial hydrolysis of a methanolysis-resistant moiety obtained from the main polysaccharide fraction from this seaweed afforded sets of the disaccharides [β-DGal(1→4)L-Gal, β-D-Gal(1→4)D-Gal, α-L-Gal(1→3)D-Gal, α-D-Gal(1→3)D-Gal] and
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trisaccharides [α-L-Gal(1→3)β-D-Gal(1→4)L-Gal, α-D-Gal(1→3)D-Gal(1→4)D-Gal, β-DGal(1→4)α-L-Gal(1→3)D-Gal, β-D-Gal(1→4)α-D-Gal(1→3)D-Gal], indicating that both agaran and carrageenan cooccur, even if the latter structures are small in quantity. Structural analysis showed that the 4-linked α-galactose units were partially methylated on C-2 and sulfate groups were on C-6 of these units, C-2 of the cyclized derivatives, and on C-3 of the α-D-galactose units. The 3-linked galactose units were partially sulfated on C-6 (Takano, Yakoi, Kamei, Hara, & Hirase 1999). Fractionation of the galactans from the agarophyte from Digenea simplex (Rhodomelaceae) by ion-exchange chromatography led to the isolation of a sulfated product, which afforded, when subjected to partial methanolysis, disaccharide derivatives characteristic of agaran and carrageenan structures. The substitution pattern was similar to that of R. larix, although some 3-linked galactose units were sulfated on C-4 (Takano, Shiomoto, Kamei, Hara, & Hirase 2003). Both, R. larix and D. simplex have been considered as potential sources of agar or agarose (Chiovitti, Liao, Kraft, Munro, Craik, & Basic 1996). On the other hand, the sulfated galactan systems from other Rhodomelaceae, i.e. Bostrychia montagnei (Duarte, Noseda, Cardoso, Tulio, & Cerezo 2002), Polysiphonia nigrescens (Prado, Ciancia, & Matulewicz 2008), Acanthophora spicifera (Duarte, Cauduro, Noseda, Noseda, Gonçalves, Pujol, Damonte, & Cerezo 2004) showed to be composed by agarans with different substitution patterns, but, in spite of the fact that extensive fractionation procedures were carried out, results did not show the existence of carrageenan structures in significant quantities.
5. DL-GALACTAN SYSTEMS FROM AGAROPHYTES OF THE ORDER RHODYMENIALES Sulfated galactans were isolated from the red seaweed Lomentaria catenata (Takano, Nose, Hayashi, Hara, & Hirase 1994) by precipitation with cetylpyridinium salt. Redissolution with KCl solutions allowed to isolate a product that, by acid hydrolysis and fractionation gave the oligosaccharides indicated in Table 13. All these oligosaccharides had carrageenan or agaran structures, but in no case a mixed product was found. The ratio of agarose- to carrageenan-chains was estimated to be 1:0.56 for this fraction. A particular structural feature of these polysaccharides was the presence of D-glucose and D-glucuronic acid side chains, which were deduced to be linked as single stubs to C-3 of the 4-linked Dgalactose units and to C-4 of the 3-linked D-galactose units, respectively in the carrageenan backbone. Although these polysaccharides were highly sulfated, the sulfation pattern was not analyzed. On the other hand, galactans from Champia novae-zelandiae are also agar-rich DLhybrids, being mainly sulfated on C-2 of both alternating units, as well as on C-3 of the 4linked residues (Miller, Falshaw, & Furneaux 1996). Extracts obtained from samples of Hymenocladia sanguinea from different locations and life stages were found to have essentially equivalent polysaccharides by 13C NMR spectroscopy. They contained 3-linked βD-galactose units sulfated on C-2; the B-units (D-galactose, L-galactose and 3,6-anhydro-Lgalactose, in ratio 0.57:0.38:0.05) were mainly 4-linked D-galactose 3-sulfate and 2,3-
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disulfate, together with L-galactose units, sulfated in part on C-6 (Miller 2001), showing important substitution similarities with polysaccharides from C. novae-zelandiae. Table 13. Neutral products obtained by partial acid hydrolysis of PS-2 from Lomentaria catenata1,2. Isolated fraction DP1 PD2a DP2b DP2e DP2d DP2c
Product identified Relative yield 1 Gal+Xyl+Glc 63.3 15.8 2 β-D-Gal(1→4)L-Gal 4.3 3 β-D-Gal(1→4)D-Gal 1.9 4 α-L-Gal(1→3)D-Gal 0.5 5 α-D-Gal(1→3)D-Gal 1.9 6 -D-Glc(1→3)D-Gal 3.4 7 α-L-Gal(1→3)β-D-Gal(1→4)L-Gal DP3a3 0.5 8 α-D-Gal(1→3)β-D-Gal(1→4)D-Gal DP3b-1 2.4 9 β-D-Gal(1→4)α-L-Gal(1→3)D-Gal 0.3 DP3b-24 10 β-D-Gal(1→4)α-D-Gal(1→3)D-Gal DP4a 2.1 11 β-D-Gal(1→4)α-L-Gal(1→3) β-D-Gal(1→4)L-Gal DP4b 12 Unidentified tetrasaccharide(s) 1.8 13 Higher oligosaccharides 3.6 1 From Takano, Nose, Hayashi, Hara & Hirase 1994. 2Two acidic oligosaccharides were identified as: D-GlcA(β1→4)D-Gal and D-GlcA(1→4)D-Gal(1→4)D-Gal. 3Obtained as a mixture of trisaccharides 7 and 8. 4Obtained as a mixture with trisaccharide 9.
6. DL-GALACTAN SYSTEMS FROM SEAWEEDS OF THE ORDER BONNEMAISONIALES Galactans from gametic, carposporic and tetrasporic stages of red seaweed Asparagopsis armata (Bonnemaisoniaceae) are mainly non-cyclized carrageenans with heterogeneous sulfation pattern and certain degree of branching. In addition, agarans were also detected, as well as small amounts of uronic acids, only in significant quantities (15.9 %) in the carposporophytes (Haslin, Lahaye, & Pellegrini 2000).
7. DL-GALACTAN SYSTEMS DETECTED IN TYPICAL AGAROPHYTES Galactans biosynthesized by seaweeds of the genus Porphyra (Bangiales) are usually known as porphyrans. They are agarans whose main structural characteristic is the presence of important amounts of precursor units (α-L-galactose 6-sulfate) (Morrice, McLean, Long, & Williamson 1983), which by alkaline treatment give 3,6-anhydro-L-galatose; in this way, an agarose structure is obtained from porphyran (Fig. 2). The other structural characteristic of these polysaccharides is a certain degree of methoxylation on C-6 of the β-D-galactose units. Unexpectedly, the presence of a small quantity (3 %) of α-D-galactose 6-sulfate units was detected in the crude extract from Porphyra columbina, indicating the presence of a carrageenan structure (Navarro & Stortz 2003).
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After extensive fractionation procedures of the room temperature and 50 ºC water extracts from Pterocladiella capillacea, small amounts of galactans containing 4-linked α-Dgalactose substituted on C-3 possibly with sulfate, were found, showing the presence of DLhybrid galactans for the first time in a seaweed of the order Gelidiales (Errea & Matulewicz 2003).
DEVIATION OF THE STRUCTURE OF CARRAGEENANS (OR CARRAGEENAN BLOCKS) AND AGARANS (OR AGARAN BLOCKS) IN THE DL-GALACTAN SYSTEMS Structure of the backbone. The presence of unusual units, as well as that of branches of xylose, galactose and/or glucose, in carrageenan and agaran molecules, which are supposed to be lineal, suggested that the structure of carrageenan blocks (or molecules) and/or agaran blocks (or molecules) in DL-galactan systems may be somewhat different to that of the “classical” carrageenans or agarans (Zibetti, Noseda, Cerezo, & Duarte 2005). Nevertheless, it should be considered that: a) some of the samples, in which the unusual units or the side chains appear have been found after extensive fractionation and in very low yield, and they cannot be considered as representative of the galactan system and b) no enantiomeric analysis on the partially methylated galactoses from methylation analysis were carried out in the earlier research of galactans from typical carrageenophytes or agarophytes, difficulting the comparison with more recent studies on other seaweeds. A fraction (Fs) from the galactans of cystocarpic G. skottsbergii (Ciancia, Matulewicz, Cerezo 1993) showed major amounts of β-D-galactose 4-sulfate units and minor amounts of β-D-galactose 2-sulfate units, these are linked to the usual α-D-units in carrageenans, but also to non-substituted α-L-galactose residues. 3-linked 6-sulfated β-D-galactose units were found in the agaran moiety of the galactans from cystocarpic (Flores, Cerezo, & Stortz 2002) and tetrasporic stages of I. undulosa, together with 4-linked 3-substituted galactose residues (Stortz, Cases, & Cerezo 1997b), a pattern common to corallinans (Cases, Stortz, & Cerezo 1994) and other red seaweeds galactans obtained from several species of the Halymeniales (Miller, Falshaw, & Furneaux 1995, Miller, Falshaw, Furneaux, & Hemmingson 1997). The agaran moiety of tetrasporic G. skottsbergii contains, as above, 3-linked 6-substituted β-Dgalactose units and 4-linked 3-substituted α-L-galactose residues, but in smaller amounts (Noseda 1994). Galactans extracted with water at room temperature from K. alvarezii have a basic κcarrageenan pattern, with small amounts of unusual residues, as non-sulfated 3-linked β-Dgalactose and its 2,4- and 4,6-disulfated forms and important amounts of 6-O-methyl β-Dgalactose (4-sulfate) in the polysaccharide backbone. Agarans are shown by the presence of 3-linked 2- and 6-substituted galactose residues and 4-linked non-substituted and 3substituted α-L-galactose units (Estevez, Ciancia, & Cerezo 2000). Those extracted with hot water are partially substituted on C-2 or C-4, or disubstituted in both positions of the β-Dgalactose units, and on C-3 or C-2 and C-3 of the α-L-galactose residues with sulfate groups or single stubs of β-D-xylopyranose, D-glucopyranose and galactose or with Dglucopyranosyl-(1→4)-D-glucopyranose side chains. Significant quantitites of 2-O-methyland 3-O-methyl-L-galactose units were also present (Estevez, Ciancia, & Cerezo 2004).
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Substitution on C-2 and C-3 of the 4-linked α-L-galactose units was also found in the agarrich DL-hybrids, Champia novae-zelandiae (Miller, Falshaw, & Furneaux 1996). Agarans that retrograded during fractionation of the hot-water extracts of K. alvarezii (F3i) or after alkaline treatment of the soluble fraction (T1), showed great structural dispersion, as the 3-linked β-D-galactose units are substituted in part on C-4 or C-2, or disubtituted on C-2 and C-4. The major B-units are 4-linked α-L-galactose substituted on C-3, nonsubstituted L-galactose, D/L-galactose substituted on C-2 and C-3 and 3,6-anhydro-Land D-galactose and their derivative substituted on C-2. Agaran structures from G. torulosus have small amounts of β-D-galactose units nonsubstituted, sulfated on C-2, and substituted on C-6 with single stubs of xylose, together with small percentages of α-L-galactose 3-sulfate. Those extracted with hot water comprised 3sulfated α-L-galactose units and 2-sulfated 3,6-anhydro-α-L-galactopyranose residues in the agaran moieties. A small amount of β-D-galactose substituted on C-6 with stubs of β-Dxylose was found in both extracts. The presence of alkali-stable L-galactose 3-sulfate units, xylosyl side chains and traces of 3-O-methylgalatose were previously found in the galactans extracted from Anatheca dentata (Nunn, Parolis, & Rusell 1971, 1981). Galactans of C. variegata are mainly cyclized λ-carrageenans (θ-carrageenans), together with highly sulfated units, but they also comprise small amounts of non-substituted β-Dgalactose units, linked to 2,3-disulfated and non-substituted α-L-galactose residues (Rodriguez, Merino, Pujol, Damonte, Cerezo, & Matulewicz 2005). These are some examples, but similar structures were reported for other carrageenophytes. On the other hand, the carrageenan blocks of galactans from the agarophyte Cryptonemia crenulata are constituted by the major quantities of β-D-galactose, partially sulfated on C-2, linked to α-D-galactose 6-sulfate and their cyclized derivative, in the carrageenan- as well as in the agaran-moiety. The A-units contained also non-substituted, 2,6-disulfated and 4,6pyruvic acid ketal substitution. The agaran B-units appeared substituted on C-6 by single stubs of xylose and galactose and methylated on C-2 (Zibetti, Noseda, Cerezo, & Duarte 2005). 4-Linked α-D-galactose units substituted on C-3 were found in the carrageenan moiety from the agarophyte Pterocladiella capillacea (Errea & Matulewicz 2003). In summary, the unusual A-units are characterized by different sulfation patterns (nonsulfated or sulfated on C2 or C6), by higher sulfation (sulfated on 2,4- or 2,6-), and by methylation or branching (mainly single stubs of xylose on C-6). Unusual B-units are unsubstituted, 3-substituted with sulfate, xylose or galactose and 2,3-disulfated or substituted by sulfate and single stubs of xylose in the same way; methyl groups appear on C-2 or C-3 of the α-L-galactose residues. All these “unusual” structural units do not appear in carrageenans and/or agarans from “classical” carrageenophytes or agarophytes, but they are often found in carrageenan-like or agaran-like galactans from other seaweeds (i.e. Halymeniales, Ceramiales, Rhodimeniales). Counterions. Ca2+ showed high affinity for sulfated L-galactans from different ascidian species, and its concentration increased with increasing amounts of nonsulfated sugar branches. This affinity also increased as the mean distance between charged groups decreased, suggesting that Ca2+ binding requires more than one sulfate group per each calcium atom (Ruggiero, Fossey, Santos, & Mourao 1998). Similar results were reported for
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glycosaminoglycans; their sulfate groups are capable of binding Ca2+ with stronger affinity than that expected from simple salt formation (Hunter, Wong, & Kim 1988). The raw extract from K. alvarezii contains major amounts of divalent (Ca2+ and Mg2+) cations in agreement with the known capacity of the seaweeds to concentrate these salts (Kloareg & Quatrano 1988). These counterions were maintained in high percentages in fractions that were obtained through fractionation procedures involving contact with massive amounts of potassium chloride or hot and concentrated sodium hydroxide solutions, or sequences of both. It is worth noting that permethylation of the polysaccharides involved as first step the preparation of the triethylammonium salts by ion-exchange chromatography, nevertheless the methylated derivative contains as much inorganic cations as the parent polysaccharide. Similar difficulties to exchange the divalent counterions were found in a commercial sample of a κ-carrageenan from the same seaweed, which originally contained about 40 % of Ca2+. After cation-exchange chromatography at 80º C on a sodium preregenerated resin, it still showed about 15 % of Ca2+ (Chen, Liao, & Dunstan 2002). Consequently, Ca2+ binding in these polymers is not a simple function of availability of anion binding sites, but a more complex Ca2+-polysaccharide interaction (Chandrasekaran, Radha, & Lee 1994, Tibbits, MacDougall, & Ring 1998). Side chains. “Classical” carrageenans and agarans are linear molecules with no branching. Nevertheless, xylose, glucose and galactose have been found as single stubs or short chains in several agarans (Kolender & Matulewicz 2002, Duarte, Noseda, Cardoso, Tulio, & Cerezo 2002, Usov, Bilan, & Shashkov 1997, Cases, Stortz, & Cerezo 1994) or in the agarans (or agaran blocks) of DL-galactan systems (Ciancia, Matulewicz, & Cerezo 1993, Estevez, Ciancia, & Cerezo 2001, Takano, Shiomato, Kamei, Hara, & Hirase 2003, Estevez, Ciancia, & Cerezo 2000). Terminal galactose units were found in the soluble fraction of tetrasporic (Stortz, Cases, & Cerezo 1997b) and cystocarpic (Fs) galactans of G. skottsbergii and I. undulosa. In Fs from cystocarpic G. skottsbergii alternating 3- and 4-linked side chains and single stubs of xylose were also found (Ciancia, Matulewicz, & Cerezo 1993). Several fractions of the DL-galactan system of K. alvarezii (Table 6) contain small-tosignificant amounts of xylose, glucose and galactose as short chains or single stubs, linked to C-2 and/or C-4 of the 3-linked β-D-galactose and C-3 and/or C-2 and C-3 of the 4-linked αD- or α-L-galactose residues. In the galactan system of Bostrychia montagnei (Ceramiales), terminal β-D-xylose units are linked to C-6 of the β-D-galactose units and C-3 of the α-galactose residues (Duarte, Noseda, Cardoso, Tulio, & Cerezo 2002). They have also been found linked to C-6 of the βD-galactose units in the “corallinans” (Usov, Bilan, & Shashkov 1997, Cases, Stortz, & Cerezo 1994). On the contrary, the system of Cryptonemia crenulata showed no branching in the β-D-galactose units, but the α-galactose residues carry single stubs of β-D-galactose and β-D-xylose (Zibetti, Noseda, Cerezo, & Duarte 2005).
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ISOLATION OF “PURE” CARRAGEENANS AND/OR “PURE” AGARANS FROM DL-GALACTAN SYSTEMS DL-galactan systems have never been totally resolved into carrageenans and agarans. Nevertheless, the use of solutions of high ionic strengh (KCl solutions), alkaline solutions or highly chaotropic media (LiCl/DMSO) at high temperature allowed isolation of “pure” carrageenans or agarans. Furthermore, carrageenan or agaran structures, sometimes to percentages as high as 95 %, have been obtained by spontaneous insolubilization or by ionexchange chromatography in systems where the amounts of sulfate in both types of structures were quite different. Alkaline treatment of the raw galactan obtained from K. alvarezii (C) and further dialysis in the same alkaline medium gave a “pure” kappa/iota-carrageenan (C’) (Estevez, Ciancia, & Cerezo 2004). Similar treatment of fractions A1 (Estevez, Ciancia, & Cerezo 2008) and C1 (Estevez, Ciancia, & Cerezo 2004) from G. torulosus also produced “pure” kappa/iotacarrageenan (Tables 6 and 7), suggesting that the kappa/iota-carrageenans biosynthesized by both seaweeds were forming complexes with agarans. In addition, during fractionation of the raw extract obtained with hot water of K. alvarezii some products retrograded spontaneously (F3i), or after alkaline treatment of the soluble fraction F3s (T1) (Estevez, Ciancia, & Cerezo 2004). These insoluble products were almost “pure” agarans (∼ 90 %) and had characteristics similar to those of the insoluble fraction obtained in comparable situation from the alkali-treated, KCl-soluble fraction of G. torulosus (F3T1), suggesting that these agarans are usual minor components of the polysaccharide system of the carrageenophytes. The above mentioned alkali-treated product from K. alvarezii gave, after precipitation of T1, two gelling fractions and a soluble one (T4), which is mostly an agaran with a substitution pattern similar to that of T1 and F3i. (Estevez, Ciancia, & Cerezo 2004). Ion-exchange chromatography on Sephadex DEAE A-25 of T4 gave six fractions eluted according their sulfate content. Even though 3,6-anhydrogalactose was not resolved into the D- and L-enantiomers in this work, all the data suggest that the first fraction (FI , 35.0 %, sulfate content, 5.8 %) was in ∼ 97 % an agaran, while the last one (FVI, 8%, sulfate content 27.8 %) was in ∼ 70-90 % a carrageenan. The fibrillar material from G. torulosus (RC4) contained major amounts of (glyco)proteins, galactans and cellulose, together with small amounts of xylose and mannose. Small amounts of L-galactose, together with traces of 3,6-anhydro-L-galactose units were detected. Extraction of RC4 with LiCl/DMSO at high temperature solubilized 34 % of the cell wall, from which only 16.5 % was isolated after dialysis. This product was a kappa/iotacarrageenan with a molecular weight of ∼ 11 kDa. Thus, about half of the extracted material was lost during dialysis in the chaotropic medium including the possible L-galactose containing polysaccharides. This result could be attributed to their low molecular weight (Estevez, Ciancia, & Cerezo 2008). It is not known whether these low Mw fragments are real or they are artifacts produced during the extraction procedure, even thought LiCl/DMSO reagent was reported not to cleave covalent linkages (Petrus, Gray, & BeMiller 1995).
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FORMATION OF CARRAGEENAN, AGARAN AND CARRAGEENAN/AGARAN COMPLEXES In early investigations, it had been suggested (Pernas, Smidsrod, Larsen, & Haug 1967) that the solubility behaviour of carrageenan molecules in mixtures was the same as that in homogeneous fractions. In other words, that there were no intermolecular interactions and therefore, the KCl-precipitation range should define the chemical structure of the carrageenan. Later data demonstrated that carrageenan molecules associate forming transients, non-stequiometric complexes, and the solubility of these complexes proved to be different from that of “pure” fractions (Stortz & Cerezo 1988, Ciancia, Matulewicz, & Cerezo 1993, 1995). Thus, three fractions of the carrageenan from unsorted I. undulosa precipitated at very sharp concentration ranges of KCl (1.20-1.25; 1.35-140; 1.55-1.65 M), nevertheless, they were mixtures of μ/ν- and λ-carrageenans. Both types of molecules, which isolated differ in solubility (μ/ν-carrageenans are totally soluble in 2M KCl, while λ-carrageenans precipitate at high concentrations of this salt), but have similar structures, differing only in the position of sulfation of the 3-linked units (Fig. 2), could form some type of aggregates (Stortz & Cerezo 1988). The effects of complexation can be seen comparing yields, precipitation ranges, and analysis of the fractions obtained from cystocarpic and tetrasporic plants of I. undulosa (Stortz & Cerezo 1993) with those obtained in the same way from unsorted samples (Matulewicz & Cerezo 1980) of the same seaweed. Taking into account the amount of κ/ιstructure, the carrageenan system from the unsorted sample seems to be composed of a mixture of about two thirds of cystocarpic carrageenans and one third of tetrasporic carragenans. However, these carrageenans precipitated at higher KCl concentrations than those observed for samples separated according to the stage of the life-cycle, 0.75-1.05 M for κ/ι-structures in mixed samples vs. 0.50-0.70 M for those of isolated cystocarpic plants, and 1.20-1.65 for λ-carrageenan in mixed systems vs. 1.00-1.20 M for the tetrasporic samples. Another concern is that the potassium chloride-soluble material in the mixture is much less than that accounted for in the sorted samples, suggesting that, in the first case, part of the soluble structures had been precipitated with the KCl-insoluble fractions (Stortz & Cerezo 1993). Alkaline treatment of a partially cyclized μ/ν-carrageenan from cystocarpic G. skottsbergii and further fractionation of the alkali-treated derivative with potassium chloride yielded a fraction soluble in 2M KCl with negative rotation (Fs). Enantiomeric analysis showed the presence of about 88% of agarans. These agarans were present together with carrageenans through the extraction and all the fractionation and alkaline treatment steps and are only evident after an elaborate fraccionation procedure (Ciancia, Matulewicz, & Cerezo 1993). Comparison of the KCl-fractionations for alkali-modified derivatives of the above mentioned partially cyclized μ/ν-carrageenan obtained in three different batches showed different precipitation patterns, the corresponding recoveries were low and yields of the fractions and arbitrary subfractions, as well as their composition were different. The low recoveries suggest the presence in the alkali-treated derivatives of low molecular weight products forming composition-dependent complexes, which are dissociated during the
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fractionation with potassium chloride, and lost in the subsequent dialysis (Ciancia, Matulewicz, & Cerezo 1995). Hence, the variability of the results appears to be associated with temperature- , time- , and composition of molecular associations (Manzi, Mazzini, & Cerezo 1984, Blake, Murphy, & Richards 1971, McCleary, Amado, Waibel, & Neukon 1981). The product (C) extracted with hot water from K. alvarezii, after separation the fraction obtained at room temperature, showed clear differences with a commercial sample of κcarrageenan (K) obtained from the same seaweed (Table 6), namely: (a) it was soluble in 75 % iso-propanol; (b) it had much lower number-average molecular weight ; (c) it contained significant amounts of L-galactose and 3,6-anhydro-L-galactose units; (d) it also contained higher quantities of xylose and glucose and (e) it contained 77.9 % of divalent ions (Ca2+ and Mg2+), against 35.3 % in the commercial κ-carrageenan (Estevez, Ciancia, & Cerezo 2004). The most significant preparative difference between this sample (C) and the commercial κcarrageenan, was that the latter was extracted from a seaweed previously treated with alkali (Glicksman, M. 1993). On this basis, C was submitted to a short, non-degrading, alkaline treatment producing C´ (Table 6), which was similar to the commercial sample. The changes C → C´ were explained in terms of complexes of κ-carrageenans and small fragments of agarans formed, possibly through Ca2+ bridging two sulfate groups from different molecules. This arrangement would be stabilized by further complexation of the cation (Estevez, Ciancia, & Cerezo 2004). These complexes were broken, at least partially, by the alkaline treatment, and the agaran fragments were lost during alkaline dialysis. This scheme of aggregation of small agaran molecules with higher molecular weight carrageenans would be a general phenomenon between the galactan sulfates extracted from carrageenophytes belonging to the Phyllophoraceae and Solieriaceae, and it could be generalized to other carrageenophytes from the Gigartinales.
CONCLUSION DL-Galactan Hybrids or Molecular Complexes? The presence of both, carrageenan and agaran, structures in galactan systems of red seaweeds is not exceptional but a general fact, showing that red marine algae biosynthesize what has been called a DL-galactan system of polysaccharides. No neat separation of these systems into “pure” agaran and carrageenan polymers has been obtained, however, no proof of D/L-hybrid galactan structures has been found. Considering the fact that partial hydrolysis has up-to-date produced only agaran or carrageenan fragments, the possibility of carrageenan and agaran mixed diads in the “hybrid” molecule (Fig 3) is very small and so the agaran-carrageenan domains (if they exist) should correspond to juntion zones of a block copolymer. It is not actually known whether the above ‘block DL-hybrid galactan’ hypothesis is correct or whether the polysaccharide extracts constitute a mixture of carrageenan-type and agaran-type molecules. Attempts to fractionate of the raw extracts and/or different fractions or subfractions obtained by KCl precipitation, ion-exchange or gel-permeation chromatography, as well as by several other fractionation methodologies showed the
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complexity of the systems (Lechat, Amat, Mazoyer, Buleon, & Lahaye 2000), but failed to produce a neat carrageenan/agaran separation. Whether this is a proof of the existence of DLhybrid galactan molecules, or only shows the failure of present-day techniques to separate mixtures of ‘diastereoisomeric’ polysaccharides, is not known, but if the classical definition of homogeneity (Aspinall 1982) is applied, these products should be considered as DL-hybrid galactans until a successful fractionation demonstrates the contrary. No matter whether DL-hybrid galactans or DL-galactan mixtures are considered, the DLgalactose variation should be visualized as another form of structural dispersion in the family of red seaweed galactans, in which the existence of only carrageenans and agarans would be just representatives of extreme structure systems.
ACKNOWLEDGMENTS This work was supported by grants from CONICET (PIP 5699) and the University of Buenos Aires (X016 and G048).
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Eucheuma uncinatum, Aghardhiella tenera, Pachimenia hymantophora, and Gloiopeltis cervicornis. Journal of the Chemical Journal of the Chemical Society, Perkin I, 21772181. Lechat, H., Amat, M., Mazoyer, J., Buleon, A., & Lahaye, M. (2000). Structure and distribution of glucomannan and sulfated glucan in the cell walls of the red alga Kappaphycus alvarezii (Gigartinales, Rhodophyta). Journal of Phycology, 36, 891-902. Leontein, K., Lindberg, B., & Lőnngren, J. (1978). Assignment of absolute configuration of sugars by g.l.c. of their acetylated glycosides formed from chiral alcohols. Carbohydrate Research, 62, 359-362. Little, M. R. (1982). Separation, by G.L.C., of enantiomeric sugars as diastereoisomeric dithioacetals Carbohydrate Research, 105, 1-8. Lindqvist, L. & Jansson, P. E. (1997). Determination of the absolute configuration of sugar residues using gas chromatography: Method with potential for elimination of references. Journal of Chromatography A, 769, 253-261. Manzi, A., Mazzini, M. N., & Cerezo, A.S. (1984). The galactomannan system from the endosperm of the seed of Gleditsia triacanthos. Carbohydrate Research, 125, 127-143. Matsuhiro, B., Conte, A.F., Damonte, E.B., Kolender, A.A., Matulewicz, M.C., Mejías, E.G., Pujol, C.A., Zúñiga, E.A. (2005). Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta). Carbohydrate Research, 340, 2392-2402. Matulewicz, M. C., & Cerezo, A. S. (1980). The carrageenan from Iridaea undulosa B.; Analysis, fractionation and alkaline treatment. Journal of the Science of Food and Agriculture, 31, 203-213. McCandless, E. L., West, J. A., & Guiry, M. D. (1982). Carrageenan patterns in Phyllophoraceae. Biochemical Systematics and Biology, 10, 275-284. McCandless, E. L., Okada, W. T., Lott, J. N. A., Volmmer, C. M., & Gordon-Mills, E. M. (1977). Structural studies of Chondrus crispus: the effect of extraction of carrageenan. Canadian Journal of Botany, 55, 2053-2064. McCleary B.V., Amado R., Waibel R., & Neukon H. (1981). Effect of galactose content on the solution and interaction properties of guar and carob galactomannans. Carbohydrate Research, 92, 269-285. Miller, I.J. (1997). The chemotaxonomic sugnificance of the water-soluble red algal polysaccharides. Recent Research Developments in Phytochemistry, 1, 531-537. Miller, I. J. (2001). The structure of the polysaccharides from Hymenocladia sanguinea through 13C NMR spectroscopy. Botanica Marina, 44, 245-251. Miller, I. J., Falshaw, R., & Furneaux, R. H. (1995). Structural analysis of the polysaccharide from Pachymenia lusoria (Cryptonemiaceae, Rhodophyta). Carbohydrate Research 268, 219–232. Miller, I. J., Falshaw, R., & Furneaux, R. H. (1996). A polysaccharide fraction from the Red Seaweed Champia novae-zelandiae Rhodimeniales, Rhodophyta. Hydrobiologia, 326/327, 505-509. Miller I.J., Falshaw R.H., Furneaux R.H., & Hemmingson J.A. (1997). Variation in the constituent sugars of the Polysaccharides from New Zealand Species of Pachimenia (Halymeniaceae). Botanica Marina, 40, 119-127.
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Morrice, L. M., McLean, M. W., Long, W. F., & Williamson, F. B. (1983). Porphyran primary structure. An Investigation using β-agarase I from Pseudomonas atlantica and 13 C NMR spectroscopy. European Journal of Biochemistry, 133, 673-684. Navarro, D. A. & Stortz, C. A. (2003). Determination of the configuration of 3,6anhydrogalactose and cyclizable α-galactose 6-sulfate units in red seaweed galactans. Carbohydrate Research, 338, 2111-2118. Nishida, Y., Bai, C., Ohrui, H., & Meguro, H. (1994). A highly sensitive method to identify the D, L-configurations of monosaccharides based on (-)-TBMB carboxilic acid and HPLC. Journal of Carbohydrate Chemistry, 13, 1003-1008. Noseda, M. D. (1994). Polissacarídeos sulfatados da fase tetrasporofítica de Gigartina skottsbergii (Rhodophyta, Gigartinales). Ph. D. Thesis, Universidade Federal do Paraná, Curitiba, Brasil. Nunn, J. R. & Parolis, H. (1968). A polysaccharide from Aeodes orbitosa. Carbohydrate Research, 6, 1-11. Nunn, J. R. & Parolis, H. (1969). Sulphated polysaccharides of the grateloupiaceae family : Part III. A polysaccharide from Phyllymenia cornea. Carbohydrate Research, 9, 265-276. Nunn, J. R., Parolis, H., & Russell, I. (1971). Sulphated polysaccharides of the Solieriaceae family. Part I. Polysaccharide from Anatheca dentata. Carbohydrate Research, 20, 205215. Nunn, J. R., Parolis, H., & Russell, I. (1981). Desulphated polysaccharide of Anatheca dentata. Carbohydrate Research, 95, 219-226. Oliveira, M. C. & Battacharya, D. (2000). Phylogeny of the Bangiophyceae (Rhodophyta) and the secondary endosymbiotic origin of algal plastids. American Journal of Botany, 87, 482–492. Parolis, H. (1978). The structure of the polysaccharide of Pachymenia carnosa. Carbohydrate Research, 62, 313-320. Parolis, H. (1981). The polysaccharides of Phyllymenia hieroglyphica (≡ P. belangeri) and Pachymenia hymantophora. Carbohydrate Research, 93, 261-267. Penman A.& Rees D.A. (1973). Carrageenans. Part IX. Methylation analysis of galactan sulphates from Furcellaria fastigiata, Gigartina canaliculata, Gigartina chamissoi, Ga atropurpurea, Ahnfeltia durvillaei, Gymnogongrus furcellatus, Eucheuma cottonii, Eucheuma spinosum, Eucheuma isiforme, Eucheuma uncinatum, Aghardhiella tenera, Pachimenia hymantophora, and Gloiopeltis cervicornis. Structure of ξ-carrageenan. Journal of the Chemical Society, Perkin I, 2182-2187. Pernas, A. J., Smidsrod, O., Larsen, B., & Haug, A. (1967). Chemical Heterogeneity of Carrageenans as Shown by Fractional Precipitation with Potassium Chloride. Acta Chemica Scandinavica, 21, 98-110. Petrus, L., Gray, D. G., & BeMiller, J. N. (1995). Homogeneous alkylation of cellulose in lithium chloride/dimethyl sulfoxide solvent with dimsyl sodium activation. A proposal for the mechanism of cellulose dissolution in LiCl/Me2SO. Carbohydrate Research, 268, 319-323. Prado, H. J., Ciancia, M. & Matulewicz, M. C. (2008). Agarans from the red seaweed Polysiphonia nigrescens (Rhodomelaceae, Ceramiales). Carbohydrate Research, 343, 711-718.
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Rees, D. A., Williamson, F.,B., Frangou, S. A., & Morris, E.R. (1982). Fragmentation and Modification of r-Carrageenan and Characterisation of the Polysaccharide OrderDisorder Transition in Solution. European Journal of Biochemistry, 122, 71-79. Rochas, C., Rinaudo, M., & Landry, S. (1989). Relation between the molecular structure and mechanical properties of carrageenan gels. Carbohydrate Polymers, 10, 115-129. Rodríguez, M. C., Merino, E. R., Pujol, C. A., Damonte, E. B., Cerezo, A. S., & Matulewicz M. C. (2005). Galactans from cystocarpic plants of the red seaweed Callophyllis variegata (Kallymeniaceae, Gigartinales). Carbohydrate Research, 340, 2742-2751. Rudolph, B. (2000). Seaweed product. Red algae of economic significance. In R. E. Martin, E. P. Carter, L. M. Davis, & G. J. Flich (Eds.). Marine and freshwater products handbook (pp. 515-529), Lancaster: Technomic Publishing Company Inc. Ruggiero, J., Fossey, M. A., Santos, J. A., & Mourão, P.A. S. (1998). Charge distribution and calcium affinity of sulfated - -galactans from ascidians. Comparison between linear and highly branched polymers. Carbohydrate Research, 306, 545-550. Saito, R.M. & Oliveira, E.C. (1990). Chemical screening of Brazilian marine algae producing carrageenans. Hydrobiologia, 204/205, 585-588. Sen, A. K., Das, A. K., Sarkar, K. K., Siddhanta, A. K., Takano, R., Kamei, K., & Hara S. (2002). An agaroid-carrageenan hybrid type backbone structure for the antithrombotic sulfated polysaccharide from Grateloupia indica boergensen (Halymeniales, rhodophyta). Botanica Marina, 45, 331-338. Stortz C.A., Cerezo A.S. (1988). The λ-components of the “intermediate” fractions of the carrageenan form Iridaea undulosa. Carbohydrate Research, 172, 139-146. Stortz, C. A., & Cerezo, A. S. (1993). The system of carrageenans from cystocarpic and tetrasporic stages of Iridaea undulosa. Carbohydrate Research, 242, 217-227. Stortz, C. A., Cases, M. R., & Cerezo, A. S. (1997a), Red Algal Galactans. Methodology for the structural determinationof corallinan, a different agaroid. In R. R. Townsen & A. T. Hotghkiss Jr. Techniques in Glycobiology (pp. 567-593). New York: Marcel Decker, Inc. Stortz, C. A., Cases, M. R., & Cerezo, A. S. (1997b). The system of agaroids and carrageenans from the soluble fraction of the terasporic stage of the red seaweed Iridaea undulosa. Carbohydrate Polymers, 34, 61-65. Stortz, C. A. & Cerezo, A. S. (1993). The systems of carrageenans from cystocarpic and tetrasporic stages from Iridaea undulosa: fractionation with potassium chloride and methylation analysis of the fractions. Carbohydrate Research, 242, 217-227. Stortz, C. A. & Cerezo, A. S. (2000). Novel findings in carrageenans, agaroids and “hibrid” red seaweed galactans. Current Topics in Phytochemistry, 4, 121-134. Talarico, L. B., Zibetti, R. G. M., Faria, P. C. S., Scolaro, L. A., Duarte, M. E. R., Noseda, M. D., Pujol, C. A., & Damonte, E. B. (2004). Anti-herpes simplex virus activity of sulfated galactans from the red seaweeds Gymnogongrus griffithsiae and Cryptonemia crenulata. International Journal of Biological Macromolecules, 34, 63–71. Takano, R., Iwane-Sakata, H., Hayashi, K., Hara, S., & Hirase, S. (1998). Concurrence of agaroid and carrageenan chains in funoran from the red seaweed Gloiopeltis furcata Post. Et Ruprech (Cryptonemiales, Rhodophyta). Carbohydrate Polymers, 35, 81-87. Takano, R., Hayashi, K., Hara, S., & Hirase, S. (1995). Funoran from the red seaweed, Gloiopeltis complanata: polysaccharides with suphated agarose structure and their precursor structure. Carbohydrate Polymers, 27, 305-311.
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Takano, R., Hayashi, K., & Hara, S. (1997), Variety in backbone modification of red algal galactan sulphates. Recent Research Development in Phytochemistry, 1, 195-201. Takano, R., Kamei-Hayashi, K., Hara, S., & Hirase, S. (1993). Assignment of the Absolute Configuration of Partially Methylated Galactoses by Combined Gas-liquid Chromatography-Mass Spectrometry. Bioscience, Biotechnology, and Biochemistry, 57, 11951197. Takano, R., Matsuo, M., Kamei-Hayashi, K., Hara, S., & Hirase, S. (1992). A Novel Regioselective Desulfation Method Specific to Carbohydrate 6-Sulfate Using Silylating Reagents. Bioscience, Biotechnology, and Biochemistry, 56, 1577-1580. Takano, R., Nose, Y., Hayashi, K., Hara, S., & Hirase S. (1994). Agarose-carrageenan hybrid polysaccharides from Lomentaria catenata. Phytochemistry, 37, 1615-1619. Takano, R., Shiomoto, K., Kamei, K., Hara, S., & Hirase, S. (2003). Occurrence of carrageenan structure in an Agar from the red seaweed Digenea simplex (Wulfen) C.Agardh (Rhodomelaceae, Ceramiales) with a short review of carrageenan-agarocolloid hybrid in the Florideophycidae. Botanica Marina, 46, 142-150. Takano, R., Yakoi, T., Kamei, K., Hara, S., & Hirase S. (1999). Coexistence of Agaroid and Carrageenan structures in the Polysaccharide from the Red Seaweed Rhodomela larix (Turner) C.Ag. Botanica Marina, 42, 183-188. Tibbits, C. W., MacDougall, A. J., & Ring, S. G. (1998). Calcium binding and swelling behaviour of a high methoxyl pectin gel. Carbohydrate Research, 310, 101-107. Usov A. I. (1984). Botanica Marina, 27, 189. Usov, A. I. (1992). Sulfated polysaccharides of red seaweeds. Food Hydrocolloids, 6, 9-23. Usov, A. I. & Barbakadze, E. (1978). Polysaccharides of algae. XXVI. Partial acetolysis of the sulfated galactan from the red seaweed Grateloupia divaricata Okam. Bioorganicheskaia Khimia, 4, 1107-1115. Usov A.N., Bilan M.I., & Shashkov A.S. (1997). Structure of a sulfated xylogalactan from the calcareous red alga Corallina pilulifera P. et R. (Rhodophyta, Corallinaceae). Carbohydrate Research, 303, 93-102. Usov, A. I. & Ivanova, E. G. (1975). Polysaccharides of algae. XIX. Partial methanolysisof sulphated polysaccharides of the red seaweed Rhodomela larix (Turn.)C.Ag.. Bioorganichescaia Khimia, 1, 665-671. Usov, A. I. & Ivanova, E. G. (1992). Polysaccharides of algae. 46. Studies on agar from the red seaweed Gelidiella acerosa. Bioorganichecaia Khimiia, 18, 1108-1116. Usov A.I., Ivanova E.G., & Shashkov A.S. (1983). Polysaccharides of algae. XXXIII: Isolation and 13C NMR study of some new gel-forming polysaccharides from Japan Sea Red Seaweeds. Botanica Marina, 26, 285-294. Usov, A. I. & Klochkova, N. G. (1992). Polysaccharides of algae. 45. Polysaccharide composition of red seaweeds from Kamchatca Costal Waters (Northwestern Pacific) studied by reductive hydrolysis of the biomass. Botanica Marina, 35, 371-378. Usov, A. I., Miroshnikova, L. I., & Barbakadze, V. V. (1975). Polysaccharides of algae. XVII. Water soluble polysaccharide of the red algae Grateulopia divaricata Okamura and Grateulopia turuturu Yamada. Zhuranal Obshei Khimii, 45, 1618-1624. Usov A. I., Yarotskii S.V., & Shashkov A. S. (1980). 13C-nmr spectroscopy of red algal galactans. Biopolymers, 19, 977-990. Wang, S. C., Bligh, S.W.A., Shi, S. S., Wang, Z. T., Hu, Z. B., Crowder, J., Branford-White, C., & Vella, C. (2007). Structural features and anti-HIV-1 activity of novel
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polysaccharides from red algae Grateloupia longifolia and Grateloupia filicina. nternational Journal of Biological Macromolecules, 41, 369-375. Whyte, J. N. C., Foreman, R. E., & De Wreede, R. E. (1984). Phycocolloid screening of British Columbia red algae. Hydrobiologia, 116/117, 537–541. Whyte, J. N. C., Hosford, S. P. C., & Engar, J. R. (1985). Assignment of agar or carrageenan structures to the red algal polysaccharides. Carbohydrate Research, 140, 336-341. Zibetti, R.G.M., Noseda, M.D., Cerezo, A.S., & Duarte, M.E.R. (2005). The system of galactans of Cryptonemia crenulata (Halymeniaceae, Halymeniales) and the structure of two major fractions. Kinetic studies on the alkaline cyclization of the unusual diad G(2S)→D(L)6S. Carbohydrate Research, 340, 711-722 York, W. S., Hantus, S., Albersheim, P., & Darvill, A.G. (1997). Determination of the absolute configuration of monosaccharides by 1H NMR spectroscopy of their per-O-(S)2-methylbutyrate derivatives. Carbohydrate Research, 300, 199-206.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 36
DEVELOPMENT OF POLYSACCHARIDE NETWORKS FOR L-(+)-ASCORBIC ACID STABILIZATION INTO ANTIOXIDANT/NUTRITIONAL SUPPLIER-EDIBLE FILMS Ana M. Rojas1,2*, Paula G. León1,3, Silvia K. Flores1,2, Carolina D. Pérez1,4 and María D. De’Nobili1,4 1
Industry Department, School of Exact and Natural Sciences (FCEN), University of Buenos Aires (UBA). Ciudad Universitaria. Intendente Güiraldes 2620, (1428) Ciudad Autónoma de Buenos Aires, Argentina. 2 Member of the National Scientific and Technical Research Council of Argentina (CONICET). 3 Fellow of the University of Buenos Aires. 4 Fellow of CONICET.
ABSTRACT In the past half century, synthetic petroleum-based polymers have been widely used in a variety of packaging materials but have become a major source of waste disposal problems due to their poor biodegradability. With the increasing demand by consumers for high-quality foods and concerns about limited natural resources and the environment, the use of renewable resources to produce edible and biodegradable packaging materials that can improve product quality and reduce waste disposal problems are being explored. Biopolymer-based edible films and coatings are intended to function as barriers against moisture, oxygen, flavor, aroma and oil, as well as carriers of additives thereby improving food quality and enhancing the shelf life of food products. Water-soluble polysaccharides are commonly used in food related applications as thickeners due to their increase in viscosity when hydrated. Polysaccharides can also constitute edible films, an important quality since these polymers are of natural origin and *
Corresponding author: E-mail:
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come from renewable sources and are biodegradable as well. Though it is recognized that polysaccharide edible films are not good barriers against water vapour, they can find interesting applications as food interfaces, as carriers of active compounds or preservatives, constituting delivery systems with local activity. The present chapter deals with the study of edible films based on deacylated and/or acylated forms of gellan gum to support L-(+)-ascorbic acid (AA) in view of natural antioxidant protection of foods, by leveraging its activity as a vitamin in the human metabolism. Kinetics of AA-destruction and subsequent non enzymatic browning development were studied in the films stored at constant temperature (25ºC) and relative humidity (33.3, 57.7 or 75.2%) and their relationship with the microstructure, at the macromolecular and molecular levels, was also analyzed with the purpose of ensuring a better AA retention as well as lower browning rate as a consequence of controlled water mobility in the polymeric networks.
1. INTRODUCTION Humans evolved on earth with its atmosphere, land and water systems and type of climates in a way that permits us to cope reasonably well with this type of environment. One vital problem (among many) that we must solve is how to continue our technologically based civilization without, at the same time, irreversibly damaging the environment in which we evolved. Two humans’ activities that are strongly connected with environmental chemistry in general and pollution in particular are energy production and waste disposal. The latter is a growing problem (Bailey et al, 2002). A shining example of waste disposal involves the usage of non-degradable plastics for packaging and disposable consumer goods. During the last decade joint efforts by the packaging and the food industries have reduced the bulk of food packaging materials. Nonetheless used packaging materials are still very visible to the consumer in the context of disposal (Petersen et al., 1999). This has led to intense research to develop biodegradable packaging materials in conjunction with the introduction of new purposes to food packaging systems. The use of edible films and coatings is an application of active food packaging, since the edibility and biodegradability of the films are extra functions that are not present in conventional packaging systems (Han, 2002; Cutter, 2006; Dutta et al., 2009). With the increasing demand by consumers for high-quality foods and concerns about limited natural resources and the environment, the use of renewable resources to produce edible and biodegradable packaging materials that can improve product quality and reduce waste disposal problems are being explored. The application of edible films and coatings is an easy way to improve the physical strength of the food products, reduce particle clustering, and enhance the visual and tactile features of food product surfaces (Cuq et al, 1995; Matuska et al., 2006). They can also protect food products from oxidation, moisture absorption/desorption, microbial growth, and other chemical reactions (Kester and Fennema, 1986; Matuska et al., 2006; Rojas-Graü et al., 2006; Seydim and Sarikus, 2006; Sanjurjo et al., 2006; Güçbilmez et al 2007; Sivarooban et al., 2008; Vásconez et al, 2009; SaucedoPompa et al., 2009). Protection from mass transfers through waxy and resins coatings is naturally found in vegetables but they are in part removed as a consequence of the washing to which the produce is submitted before its commercialization. Edible films and coatings have long been used empirically for food protection and shelf-life prolongation (Guilbert & Gontard, 1995).
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Ancient Egyptians, Chinese, Greeks and Romans used casein in glues among other proteinaceous substances, because of its water resistance (Gennadios et al., 1994). In fact, sausage casings and candy coatings, while not ‘packaging’ in the strict sense, have been successful forerunners for the concept of edible films. Interest in edible film and coating development has increased because evidence was obtained about their beneficial effects on fresh and processed foods (Baker, Baldwin, & Nisperos-Carriedo, 1994; Mei, Zhao, Yang, & Furr, 2002; Cutter, 2006; Dutta et al., 2009; Saucedo-Pompa et al., 2009). They are capable of providing many functional advantages like modifying vegetable tissue metabolism by affecting respiration; being carriers of antimicrobials, antioxidants and other preservatives; enriching product formulation while acting as carriers of vitamins and minerals; improving structural integrity or handling; controlling location or rate of release of additives, retaining volatile flavour, and they can also reduce packaging waste associated with processed foods (Greener Donhowe & Fennema, 1994; Franssen, Rumsey, & Krochta, 2002; Buonocore et al., 2002; Bautista-Baños et al., 2006; Ragaert et al, 2007; Sothornvit and Rodsamran, 2008; Geraldine et al., 2008). The potential of edible film usage for special applications is a very promising alternative. In micronutrient fortification of foods, edible films and coatings have been used to prevent the leaching of vitamins during washing and cooking (Shrestha, Arcot, & Paterson, 2003), having also been shown their ability to reduce the rate of oxidative reactions and extend the shelf life of mushrooms and cauliflower (Ayranci & Tunc, 2003). At the same time, the barrier properties associated to some edible film applications are of health concern. The important decrease of oil absorption in potato strips during deep fat frying was early determined by Williams and Mittal (1999) in gellan, methylcellulose (MC) or hydroxypropyl methylcellulose (HPMC) based edible films, followed by the research work of Albert and Mittal (2002), where edible films were directly constituted on the surface of potato strips from solutions containing different polymers based either on polysaccharides or proteins. Garcia et al. (2002) also studied MC and HPMC as edible coatings to reduce oil uptake in deep fried potato strips. They found that a mixture of MC with sorbitol as plasticizer was effective in reducing oil absorption by 40% compared to uncoated potato strips. On the other hand, the increasing demand by consumers for minimally processed fruits and vegetables leads to investigate about different ways to increase their stability. Polysaccharide films are used in Japan for meat products, ham and poultry packaging before smoking and steaming processes. The film is dissolved during the process and the coated meat exhibits improved yield, structure, and texture, as well as reduced moisture loss (Cutter, 2006). It was determined that edible coatings based on carrageenan or whey protein concentrate in combination with antibrowning agents effectively extended the shelf-life of minimally processed apple slices by two weeks when stored in packed trays at 3ºC. A synergistic effect was found on colour of the apple slices by mixing ascorbic, citric and oxalic acids in combination with CaCl2 (Lee et al., 2003). Edible films and coatings are useful materials produced mainly from edible biopolymers and food-grade additives. Most biopolymers are naturally existing polymers, including proteins, polysaccharides and lipids (Gennadios et al, 1997). For health and safety as well as edibility, the solvents used have to be water and/or ethanol. Plasticizers are necessarily included with the film-forming biopolymers in order to modify film physical properties, as well as other additives to create extra functionalities. Specifically, edible films are freestanding structures, first formed and then applied to foods. They are developed by casting and drying of film-forming solutions on a levelled surface, or by using a drum drier or other
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traditional plastic processing techniques such as extrusion. Edible films can also be formed by applying the coating solutions directly on a food product either by dipping, brushing or spraying (Krochta and De Mulder-Johnston, 1997; Bravin et al., 2006; Vásconez et al, 2009). Therefore, formulation of edible as well as of bio-plastic (not edible but biodegradable) films and coatings, implies the use of at least one component able to form a network, having sufficient cohesion and continuity which under preparation conditions, have the property to form crystalline and/or amorphous continuous structures. The film-forming mechanisms of biopolymers include inter-macromolecular forces such as covalent (e.g., disulfide crosslinking) and/or physical bonds like electrostatic or hydrophobic interactions and hydrogen bonds. Only polyesters (exclusively for bio-plastic films), polysaccharides or proteins are used for making "materials". Film formation is also dependent on adhesion (attractive forces between film and substrate), which is of great importance when films are directly constituted on foods (Han, 2005). Manufacture procedures initiate these film-forming mechanisms. For the resulting films or coatings to be edible, the film-forming mechanism involved should be an appropriate food process in relation, for example, to pH value, acidifying agent used, salts, plasticizing substances and drying process conditions (Han and Gennadios, 2005). This consideration makes very difficult to find adequate chemical reactions to be applied for modifying the macromolecular network in order to explore new material structures for edible film or coating constitution. On the other hand, filmmaking conditions have to be exhaustively controlled because their changes can alter the kinetics and reaction mechanisms involved in the development of film networks, since the latter are transient systems (Ferry, 1980; Yang and Paulson, 2000a; Han and Gennadios, 2005; Flores et al., 2007; Kasapis, 2008; Leon et al, 2009). At the same time, functional additives present in the film formulation to accomplish, for example, antimicrobial activity (e.g. sorbates) may also modify the film microstructure, contributing with additional plasticization (Flores et al., 2007). Uncontrolled procedures and/or additive effects are finally manifested through changes in the mechanical performance and permeability of the edible films and coatings (Famá et al., 2005). The polysaccharides are an amazing group of biopolymers that have evolved to serve a whole range of functions in the host organism such as energy storage, structure, defence, pathogenicity, wound healing. Very often, a subtle change in the structure of the monomer or the type of glycosidic linkage employed has a profound effect on the properties and function of the resultant polysaccharide (Stick, 2001). The important functional properties that polysaccharides present in pharmaceutical and food formulations are of rheological nature, which derives from the three-dimensional structure that the macromolecule adopts in a given solvent, in conjunction with their molecular weight (Lapasin and Pricl, 1995). Starch, pectins and cellulose, all of them extracted from evolved vegetables, as well as polysaccharides obtained from algae (carragenan, alginates) or produced by the metabolism of bacteria (gellan, pullulan, wellam) show important thickening to gelling effects at low concentrations, while are insipid and with low caloric value (Scott-Thomas, 2008). These rheological characteristics indicate the development of interactions between the solvated macromolecules as polysaccharide concentration increases, hindering the water (solvent) flow. A network can be finally formed in some concentrated polysaccharide solutions like xhantan or when gelling occurs as a consequence of using a polysaccharide concentration higher than the critical one (C*) together with an adequate ionic force if poly-anions (pectins, alginates, gellan) were considered (Jampen et al., 2000; Doublier et al., 1992). The viscous film-forming solution,
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basically constituted by the polysaccharide/s and the plasticizing/s agent/s, turns firstly to a gel and, finally, to the solid-like network of the edible film as a consequence of the increasing polymer interactions because of water (solvent) removal during drying, when casting technology is used. However, the plasticizing agent persists after drying, avoiding complete polymer-polymer interactions after dehydration, and films of adequate flexibility are then obtained, bringing the system to the rubbery state. A plasticizer is defined as the substance incorporated in a material to increase the material´s workability, flexibility, or extensibility. Proteins or polysaccharides are plasticized by low molecular weight diluents. Here, the glass transition temperature (Tg) of the biopolymer is considerably higher than that of the diluent so as the concentration of the diluent increases the Tg of the molecularly miscible mixture decreases (Kasapis, 2008). Since polysaccharides used are hydrophilic in nature, plasticizing substances have to be compatible with them, as water and/or etanol are used like solvents. Glycerol, sorbitol and polyethylene glycols are commonly employed for making homogeneous edible films by casting because they produce plasticization at reasonable low concentrations (Yang and Paulson, 2000a; Kristo and Biliaderis, 2006). Due to the hydrophilic nature, polysaccharide-based films exhibit limited water vapour barrier ability. Cross-linking of the polymeric network leads to permeability decrease (Rhim, 2004; Jo et al. 2005; Kang et al., 2005). On the other hand, films based on polysaccharides such as alginate, cellulose ethers, chitosan, carrageenan or pectins exhibit good gas-barrier properties (Williams et al., 1978; Baldwin et al., 1995). The water vapour and gas permeabilities of hydrophilic polymers are dependent on the relative humidity (RH) of storage as well as on plasticizing content (Yang and Paulson, 2000a; Mali et al., 2004). The gas-permeability properties of hidrophilic films result in desirable modified atmospheres, thereby increasing the product shelf life without creating anaerobic conditions (Baldwin et al., 1995). The required specific permeability properties of the films can be obtained by inclusion of inert impermeable barriers (lipids) and/or reactive compounds (reducing substances) in the polymer matrix (Yang & Paulson, 2000b; Bravin et al., 2006). The inert barriers can reduce permeability by increasing either the diffusion path and/or gas-solubilization, while the reactive compounds can in part interact selectively with the diffusing species increasing the time before a significant permeability occurs (Alves et al., 2006). Biological systems are subjected to a constant oxidative stress by reactive oxygen species under normal physiological conditions, as a result of respiration. The balance between prooxidant forces and antioxidant defence barriers influences the susceptibility to prooxidant damage (Morrisey & O’Brien, 1998; Weisburger, 1999). Vitamin C is one of the micronutrients involved in this prooxidant–antioxidant balance in humans. The principal biologically active form of vitamin C is L-(+)-ascorbic acid (AA) but its oxidation product, Ldehydroascorbic acid (DHA), is also active (Levine & Morita, 1985; Villota & Karel, 1980). Since AA-effects are based on its reducing activity, it is also adopted as a water soluble antioxidant for food preservation. In recent years, there has been an enormous demand for natural antioxidants mainly because of adverse toxicological reports on many synthetic compounds (Miková, 2001). The importance of antioxidants contained in foods is well appreciated for food preservation and supplying essential antioxidants in vivo (Shi, 2001). According to the aforementioned facts, AA carried on edible films for antioxidant food protection may constitute other alternative of active packaging. However, AA stability is affected by processing and storage conditions because depends on a large number of factors such as temperature, equilibrium RH, oxygen partial pressure, light, package permeability and
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package configuration (Kitts, 1997). It is lost by oxidation when oxygen is present, catalysed by metal ions and light (Bissett & Berry, 1975). Anaerobic degradation proceeds simultaneously when oxygen is present, but much slower (Kurata & Sakurai, 1967). On the other hand, non-enzymic browning (NEB) is related to AA loss since products of the reactions that follow the first step of AA destruction are also part of the NEB reaction-chain (Rojas and Gerschenson, 2001). Among the polysaccharides used in the food industry, gellan is a relatively new gum in relation to its usage for food formulation. Acylated gellan is produced by Sphingomonas elodea through fermentation in culture media. Gellan (deacylated) gum, is obtained further after chemical treatment for commercialization. While the acylated form produces non-brittle and elastic gels, deacylated gum yields brittle and rigid gels. The presence or absence of glicerate and acetate residues in high acyl and low acyl forms, respectively, offers a wide range of textural possibilities to food technology. The addition of cations, such as calcium or potassium, increases the bond association between the polymer components, giving as a result, a physical cross-linked network (Chandrasekaran et al., 1988; Hamcerencu et al., 2008). Gellan has increasing demand because of its thermal stability and relative resistance to acid medium, as well as sparkling clarity, in addition to its high efficiency like gelling agent. Critical concentration of deacylated (low acyl form)-gellan aqueous solutions was found to be low (C* = 0.064%), while gellan concentrations around 0.5% (w/v) just led to partial and localized gelation as it was cooled (Jampen et al., 2000). Since it can form stable and true gels (Lapasin and Pricl, 1995) in the presence of salts (which are naturally contributed by the Kelcogel F gellan polymer, for example), it was then chosen as an interesting matrix to support AA for edible film constitution after gel drying. A film network made with a 1:1 (w/w)-mixture of acylated and deacylated gellan polymers was also developed from structural considerations in relation to AA stability and restriction of water mobility. From preliminary results obtained for AA loss and browning development into gellan film, it was determined that water availability would be a key factor in the AA degradation reaction. Consequently, it was hypothesized that a less rigid network could better immobilize water and, then, stabilizes AA in conjunction with the necessity of a lower glycerol concentration for plasticization. Otherwise, the effect of additional calcium presence in gellan (deacylated) films was investigated as an alternative strategy to control AA degradation and NEB rate. In this context, the present chapter examines the effect of film microstructure, at the macromolecular and molecular levels, on the AA stability and subsequent NEB development, in order to succeed in finding natural antioxidant protection of foods through these edible film interfaces.
2. EXPERIMENTAL SECTION 2.1. Chemicals Food grade gellan gum (deacylated polymer; Kelcogel F) as well as its high acylated form (Kelcogel LT100) were from CP Kelco (J. M. Huber Corporation, USA). Chemicals of analytical quality were used: glycerol, L-(+)-ascorbic (AA), citric, acetic and oxalic acids, hydrogen peroxide, sodium acetate, magnesium perchlorate, sodium bromide, magnesium,
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sodium and calcium chloride, from Anedra (Buenos Aires, Argentina), as well as potassium sorbate and 2,6-dichlorophenol-indophenol from Sigma (St. Louis, MO, USA).
2.2. Film Preparation The 1% (w/w)-polymer film-forming solutions were prepared by swelling either 3 g of gellan gum powder (A-system) or 1.5 g of gellan followed by 1.5 g of its high-acylated form (1:1 weight proportion), in 260 g of continuously stirred deionized water, under high speed shear (vertical stirrer model LH, Velp Scientifica, Italy), in order to accomplish homogeneous hydration of the powder, avoiding lump formation (Leon and Rojas, 2007; Leon et al., 2008). A homogeneous, transparent and viscous system was obtained while stirring at the same initial constant velocity. This was followed by heating to 90ºC at a constant heating speed (5ºC/min) on a hot plate, with simultaneous recording of the temperature every 20 seconds through a thermocouple connected to a Consort millivoltimeter (P 901, CE Belgium). Glycerol was added as a plasticizer at a concentration of 1.5% w/w, excepting for C-system where a 0.75% w/w-concentration was used. Potassium sorbate (0.03% w/w) and AA (0.100% w/w), both pre-dissolved in a minimum volume of deionized water, were then added. Glycerol was generally used at a 60% w/w [plasticizer/(plasticizer + gellan)] proportion for film development in A, B and D systems (Yang and Paulson, 2000a), considering that other substances like potassium sorbate, which are able to exert additional plasticizing effect (Famá et al., 2005) were present in the formulation used. Enough citric acid was added to adjust the pH to a value of ≈ 4.0, while stirring and heating. Calcium chloride, previously dissolved in deionized water, was also aggregated only in the case of B-system solution, when it was at 80ºC. The total weight of each prepared system was then made to 300.00 g by adding enough deionized water while stirring to homogenize (Table 1). The hot solution was placed under vacuum for 20 s to remove air-bubbles and immediately poured on to levelled glass plates. They were cooled at room temperature for 20 min to allow gel setting, after that they were placed into an air convection-oven at 60ºC for 2.5 h. After cooling to room temperature, films were peeled from the glass plates and stored at 25ºC and constant relative humidity given by saturated solutions of MgCl2 (water activity, aW = 0.333), NaBr (aW = 0.577) or NaCl (aW = 0.752). Equilibration was assessed by measurement of aW of film samples every day till attaining the RH/100 of the saturated solution used. Afterwards, sample thickness was measured to the nearest 0.001 mm using a digital micrometer (Mitutoyo, Japan) at six different locations in each of ten specimens. For kinetic studies, samples were collected along storage. Three batches of films (replicates) were prepared as indicated and stored at each relative humidity (RH) to allow considering in the study, the influence of film making. The following analyses were performed on samples of the three batches for each time of interest.
2.3. Dosage of L-(+)-ascorbic Acid Each film sample was first cut into pieces smaller than 1-mm size, weighed on an analytical scale (0.0001 g) and extracted for 1 h at 5ºC with a 1% (w/v)-oxalic acid solution under magnetic stirring into a 25.00 ml-volumetric flask. An aliquot was taken and AA
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concentration was determined through the 2,6-dichlorophenol indophenol (2,6-DPIP) spectrophotometric method as indicated by Rojas and Gerschenson (1991) but without formaldehyde blank and xylene for organic extraction as previous assays showed that they were not necessary due to system characteristics. Absorbance measurements were conducted at 540 nm. Two determinations were made for each film sample.
2.4. Measurement of pH A bulb-combined glass electrode (Phoenix, USA) as well as a flat surface combination electrode (Phoenix, USA) respectively connected to a pH meter (Consort P901, ECC), were used for pH measurement of gel-forming solutions or films after their equilibration at the corresponding relative humidity of interest, respectively (Burin & Buera, 2002). Calibrations were carried out with standard buffers of pH 4.00 and 7.02.
2.5. Water Activity It was determined with a Decagon’s Aqualab (Series 3 water activity meter, USA) at 25ºC-constant temperature, using a calibration curve made with standard saturated salts (MgCl2, NaBr and NaCl) of known aW values (Greenspan, 1977; Favetto, Resnik, Chirife, & Ferro Fontán, 1983). The reported true aW (aWº) of film samples was extrapolated from the fitted calibration curve for aW determined for each sample in the equipment. Cell volume of Decagon´ s Aqualab was half-reduced by inserting a TeflonTM cylindrical piece, in order to assure a cell atmosphere conditioned by the RH of the equilibrium vapour pressure coming from the film sample.
2.6. Color Film disks of 20 mm-diameter were used for colour measurement in a Minolta colorimeter (Minolta CM-508d, Tokyo, Japan) using and aperture of 1.5 cm-diameter. The exposed area was sufficiently great in relation to the illuminated area to avoid any edge effect. Film samples were first rested on white background standard (Trezza and Krochta, 2000). The Hunterlab parameters: L, a, and b were measured according to a standard test method (ASTM E1925, 1995), in at least five positions randomly selected for each sample. Colour parameters range from L = 0 (black) to L = 100 (white); -a (greenness) to +a (redness), and -b (blueness) to +b (yellowness). Standard values considered were those of the white background. Calculations were made for D-65 illuminant and 2º observer. The yellow index (YI) parameter was measured for reporting the NEB developed in film samples, according to ASTM D-1925 (21), which defines YI as,
YI =
100 (1,2769 X − 1,0592Z ) Y
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where X, Y, Z are the CIE tristimulus values of the sample measured under conditions of C illuminant and 2º observer. A positive value of YI describes the presence and magnitude of yellowness, whereas a negative one indicates the same with reference to blueness.
1.7. Moisture Content Films were sampled at various time intervals during storage and dried in a vacuum oven at 70ºC until constant weight (around 30-35 days). Before weighing, samples were cooled to room temperature into a desiccator over P2O5. Determinations were performed on six film specimens taken from each storage condition.
2.8. Glass Transition Temperature (Tg) It was determined at a heating rate of 10ºC/min through differential scanning calorimetry using a DSC 822e Mettler Toledo calorimeter (Schwerzenbach, Switzerland). The instrument was calibrated with a sapphire disk in the whole temperature range used for measurements (-160 to 550ºC) as well as with the melting points of indium (156.6ºC), lead (327.5ºC) and zinc (419.6ºC). An amount of 10–15 mg of equilibrated film sample was placed into a hermetically sealed 40 μl-aluminium-pressure pan, using an empty pan as reference. The first scan performed from -140ºC to 40ºC was followed by a second scan from -140ºC to 125ºC, after cooling at 15ºC/min. Thermograms were evaluated using the Mettler Stare program (Switzerland). An average value of Tg (onset) was reported from the second scan of two replicates.
2.9. NMR Proton Mobility All the experiments were performed on equilibrated film samples using a Bruker Avance II spectrometer operating at 300 MHz for 1H. The probe is a Bruker high power CPMAS and was used under static conditions. The rotor sizes were 18-mm long with a 4-mm outer diameter. All the experiments were conducted on-resonance at room temperature. Typically, the p/2 pulse was set as 3.3 ms. To measure the transverse magnetization decay (T2), a Carr Purcell Meiboom Gill (CPMG) multipulse sequence was used, where data were acquired at the top of the eco between π-pulses every 2τ. A biexponential magnetization decay curve vs time was then obtained. This experiment was performed twice for each sample, once for t = 50 ms and the other with t = 10 ms. Exponential curve fittings were performed for τ = 50 and τ = 10 μs using a nonlinear fitting program (OriginPro 7.5 SRO, Origin Lab Corporation, Northampton, MA 01060, USA) and a Maxwell-two component-curve was the best fit model: ⎛ ⎞ ⎛ ⎞ M (t ) = M1 exp⎜⎜ − t ⎟⎟ + M exp⎜⎜ − t ⎟⎟ 2 ⎜T ⎟ ⎜T ⎟ 2a 2b ⎝
All analyses were performed in triplicate.
⎠
⎝
⎠
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2.10. X-ray diffraction A Philips X-ray diffractometer with vertical goniometer was used (Cu Kα radiation λ = 1.542 Å). Operation was performed at 40 kV and 30 mA. Samples mounted on a glass and conditioned at a aW of 0.575 were attached to the equipment holder and X-ray intensity was recorded with a scintillation counter in a scattering angle (2θ) range of 6–33º with a scanning speed of 1º/min. Distances between the planes of the crystals, d (Å), were calculated from the diffraction angles (º) obtained in the X-ray pattern, according to Bragg’s law: n λ = 2d sin(θ) wherein λ is the wavelength of the X-ray beam and n is the order of reflection, which was considered as 1 for calculation.
2.11. Tensile Assay Tensile strength (N/m) was calculated as the ratio between the tensile force (N) at failure and the corresponding extension (m) determined from the force-elongation curves recorded at 5 mm/min-constant cross-head velocity in an Instron Testing Machine (model 3345, Norwood, Ma, USA), equipped with a load cell of 100 N and pneumatic grips with flat rubber coated faces. Rectangular specimens of 6 mm width x 60 mm-total length were cut and used in the tensile test, in order to be sure that only uniaxial tension was accomplished along testing, as it was determined through previous assays carried out with specimens of different width. A gage length of 20 mm was used along assays. At least seven specimens of a given formulation and storage condition, each of them cut from a different film sample, were assayed.
2.12. Statistical Analyses of Data Results are reported as the average and standard deviation. Rate constants were calculated by linear regression where AA concentration was in terms of g AA/weight (g) of the corresponding film sample assayed. Analyses of covariance (ANCOVA) were applied for comparison of rate constants derived from slopes, as indicated by Sokal and Rohlf (1969). Statistical analyses of results were performed through ANOVA with α: 0.05, followed by pair wise multiple comparisons evaluated by Tukey’s significant difference test (Sokal & Rohlf, 1969). Significance of correlations was evaluated according to Bancroft (1960). The GraphPad Prism (statistical) software (version 5.00, 2007, GraphPad Software Inc., USA) was used for all analyses previously detailed.
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3. RESULTS AND DISCUSSION 3.1. Characteristics of the Casted Films Gellan based gelling solutions produced robust films of 75-90 μm-thickness just at 1% w/w of polymer concentration, in presence of glycerol as plasticizer (30 or 60%w/w [plasticizer/(plasticizer + polymer/s)] proportion). Films were colourless and transparent, as indicated by the initial values of the colour parameters (YI = 4.0-8.7; b = 3.8-4.35 and lightness, L ≈ 85%). A film pH of ≈ 3.8 was initially determined. Citric acid was added to the formulations since pH values < 4.1-4.5 are recognized to be necessary for maximal AA stability (Clegg, 1964; Rojas and Gerschenson, 1997), as well as for comparison with aqueous model systems studied by us in a previous work (Rojas and Gerschenson, 2001). The initial AA concentration coincided with that considered for film formulation (≈ 3.2% w/w on film basis) for all systems studied, which accounted for retention of ≈ 100% after film casting. Hence, the film composition as well as deformation and thermal history led to develop polymeric film networks that successfully retained all AA added. In order to determine if they were also able to stabilize AA during film storage in view of future technological applications as antioxidant interface/nutritional supplier edible films, it was studied the kinetics of AA degradation as well as of the subsequent browning development, the latter due to organoleptical implications. Hence, casted films were stored at constant temperature (25ºC) and RH (33.3, 57.7 or 75.2%) into chambers and under vacuum, which is needed to rapidly reach the equilibrium-aW with the RH of the chamber-atmosphere and, at the same time, permitted that a specific chain of initial reactions of AA degradation occurred.
3.2. Kinetics of Ascorbic Acid Destruction and Browning Development “Equilibration” of the casted films at each RH occurred at about the 4th storage day, with the exception of the gellan film (A-system) stored at 75.2% RH, which equilibrated rapidly, at 24 h of storage. However, it was observed that the decrease of the ratio between AA concentration and initial one with time, statistically fitted (p 3). It was measured as yellow index (YI) increase against time of storage. 3 Values reported are the mean and standard deviations corresponding to all values recorded during the total storage period. 2
The increase of the yellow index (YI), that is, of film browning with storage time statistically fitted (p glucose (5.62%) > arabinose (2.28%) > xylose (1.29%) > methylated galactose (1.19%) ribose (1.06%)]. However, the charged fraction consisted of principally three sugar units: (mol%) Galactose (78.53%) > mannose (18.06%) > glucose (2.08%) > methylated galactose (1.33%), whereas the neutral fraction consisted of three sugar units with predominantly glucose: (mol%) Glucose (73.11%) > mannose (15.23%) > galactose (11.66%). It is of interest to note that only neutral GIsps contained glucose (major) and mannose, galactose (minor) merely three sugar residues unlike the charged fraction of GIsps. It is highly likely that these heteropolymers have branched structures in various degrees, which may be the reason for their (parent and cross linked polysaccharides) inability to swell as much unlike their galactan (agarose and carrageenan) counterparts. The favorable network geometry in the latter is certainly absent in the genipin cross linked GIsps. This aspect remains to be studied further.
Water Soluble Polyuronic Acid-biaryldiamine Conjugate The yield of the conjugates increased on increasing the proportion of o-tolidine up to 50 wt% (with respect to alginic acid), and it decreased on increasing the proportion of o-tolidine above 50 wt%. The maximum yield (70%) of the conjugate was obtained with 50 wt% of otolidine. Hence, in this investigation the product obtained with 50 wt% o-tolidine was subjected to detailed characterization. The parent alginic acid does not have nitrogen in its polymeric backbone, while complex of alginic acid with 50 wt% o-tolidine was estimated to contain 3.48% N indicating addition of o-tolidine to alginic acid (Table 4). The proposed mechanism for preparation of conjugate and other possible structure of the conjugate have been given in Figure 7 and Figure 8, respectively. The conjugate obtained under optimum conditions (e.g. ratio of alginic acid and o-tolidine, 1:1 w/w) was easily soluble in water at room temperature, which did not produce fibrous precipitation of alginic acid at pH 2; the one obtained with alginic acid/o-tolidine (2:1 w/w) was not water soluble even at higher temperatures and the one obtained with alginic acid/o-tolidine (1:2 w/w) was partially soluble leaving behind unreacted o-tolidine (superimposable IR spectra with o-tolidine). Bulk and true densities, and porosity of the parent alginic and conjugate prepared under optimum conditions are presented in Table 4. The bulk and true density of the conjugate was greater (0.94g/ml and 1.232g/ml, respectively) than that of the parent alginic acid (0.89g/ml nd 1.212g/ml, respectively), while the porosity of parent alginic acid was greater (0.85) than that of the conjugate (0.67) (Table 4).
Development of Carbohydrate Polymer Based New Hydrogel Materials… 5
H3C
O
O A
3 O HOOC
OH
O
C OH O
OH
O O A OH
2
o-tolidine
OH
+
1
C
4
6
6
5
H3C
1
O A
3 O HOOC
O
C
6
O 6 O HO HO C OH C OH OH 4 HO HO 5 O O O O B O B2 B B O O O O HO 1 2 A O HO 3 3 1 O OH OH O 2 1 OH OH OH 5
4
OH
O
O A OH
3
O 1 OH HOOC
A2
H2N
OH
C
OH OH O
CH3
4 3
H2N OH
O
NH2
5
D 2
2
3
OH
CH3
pH 4 MW 90 oC
Alginic acid
O
4
3
HO 6 O O HO C OH C OH OH 4 HO HO 5 O O O O O B2 B B B O O O O HO 1 2 A O HO 3 3 1 OH OH O 2 1 O OH OH OH
3
O 1 OH HOOC
A2
5
D
5
4
O
1
H2N 6
C
OH
1 2
3
OH
6
6
C
4
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Intermediate
-2 H2O
6
5
H3C
4 3
6 1
C 2
1
5
D 2
HN O C
OH O
O
C
OH
O
OH O
CH3
HN
6
O
O A1 OH O OH HOOC
A2
O 6 O HO C OH C OH OH 4 HO HO 5 O O O O O B2 B B B O O O O HO 1 2 A O HO 3 3 1 OH OH O 2 1 O OH OH OH 5
4
A3 OH
O HOOC
OH
4 3
3
Figure 7. Formation of Alginic acid/o-tolidine-A6,C3-B16,D3-diamide conjugate (Steric energy 170.43 kcal/mol).
Table 4. Properties of alginic acid/o-tolidine conjugate. Properties Optical rotation [α]D (c 0.25; 25oC) Total Nitrogen (%) Viscosity (cP) (1.5 wt% ; 70oC) Bulk density (g/ml) True density (g/ml) Porosity peak/trough ratio
Alginic acid -30.82o a 0.05 120 ±4.30 a 0.89 1.212 0.85 0.70
o-tolidine Alginic acid/o-tolidine +3.12o b +39.48 o c 13.48 3.48 --80 ± 5.0 c 0.96 0.93 1.271 1.232 0.82 0.67 -0.97 a Measured in 0.5 M aqueous Na2CO3 solution; bMeasured in methanolic solution; c Measured in DM water solution.
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H3C
C
4 3
6
6
5
1 2
6
C
OH O O OH A
3 O HOOC
O
4
OH 5
3
OH O O
2
A2
O O OH A 1
1 OH HOOC
4
D
1 2
NH O
5
CH3
3
HN
OH
C
O 6 C OH 5 O
OH 4 HO O O O B O HO B1 A 3 OH O OH OH
2
HO
1
O C OH O
HO O O B2 O HO B3 OH O OH
O
Figure 8. Alginic acid/o-tolidine-A26,C3-B16,D3-diamide conjugate (Steric energy 150.76 kcal/mole).
The parent alginic acid did not exhibit any absorption maxima in the UV-Vis region in 0.5 M sodium carbonate solution in water, while alginic acid/o-tolidine conjugate showed λmax at 282 nm in water. A reversal was observed in the optical rotation value. Specific rotation [∝]D values of parent alginic acid was -30.82o (c 0.25%, 0.5M aq Na2CO3 at 30oC, 589 nm), while that of alginic acid/o-tolidine conjugate was +39.48o (c 0.25%, H2O at 30oC, 589 nm) (Table 4). o-Tolidine is not optically active due to the presence of plane of symmetry. Steric energies of possible alginic acid/o-tolidine conjugates isomers were calculated using ChemBiooffice software, 2008 Chem3D, Molecular mechanics II (MM II) (Table 5). The most probable appropriate sites for the addition of two amino groups of otolidine to the carboxylic acid groups of M- and G-residues of one repeating unit of alginic acid, designated as A6,C3-B16,D3 (Figure 7) or A26,C3-B16,D3 (Figure 8) conjugates, having steric energies 170.43 and 150.76 kcals/mole respectively (Table 5). It was observed in the molecular model that to attain the structure A26,C3-B16,D3, there would be extensive folding. Other possibilities for the conjugate formation are that the addition could take place involving the carboxylic acid groups of two adjacent repeating units of alginic acid e.g. GG, GM, MG and MM. Steric energies of these possible conjugates have been given in Table 5, which are higher than those of the proposed above structures and hence less likely to be formed [cf. 6566]. To calculate the steric energies it was assumed that alginic acid would have a basic triblock GGG-MMM repeating units. Table 5. Calculated steric energy values of various possible structures of alginic acid/otolidine-diamide conjugatesa. Sr. No 1 2 3 4 5 a
Possible products A6,C3-B16,D3 A26,C3-A6,D3 B16,C3-B36,D3 A26,C3-B16,D3 A6,C3-B36,D3
Iterations 411 478 511 574 385
Steric Energy (kcal/mol) 170.43 191.55 179.24 150.76 197.36
Steric energy of alginic acid having the backbone as shown in Figure 4c, with four each of M- and Gacid residues, is 180.04 (Iterations 370). The steric energy was calculated using ChemBiooffice (v 10.0) Chem3D, Molecular mechanics 2 (MM 2) software.
Development of Carbohydrate Polymer Based New Hydrogel Materials…
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The FT-IR spectra of the alginic acid, o-tolidine and alginic acid/o-tolidine conjugate are shown in the Figure 9. The strong IR bands at 1642 cm-1 and 1414 cm-1 in the alginic acid /otolidine conjugate confirmed the formation of amide linkages between alginic acid and otolidine after MW irradiation. Furthermore, near disappearance of IR band at 1700 cm-1 also confirmed addition of o-tolidine onto the carboxylic groups of the alginic acid. 13C-NMR spectra of the alginic acid, o-tolidine and conjugate samples are presented in Figure 10a-c, respectively. The chemical shifts of the 12 carbon atoms (Figure 10a) of the alginic acid repeating unit were comparable with those reported in our previous work [34] and o-tolidine showed chemical shifts for the 7 carbon atoms (Figure 10b), while alginic acid/o-tolidine conjugate showed five additional chemical shifts (Figure 10c) for carbon atoms with fresh aniosotropy. Chemical shift at δ 179.90 indicates formation of amide (-CONH2) linkage between alginic acid and o-tolidine, and the carbon resonance at δ 22.81 was assigned to the methyl carbon of o-tolidine unit. The carboxyl carbon of alginic acid resonated at an upfield value of 174.41 ppm. Three carbon resonances appeared at 127.26, 123.73 and 115.86, which were attributed to o-tolidine carbons, C-4/C-5, C-6 and C-2 respectively by comparison with the values of pure o-tolidine (Table 6). Nine carbon resonances at 79.23, 77.22, 75.47, 70.74, 69.86, 69.33, 68.44, 66.50 and 64.38 were attributed to the carbons of M- and G-units of the alginic acid residue. All the observed chemical shift values as well as their probable assignments are given in the Table 6. The thermograms of parent alginic acid, o-tolidine and conjugate are shown in the Figure 11. In the case of parent alginic acid and o-tolidine the degradation took place in two steps, while conjugate showed a three-step mass loss pattern. The initial weight loss (ranged between 3-16%) in the parent and conjugate may be due to the loss of bound water. On the other hand, the mass losses of 68%, 82% and 50% for the parent alginic acid, o-tolidine and the conjugate at 700oC (Figure 11), indicated that the thermal stability of conjugate was significantly greater than the parent alginic acid and o-tolidine.
984 1037
a 3469 3374 3410
1314 1379
3015 2934
1570
735 1149
1450
683 443 657575 606
881 1262
3338
520
1624 1490
824
%T
b
814 2360
1629 1037
3433
875
669
1727
c
2358
812 1024 642 1559
3469
4000.0
17051646
3000
2000
1414
1500
1000
400.0
cm-1
Figure 9. FT-IR spectra of (a) o-tolidine (b) alginic acid and, (c) Alginc acid/o-tolidine conjugate.
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a
b
c
Figure 10 (a)-(c). 13C NMR of (a) alginic acid (b) o-tolidine, and (c) alginic acid/o-tolidine conjugate.
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110 100 90
Alginic acid/o-tolidine conjugate % Weight retention
80 70 60 50
Alginic acid
40 30
o-Tolidine
20 10 0
100
200
300
400
500
600
700
o
Temperature ( C) Figure 11. TGA graphs of o-tolidine, alginic acid and Alginc acid/o-tolidine conjugate.
The CD spectrum of non-modified alginic acid was fully in the negative region and observed peak and trough values of [θ] -509.12 and [θ] -1652.11 at 200 nm and 214 nm, respectively, were observed. The trend of the spectrum for alginic acid /o-tolidine conjugate was similar having smaller peak (-9.66) and trough (-375.97) values at 197.5 nm and 207.7 nm. The CD spectrum of o-tolidine showed both positive and negative trends (Figure 12a-c). These results clearly indicated variation in the chiroptical properties presumably due to the addition of o-tolidine restricting free rotation of G acid, which have negative [θ] values [cf. 34], in the conjugate. Furthermore, it can also be explained on the basis of peak/trough ratios of the parent and modified polymeric materials. If peak height to trough depth in CD spectrum was 1). The use of the peak-to-trough analysis has been gainfully used to describe conformational changes that occur within polymers or modified polymers with varied chain lengths [64]. The peak-to-trough ratios for the alginic acid and o-tolidine were 0.70 and zero (nil), respectively, while alginic acid / otolidine conjugate showed 0.97, i.e., peak/trough < 1 in all cases (Table 4). The fluorescence emission spectra of the parent alginic acid, o-tolidine and alginic acid /o-tolidine conjugates were recorded using different molar concentrations (10-3 to 10-5M), in 0.5 M Na2CO3, methanol and DM water, respectively. Sharp emission maxima were observed for both o-tolidine and alginic acid/o-tolidine at ca. 388 nm, with significantly lower fluorescence intensity in the latter, indicating diminished efficiency of the fluorophore therein, presumably as a result of quenching through the formation of amide inhibiting transition processes involving the nitrogen π-electrons (Figure 13).
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A. K. Siddhanta, Ramavatar Meena, Gayatri Prasad et al. Table 6. 13C NMR chemicals shift values for alginic acid, o-tolidine and alginic acid/o-tolidine conjugate. Entry Alginic acid
o-Tolidine
Alginic acid / o-tolidine conjugate
a
cf. Meena et al. 2009; bData interchangeable.
δ (ppm) 100.69 71.46 72.45 70.18 76.69 72.49 176.0 100.36 67.35 80.81 72.31 176.38 143.72 132.55 129.10 125.49 123.08 115.86 18.05 179.90 174.41 127.26 123.73 115.86 99.92b 99.31b 79.23 77.22 75.47 70.74 69.86 69.33 68.44 66.50 64.38 22.81
Assignmentsa C- 1 of M-unit C-2 of M-unit Not assigned C-3 of M-unit C- 4 of M-unit C- 5 of M-unit C-6 of M-unit C- 1 of G-unit C- 3 of G-unit C-4 of G-unit C- 5 of G-unit C- 6 of G-unit C-3 C-1 C-5 C-4 C-6 C-2 -CH3 at C-4 -C=O-NH2 -COOH of alginic acid o-tolidine C-4/C-5 o-tolidine C-6 o-tolidine C-2 C-1 of M unit C-1 of G unit Alginic acid C -do-do-do-do-do-do-do-doCH3 at o-tolidine C-4
The X-ray diffraction patterns of parent alginic acid, o-tolidine and alginic acid /otolidine conjugates were depicted in Fig.14. The X-ray diffraction pattern of alginic acid showed that it was amorphous in nature, while that of alginic acid/o-tolidine conjugate
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showed eight distinct sharp peaks at 11o, 17o, 19o, 22o, 25o, 30o, 35o and 37o, indicating induced crystalinity (C.I. 0.53) on the parent polymer, as a result of addition of crystalline otolidine (Figure 14) affording ordered molecular arrangements. Similar observations have been reported for hydrogels based on grafting in our previous work [26, 35]. The scanning electron micrographs (SEM) of o-tolidine, the modified and non-modified alginic acid are depicted in Fig.15a-c. o-Tolidine showed a surface morphology with randomized particulate surface structure. The morphology of non-modified polysaccharide in the SEM image appeared like a sheet dotted with cloud-like mounds (Figure 15a), which can be distinguished easily from the modified polysaccharide conjugate that looked composed, integrated and porous. The pore size of the latter was in the range 1.5-2.0 µm. To our knowledge, this constitutes the first report of the synthesis of a water soluble polyuronate-biaryldiamine conjugate with porous structure, which can be developed into a vehicle for delivery systems as well as for catalytic applications. More studies are required to understand the performance of this compound in solution.
Figure 12 (a)-(c). Circular dichroism (CD) spectra of (a) alginic acid, (b) alginic acid/o-tolidine, and (c) o-tolidine.
A. K. Siddhanta, Ramavatar Meena, Gayatri Prasad et al.
Flourescence Intensity
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Figure 13. Fluorescence spectra of o-tolidine and the conjugate at different concentrations.
Figure 14 (a)-(c). XRD of (a) alginic acid and (b) o-tolidine, and (c) alginic acid/o-tolidine conjugate.
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Figure 15 (a)-(c). SEM images of (a) o-tolidine,(b) alginic acid, and (c) alginic acid/o-tolidine conjugate.
FUTURE PROSPECTS Galactans, arabinogalatan, rhamno-arabino-ribo-galactan, polyuronates and other heteropolymers as well as polysaccharide blends were cross linked with genipin in a waterbased eco-friendly process yielding new polymeric hydrogel materials. Galactan-polyuronic acid blend was grafted with a polymeric substrate to yield a robust hydrogel. The heteropolysaccharides arabinogalactan and rhamno-galacto-ribo-arabinan have yielded genipin crosslinked products with improved swelling functions. The genipin crosslinked heteropolysaccharides of Grateloupia indica were moderately swellable. Nevertheless, on the basis of other efforts in our laboratory (unpublished work), it appears that the genipin cross linked polymeric products with significantly improved properties are best prepared with homopolymers, the heteropolymers are comparatively less amenable to this modification strategy. Further studies with heteropolysaccharides, however, are in order to ascertain this premise. Polysaccharide blends are also a good starting material of choice for the modification work. The cross linking reaction brings about a significant makeover in the chiroptical properties of the polysaccharides. The modified products exhibited considerable thermal stability and pH-responsiveness in aqueous media. The body of work on the development of new materials, described in this article is based on seaweed based carbohydrate polymers obtained from a renewable resource and are biodegradable. These merits coupled with the
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strength of an array of functional properties predispose the materials to sustainable applications in various domains including the ingestible and non-ingestible ones.
ACKNOWLEDGMENT The authors are grateful to the Ministry of Earth Sciences (MoES/9-DS/6/2007-PC-IV), and the Council of Scientific & Industrial Research (CSIR), New Delhi, for funding.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 43
CHITOSANS: NEW VECTORS FOR GENE THERAPY Riccardo A.A. Muzzarelli* Muzzarelli Consulting, Via Volterra 7, IT-60123 Ancona, Italy.
ABSTRACT During the last quinquennium, chitosan has suddenly gained importance as a plasmid vector to be used in gene therapy in alternative to unsafe viral vectors. The main characteristics that qualify chitosan for this role are its spontaneous reactivity with DNA and oligonucleotides in general, the protection of DNA against enzymatic destruction by nucleases, the slow release of DNA, the capacity to penetrate into tissues by opening tight junctions of cells, besides its full biocompatibility with human cells and its safe assimilation in the body. The chitosan chemistry has been refined in order to impart to chitosan better solubility in physiological media, by acting on its cationicity and hydrophilicity / hydrophobicity, or combining it with poly(ethyleneimine), polyethylene glycol and derivatives, or conjugating it with cell-specific ligands. The transfection efficacy of said modified chitosans was greatly improved compared to plain chitosan. In order to understand the fate of internalized vector-DNA complexes within cells, fluorophores including the green fluorescent protein have been conjugated with DNA or with chitosan. After the exceptionally good performances of chitosans as functional wound dressing materials and drug carriers, it appears that the involvement of chitins and chitosans in gene therapy will develop into an additional exceptional achievement for this class of polysaccharides.
INTRODUCTION The Human Genome Project has greatly increased the knowledge about cancer and other gene-based diseases. Changes in the genes were found to be associated with various disorders, some of which were deemed to be the cause of the onset of certain diseases. Gene therapy is considered as a valid approach for the treatment of congenital and acquired *
Corresponding author: E-mail:
[email protected], www.chitin.it, Tel. & Fax +39-071-36206
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diseases by producing bioactive agents or preventing dysfunction of the cells such as uncontrollable proliferation (Austin, 2004;, Cullen & Marshall, 2006; O’Connor & Crystal, 2006; Vogelstein & Kinzler, 2004; Kim & Rossi, 2007; Gao et al., 2008; Roy et al., 2008). The application of gene therapy in the suppression or replacement of malfunctioning genes promises progress in treating diseases more effectively. The non-viral gene transfer methods permit targeting to specific cells. Ultimately, gene therapy will evolve into improvements for a broad range of diseases presently untreatable or poorly managed. While being conceptually straightforward, the efficient expression of foreign genes is a crucial aspect for the success of in vivo gene manipulation or therapy. The first step of gene therapy is the delivery of the therapeutic genetic material to the interior of a living cell for its subsequent expression or for inhibition of a specific gene function, taking into account that the negatively charged cellular membrane repels the genes due to their anionic nature. The complexity of the biological system represents an obstacle to successful gene delivery: the natural immune system is ready to degrade the foreign gene as soon as it is introduced. Once within the cell, the genetic material has to escape intracellular degradation to reach and enter the nucleus (Medina-Kauwe et al.,2005; Rolland, 2006; Davis, 1997). Thus, the choice of a vector, that will protect the genetic material and convey it to the interior of the intended cell type is paramount, particularly in the case of inherited diseases: the goal of applying gene therapy remains a very difficult one to achieve. Nevertheless good perspectives exist even in the reversal of course of acquired disorders of the central nervous system, such as spinal cord injury, stroke and degenerative diseases i.e. Alzheimer's and Parkinson's: dormant cells like stem and progenitor cells can be genetically induced to differentiate into various neuronal cell types that could replenish areas impaired or undergoing neurodegeneration. The gene transfer in animals can be achieved either by directly introducing the gene-vector conjugate into the animal, or after neuronal cells in culture are transfected with the desired gene in a preliminary step and then the transfected cells are reintroduced into the animal.
NON-VIRAL VECTORS The gene therapy depends on the availability of delivery vectors. Although viral vectors display rather good transfection properties, several drawbacks, including the lack of specificity toward target cells, limitation in the size of inserted DNA, high production costs, and safety concerns (risk of immunogenicity and chromosomal insertion of viral genome) greatly limit their practical applications. Early non-viral techniques involved the direct injection of naked DNA through the use of electrical impulses (electroporation), or bombardment with gold particles (gene gun), to force them across cellular membranes. However, these methods were inefficient, localized, and scarcely versatile. Non-viral carriers were developed using cationic lipids, polymers, carbon nanotubes, metal nanorods, and ceramic- and silica-based nanoparticles (Nishikawa & Huang, 2001; Roy et al., 2003, 2005, 2008; Mintzer & Simanek, 2009). The non-viral gene vectors have been proposed as safer alternatives to viral vectors because they can be administered repeatedly with minimal host immune response, are stable in storage, and can be produced in large quantities. Cationic polymers have gained increasing
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attention because they mediate transfection via condensing DNA into particles that protect DNA from enzymatic degradation and facilitate cell uptake and endolysosomal escape (Dang & Leong, 2006). They include poly(ethyleneimine), poly(L-lysine), polybrene, gelatin, tetraminofullerene, and poly(L-histidine)-g-poly(L-lysine). Although their polyelectrolyte complexes have some advantages over viral vectors, e.g. low immunogenicity and easy manufacture, several negative aspects such as toxicity, incomplete biodegradability, scarce biocompatibility and low tranfection efficiency delay their practical use (Liu & Yao, 2002; Kumar et al., 2004; Mintzer & Simanek, 2009). With the anionic genetic materials, these vectors form polyelectrolyte complexes, sometimes endowed with modest positive charge, that greatly reduce the enzymatic degradation of the genetic materials and are often readily taken up by cells. The biochemistry of the cellular uptake of such complexes is probably mediated by the electrostatic interaction of the nanocomplex with the anionic cellular membrane (Mounkes et al., 1998). Once inside the cell the complex unwinds, setting the genetic material free to execute its function. The lack of toxicity being the major demand in the design of new gene delivery systems, the development of vectors has been focused on biocompatible systems and natural polymers. As a non-viral vector for gene delivery, chitosan offers outstanding advantages such as absence of virally induced inflammation, absence of cytotoxicity, absence of immunological reactions, absence of oncogenic effects, compatibility with soft tissues, controllable biodegradability, high stability and reasonable cost (Muzzarelli, 1977, 2009a,b,c,d,e; Muzzarelli & Muzzarelli, 2005, 2006; Hejazi & Amiji, 2003; Li & Zhang, 2005). Of course, when the vector is cationic per se, as in the case of chitosan, the advantages gained are most evident, because the cationicity is accompanied by other favorable characteristic properties: chitosan protects DNA from degradation by DNase I and II and transfects into different cell types. A further attractive feature of chitosan is the capacity to assume various forms generally suitable for supporting the DNA action in gene delivery: microspheres and nanoparticles are convenient due to their uniform morphology and high transfection of cells. Moreover, there has been growing interest recently in the use of porous materials provided that they offer stability and uniformity of porous structures, tunable pore size and well defined surface properties. These desirable properties allow the encapsulation or adsorption of a particular gene and its release in a reproducible and more predictable manner. Moreover, a porous structure can support cell attachment and can hold/release DNA to induce the formation of new tissue. Consequently, it degrades (or even better, it is resorbed) gradually and provides 3-dimensional structural guide in the formation and regeneration of tissue, therefore, combining the concepts of gene delivery and tissue engineering. It should be noted, however, that the intrinsic properties of a particular chitosan sample depend on structural variables such as the degree of acetylation, the degree of substitution and the average molecular weight. Compared with other amines and polyamines such as polyethylenimine and poly(L-lysine), the primary amino group of the glucosamine unit has an exceptionally low pKa value of 6.5, that means that chitosan at pH 7.4 tends to become insoluble because its protonation is low: thus, the pH of the transfection medium is important because it affects the transfection efficiency of the chitosan-DNA complexes. In said complexes the N/P ratio (molar ratio of chitosan nitrogen to DNA phosphorous) is also an important parameter. Various articles described the preparation and in vitro efficiency of chitosan gene transfection systems, and illustrated the capacity of plain chitosan itself to act as a vector. For
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example, plain chitosan nanoparticles with size ranges of 40-200 nm and 40-80 nm were observed to complex DNA: plasmid DNA pGL3 encoding firefly luciferase and pCH110 encoding beta- galactosidase were used as reporter genes. For transfection, human embryonal kidney cells and Chinese hamster ovary cells were used. Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection showed a sustained expression of the luciferase gene for about 10 days, whilst commercial transfection reagents lasted only 2 days. No toxic effect on the mammalian cells was seen with chitosan (Li et al., 2003). In fact, early reports underlined that chitosan is useful as a vector for gene delivery. The level of transfection with plasmid/chitosan complexes was found to be highest when the molecular mass of chitosan was 40 or 84 kDa, the N/P ratio was 5, and the transfection medium contained 10% serum at pH 7.0 (Ishii et al., 2001). Similarly, Romoren et al. (2003) found that the in vitro transfection in Epithelioma papulosum cyprini cells was affected by the DNA concentration in the complexes as well as the molecular weight and degree of acetylation of the chitosans. Also Weecharangsan et al. (2006) proposed the chitosan lactate salt for in vitro gene transfection, considering the ease of preparation of polymer/DNA complexes and their low cytotoxicity. Reference data on common chitosan salts are useful to assess the characteristics of complexes made by modified chitosans and DNA. Complexes formulated with several chitosan salts were assessed for complexing ability toward DNA, transfection efficiency in Chinese hamster ovary cells, and their effect on cell viability. Chitosan hydrochloride, lactate, acetate, aspartate and glutamate (chitosan MW 45 kDa) formed complexes with pcDNA3CMV-Luc. Gel electrophoresis documented that complexes formed at N/P ratios above 2 with all chitosan salts. The transfection efficiency depended on the anion, on the chitosan MW, and on the N/P ratio: maximum transfection efficiencies were at N/P ratio of 12, 12, 8, 6 and 6 for said salts, respectively. The MTT assay indicated that all chitosan/DNA complexes had low cytotoxicity (Weecharangsan et al., 2008).
ENHANCED PERFORMANCES OF CHITOSAN VECTORS The following are alternative approaches intended to enhance the performances of chitosan as a vector and its transfection efficiency as well: •
Quaternization of chitosan via trimethylation of amino groups or reaction with compounds containing the pre-formed quaternary ammonium. These derivatives were originally developed for the enhancement of epithelial penetration, particularly in the drug delivery field (Thanou et al., 2002). According to DiColo et al. (2008) they belong to a class including polycations [chitosan and its quaternary ammonium derivatives, poly(L-arginine), aminated gelatin], polyanions [N-carboxymethyl chitosan, poly(acrylic acid)], and thiolated polymers [carboxymethyl cellulosecysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates (Lee et al., 2007)]. The methylated / ethylated quaternary chitosans were studied for the delivery of insulin by Sadeghi et al. (2008). Other quaternary ammonium chitosan derivatives were proposed for the promotion
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of intraocular drug absorption (Zambito et al., 2007). A method for obtaining N,N,Ntrimethylchitosan has been developed using dimethylsulfate as the methylation agent (DeBritto et al., 2007). Pegylation, i.e. reactions with polyethylene glycol and derivatives. In general, PEG facilitates the formation of polyplexes with improved solubility, lower surface charge, diminished aggregation, lower cytotoxicity (Park et al., 2001). With polyethylene glycol-graft-trimethyl chitosan, cellular uptake was increased 8.5-fold compared to chitosan polyplexes resulting in up to 678-fold increased transfection efficiency in NIH/3T3 cells. Apart from reduction of the cytotoxicity, PEGylation led to improved colloidal stability of polyplexes and significantly increased cellular uptake compared to plain trimethyl chitosan. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan (Germershaus et al., 2008). A biodegradable graft copolymer of chitosan-graft-poly(ethyleneimine)-graftpolyethylene glycol having a hydroxyl group at the PEG chain end was prepared by grafting chitosan, poly(ethyleneimine) and commercially available heterobifunctional PEGs for constructing amphiphilic copolymers (Wu et al., 2008). Stearic acid-grafted oligomeric chitosan was found suitable as an intracellular drug carriers in the form of micelles, and was further modified with polyethylene glycol (Hu et al., 2008). Macrophages stimulated with vaccine-loaded pegylated chitosan microspheres gradually secreted tumor necrosis factor alpha and nitric oxide, suggesting that pegylated chitosan microspheres are very good vaccine delivery systems (Jiang et al,. 2007). Chitosan derivatives carrying either the octyl group or the PEG moiety on the amino group, and sulfate on the hydroxyl groups, formed 100-130 nm micelles potentially suitable for brain-targeting (Yao et al., 2007). Methoxy-omegasuccinimidylpolyethylene glycol was conjugated to the surface of chitosan-DNA complexes so that the PEGylated complexes mediated higher gene expression in tumors than plain chitosan-DNA. Analogous data were obtained with the aid of poly(lactic acid) particles (Zhang et al., 2007; Chen et al., 2007). Linking or conjugating cell-specific ligands (Kim et al., 2004). To overcome the limitations of chitosans related to low cell specificity, transferrin-, folate-, mannoseand galactose-conjugated chitosans have been designed and evaluated for receptormediated endocytotic gene delivery (Mansouri et al., 2006; Mao et al., 2001; Zhang et al., 2006; Chan et al., 2007; Gao et al., 2003; Kim et al., 2004, 2005, 2006). Galactosylated chitosans have been reported as gene carriers targeting hepatocytes, due to specific ligand–receptor interactions between galactose moieties and asialoglycoprotein receptors. The latter are present with high density only on hepatocytes, and are retained on several human hepatoma cell lines. Folate conjugation may improve gene transfection efficiency due to promoted uptake of folate by receptor bearing tumor cells. Chan et al. (2007) synthesized and characterized folate-polyethylene glycol-grafted chitosan for targeted plasmid DNA delivery to tumor cells. The folate-bearing chitosan was deemed to be a good gene carrier due to its solubility at physiological pH, efficiency in condensing DNA, targeting ability, and low cytotoxicity. When the graft degree of folate was 12.5 %, the chitosan folate conjugate exhibited high storage capacity for nitric oxide (up to
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Riccardo A. A. Muzzarelli 84.3 nanomol NO per mg, that made it interesting fro selective delivery of NO to cancer cells (Wan et al., 2008) Deoxycholic acid covalently linked to chitosan permitted to prepare dispersions and to reduce the aggregation of chitosan complexes and to improve the interactions with cell surfaces, as confirmed with other hydrophobic moieties such as stearic acid and alkyl chains: the transfection efficiency of the latter were 5- to 7-fold higher than for plain chitosan (Kim et al., 2001; Pang et al., 2007; Pasanphan et al. 2008; Pasanphan & Chirachanchai, 2008). Other hydrophobic chitosans have been described such as the taurocholate chitosan (Muzzarelli et al., 2006) but not tested for this purpose yet. Urocanic acid-chitosan showed good DNA binding ability, high protection of DNA from nuclease attack, and low cytotoxicity. The buffering capacity and the transfection efficiency of chitosan into 293T cells were much enhanced after coupling with urocanic acid. (Kim et al., 2003). The use of urocanic acid-chitosan in aerosol was found to be compatible with noninvasive in vivo gene therapy (Jin et al., 2008). Poly(ethyleneimine) has been shown to condense plasmids into colloidal particles that can effectively transfect a variety of cells, both in vitro and in vivo, due to its buffering capacity; however, many studies raised concern regarding the toxicity of poly(ethyleneimine). Chitosan-graft-poly(ethyleneimine) exhibited enhanced solubility at physiologic pH values, low cell toxicity and superior transfection efficiency compared to chitosan, but limited cell specificity (Kim et al., 2004, 2005, 2006; Mansouri et al., 2006; Mao et al., 2001; Zhang et al., 2006; Chan et al., 2007; Gao et al., 2003; Park et al., 2001).
It should be underlined that substantial modification of the polycation structure interferes with both DNA condensation and uptake by cells; extended quaternization of chitosan might also lead to increased cytotoxicity. The sensitivity to pH and ionic conditions may be a reason for the highly variable efficacy of chitosan-based gene delivery systems reported in the literature. Although most chitosans are able to compact DNA into nanosized polyelectrolyte complexes, the stability and properties of the latter strongly depend on the structural variables of chitosans. Whereas plain high molecular-weight chitosans have been found to possess low gene delivery efficacy both in vitro and in vivo, optimization of transfection protocols, and tailoring of degree of acetylation, average MW and polydispersity yield highly efficient gene vectors in vitro and in vivo. A balance between DNA protection and intracellular DNA release should therefore be a key to satisfactory transfection with chitosans. Exceedingly high stability constants of polymer-DNA complexes appear to be a major difficulty for intracellular release of DNA. It was reported that the ability of polymer-DNA complexes to escape the endolysosomal compartment could be correlated to the buffering capacity of the polycation in the pH range of 5-7 (Lu et al., 2008). More specifically, optimal transfection efficiency of chitosan polyplexes can be achieved between pH 6.8 and 7.0. Above pH 7.5, DNA was shown to dissociate from the complex, thus preventing cellular uptake and transfection efficiency. Below pH 6.5, cellular uptake was significant but transfection efficiency was low, possibly due to hindered endosomal release (Ishii et al., 2001; Sato et al., 2001; Mintzer & Simanek, 2009).
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Certain peptide sequences known as protein transduction domains or membrane translocalization signals were introduced into chitosan for the delivery of plasmid DNA (Tung & Weissleder, 2003; Futaki, 2005). These sequences usually contain positively charged amino acid residues such as arginine and lysine, which have been reported to be able to enhance transportation into cells (Eguchi et al., 2004; Nakanishi et al., 2003). Oligoarginine conjugates demonstrated the characteristics similar to cell penetrating peptides in cell translocation, and the transfection efficiency in Hela cells could be highly improved by conjugating oligomeric arginine to PEGylated lipids (Furuhata et al., 2006).
FLUOROPHORES Many fluorophores have been linked to and conjugated with DNA or the carrier itself before or after transfection. Fluorescein isothiocyanate was one of the widely employed fluorophores for labeling cationic polymers, including polyethyleneimine and chitosan, because of high quantum yields. However, continuous exposure to light leads to fluorescence quenching, that hinders monitoring over protracted periods of time. Furthermore, those fluorophores failed to determine precisely whether fluorophore-labeled molecules were dissociated or not, because the signals from a multiplicity of fluorophore often overlapped, making it impossible to locate each molecule exactly. Luminescent semiconductor nanocrystals, or quantum dots, have received much attention because of their unique optical properties, that make them potentially applicable as imaging materials. These properties include high quantum yield, broad absorption spectra, narrow emission spectra and resistance to photo-bleaching, thus quantum dots have been conjugated to DNA and proteins, to detect the proteolytic activities of caspase-1, thrombin, collagenase and chymotrypsin against dyelabeled substrates. Fluorescence resonance energy transfer (FRET) is a phenomenon that transfers energy from an excited donor to an acceptor by a long-range dipole–dipole interaction, suitable for measuring the proximity and orientation of biomolecules. Because FRET occurs only when donor–acceptor distances are 80 °C Growth 60–80°C 15–60 °C 1g 9 Low pH-loving Cannot tolerate O2 Tolerates O2 deprivation Requires O2 Survive in high sugar environments Able to tolerate high concentrations of metal Tolerates toxic and xenobiotic chemicals like benzene
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Halophilic or halotolerant organisms can grow over a wide range of salt concentrations (2 to 5 M NaCl) by two strategies. One is based on the intracellular accumulation of salts at concentrations close to those in the outside medium, while the other is based upon exclusion of salts from the cytoplasm to a large extent and production or accumulation of organic solutes such as ectoine and glycine-betaine, to balance the osmotic pressure of the medium [46, 70]. Acidophiles and alkalophiles retain the intracellular pH at a value close to neutrality by the very low permeability of their membranes to protons. Whereas high levels of polyunsaturated fatty acids are found in the membranes of some psychrophiles, some UV radiation-resistant radiophiles either have UV-absorbing pigments (scytonemin) in the sheath that surrounds the cell, or accumulate UV-absorbing pigments (mycosporine-like amino acids) within their cytoplasm, to protect their DNA from radiation damage [13]. Hyperthermophiles that grow at temperatures above 100 °C are all Archaea since their lipids contain ether bonds that are far more stable than the ester bond-based bacterial and eukaryal lipids. Also the monolayer membrane in which the glycerol moieties at both sides of the membrane are bridged by covalent bonds confer Archaea with additional stability towards high temperatures [54]. Besides all the above mentioned adaptation strategies, microbially-produced extracellular polysaccharides are also known to be involved in protecting cells from numerous external stresses [72, 45]. Exopolysaccharide (EPS) is a term first used by Sutherland (1972) to describe high molecular weight, structurally diverse carbohydrate polymers produced by many marine bacteria [67]. Since then, EPS has also been used to indicate more broadly defined extracellular polymeric substances [45]. Different classes of EPS can be distinguished based upon the mechanism of biosynthesis and the precursors required. The first class comprises the extracellularly produced homo-polysaccharides like dextran, levan and mutan. The polymerization reaction in these cases proceeds via extracellular glycosyltransferases, which transfer a monosaccharide residue from a disaccharide to a growing polysaccharide chain. The other categories comprises homo- and hetero- polysaccharides with (ir)regular repeating units that are synthesized from intracellular sugar nucleotide precursors. Some of these sugar nucleotides serve as precursors for EPS biosynthesis. However, they are also involved in the biosynthesis of several cell wall components and can therefore be considered essential for growth [9]. EPSs can absorb water and form a highly hydrated matrix. The presence of such a gelled polysaccharide layer around the cell may affect the diffusion of compounds both into and out of the cell, and hence act as a layer of protection to cells against toxic compounds [8, 24], very high acidity [2], UV radiation [71] or digestion by other organisms [12]. The specific role of the EPS depends on the natural environment of the microorganism. Changes in pH and salinity have little effect on the viscosity and stability of the EPS layer. Besides their buffering capacity, EPSs may also reduce the rate of water loss during desiccation and assist with water uptake during rehydration. Polymers made up of fructose residues (fructans) are known to be involved in the protection of plants during drought, salt or cold stress by preventing membrane damage [57]. High concentrations of EPS with high polyhydroxyl content would decrease the freezing point of water in the low temperature, high salinity brine channels and hence act as a cryoprotectant. For example EPS from a fungal strain, Phoma herbarum, isolated from Antarctic soil provides a cryoprotective role in the harsh Antarctic environment where the availability of liquid water and temperatures are extremely low [65].
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Microbial EPSs serve countless ecological functions like stabilizing marine sediments and preventing erosion, improvement of water holding capacity of soil, detoxification of heavy metals and removal of solid matter from water reservoirs [7]. They form biofilms and aggregates facilitating intracellular interaction and biochemical exchange between organisms [47, 73]. EPSs also contribute to various physiological activities in human beings as antitumor, anti-viral, and anti-inflammatory agents, and can act as inducers for interferon, platelet aggregation inhibition, and colony stimulating factor synthesis [29].
EXTREMOPHILES AS EPS PRODUCERS Because of their many interesting physicochemical and rheological properties with novel functionality, microbial EPSs in general act as new biomaterials and find wide ranges of applications in many industrial sectors like textiles, detergents, adhesives, microbial enhanced oil recovery (MEOR), wastewater treatment, dredging, brewing, downstream processing, cosmetology, pharmacology, and food additives [29]. On the other hand, EPSs can act as an emulsifier and bioflocculant, and they have the function of sorption of inorganic ions which constitute metabolic elements for bacteria. The microorganisms used as industrial or technical producers of extracellular polysaccharides are chiefly the bacteria. Species of Xanthomonas, Leuconostoc, Pseudomonas, Alcaligenes which produce xanthan, dextran, gellan, curdlan are the best known and most industrially used. Actually, much attention is lately accorded to the EPSs produced by lactic acid bacteria which are already accepted as GRAS (Generally Recognised As Safe) and are most adequate for the food industry. Fungal polysaccharides are still somewhat limited, with pullulan from Aureobasidium pullulans and scleroglucan produced by Sclerotium glucanicum being the most known and already obtained at technical scales. While dextran (synthesized by certain lactic-acid bacteria such as Leuconostoc mesenteroides and Streptococcus mutans) was the first microbial polysaccharide to be commercialized and to receive approval for food use, several such polymers now have a variety of commercial uses [68]. Xanthan gum (the EPS from Xanthomonas campestris pv. campestris bacterium) is already well established by modern biotechnology and has a sizable market due to its exceptional qualities as a rheology control agent in aqueous systems and as a stabilizer for emulsions and suspensions [60]. Another example is pullulan (a fungal EPS produced by Aureobasidium pullulans), which has been used extensively in the food industry and as a pharmaceutical bulking agent for more than 20 years. Only recently, there is an attempt to explore the potential uses of this non-toxic, immunoceutical, non-mutagenic and non-carcinogenic EPS for various applications such as targeted drug and gene delivery and surface modification [55]. Until now, very few biopolymers have been produced on a commercial basis due to two major facts. Firstly, their production costs are much higher when compared with chemically synthesized polymers that possess similar material characteristics. Consequently, much effort has been devoted to the development of processes for biopolymer production by optimizing the upstream to downstream engineering strategies including the metabolic and cellular engineering of host cells, efficient fermentation and recovery processes, and post-production modification of the biopolymers obtained [30]. The second fact is that from a variety of
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microbial polysaccharides characterized so far, only those with physicochemical properties similar to those of plant (cellulose, pectin and starch) and seaweed (alginate and carrageenan) are used in industry [62]. Although many researchers claim to have found polysaccharides ‘of superior viscosity to xanthan’, in reality few match the robustness of the Xanthomonas polysaccharide and few can maintain their physical properties in the presence of salts, at higher temperatures or extremes of pH [30]. Therefore, most research is focused on the identification of EPS producing extremophiles with the idea that as these microorganisms survive environmental extremes of desiccation, temperature, pressure, salinity, acidity, heavy metals, and radiation, it is to be expected that their biopolymers will also have some unique properties to adapt to such extreme conditions. Since 1991, EPS producing extremophilic microorganisms have been isolated from deepsea hydrothermal vents characterized by extreme pressure and temperature, high concentrations of H2S and heavy metals. These include some bacteria such as Alteromonas macleodii sub spec fijiensis, Vibrio diabolicus, Alteromonas infernos [53, 58], Thermotoga maritime [56] and the psychrophile Pseudoalteromonas sp. SM9913 [31]. EPS producing bacteria from marine Mediterranean shallow vents were also described [41, 64]. EPS produced by moderately halophilic bacteria of the genus Halomonas that include Halomonas maura [4], Halomonas ventosae and Halomonas anticariensis [39], Halomonas eurihalina F2-7 [38], Halomonas cerina [21], Halomonas sp. GT-83 strain [19], the haloalkalitolerant Halomonas alkaliantarctica sp. [50] and the halophilic levan-producer Halomonas sp. AAD6 [51] strains. The two haloalkalophilic microorganisms, Bacillus sp. I-450 [29] and Salinivibrio costicola subsp. alcaliphilus [59] were also reported as good EPS producers. Additional examples include two halophilic Cyanobacteria, namely Cyanothece sp. 113 [14] and Aphanocapsa halophytica [40] and also Halobacterium sp. SM5 [34]. High molecular weight glucan produced by the thermophile Geobacillus tepidamans V264 isolated from a Bulgarian hot spring has been attributed with promising biotechnological applications due to its unusual stability and good biological activity [25]. Other EPSs isolated from thermophilic bacteria include EPS with high immunomodulatory and antiviral activity isolated from Geobacillus thermodenitrificans B3-72 strain [3], EPSs from Bacillus thermoantarcticus isolated from Mount Melbourne, Antarctica [36], Anoxybacillus amylolyticus isolated from Mount Rittmann, Antarctica [49] and Streptococcus thermophilus strains [52]. EPS production is not that common among Archaea, however, the halophilic Haloferax and Haloarcula and thermophilic Thermococcus are examples of producer genera. Whereas Haloferax gibbonsii and Haloferax denitrificans are known to produce a neutral and an acidic EPS, respectively, sulfated EPSs have been isolated from Haloferax mediterranei and Haloarcula japonica [44]. EPS production by the hyperthermophilic archaeon Thermococcus litoralis has been reported by Rinker and Kelly (2000). Although less is known about eukoaryotic life in extreme environments in comparison to prokaryotic extremophiles, advances in genomics and in comprehensive, high-throughput metabolic profiling techniques have provided new insight into the metabolic adaptations of eukaryotes living under extreme conditions. Recently, isolation and chemical characterization of EPSs produced by the halophilic micro-algea Dunaliella salina under salt stress were reported and were found to make emulsions with stability comparable to bacterial EPSs [42]. Duan et al. (2008) proposed a pathway for pullulan biosynthesis by the halotolerant
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Aureobasidium pullulans Y68 yeast strain and also reported their latest studies on the improvement of EPS yield. Monitoring the diversity, colonization rates, and seasonal variations of eukaryotic organisms in Rio Tinto, an extremely acidic environment (pH ca. 2) in Spain, revealed that biofilm formation by the two acidophiles Dunaliella and Cyanidium provided them nutritional advantages and less severe surrounding conditions [2].
APPLICATION AREAS OF EPSS FROM EXTREMOPHILES Commercially, the most important microbial EPS is xanthan gum, a complex polymer that is very efficiently produced by the phytopathogen Xanthomonas campestris and has been used for decades in food and non-food applications. Acetan is structurally related to xanthan and is produced by Acetobacter xylinum, a strain that is used in the food industry for the production of a sweet confectionery (Nata) and vinegar. Sphingans, capsular polysaccharides secreted by Sphingomonas strains, including gellan, wellan, rhamsan, and sphingan S-88, have special rheological properties and can be used in foods as gelling agents, stabilizers or suspending agents. In the dairy industry, EPS-producing lactic acid bacteria (LAB), including the genera Streptococcus, Lactobacillus and Lactococcus, are used in situ to improve the texture of fermented dairy products such as yoghurt and cheese. Numerous studies have been conducted for developing and implementing innovative technology to clean up contamination with petroleum hydrocarbons [26]. Bioemulsifiers are able of emulsifying these pollutants much more effectively than do chemical surfactants. Halophilic EPS producers are considered as an interesting source for microbial enhanced oil recovery (MEOR) where polymers act as emulsifiers and mobility controllers. Active emulsification of petroleum has been noted for six strains, close to Halobacterium salinarium, Haloferax volcanii, and Halobacterium distributum [27]. Martínez-Checa et al. (2007) studied the characteristics of the bioemulsifier V2-7 synthesized by strain F2-7 of Halomonas eurihalina and they found that it has the ability of emulsifying a wide range of hydrocarbons i.e. n-tetradecane, n-hexadecane, n-octane, xylene mineral light and heavy oils, petrol and crude oil. Flocculating agents are generally categorized into three major groups, namely, inorganic flocculants such as aluminum sulfate and polyaluminum chloride (PAC); organic synthetic polymer flocculants such as polyacrylamide derivatives and polyethylene; and naturally occurring biopolymer flocculants such as chitosan and algin. Among these, organic synthetic polymers are the most widely used flocculants, since they are the most economical and highly effective. However, the use of these flocculants sometimes causes environmental and health problems, because they are not readily biodegradable and some of their degraded monomers, such as acrylamide, are neurotoxic and even potent human carcinogens. On the other hand, naturally occurring biopolymers (bioflocculants), which are produced by microorganisms during their growth, have special advantages such as safety, produce a strong effect, are biodegradable and harmlessness to humans and the environment, so they may potentially be applied in drinking and wastewater treatment, downstream processing, and fermentation processes [33, 18]. In recent years, many bioflocculant-producing microorganisms including bacteria, fungi and actinomyces have been reported to produce extracellular polymeric substances, such as
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polysaccharides, functional proteins and glycoprotein, which function as bioflocculant. Flocculants produced by a haloakalophilic Bacillus sp. I-471 [28], a psychrophilic bacterium Pseudoalteromonas sp. SM9913 [31] are polysaccharides. Moreover, Li et al. (2008) reported that EPS SM9913 showed better flocculation performance than an inorganic aluminum based flocculant at low temperature (5 – 15 °C) or in high-salinity (5 –100 %) water suggesting its probable use as an effective flocculant for wastewater treatment at low temperature and/or salinity. However, only few of these bioflocculants have been practically applied in industry because of their low flocculating capability and large dosage requirement. To utilize bioflocculants widely in industrial fields, it is desirable to find various microorganisms with high bioflocculant-producing ability and improve the flocculating efficiency of the bioflocculant [74]. EPSs also have the function of sorption of inorganic ions which constitute metabolic elements for bacteria. Their composition is complex but EPS are mainly composed of polysaccharide, protein, humic substances, uronic acids, nucleic acids and lipids, containing ionizable functional groups such as carboxyl, phosphoric, amine, and hydroxyl groups. These functional groups represent potential binding sites for the sequestration of metal ions. It is assumed that metal biosorption involves a physicochemical interaction between the metal and functional groups on the cell surface, based on physical adsorption, ion exchange, complexation and precipitation. Moreover, metal biosorption performance depends on external factors, such as pH, other ions in bulk solutions (which may be in competition), organic material in bulk solution and temperature. These properties are potentially of great importance in sewage treatment processes for the removal of toxic heavy metal pollutants [15, 32, 22, 69]. Though highly unusual in bacterial polysaccharides, EPSs of halophilic bacteria Halomonas ventosae and Halomonas anticariensis were found not only to have a high capacity for binding cations but also to incorporate considerable quantities of sulfates [39]. Removal of vanadate by biosorption with Halomonas sp. GT-83 [43] and considerably high heavy metal binding capacity of biofilms produced by eukaryotic extremophiles isolated from acidic environments in Rio Tinto (SW, Spain) were also reported [2]. The importance of various EPSs for pharmaceutical purposes has a long historical background and increased considerably during the last decades. Many interesting areas have opened in the past which include their role in drug delivery, in wound treatment, in cancer therapy, and the diagnosis, prevention, and treatment of bacterial and viral diseases. Some polysaccharides form integral components of vaccines, usually when coupled to a suitable protein. Thus, meningitis vaccines have been prepared in this way and multivalent polysaccharide vaccines have been formulated against Streptococcus pneumoniae and Klebsiella spp. However, these are expensive to prepare and only use very small amounts of material. Possibly of much greater significance is the role of certain microbial EPSs in tumour suppression and immunostimulation. A homopolymer named ‘scleroglucan’ or schizophyllan, from several fungal species, adopts a triple helix conformation that strongly influences biological activity and appears to be very effective against some types of cancers when applied in the ordered, triple helical form. These β-linked glucans are therefore the subject of much current study and have already been tested clinically in Japan, proving effective against certain types of tumors [20, 68]. Dextran, although no longer used as a food ingredient, is the base from which the ‘Sephadex’ range of biochemical adsorbents is prepared. Dextran solutions can also be used as a plasma substitute, being very poorly antigenic and having the correct physical properties
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[68]. Polysaccharides also contribute to various physiological activities in human beings as antitumor, antiviral, and anti-inflammatory agents, and can act as inducers for interferon, platelet aggregation inhibition, and colony stimulating factor synthesis [29]. Recently there is an attempt to explore pullulan for various biomedical applications including targeted drug and gene delivery and tissue engineering due to its non-toxic, nonimmunogenic, non-mutagenic and non-carcinogenic nature. The early observation on this exopolymer was made by Bauer in 1938 and this exopolysaccharide was named pullulan by Bender et al in 1959 [5, 6]. Pullulan and its derivatives can be used as a denture adhesive. It can also be used for pharmaceutical coatings, including sustained release formulations. Novel preparations such as tablets, pills, granules or the like, which contain pullulan in the sugar layer serve the purpose of preventing brownish color change of the composition. Oral care products have been commercialized based on pullulan films. The colorless, transparent and edible pullulan film has also attracted a great deal of interest for other uses such as a nonpolluting wrapping material [66, 55].
CONCLUSION It is widely accepted that extremophiles offer important insights into biology and evolution of many organisms, and they provide a valuable resource for exploitation in novel biotechnological processes. Despite the vast amount of extremophiles that are being isolated and identified so far, literature reports concerning their EPSs are still very limited. In recent years, significant progress has been made in discovering and developing novel and functional EPSs from extremophilic producer strains. These natural, non-toxic and biodegradable polymers show considerable diversity in their composition and structure. There is a huge gap between our knowledge of the relationship between the composition and structure of EPSs and the ability to predict their physical and health-promoting properties and thus their potential applications. Such knowledge is essential to increase the range of biopolymers with desirable functions and the present gap seems to be filled in the near future due to the current interest in glycobiology and the application of new analytical methods. Microbial EPS production and the structure, composition and viscosity of EPSs are greatly influenced by fermentation conditions, such as the type of strain, composition of the nutrient medium, pH, temperature, oxygen concentration and agitation. Hence, by manipulating the producer microorganism, feedstock and process conditions, fermentation allows a wide and reproducible range of different biomaterials with very good control over their characteristics. Currently, EPSs can be produced under controlled conditions to the specifications required at industrial levels. For example, xanthan, dextran and pullulan are well known industrial microbial polysaccharides with numerous applications and a considerable and sizable market. However, when compared with the synthetic polymers, natural origin polymers still represent only a small fraction of the current polymer market, mostly due to their costly production processes. Therefore, much effort has been devoted to the development of cost-effective and environmentally-friendly production processes such as investigating the potential use of cheaper fermentation substrates. Considering the wide spread use of these biopolymers in various industries as well as their potential applications in novel biotechnological processes, more studies need be
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conducted not only to minimize the production costs, but also to elucidate their production mechanisms, chemical structure and rheological properties.
ACKNOWLEDGMENTS Hande Kazak gratefully acknowledges TUBITAK (Project 108M193) for a Fellowship.
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Andrade, C. M. M. C., Pereira, N. Jr., & Antranikian, G. (1999). Extremely thermophilic microorganisms and their polymerhydrolytic enzymes. Rev Microbiol, 30, 287-298. [2] Aguilera, A., Souza-Egipsy, V., Martin-Uriz, P. S. & Amils, R. (2008). Extracellular matrix assembly in extreme acidic eukaryotic biofilms and their possible implications in heavy metal adsorption. Aquat Toxicol, 88, 257-266. [3] Arena, A., Gugliandolo, C., Stassi, G., Pavone, B., Iannello, D., Bisignano, G. & Maugeri, T. L. (2009). An exopolysaccharide produced by Geobacillus thermodenitrificans strain B3-72: Antiviral activity on immunocompetent cells. Immunol Lett, 123, 132-137. [4] Arias, S., Ferrer, M. R., del Moral, A. E. & Quesada V Béjar Mauran (2003). An exopolysaccharyde produced by the halophilic bacterium Halomonas maura, with a novel composition and interesting properties for biotechnology. Extremophiles, 7, 319324. [5] Bauer, R. (1938). Physiology of Dematium pullulans de Bary. Zentralbl Bacteriol Parasitenkd Infektionskr Hyg Abt2, 98, 133-167. [6] Bender, H., Lehmann, J. & Wallenfels, K. (1959). Pullulan, an extracellular glucan from Pullularia pullulans. Biochim Biophys Acta, 36, 309-316. [7] Bender, J. & Phillips, P. (2004). Microbial mats for multiple applications in aquaculture and bioremediation. Bioresour Technol, 94, 229-238. [8] Bitton, G. & Friehofer, V. (1978). Influence of extracellular polysaccharides on the toxicity of copper and cadmium towards Klebsiella aerogenes. Microb Ecol, 4, 119125. [9] Boels, I. C., van Kranenburg, R., Hugenholtz, J., Kleerebezem, M. & de Vos, W. M. (2001). Sugar catabolism and its impact on the biosynthesis and engineering of exopolysaccharide production in lactic acid bacteria. Int Dairy J, 11, 723-732. [10] Brock, T. D. (1978). Thermophilic microorganisms and life at high temperatures, NewYork, USA, Springer-Verlag. [11] Buenger, J. & Driller, H. (2004). Ectoin: An Effective Natural Substance to Prevent UVA-Induced Premature Photoaging. Skin Pharmacol Physiol, 17, 232-237. [12] Caron, D. A. (1987). Grazing of attached bacteria by heterotrophic microflagellates. Microb Ecol, 13, 203-218.
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[13] Castenholz, R. W. & Garcia-Pichel, F. (2000). Cyanobacterial responses to UVradiation. In Ecology of Cyanobacteria: Their Diversity in Time and Space. B. A. Whitton, & M. Potts, (Eds.), Kluwer Academic Publishers, Dordrecht. [14] Chi, Z., Su, C. D. & Lu, W. D. (2007). A new exopolysaccharide produced by marine Cyanothece sp. 113. Bioresour Technol, 98, 1329-1332. [15] Comte, S., Guibaud, G. & Baudu, M. (2008). Biosorption properties of extracellular polymeric substances (EPS) towards Cd, Cu and Pb for different pH values. J Hazard Mater, 151(1), 185-193. [16] Demirjian, D. C., Morís-Varas, F. & Cassidy, C. S. (2001). Enzymes from extremophiles. Curr Opin Chem Biol, 5(2), 144-51. [17] Duan, X., Chi, Z., Wang, L. & Wang, X. (2008). Influence of different sugars on pullulan production and activities of α-phosphoglucose mutase, UDPGpyrophosphorylase and glucosyltransferase involved in pullulan synthesis in Aureobasidium pullulans Y68. Carbohydr Polym, 73, 587-593. [18] Gao, J., Bao, H. Y., Xin, M. X., Liu, Y. X., Li, Q. & Zhang, Y. F. (2006). Characterization of a bioflocculant from a newly isolated Vagococcus sp. W31. J Zhejiang Univ Sci B, 7(3), 186-192. [19] Ghazvini, P. T. M. & Mashkani, S. G. (2009). Effect of salinity on vanadate biosorption by Halomonas sp. GT-83: Preliminary investigation on biosorption by micro-PIXE technique. Bioresour Technol, 100, 2361-2368. [20] Giese, E. C., Dekker, R. F. H., Barbosa, A. M. & da Silva, R. (2008). Triple helix conformation of botryosphaeran, a (1→3;1→6)-β-d-glucan produced by Botryosphaeria rhodina MAMB-05. Carbohydr Polym, 74, 953-956. [21] González-Domenech, C. M., Martínez-Checa, F., Quesada, E. & Béjar, V. (2008). Halomonas cerina sp. nov., a moderately halophilic, denitrifying, exopolysaccharideproducing bacterium. Int J Syst Evol Microbiol, 58, 803-809. [22] Guibaud, G., Tixier, N., Bouju, A. & Baudu, M. (2003). Relation between extracellular polymer’ composition and its ability to complex Cd, Cu and Pb. Chemosphere, 52, 1701–1710. [23] Horikoshi, K. & Grant, W. D. (1998). Extremophiles - Microbial Life in Extreme Environments, Wiley-Liss (eds), New York. [24] Jeanthon, C. & Prieur, D. (1990). Susceptibility to heavy metals and characterization of heterotrophic bacteria isolated from two hydrothermal vent polychaete annelids, Alvinella pompejana and Alvinella caudata. Appl Env Microbiol, 56, 3308-3314. [25] Kambourova, M., Mandeva, R., Dimova, D., Poli, A., Nicolaus, B. & Tommonaro, G. (2009). Production and characterization of a microbial glucan, synthesized by Geobacillus tepidamans V264 isolated from Bulgarian hot spring. Carbohydr Polym, 77, 338-343. [26] King, R. B., Long, G. M. & Sheldon, J. K. (1998). The Field Guide, CRC Press LLC, Florida. [27] Kulichevskaya, I. S., Milekhina, E. I., Borzenkov, I. A., Zvyagintseva, I. S. & Belyaev, S. S. (1998). Oxidation of petroleum hydrocarbons by extremely halophilic archaeabacteria. Microbiology, 60, 596-601. [28] Kumar, C. G., Joo, H. S., Kavali, R., Choi, J. W. & Chang, C. S. (2004a). Characterization of an extracellular biopolymer flocculant from a haloalkalophilic Bacillus isolate. World J Micro Biotech, 20(8), 837-843.
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[29] Kumar, C. G., Joo, H., Choi, J., Koo, Y. & Chang, C. (2004b). Purification and characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp. I450. Enzyme Microb Technol, 34, 673-681. [30] Lee, S. Y., Park, S. J., Park, J. P., Lee, Y. & Lee, S. H. (2002). Economic aspects of biopolymer production. Biopolymers, 10(2), 307-337. [31] Li, W. W., Zhou, W. Z., Zhang, Y. Z., Wang, J. & Zhu, X. B. (2008). Flocculation behavior and mechanism of an exopolysaccharide from the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913. Bioresour Technol, 99, 6893-6899. [32] Liu, Y., Lam, M. C. & Fang, H. H. P. (2001). Adsorption of heavy metal by EPS of activated sludge. Water Sci Technol, 43, 59-66. [33] Lu, W., Zhang, T., Zhang, D. Y., Li, C. H., Wen, J. P. & Du, L. X. (2005). A novel bioflocculant produced by Enterobacter aerogenes and its use in defecating the trona suspension. Biochem Eng J, 27(1), 1-7. [34] Lungmann, P., Choorit, W. & Prasertsan, P. (2007). Application of statistical experimental methods to optimize medium for exopolymer production by newly isolated Halobacterium sp. SM5. Electron J Biotechnol, 10(1). [35] MacElroy, R. D. (1974). Some comments on the evolution of extremophiles. Biosystems, 6, 74-75. [36] Manca, M. C., Lama, L., Improta, R., Esposito, E., Gambacorta, A. & Nicolaus, B. (1996). Chemical composition of two exopolysaccharides from Bacillus thermoantarcticus. Appl Environ Microbiol, 62, 3265-3269. [37] Margesin, R. & Schinner, F. (2001). Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles, 5, 73-83. [38] Martínez-Checa, F., Toledo, F. L., El Mabrouki, K, Quesada , E. & Calvo, C. (2007). Characteristics of bioemulsifer V2-7 synthesized in culture media added of hydrocarbons: Chemical composition, emulsifying activity and rheological properties. Bioresour Technol, 98, 3130-3135. [39] Mata, J. A., Béjar, V., Llamas, I., Arias, S., Bressollier, P., Tallon, R., Urdaci, M. C. & Quesada, E. (2006). Exopolysaccharides produced by the recently described halophilic bacteria Halomonas ventosae and Halomonas anticariensis. Res Microbiol, 157, 827835. [40] Matsunaga, T., Sudo, H., Takemasa, H. & Wachi, Y. (1996). Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytica immobilized on light-diffusing optical fibers. Appl Microbiol Biotechnol, 45, 24-27. [41] Maugeri, T. L., Gugliandolo, C., Caccamo, D., Panico, A., Lama, L., Gambacorta, A. & Nicolaus, B. (2002). A halophilic thermotolerant Bacillus isolated from a marine hot spring able to produce a new exopolysaccharide. Biotechnol Lett, 24, 515-519. [42] Mishra, A. & Jha, B. (2009). Isolation and characterization of extracellular polymeric substances from micro-algae Dunaliella salina under salt stress. Bioresour Technol, 100, 3382-3386. [43] Mohammad Ghazvini, P. T. & Mashkani, S. G. (2009). Effect of salinity on vanadate biosorption by Halomonas sp. GT-83: Preliminary investigation on biosorption by micro-PIXE technique. Bioresour Technol, 100, 2361-2368. [44] Nicolaus, B., Schiano Moriello, V., Lama, L., Poli A. & Gambacorta, A. (2004). Polysaccharides from extremophilic microorganisms. Orig Life Evol Biosph, 34, 159169.
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[45] Nichols, C. A. M., Guezennec, J. & Bowman, J. P. (2005). Bacterial polysaccharides from extreme marine environments with special consideration of the Southern Ocean, sea ice, and deep-sea hydrothermal vents: a review. Mar Biotechnol, 7(4), 253- 271. [46] Oren, A. (2000).Life at high salt concentrations. In Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K. H. & Stackebrandt, E. (Eds.), The Prokaryotes:A Handbook on the Biology of Bacteria:Ecophysiology and Biochemistry, (3rd edn.,263282) New York, Springer-Verlag. [47] Paerl, H. W. (1976). Specific associations of the bluegreen algae Anabaena and Aphanizomenon with bacteria in fresh water blooms. J Phycol, 12, 432-435. [48] Podar, M. & Reysenbach, A. L. (2006). New opportunities revealed by biotechnological explorations of extremophiles. Curr Opin Biotechnol, 17, 250-255. [49] Poli, A., Esposito, E., Lama, L., Orlando, P., Nicolaus, G., de Appolonia, F., Gambacorta, A. & Nicolaus, B. (2006). Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica). Syst Appl Microbiol, 29, 300-307. [50] Poli, A., Esposito, E., Orlando, P., Lama, L., Giordano, A., de Appolonia, F., Nicolaus, B. & Gambacorta, A. (2007). Halomonas alkaliantarctica sp. nov., isolated from saline lake Cape Russell in Antarctica, an alkalophilic moderately halophilic, exopolysaccharide-producing bacterium. Syst Appl Microbiol, 30, 31-38. [51] Poli, A., Kazak, H., Gürleyendağ, B., Tommonaro, G., Pieretti, G., Toksoy Öner, E. & Nicolaus, B. (2009). High level synthesis of levan by a novel Halomonas species growing on defined media. Carbohydr Polym, 78, 651-657. [52] Purwandari, U., Shah, N. P. & Vasiljevic, T. (2007). Effects of exopolysaccharideproducing strains of Streptococcus thermophilus on technological and rheological properties of set-type yoghurt. Int Dairy J, 17, 1344-1352. [53] Raguénès, G. H. C., Peres, A., Ruimy, R., Pignet, P., Christen, R., Loaec, M., Rougeaux, H., Barbier, G. & Guezennec, J. G. (1997). Alteromonas infernus sp. nov., a new polysaccharide-producing bacterium isolated from a deep-sea hydrothermal vent. J Appl Microbiol, 82, 422-430. [54] Rainey, F. A. & Oren, A. (2006). Methods in Microbiology, vol. 35, Extremophiles. New York: Academic Press. [55] Rekha, M.R. & Sharma, C.P. (2007). Pullulan as a Promising Biomaterial for Biomedical Applications: A Perspective. Trends Biomater Artif Organs, 20(2). [56] Rinker, K. D. & Kelly, R. M. (2000). Effect of carbon and nitrogen sources on growth dynamics and exopolysaccharide production for the hyperthermophilic archaeon Thermococcus litoralis and bacterium Thermotoga maritima. Biotechnol Bioeng, 69, 537-547. [57] Ritsema, T. & Smeekens, S. (2003). Fructans: beneficial for plants and humans. Curr Opin Plant Biol, 6, 223-230. [58] Roger, O., Kervarec, N., Ratiskol, J., Colliec-Jouault, S. & Chevolot, L. (2004). Structural studies of the main exopolysaccharide produced by the deep-sea bacterium Alteromonas infernus. Carbohydr Res, 339(14), 2371-2380. [59] Romano, I., Gambacorta, A., Lama, L., Nicolaus, B. & Giordano, A. (2005). Salinivibrio costicola subsp. alcaliphilus subsp. nov., a haloalkaliphilic aerobe from Campania Region (Italy). Syst Appl Microbiol, 28(1), 34-42.
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[60] Rosalam S. & England R. (2006). Review of xanthan gum production from unmodified starches by Xanthomonas comprestris sp. Enzyme and Microb Tech, 39(2), 197-207. [61] Rothschild, L. J. & Mancinelli, R. L. (2001). Life in extreme environments. Nature, 409, 1092-1101. [62] Ruas-Madiedo, P. & de los Reyes-Gavilan, C. G. (2005). Invited Review: Methods for the Screening, Isolation, and Characterization of Exopolysaccharides Produced by Lactic Acid Bacteria. J Dairy Sci, 88, 843-856. [63] Satyanarayana, T., Raghukumar, C. & Shivaji, S. (2005). Extremophilic microbes: Diversity and perspectives. Curr Sci, 89(1), 78-90. [64] Schiano Moriello, V., Lama, L., Poli, A., Gugliandolo, C., Maugeri, T. L., Gambacorta, A. & Nicolaus, B. (2003). Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas. J Ind Microbiol Biotechnol, 30, 95-101. [65] Selbmann, L., Onofri, S., Fenice, M., Federici, F. & Petruccioli, M. (2002). Production and structural characterization of the exopolysaccharide of the Antarctic fungus Phoma herbarum CCFEE 5080. Res Microbiol, 153, 585–592. [66] Singh, R. S., Saini, G. K. & Kennedy, J. F. (2008). Pullulan: Microbial sources, production and applications. Carbohydr Polym, 73, 515-531. [67] Sutherland, I. W. (1972). The exopolysaccharides of Klebsiella serotype 2 strains as substrates for phage-induced polysaccharide depolymerases. J Gen Microbiol, 70(2), 331-8. [68] Sutherland, I. (2002). A sticky business. Microbial polysaccharides: current products and future trends. Microbiol Today, 29, 70-71. [69] Thomas, A.D., Volesky, B. & Mucci, A. (2003). A review of the biochemistry of heavy metal biosorption by brown algae. Water Res, 37, 4311-4330. [70] Ventosa, A., Nieto, J. J. & Oren, A. (1998). Biology of Moderately Halophilic Aerobic Bacteria. Microbiol Mol Biol Rev, 62, 504-544. [71] Wang, H., Jiang, X., Mu, H., Liang, X. & Guan, H. (2007). Structure and protective effect of exopolysaccharide from P. Agglomerans strain KFS-9 against UV radiation. Microbiol Res, 162(2), 124-129. [72] Wharton, D. A. (2002). Life at the Limits: Organisms in extreme environments, Cambridge University Press, Cambridge. [73] Wotton, R. S. (2004). The utiquity and many roles of exopolymers (EPS) in aquatic systems. Sci Mar, 68, 13-21. [74] Yükselen, M. A. & Gregory, J. (2004). The reversibility of floc breakage. Int J Miner Process, 73(2-4), 251-259.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 45
ALGINATE: A PROMISING POLYSACCHARIDE FOR DELIVERY OF ESSENTIAL OILS Pranee Lertsutthiwonga,*, Ian S. Haworthb and Pornchai Rojsitthisakc a
Metallurgy and Materials Science Research Institute, Chulalongkorn University, Bangkok, 10330, Thailand. b Department of Pharmacology & Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, 90089-9121, USA. c Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
ABSTRACT Naturally occurring polysaccharides are of interest in pharmaceutical and cosmetic applications due to their biodegradability, biocompatibility and non-toxicity. Among these polysaccharides, alginate, a linear polysaccharide that is extracted from marine brown algae, is widely used as a particulate carrier for encapsulation, controlled release and delivery of a variety of bioactive compounds to target organs. Due to its bioadhesive, anionic and colloidal properties, alginate can interact with divalent cations or cationic polymers to form hydrogel micro- or nanoparticles that have specificity for mucosal tissues. A particular area of interest is the use of alginate as a shell or matrix material for encapsulation of essential oils. These oils have therapeutic properties that include antibacterial, antifungal, antiviral, antioxidant, and insect-repellent effects, but are insoluble in water, volatile and unstable in certain environments. This has limited development of new formulations and encapsulation of the oil in carriers is required to overcome these problems. Size reduction of the carriers to the nanometer scale greatly improves the efficacy of the delivery system and may allow targeting of the pharmacological activity of the oil to specific organs. In this review, we focus on an overview of alginate and essential oils, the preparation of alginate matrices from large to nanometer scale, and encapsulation of essential oils in these matrices. The prospects for *
Corresponding author: Tel.: +662 218 4221; Fax: +662 611 7586; E-mail address:
[email protected] 1312
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development of improved alginate nanoparticles as delivery systems for essential oils are discussed at the end of the chapter.
1. INTRODUCTION Naturally occurring polymers have attracted interest in the field of pharmaceutics due to their non-toxicity, biocompatibility, biodegradability, bioadhesion and good film formation [1-5]. These characteristics are useful for pharmaceutical applications such as dosage formulation and drug delivery systems. The natural polymers are mainly polysaccharides or carbohydrates such as starch, methylcellulose, dextran, arabinogalactan, pullulan, cyclodextrin, mannan, heparin, hyaluronic acid, pectin, carboxymethylcellolose, alginate and chitosan [6-9]. These molecules are classified into three categories: non-ionic, anionic and cationic polymers, as shown in Figure 1. In formulation development, polysaccharides have long been used as excipients in dosage forms, including as binders, disintegrants and film coating agents in tablets. In liquid dosage forms, they serve as viscosity and flow control agents in solutions, suspensions and emulsions, and as suspending agents in suspensions. Polysaccharides and their derivatives have also been used for delivery of drugs, cosmeceuticals and neutraceuticals in the form of beads, microparticles and nanoparticles. Polysaccharides behave as drug carriers by coating, encapsulating or embedding active compounds in their matrices. Among the polysaccharides, alginate has considerable potential as a polymeric material for development of drug delivery systems. Alginate is widely used as a particulate carrier for encapsulation, controlled release and targeted delivery of bioactive compounds due to its bioadhesive, anionic and colloidal properties. Alginate can be crosslinked with divalent cations or cationic polymers to form hydrogels that have many advantages, including development of micro- or nanoparticles to allow controlled release of active compounds and avoidance of use of hazardous organic solvents. In this review, we focus on the use of alginate matrices for encapsulation of essential oils. Essential oils or complex mixtures of volatile and lipophilic substances extracted from different parts of plants have been extensively used in food, pharmaceuticals and cosmetics due to their antibacterial, antifungal, antiviral, antioxidant, and insect-repellent activities [1012]. However, essential oils are insoluble in water, volatile, and unstable in some environments such as in the presence of light or oxygen [13]. Therefore, a suitable delivery system to transport essential oils to biological targets is required to maintain or enhance their therapeutic properties, and use of a polymeric matrix for entrapment and delivery of essential oils has potential in this respect. Essential oil delivery systems focused on naturally occurring polysaccharide-based carriers are of increasing interest due to their excellent in vitro and in vivo characteristics [14-21]. The carriers are more attractive if their size is reduced to a nanometer scale, since this significantly improves the efficiency of the delivery system and may allow the encapsulated materials to reach the biological targets.
Alginate: A Promising Polysaccharide for Delivery of Essential Oils CH2OH
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CH2OH O
O
OH
OH
O
O OH
OH O CH2OH
CH2 O
CH2OH O
OH
O
O
O
OH
OH
OH
O
O
OH
OH OH
Starch O
HO
O O
OH CH2OCH3
HO OH
CH2OCH3
O
O O
OH
O
HO O
O
OH
HO
OH
OH OH
O
OH
O
Methylcellulose OH
HO OH
O O
OH
O OH
CH2 O OH O
OH
CH2
OH
O
O
OH
OH O
O
OH OH
OH
Dextran O
OH
O
CH2OH O O
CH2
O
O
O
OH O
OH
Cyclodextrin
HO
OH
OH
Arabinogalactan
OH
OH
O CH2
O
CH2
CH2OH O
O
OH
O
OH
OH
OH
OH
O
OH
OH
OH
O
OH
O
CH2OH
O
O
H2C
OH
O
Pullulan
OH
OH
OH
Mannan
(a) Figure 1. Chemical structures of polysaccharides used in the pharmaceutical field: (a) non-ionic, (b) anionic and (c) cationic polymers.
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(b)
(c) Figure 1. (Continued)
Examples of polysaccharide applications for preparing beads, microparticles and nanoparticles containing essential oils are summarized in Table 1. It is noteworthy that alginate is the most popular polysaccharide for delivery of essential oils. Beads produced from alginate with an appropriate crosslinking agent can be used for encapsulation of Artemisea arborescens L essential oil with good yields, and allow controlled release of the oil [22]. Chang and Dobashi [15] suggested that a maximal eucalyptus oil content of 90-92% in alginate complex capsules could be obtained using preparation parameters above critical values of 1% w/v sodium alginate, 2% w/v calcium chloride, and a 20-min crosslinking time. We have achieved encapsulation of turmeric oil in alginate nanocapsules prepared by a threestep procedure of emulsification, crosslinking with calcium chloride, and solvent removal [3]. Malone and Appelqvist [23] showed that encapsulation of oil droplets within biopolymergelled particles in the size range of 70-5000 μm could be used to control release of lipophilic volatile compounds, with the rate of release depending on the oil phase volume, particle size and partition coefficient of the volatile compound. Landfester et al. [24] suggested that size reduction with uniformity could be achieved by sonication.
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These studies indicate that naturally occurring polymers are outstanding materials for formulation of oily compounds. Within the scope of this review, we focus on an overview of alginate and essential oils, and various techniques for preparation of alginate matrices from large to nanometer in scale. Encapsulation of essential oils in alginate matrices is also reviewed, and the prospects for use of alginate for development of essential oil delivery systems are highlighted at the end of the chapter. Table 1. Formulation of essential oils in polysaccharide-based matrices. Essential oils Artemisia arborescens L. essential oil Eucalyptus oil
Turmeric oil
Volatile citronella essential oil Zanthoxylum limonella essential oil
Major components Camphor β-Thujone Chamazulene 1,8-Cineole α-Pinene p-Cymen ar-Turmerone α- Turmerone β-Turmerone d-Limonene Citronellal Sabinene, Limonene, Terpinen-4-ol
Formulations
Activities
References
alginate bead
antiviral
[22]
alginate complex capsules
antibacterial
[15]
alginate nanocapsules, chitosan-alginate nanocapsules
antibacterial, antifungal, insect repellent
[3,69]
chitosan microcapsules
bug repellent, deodorizing
[16]
glutaraldehyde crosslinked gelatin microcapsules
mosquito repellent
[12]
2. ALGINATE Alginate is a naturally occurring anionic polymer that is mainly isolated from marine brown algae. Alginate is a family of linear polysaccharides composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M) residues joined by 1,4-glycosidic linkages (Figure 2) [2526]. The M and G residues are arranged in a pattern of blocks along polymer chains that vary in composition and sequence [27-30]. For example, alginate prepared from stipes of old Laminaria hyperborea kelp contains large numbers of G residues whereas alginate extracted from Ascophyllum nodosum has more M residues [28-29]. In molecular terms, the pattern of blocks along the chain of alginate can be MM… and GG… homopolymer blocks or mixed blocks containing irregular sequences of M and G units (Figure 2) [27]. Alginate forms soluble salts with monovalent metal ions, of which sodium alginate is the major form in current use. On the other hand, alginate forms gels or precipitates with divalent or multivalent cations such as Ca2+, Ba2+ and Sr2+, but not with Mg2+ [21]. Although Ba2+ and Sr2+ are able to produce stronger alginate gels than Ca2+ [27], they are rarely used because of their toxicity. Due to the stronger affinity of G residues for divalent ions compared to M residues, the gel produced from alginate that is rich in G residues is more rigid, brittle and porous, and much less opaque, whereas the gel that is rich in M residues is weak with high
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turbidity and greater extensibility, but less porosity. These physical properties cause the alginate gel with a higher G content to adopt an “egg-box structure”, whereas that with the higher M content is more randomly packed [31]. Alginate gels of different morphologies can be prepared, including large beads (> 1 mm in diameter), microparticles (< 0.2 mm in diameter) [28] and nanoparticles (10-1000 nm in diameter) [8,32]. Alginate is of interest as a drug delivery system owing to its excellent biocompatibility, mucoadhesion, non-immunoginicity, non-toxicity, gelation and film formation properties [3334]. Alginate also has other unique properties that have enabled its use as a matrix for encapsulation or delivery of a variety of active compounds. These properties include a high gel porosity that can be controlled by simple coating procedures; no requirement for organic solvents during the encapsulation process; and dissolution and biodegradation under normal physiological conditions [28].
Figure 2. Chemical structures of alginate: homopolymer blocks (GGGG and MMMM) and mixed blocks (MGMM and GGMG).
3. ESSENTIAL OILS Essential oils are limpid, volatile and lipophilic mixtures of natural compounds with strong and unique odors that are biosynthesized by aromatic plants as secondary metabolites [35]. Essential oils are soluble in organic solvents and immiscible with water, and generally have a lower density than that of water [10]. The compounds found in essential oils are mainly alcohols, ketones, aldehydes and esters, which account for their physicochemical
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properties, and are categorized into monoterpenes (e.g. citronellol, thymol), sesquiterpenes (e.g. farnesol, caryophyllene), aromatic compounds (e.g. cinnamaldehyde, eugenol, safrole) and terpenoides (e.g. ascaridole, menthol). The chemical structures of compounds found in essential oils are shown in Figure 3.
Figure 3. Chemical structures of compounds found in essential oils.
The quality, quantity and composition of essential oils are dependent upon the plant source and the preparation method. The amounts of essential oils found in plant raw materials vary with the plant organ, age, climate and soil composition [10,36]. The preparation methods include steam distillation, solvent extraction or cold pressing of different parts of aromatic plants, including flowers, leaves, stems, buds, seeds, twigs, fruit, roots, bark or seed. In addition to the use of volatile organic solvents for extraction, liquid carbon dioxide, supercritical carbon dioxide (SC-CO2), and SC-CO2 combined with co-solvent addition have been used [35,37-38]. In nature, essential oils contain 20-60 components at different concentrations and only 2-3 major components at high concentrations [10]. For example, turmeric oil extracted from Curcuma longa L. by SC-CO2 is composed of ar-turmerone, αturmerone and β-turmerone as major components, all of which play a role in its therapeutic properties [38]. The major components found in the important essential oils and their chemical structures are presented in Table 1 and Figure 4.
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O
-Thujone
Camphor
Chamazulene
O
-Pinene
1,8-Cineole
O
O
O
ar-Turmerone
p -Cymene
CH2
-Turmerone
-Turmerone
O
Citronellal d -Limonene
OH
Sabinene
Terpinen-4-ol
Limonene
Figure 4. Chemical structures of major components of important essential oils.
As secondary plant metabolites, essential oils possess biological activities that include antibacterial [39-41], antifungal [42-44], antiviral [45], antiparasitic [46-47], insect-repellent [48-49], antioxidant, antimutagenic [50] and anticarcinogenic [51] properties. The specific advantage of essential oils appears to be the synergistic effects of their compounds, based on the increased activity when applied as the natural essential oil compared to individual components. The activity of the main components may also be modulated by minor components [10], and Jaki et al. [52] found a “purity-activity relationship” in showing that ursolic acid of higher purity has less activity against Mycobacterium tuberculosis. Essential oils show cytotoxicity against many organisms that decompose agricultural and food products, and against human and animal pathogens and parasites. In bacteria, essential oils can pass through the cell wall and cytoplasmic membrane, and destroy the structure of
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different layers of polysaccharides, phospholipids and fatty acids, leading to leakage of macromolecules or fluid from the cell and resulting in cell death. In eukaryotic cells, essential oils can damage the cellular and organelle membranes and act as prooxidants on proteins [10]. Some essential oils, such as Citrus bergamia essential oil, contain photoactive molecules that can be activated under light exposure, and consequently these oils have potential phototoxicity [53]. The mechanism of phototoxicity differs from that of cytotoxicity because phototoxic essential oils penetrate the cell without destroying membranes or biomolecules, and radical reactions only occur when the cells are exposed to light, resulting in production of phototoxic substances. However, cytotoxicity and phototoxicity are both dependent on the molecules in the essential oils and their compartmentalization in cells, with production of different types of radicals with or without light exposure [10]. These properties make essential oils of importance in pharmaceutical and cosmetic applications, particularly in antifungal and antibacterial applications of the oils or component molecules. However, formulation of essentials oils is required to achieve efficacy and stability, and alginate provides a basis for delivery of these oils.
4. PREPARATION OF ALGINATE MATRICES 4.1. Alginate Beads As discussed above, alginate can be crosslinked with divalent or multivalent cations to form particulate gels. In general, alginate beads with a diameter > 1 mm can be prepared by transferring sodium alginate solution into a burette or syringe, followed by dropping through a needle into a divalent crosslinking solution under gently agitation [54-56]. The formed beads are then cured in the crosslinking solution for a short period of time (usually minutes) before rinsing with distilled water [28]. For encapsulation, an active compound is mixed with the alginate solution before dropping into the crosslinking solution [15,28]. The size of the beads depends on the stirring rate of the reactor and the viscosity or concentration of alginate: the higher the speed, the lower the average bead size [57]. To obtain beads that are larger and more spherical, a higher concentration of alginate must be used in the process [58]. In addition, water loss during the drying process at room temperature can lead to a decrease in bead size [22]. Martinsen et al. [59] suggested that the physical properties of alginate beads are also dependent on the composition, sequential structure and molecular size of the alginate. Beads with the lowest shrinkage, highest mechanical strength and highest porosity are made from alginate with a G-content > 70% and an average length of G blocks of higher than 15. The gel strength is independent of the molecular weight for alginate with a molecular weight > 240,000 Da. Anal and Stevens [1] proposed a multi-coating technology for prolongation of drug release from the alginate bead. Multilayer beads produced by dropping an ampicillinalginate mixture into a calcium-chitosan coagulation fluid, followed by incubation in chitosan, sodium alginate and calcium chloride, respectively, resulted in prolongation of the release of ampicillin by about 60% compared to single layer beads in simulated gastric fluid
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for 4 h. The rate of release can also be controlled by changing the chitosan concentration in the coagulation fluid. Lai et al. [22] incorporated essential oil in alginate beads using calcium chloride or glutaraldehyde as a crosslinking agent, and found that the type of crosslinking agent and the drying process affected the stability and loading efficiency. To get a high loading capacity and good stability, the crosslinking agent must not react with the incorporated essential oil. Chang and Dobashi [15] discovered that the oil leached from the capsules during the hardening process at 37°C, and that the rate of release of oil depended on the concentrations of sodium alginate and calcium chloride, and the crosslinking time. The optimal conditions for preparation of capsules containing a maximum content of eucalyptus essential oil of about 90% were found to be 1% w/v sodium alginate, 2% w/v calcium chloride, and a 20-min crosslinking time. Calcium alginate beads are degraded by removal of calcium ions using a chelating agent such as lactate, citrate and phosphate or by a high concentration of ions such as Na+ or Mg2+ [60]. As the Ca2+ ions are removed, the crosslinking in the gel decreases and the gels are destabilized, which can lead to leakage of entrapped material.
4.2. Alginate Microparticles Microencapsulation of bioactive compounds is of growing importance in pharmaceutical and cosmetic applications. Formulations based on microparticles have the goal of masking of odors and unpleasant tastes, controlled release of drugs, and protection of drugs from body fluids such as gastric juice [18,61]. Microparticles are usually prepared using polymers as the shells or matrices, and the polymers of choice must be biodegradable and biocompatible [62]. Alginate microparticles including microbeads, microcapsules and microspheres can be prepared using several techniques as follows.
Atomization Atomization or spray-drying is a common technique for microparticle preparation, in which sodium alginate containing an active compound is loaded into a syringe mounted on a syringe pump and delivered through an atomization device with an orifice of defined diameter (∼ 1 mm) at the tip. Much smaller diameter orifices can be used, but may be clogged by the high viscosity alginate solution. The size of particles depends upon the size of the nozzle, the flow rate of the syringe pump, atomization pressure, inlet air temperature, and the distance between the orifice and the surface of the crosslinking solution [28,63]. Microparticles form as soon as the alginate is crosslinked with divalent ions in the solution. Ionotropic gelation In this method, an alginate polymer solution containing drug is extruded dropwise through a needle with an internal diameter of 0.2 mm into a crosslinking agent such as CaCl2 or CaCl2-chitosan solution under magnetic stirring. The resultant microparticles are stirred for an additional 15 min to allow the alginate gel to stabilize, and are then washed with deionized water. Particles can be stored in a desiccatior under vacuum and dried until constant weight [64].
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Coacervation Coacervation has become a promising microencapsulation technology and is typically used for encapsulation of flavor oils because of the high achievable payloads of up to 99% [65-66]. In principle, coacervation is a basic process of capsule-wall formation in a three-step procedure: phase separation of hydrocolloids from the initial solution, deposition of the coacervate phase around the active compound that is emulsified or suspended in the same reaction, and hardening of the shell of the hydrocolloid with an appropriate chemical [67]. Coacervation is a colloidal phenomenon that starts with a solution of a colloid in an appropriate solvent, and then changes in temperature, pH or addition of a second substance result in reduction of the solubility of the colloid and separation of a large part of the original colloid into a new phase [65]. The original one-phase system becomes two phases: one richer and the other poorer in colloid concentration. The colloid-rich phase in a dispersed state appears as amorphous liquid droplets that can then be solidified by deposition of wall materials, followed by isolation of the microcapsules. Other techniques Drug leaching occurs during preparation of alginate microparticles, and many techniques have been developed to overcome this problem [25]. Crosslinked alginate has more capacity to retain the entrapped drug and mixing of alginate with other polymers such as pectin, chitosan and neutral gums has been found to solve the problem of drug leaching. Alginate forms strong complexes with polycations such as chitosan, glutaraldehyde, and poly-L-lysine [68-71], and can also be blended with methylcellulose to form microspheres using water-inoil emulsification and crosslinking with glutaraldehyde [14]. Ribeiro et al. [72] incorporated lipophilic drugs into alginate microspheres using a multiple-phase emulsion technique, in which the drug is dissolved in oil such as soybean oil and the resulting alginate microspheres contain immobilized drug-containing oil microdroplets. The microspheres can be further coated to facilitate pH-dependent release. The smaller microparticles show the higher release rate because of their large specific surface area [16].
4.3. Alginate Nanoparticles Favorable properties of biocompatibility, biodegradability, pH sensitivity, and mucoadhesion make alginate biopolymers a good choice as carriers for a drug delivery system. These properties are most interesting if the size of the carriers is in the nanometer range. By general consensus, nanoparticles vary in size from 10 to 1000 nm [8], and this size makes them very useful for delivery of an encapsulated or entrapped drug to a target organ. Rajaonarivony et al. [70] prepared alginate nanoparticles by adding calcium chloride to sodium alginate solution, followed by poly-L-lysine. The concentrations of sodium alginate and calcium chloride were lower than those required for gel formation, and corresponded to formation of a pre-gel state. The size of the particles was strongly dependent on the order of addition of calcium and poly-L-lysine to the sodium alginate solution. This phenomenon can be attributed to the difference in the nature of interactions between calcium and alginate and between poly-L-lysine and alginate, since formation of the nanoparticles probably occurs during addition of the first component to the sodium alginate solution.
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De and Robinson [68] suggested that formation of nanospheres requires a low calcium chloride to alginate mass ratio (< 0.2) to form a negatively charged, calcium alginate pre-gel that is subsequently enveloped by the positively charged poly-L-lysine or chitosan. The cationic polymers further restrict cooperative binding between calcium and alginate ions. When the calcium concentration exceeds the pre-gel concentration, the calcium ions cooperatively bind the alginate molecules, thereby preventing sufficient envelopment of the alginate polymer by cationic poly-L-lysine. We have proposed a method for preparation of alginate nanocapsules for encapsulation of essential oils [3]. In this study, turmeric oil was used as a model essential oil and the formulation was prepared in a three-step procedure using emulsification, cross-linking with calcium chloride, and solvent removal. The size of the turmeric oil-loaded nanocapsules depended on the type of solvent, oil concentration, oil/alginate mass ratio, and sonication. In the optimal preparation process, turmeric oil was dissolved in ethanol and Tween 80® with an appropriate time of sonication. Bouchemal et al. [73] also found that oil droplets are unstable unless the surfactant has adsorbed to a sufficient extent. In addition, the formation of a polymer membrane at the surface of the oil droplets improves the colloidal stability of the suspension, in agreement with Fallouh et al. [74]. Although the turmeric oil-loaded alginate nanocapsules showed good physical stability at 4°C, they aggregated at 25°C [3]. This problem was solved by solidifying the surface of the alginate nanocapsules using chitosan [69]. Additionally, chitosan with a low molecular weight was required to produce small nanocapsules. At a fixed chitosan/alginate mass ratio of 0.1:1, addition of chitosan after calcium chloride was optimal for improving the physical stability and recovery of turmeric oil.
5. ENCAPSULATION OF ESSENTIAL OILS IN ALGINATE MATRICES Lai et al. [22] suggested that a high loading capacity of essential oil into alginate beads could be obtained through application of appropriate techniques and crosslinking agents, including the condition that the crosslinking agent must not react with the essential oil. In this work, calcium chloride and glutaraldehyde were used as crosslinking agents. Over a period of 3 months, glutaraldehyde reacted with some components of Artemisia arborescens L essential oil, with a consequent change in the composition of the oil. Use of glutaraldehyde as a crosslinking agent was concluded to be inappropriate due to an interaction with azulene derivatives and a low degree of matrix crosslinking. In contrast, calcium alginate beads showed in vitro controlled release of the essential oil for 24 h. As discussed above, Chang and Dobashi [15] found that a 90% eucalyptus oil content could be obtained in formulations with 1% w/v sodium alginate, 2% w/v calcium chloride and a 20-min of crosslinking time. Calcium alginate beads can be prepared under simple and mild conditions, but drug loss can occur during bead preparation by leaching through the pores of the beads [75], as also found by Bouchemal et al. [73], in which the loss of α-tocopherol from nanocapsules corresponded to oil leaching at the washing step. We have found that 45% of turmeric oil is lost during formation of turmeric oil-loaded nanocapsules, with 10% and 2225% of the total oil lost during the solvent removal and separation processes, respectively [3]. This loss causes a low loading capacity of turmeric oil in alginate nanocapsules. Teixeira et al.
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[76] have suggested that a drug with low affinity for the polymer might diffuse from the organic phase to the external aqueous medium during formation of nanoparticles, resulting in a low drug loading capacity. This limitation can be overcome by process modifications such as polyelectrolyte complexation and polymeric emulsification, with consequent enhancement of the encapsulation efficiency [25].
6. CONCLUSION AND PROSPECTS FOR DEVELOPMENT OF ALGINATE NANOPARTICLES AS DELIVERY SYSTEMS FOR ESSENTIAL OILS In this review, we have illustrated the developments in knowledge and technology associated with formulation of essential oils using alginate. We anticipate that future applications will be guided and improved by rational design of alginate-based nanoparticles for therapeutic applications. There have been major advances in computational modeling of polysaccharides in recent years [77] and this provides the potential to define the specific polymer structures and cation binding sites in the alginate matrix. Braccini et al. [78] provided an early example of this kind of calculation for binding of calcium to acidic polysaccharides, and similar approaches may allow optimization of the properties of crosslinked alginate gels. Modeling at the atomic level can complement mathematical models of drug release, such as that recently describe for alginate by Ciofani et al. [79], and this combination of approaches may permit improved design of drug delivery vehicles. Thus, we conclude that alginate-based formulations will have an important role in future pharmaceutical and cosmetic products based on essential oils.
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[72] Ribeiro, A. J., Neufeld, R. J., Arnaud, P. & Chaumeil, J. C. (1999). Microencapsulation of lipophilic drugs in chitosan-coated alginate microspheres. International Journal of Pharmaceutics, 187, 115-123. [73] Bouchemal, K., Briançon, S., Fessi, H., Chevalier, Y., Bonnet, I. & Perrier, E. (2006). Simultaneous emulsification and interfacial polycondensation for the preparation of colloidal suspensions of nanocapsules. Materials Science and Engineering C, 26, 472-480. [74] Fallouh, N. A. K., Roblot-Treupel, L., Fessi, H., Devissaguet, J. P. & Puisieux, F. (1986). Development of a new process for the manufacture of polyisobutylcyanoacrylate nanocapsules. International Journal of Pharmaceutics, 28, 125-132. [75] Liu, P. & Krishnan, T. R. (1999). Alginate-pectin-poly-L-lysine particulate as a potential controlled release formulation. Journal of Pharmacy and Pharmacology, 51, 141-149. [76] Teixeira, M., Alonso, M. J., Pinto, M. M. M. & Barbosa, C. M. (2005). Development and characterization of PLGA nanospheres and nanocapsules containing xanthone and 3-methoxyxanthone. European Journal of Pharmaceutics and Biopharmaceutics, 59, 491-500. [77] DeMarco, M. L. & Woods, R. J. (2008). Structural glycobiology: a game of snakes and ladders. Glycobiology, 18, 426-440. [78] Braccini, I., Grasso, R. P. & Pérez, S. (1999). Conformational and configurational features of acidic polysaccharides and their interactions with calcium ions: a molecular modeling investigation. Carbohydrate Research, 317, 119-130. [79] Ciofani, G., Raffa, V., Pizzorusso, T., Menciassi, A. & Dario, P. (2008). Characterization of an alginate-based drug delivery system for neurological applications. Medical Engineering & Physics, 30, 848-855.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 46
OBTENTION OF PRODUCTS ENRICHED IN CARBOHYDRATE POLYMERS FROM THE FOOD INDUSTRY LEFTOVERS Lía N.Gerschenson1,2*, Ana M. Rojas1,2, Marina de Escalada Pla1,2, Eliana Fissore1,3 and María E. Latorre1,4 1
Industry Department, School of Natural and Exact Sciences (FCEN), Buenos Aires University (UBA).Ciudad Universitaria. Intendente Güiraldes 2620, (1428) Ciudad Autonoma de Buenos Aires, Argentina. Phone: 54 – 11 – 4576-3366 / 3397. 2 Member of the National Scientific and Technical Research Council-Argentina (CONICET) 3 Fellow of CONICET. 4 Fellow of National Agency of Scientific and Technological Promotion-Argentina (ANPCyT)
ABSTRACT Wastes from the food processing industry have some common characteristics such as large amounts of organic materials: proteins, carbohydrates and lipids. The great production of processed vegetable products is generating an increase of the amount of wastes which represent a valuable source of by products such as carbohydrate polymers. Utilization of by-products may contribute to the efficiency of the processing and also to the sustainability of the environment. Fruits and vegetables are important sources of dietary fiber although their content is lower than the one of cereals. The cell wall matrix is the major fiber source in this type of foods and these fibers are rich in pectins. The carbohydrate composition, molecular weight and physiological properties of these fibers might be influenced by conditions of extraction as well as sources, location and many other environmental factors.
*
Corresponding author: E-mail:
[email protected];
[email protected] 1330
Lía N.Gerschenson, Ana M. Rojas, Marina de Escalada Pla et al.
The object of this research was the study of products enriched in carbohydrate polymers and obtained from quince (Cydonia oblonga Miller) or red beet (Beta vulgaris L. var. conditiva). Different methods of obtention which involved dehydration, acid and/or ethanol treatment were assayed and yield, chemical composition, molecular weight, physiological and technological properties of isolated products were stated.
INTRODUCTION The benefits of fibers for the gut are amply recognized, fact that has determined its consideration as a nutrient (FDA, 1993). Besides this, fibers can be used for technological purposes because of their functional properties. As functional additives, their usage can range from bulking agent to fat substitute (Fissore et al., 2007). By-products of vegetable-food processing represent a major disposal problem for the industry (Schieber, Stintzing and Carle, 2001) and their transformation into value-added products as fibers are, may contribute to diminish the problem and to recover valuable biomass and nutrients (de Escalada Pla et al., 2007). Different sources of fiber can have different metabolic and physiological effects (Guillon and Champ, 2000). Moreover, fiber obtained from the same source can have different behavior depending on the process applied to obtain it. Effect of wet milling and washing before drying on fiber characteristics was summarized by Larrauri (1999) for orange and mango peel. Chemical treatments and drying conditions were studied by Guillon et al. (1998) for sugar beet-fibre. The effect of washing with water, cooking pressure, as well as solvent extraction on the characteristics of the dietary fiber obtained from coconut residue was reported by Raghavendra et al. (2004). More recently, Chau et al. (2007) demonstrated that physicochemical properties of carrot insoluble fiber depend not only on fiber particle size but also on the grinding method applied. Different researchers studied also pectin extraction from various food industry by-products, such as cocoa husks (Mollea et al., 2008), sunflower (Iglesias and Lozano, 2004), sugar beet (Levigne et al., 2002), acerola (Prato et al., 2005), cactus Opuntia spp (Goycoolea and Cárdenas, 2003), pumpkin peel (Jun et al., 2006), mango (Gourgue, 1992), mandarin (Kim et al., 2000), soy hull (Kalapathy and Proctor, 2001), japanese quince (Thomas and Thibault, 2002), plum (Dikeman and Fahey, 2004), strawberry (Legentil et al., 1995). Quince (Cydonia oblonga Miller) and red beet (Beta vulgaris L. var. conditiva) are raw materials used by the food industry. By-products of their industrialization are used either for animal feeding or disposed as industrial wastes. Most research on beet pectin is made on sugar beet (Beta vulgaris var. altissima) and on the by-products of the extraction of sugar. Red beet or table beet is mostly used for salads and purees, and also to extract a natural pigment which confers its characteristic red color to a variety of food products (Gasztonyi,et al., 2001). These roots are available all year-round in the market. They accumulate a considerable amount of sugars which confer their sweet taste. Their caloric content is moderate and they are a good source of fiber; in addition, they can be consumed either fresh or cooked. On the other hand, quince is the fruit of a deciduous tree of Rosaceae family, Cydonia oblonga Miller (Silva et al., 2005). Most varieties of quince are too hard, astringent and sour to be eaten raw. They are used to make jam, “marmalade” and jelly, as well as quince pudding. The quince has a long history of cultivation in the Middle East where it is native, and throughout the Mediterranean region
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(Mark Rieger, http://www.uga.edu/fruit/quince.html). Approximately 20,000 tons are annually produced in Argentina.
QUINCE (CYDONIA OBLONGA MILLER) The industrially manufactured quince jam is prepared with quince puree, sugar and additives (Silva et al., 2006). Previous to this process, peel, seed and stem are separated and discarded or used as animal feed. In our laboratory, it was evaluated the upgrading potential of quince wastes for production of fiber enriched powders with useful functional and physiological properties. Three products were obtained by applying different drying conditions without or with a previous extraction either with water or ethanol. Processes assayed were: i) Water washing and drying at 35ºC (MA product), ii) treatment with boiling ethanol (95%, v/v) and drying at 35ºC (MEQ), iii) drying at 80ºC (M). Then, the different fractions obtained were milled and sifted (sieve ASTM-USA, mesh 40). Chemical composition as well as functional properties of the isolated fractions were evaluated for the characterization of these materials.
Chemical Composition The fiber-rich product constituted by dried quince (Cydonia oblonga Miller) wastes (Mfraction), as well as those fractions essentially constituted by the water insoluble (MA) or the ethanol insoluble (MEQ) dried residues were analyzed. The latter product was mainly constituted by the cell wall polymers of quince cellular residues since it is known that the direct treatment of a vegetable sample with hot ethanol produces the isolation of the cell wall material (Stevens and Selvendran, 1980). Yielding and chemical composition of the fractions are reported in Table 1. Significantly lower yields were observed after water (MA) or ethanol (MEQ) extraction with respect to M product due to water or ethanol soluble compounds elimination. Lower free glucose contents could be observed on the dry matter of water (MA) or ethanol (MEQ) extracted powders. Drying performed for obtention of M fraction (80ºC, 4 h) seemed to be more efficient than the one performed in the case of MA and MEQ (30ºC, 24 h), as indicated by the lower moisture content of the former. Non significant differences were found between ash concentrations. MA and MEQ showed higher proportions of proteins, calculated on dry matter, than M product, though more proteins were lost through ethanol treatment. Proteins in the fiber products were mainly from the cell wall, as can be elicited from the 8.69% content remaining in the ME fraction. As can be seen in Table 1, fats were concentrated in the MA-product as a result of the extraction of the water soluble components, but were in part removed by hot alcohol. MA and MEQ products were richer in cell wall polymers, showing a content of 53 and 59 g of non-cellulosic carbohydrates and 12.2 and 9 g of cellulose per 100 g of fraction (Table 1). Since lignin and cellulose polymers are insoluble in the solvents herein used and ash is invariable for the same reason, their sum can be considered as the fixed solids for each fiber fraction. With that consideration, the protein contents of MA or MEQ could be calculated from that of M product on the respective fixed solid bases. These calculi gave the
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same proportions of proteins indicated in Table 1 for each fiber fraction. Thus, it can be concluded that proteins were not lost during water or ethanol treatments to produce either MA or ME. Proteins could then be added as another fixed solid component in conjunction with lignin, cellulose and ashes. On this basis, it could be determined that non-cellulosic carbohydrate and uronic acid contents of MA were lower than those predicted from M composition. In the same way, it could be calculated that some proportion of uronic acids were lost during ethanolic extraction to produce MEQ. The non cellulosic carbohydrates of M product were almost entirely constituted by uronic acids (HG side chains), with only ≈ 4% of neutral sugars belonging to the RG I core, whereas neutral sugar content rose to ≈ 14% in MA fiber product due to a noticeable decrease in uronic acid content (Table 1). A similar conclusion can now be inferred with respect to the characteristics of the pectins that were lost during the hot ethanolic treatment for producing MEQ, though in this case the total non cellulosic carbohydrate content was slightly affected after extraction. Therefore, MA and MEQ products are enriched in cell wall polymers belonging to the primary cell wall as indicated from their higher proportion of neutral sugars (Vincken et al., 2003) whereas M fraction has lower lignin and cellulose contents and a lower fat proportion (Table 1). Table 1. Yield and composition of enriched fiber fractions obtained from quince byproducts either by drying at 80ºC for 4 hours (M) or by performing a water (MA) or an ethanolic treatment (ME) before drying at 30ºC for 24 hours.
1
Yield
M
MA
ME
a
b
22.05 ± 0.03b
33 ± 2
Moisture2 Protein
21 ± 2
4.40 ± 0.03a**
3
6.6 ± 0.3
a**
10.11 ± 0.05b b
10.38 ± 0.04c
10.8 ± 0.7
8.69 ± 0.06c
Fat3
6.7 ± 0.9a
12.0 ± 0.3b**
5.3 ± 0.9c
Ash3
1.5 ± 0.5a
2.3 ± 0.7a
2.6 ± 0.6a
Free glucose3
5.4 ± 0.7a
1.5 ± 0.2b a
1.7 ± 0.3b
18.4 ± 0.5
26.1 ± 0.7
25.3 ± 0.7b
Cellulose3,4
8.4 ± 0.5a
12.2 ± 0.7b
9 ± 1a
Non-cellulosic carbohydrates3,4
49 ± 2a
53 ± 1a,b
59 ± 4b**
Uronic acids3,4
45 ± 3a
39 ± 5a
41 ± 2a
Lignin
3,4
Degree of methylation (DM)5 5
Degree of acetylation (DA)
11.1 ± 0.1a 7.4 ± 0.3
a
b
7.3 ± 0.1b*** 8.1 ± 0.1
b
4.8 ± 0.2c*** 8.0 ± 0.2b
1
g of rich fibre fraction/100 g of quince pressed waste. g of water/100 g of rich fibre fraction. 3 g/100 g of dry rich fibre fraction. 4 Components of AIR. 5 DM and DA were calculated as percent ratio between mols of methanol or acetyl group and mols of GalA or non- cellulosic carbohydrates, respectively, Means and standard deviations are reported (n = 3). Different letters in the same row indicate significant differences (p < 0.05; **p < 0.01). 2
Pectins of low degree of methylation and acetylation were the main components of the quince fiber extracted products (Table 1). The treatments of quince wastes either with water
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or hot alcohol before drying at low temperature-long time, seemed to produce some degree of methyl ester hydrolysis or to retain pectins with lower DM, as indicated by the significantly lower DM of MA (7.3%) and ME (4.8%) fractions than that of M product (11.1%). The degree of acetylation (DA) was slightly but significantly lower for the pectins of the fraction produced at the highest drying temperature (M fraction). It is known that thermal processing increases pectin degradation by β-elimination (Brett and Waldron, 1996). Some authors reported that pectins from quince fruits had medium and high degree of methylation and low degree of acetylation (Forni et al., 1994; Thomas and Thibault, 2002); probably, quince pectins with higher DM mainly remained in the pulp used for jam production determining the low DM herein detected. An important proportion of lignin was present in the isolated quince products (18.4-26.1 g/100g dry fraction) (Table 1). Lignin is responsible for the hydrophobic properties of the cell wall polymeric network, constituting the secondary cell wall (Brett and Waldron, 1996). It is physically associated to the cellulose framework (Besombes and Mazeau, 2005) and presents important antioxidant effects (Castignani and Carter, 1982; Vinardell et al., 2008).
Functional Properties The functional properties of quince fractions obtained were investigated because of their importance in relation to nutritional quality and potential application of these fractions as food ingredients (de Escalada Pla et al., 2007). Hydration properties of quince fractions as well as oil holding capacity were studied and results are shown in Table 2. Water holding capacity (WHC) for the three fractions was higher than water retention capacity (WRC). WHC is defined as the quantity of water retained by the fibers without the application of any external force, except gravity and atmospheric pressure (Raghavendra et al., 2004). Thus, this parameter also includes the proportion of water loosely associated to the fiber matrix and it is therefore related to the increase in stool weight (Cadden, 1987). On the other hand, WRC is defined as the quantity of water that remains into the hydrated fiber following application of an external force like pressure or centrifugation (Raghavendra et al., 2004). This strongly bound water has been found to have no effect on stool weight. In the present work, more than a half of the water was loosely absorbed by the three quince fiber fractions studied. At the same time, MEQ presented the highest value of strongly absorbed water (WRC). These parameters are interesting because they can be associated to the amount of water retained by the fiber when it is submitted to further processes, for example, during kneading of dough, stirring or emulsifying process where fiber fractions could participate as ingredients. These absorption capacities of water involve the swelling of the matrix of each material herein tested as a consequence of the macromolecule relaxation during hydration, which lead to an increment in the occupied volume by the fiber product, indicated by the swelling capacity, SC (Table 2). Generally speaking, plasticizers like water reduce intermolecular forces along the polymer chains, thus increasing free volume and chain movements (Kasapis, 2008), as occurred in the case of quince fiber materials. This phenomenon leads to an amorphous state of the hydrated material. MEQ fraction presented the highest value of swelling. All the parameter values showed by the fiber products indicate that these materials are mainly hydrophilic, which is coherent with their contents of pectins (Table 1). As expected from these results, the oil
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holding capacities (OHC) showed by the fiber matrices were low: ≈10% of the WHC values herein determined. Many authors have reported a relationship between the WHC and OHC with the bulk density or the specific volume (Chau et al., 2004; Chau et al., 2007; Prakongpan et al., 2002; Femenia et al., 1997; Guillon and Champ, 2000; de Escalada Pla et al., 2007). The OHC increased when the powder density for an applied force of 40 N (ρ40N) decreased (0.65, 0.56, 0.497 g/cm3 for M, MA and ME, respectively) , showing the influence of microstructural differences on the ability to uptake oil, while water absorption seemed to be determined by the material’s hydrophilicity, hence, by the polysaccharide composition of the isolated products. The worst functional performance in relation to water showed by M fraction, may be attributed to a lower AIR content (M: 60.7 g/100 g dry fraction; MA: 75 g/100 g dry fraction; ME: 71 g/100 g dry fraction) as well as to matrix damage produced by the drying at higher temperature in the presence of a high proportion of monosaccharides like D-glucose (Table 1). This last fact might determine the collapse of the porous matrix during drying at high temperature (Gerschenson et al., 1981). Femennia et al. (1997) also reported lower hydration parameters for cauliflower fiber which was dried at high (75ºC) temperature. For the complete characterization of the hydration properties of fibers, it is also important to know their kinetics of water absorption. The knowledge of kinetics allows, for example, to determine the time that takes fibers to absorb the quantity of water necessary for a certain process or a given formulation or to decide if they have to be hydrated during a known time before addition to a formulation. Moreover, it is very important to know the water absorption kinetics of fibers to understand the effect of their presence in dry or semidry systems which will be re-hydrated in a further step or previous to consumption. Kinetic experimental data obtained were fit to the following equation: qW =
QW t (BW + t )
(equation 1)
where qW is the water absorbed at time t, QW is the maximal water absorption capacity, also called spontaneous water binding capacity (WBC), and BW is the time needed to absorb a half of the maximal water absorption capacity (QW/2) (Pilosof et al., 1985). In Figure 1, experimental data obtained for spontaneous water absorption are shown, as well as the curve obtained by means of equation 1. WBC (QW) of ME fraction almost doubled those values respectively determined for MA and M. This trend may be attributed to shrinking and collapse of the matrix during drying at high temperature in the case of M fraction while higher fat and, probably, also cellulose contents may act decisively to determine the lower WBC of MA (Table 2), as also occurred with SC and WRC. BW parameter can be related with the velocity. Non significant differences were observed on this parameter when quince fractions were characterized and values obtained suggest that the three fractions absorbed part of the water almost instantaneously. In a previous work, de Escalada Pla et al. (2007) observed that Bw parameter of butternut fiber depended more on fiber origin than on the treatment applied: fiber from butternut peel presented a Bw value two orders greater than the one observed for butternut mesocarp fiber; at the same time, mesocarp fiber presented similar Bw values to the ones herein obtained for quince products.
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Table 2. Functional properties of fiber-rich products obtained from quince by-products, either by drying at 80ºC for 4 hours (M) or by performing a water (MA) or an ethanolic extraction (ME) before drying at 30ºC for 24 hours. Hydration Properties
1
M MA MEQ
WHC (g/g)1 13 ± 1a 15 ± 1a,b 19 ± 3b
SC (ml/g)2 6.40 ± 0.09a 7.1 ± 0.9a 11.6 ± 0.2b***
WRC (g/g)3 5.6 ± 0.3a 4.8 ± 0.3a 9.3 ± 0.6b***
OHC (g/g)6
WBC (ml/g)4 3.14 ± 0.06a 3.9 ± 0.1b** 6.6 ± 0.1c***
BW (min)5 0.29±0.04a 0.38±0.09a 0.47±0.07a
1.26 ± 0.02a 1.59 ± 0.06b 1.8 ± 0.1b,c**
Water holding capacity: gram of water per gram of dry mass. Mean and standard deviation (n = 3) are shown. 2 Swelling capacity: ml of hydrated and swelled fiber per gram of dry mass. Mean and standard deviation (n = 3) are shown. 3 Water retention capacity: gram of water retained after centrifugation per gram of dry mass. Mean and standard deviation (n = 4) are shown. 4 WBC: water binding capacity. 5 BW: time needed to absorb WBC/2. 6 Oil holding capacity: gram of sunflower oil per gram of dry mass. Mean and standard deviation (n = 3) are shown. Different letters in the same column indicate significant differences (p < 0.05; **p < 0.01; ***p < 0.001).
When polymers are completely hydrated, water amount absorbed (WHC) was considerable higher (2.9-4.3 times) than the WBC determined after spontaneous absorption (Table 2). This important difference suggests that more than 3 hours (Figure 1) were needed by the polymers to completely relax and swell, at the equilibrium of hydration, changing to an amorphous stage.
q (ml water / g dry fiber fraction)
9 8 7 6 5 4 3 2 1 0 0
20
40
60
80
100
120
140
160
180
200
time (min)
Figure 1. Kinetics of water absorption for fractions obtained from quince, indicated as volume absorbed per gram of dry fiber material (q) with time. Curves obtained by fitting of experimental points to equation 1, are shown for × — (ME), ♦ — (M) and ∆ - - - (MA) fiber-rich fractions.
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Table 3. Composition of alcohol insoluble residue (MERD) obtained from red beet (Beta Vulgaris L. var. conditiva). Composition (g/100g)1,2
Non-cellulosic carbohydrates 51.96± 3.92
Uronic acids 14.3 ± 0.7
Cellulose
Starch
18.9± 6.8
1.2 ± 0.1
Lignin 5.93±0.96
Protein 9.02 ± 0.63
1
: Mean and standard deviations are shown (n = 3). :Results are expressed in dry basis. 3 : DM (degree of methylation) and DA (degree of acetylation) were calculated as a percent ratio between moles of methanol or acetyl group and moles of GalA or non-cellulosic carbohydrates, respectively. 2
Table 4. Functional properties and kinetic parameters for fiber-rich fraction obtained from red beet (MERD). WHC (g/g)1 39 ± 2
SC (ml /g)2 39 ± 2
WRC (g/g)3 41.0 ±0.8
WBC (ml/g)4 28.0±2.8
BW (min)5 2.8±0.7
1
Water holding capacity: gram of water per gram of dry mass. Mean and standard deviation (n = 3) are shown. 2 Swelling capacity: ml of hydrated and swelled fiber per gram of dry mass. Mean and standard deviation (n = 3) are shown. 3 Water retention capacity: gram of water retained after centrifugation per gram of dry mass. Mean and standard deviation (n = 4) are shown. 4 WBC: water binding capacity. 5 BW: time needed to absorb WBC/2.
WHC values observed for MEQ, MA and M (19-13 g/g) fractions were in the same order than those reported by Grigelmo-Miguel and Martin-Belloso (1999) for orange, peach, artichoke and asparagus dietary fiber (> 11 g/g), as well as by Chau et al. (2004) for carrot insoluble fiber-rich fractions (8.73-18.7 ml/g). At the same time, WHCs of quince fiber fractions were higher than those presented by citrus husk (3.6 g/g), pineapple peel (3.5 g/g) and fiber from coconut residues (7.1 g/g) (Grigelmo-Miguel and Martin-Belloso, 1999). The values of WRC and SC were of the same order (4.8-9.3 g/g and 6.4-11.6 ml/g, respectively) than those reported by Robertson et al. (2000) for apple (WRC: 5.43 g/g; SC: 7.42 ml/g) and citrus pulp (WRC: 10.66 g/g; SC: 10.45 ml/g) and higher than those of pea hull (WRC: 3.9 g/g) and resistant starch (WRC: 2.95-3.74 g/g). OHC ranged from 1.26 to 1.8 g/g, being similar to values reported for fruit and vegetables (< 2 g/g) by Femenia et al. (1997).
RED BEET (BETA VULGARIS L. VAR. CONDITIVA) In our laboratory, different fractions obtained from Beta Vulgaris L. var. conditiva were studied: one obtained through treatment with boiling ethanol and others acid extracted, giving origin to pectin rich products. The chemical composition and functional properties of products obtained were characterized.
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Ethanol Extracted Fraction Red beet tissue was treated with boiling ethanol (95%, v/v) during 10 minutes. Insolubles obtained were freeze dried and milled.
Chemical composition As can be observed in Table 3, the ethanolic extracted fiber fraction of beet root was essentially constituted by polysaccharides (≈ 72%) with minimal contents of lignin (5.93 g /100 g dry product) and protein (9.02 g /100 g dry product) of the cell wall. Carbohydrates included starch (1.2 g /100 g dry product), cellulose (18.9 g /100 g dry product) and uronic acids (14.3 g /100 g dry product) from pectin homogalacturonan (HG) (Vincken et al., 2003). Taking this into account, it can be concluded that neutral sugar content was ≈ 37 g/100g and they came from the pectic macromolecules (RG-I and RG-II) present in red beet. All pectic polysaccharides were of high degree of methylation (DM ≈ 80 %) and acetylation (DA ≈ 79 %). Carbodiimide assays showed that uronic acids present were constituted by ≈ 94 % of galacturonic acid and ≈ 6.0 % of glucuronic acid. Functional properties As can be observed in Table 4, MERD showed good hydration properties. Values are in the order of those informed by de Escalada Pla et al. (2007) for butternut (Cucurbita moschata Duch ex Poiret) and were higher in a factor of two or three to those informed in the bibliography for apple and citrics. They were also higher than those informed, in this chapter, for quince. Kinetic parameters obtained from the fitting of experimental data to equation 1, showed that MERD spontaneously absorbed a greater water amount (WBC) than the one observed for MEQ. At the same time, it seemed not to reach the equilibrium of water absorption since WBC shows further increasing (Figure 2). This suggests that the constituent polymers may be characterized by a retarded relaxation response or, in other words, polymeric chains were relaxing while absorbing water and, consequently, more hydrophilic sites resulted exposed at every time for additional water absorption. This contrasts with the more rigid structure of quince polymers (Figure 1), for which WBC did not perceptibly changed in the time scale (200 min) studied. Hence, kinetics of water absorption is an interesting property to be determined because it also helps characterizing the rheological behavior of the macromolecular network in water. The time for absorbing half of that amount (BW) was also greater for MERD.
Acid Extracted Fractions The mesocarp tissue was separated and juice was removed using a juice extractor. The residue was washed twice with distilled water, dried (85ºC, 2 hours) in a convection oven, milled and sieved for obtaining beet powders with particles in the range 420-710 μm. The powder obtained was treated under stirring with citric acid solutions (Canteri-Schemin et al., 2005) of pH 2.0 during 2 or 3 hours maintaining the temperature at 85ºC (May, 1990). Insolubles obtained after acid extractions were separated through filtration with glass fibre
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filter paper under vacuum and cell wall polysaccharides (CWP) were finally precipitated from each supernatant through 95% (v/v)-ethanol addition. These cell wall enriched products were collected through filtration using a nylon mesh (pore: 0.06 mm2) and they were, finally, freeze-dried. Samples obtained were the following: R 2-2: sample extracted from red beet powder at pH 2 for 2 h and R 2-3: sample extracted from red beet powder at pH 2 for 3 h.
q (ml water / g dry fiber fraction)
Chemical composition Fraction yields were 11 g/100 g and 17 g/100 g after 2 and 3 hours of treatment, respectively. Total carbohydrate content was 75.9-79.4 g/100g, being constituted by noncellulosic carbohydrates. Uronic acid content was 27.3-30.2 g/100g (Table 5). Neutral sugars were ≈ 63.0% of the total carbohydrate content and they were mainly constituted by Ara, Rha, and Gal. It is important to remark that products R22 and R23, had a very high Ara content (Ara/Rha = 5.5 and 3.8, respectively). The ratio Gal/Rha (≈ 1.0) was not modified with increasing treatment time. Glucose (Glc) concentration was around 12 g/100g although starch concentration in this fraction was very low (Table 5); probably, Glc was originated from other polysaccharides present in these fractions. DM was ≈ 44 % and DA was ≈ 20 %. Only traces of RG-II were detected in these red beet fractions. It has to be mentioned that carbodiimide reaction showed that, for all red beet fractions, approximately 94.0% of uronic acids was GalA and the remaining 6.0% was glucuronic acid (GlcA). Strasser and Amadó (2002) reported the presence of GlcA in red beet tissue.
35 30 25 20 15 10 5 0 0
20
40
60
80
100
120
140
160
180
200
time [min]
Figure 2. Kinetic of water absorption for red beet ethanol extracted product (MERD), expressed as volume absorbed per gram of dry fiber material (q) with time.
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Table 5. Chemical composition of fibre enriched products acid extracted from red beet. Sample Total carbohydrates (g/100 g)1,2 Uronic acids (g/100 g)1 Protein (g/100 g)1 Starch (g/100 g)1 DM (%)3 DA (%)3
R 2-2 79.4±4.6 30.2±1.4 8.3±0.2 0.2±0.0 43.0 19.8
R 2-3 75.9±7.9 27.3±1.4 7.7±0.8 0.6±0.1 45.4 21.7
Pectin enriched product extracted with: pH 2, time 2h (R2-2) or pH 2, time 3h (R2-3). DM: degree of methylation; DA: degree of acetylation. 1 : Mean and standard deviations are shown (n = 3). 2 : Total carbohydrates are calculated as GalA. 3 DM and DA were calculated as a percent ratio between moles of methanol or acetyl group and moles of GalA or non-cellulosic carbohydrates, respectively.
Table 6. Apparent viscosity and Ostwald model-parameters calculated after fitting the flow experimental data (25ºC) for 2.00 % (w/v)-aqueous systems containing different red beet pectin enriched products. Apparent viscosity k n1 R2 -1 1 n 1 at 20 s (Pa.s) (Pa s ) R 2-2 0.027±0.009 0.04±0.01 0.85±0.06 0.9042 R 2-3 0.018±0.005 0.04±0.01 0.73±0.05 0.9189 1 Mean and standard error of fittings are shown. Pectin enriched product extracted with pH 2, time 2h (R2-2) or pH 2, time 3h (R2-3) from red beet powder. k: consistency index; n: pseudoplasticity index. R2: goodness of fitting (α: 0.05).
Product
Table 7. Cross model-parameters calculated after fitting the flow experimental data (25ºC) for 2.00 % (v/v)-aqueous systems containing different red beet pectin enriched products. Product R2-2 R2-3
η∞(Pa s)1 -
η0 (Pa s)1 9.6±0.2 12.4±0.5
τ (s)1 45.2±1.6 49.1±4.7
m1 2.50±0.20 2.29±0.43
R2 0.9846 0.9111
1
Mean and standard error of fittings are shown. Pectin enriched product extracted with pH 2, time 2h (R2-2) or pH 2, time 3h (R2-3) from red beet powder. η0 represents the zero shear rate viscosity, η∞ represents the infinite shear rate viscosity, τ is the time constant corresponding to each model, and m is a dimensionless parameters. R2: goodness of fitting (α: 0.05).
Rheological behavior For this characterization, 2.00 % (w/v) aqueous systems of the different products obtained were constituted. No insoluble material was observed showing that products isolated were constituted by “soluble” polysaccharides.
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Table 8. Glucose dialysis retardation index1 (GDRI) for different isolated fibre fractions. Product R 2-2 R 2-3
0 100±0 100±0
10 16.9±8.2 11.5±0.7
Time (min) 30 60 16.9±5.8 5.7±2.0 11.9±1.3 -
90 2.5±1.2 -
1
Mean and standard error of fittings are shown. Pectin enriched product extracted with pH 2, time 2h (R2-2) or pH 2, time 3h (R2-3) from red beet powder.
Flow behavior was modeled through Ostwald and Herschel-Bulkley equations. Experimental data fit adequately to the first one, showing no τ0 as well as apparent viscosity (ηa) (shear rate: 20 s-1) values ranging from 0.018 to 0.027 Pa.s, trend that showed a weak thickening effect of these fractions. Aqueous systems showed pseudoplastic behavior with values of exponential index of 0.85 and 0.73 for R 2-2 and R 2-3, respectively (Table 6). Table 7 shows results obtained when data were fit to Cross model,. The upper Newtonian plateau was, as usually, inaccessible. Structural relaxation times,τ, showed that hydrated polysaccharides constituted a network which relaxed after attainment of its corresponding onset value.
Glucose dialysis retardation index These fractions showed an “in vitro” retardation of glucose dialysis (Table 8). R2-2 showed a retardation that lasted for 90 min although it was significant only for the first half hour. Pectin enriched products enzymatically isolated by Fissore et al. (2007) from butternut (Cucurbita moschata Duch ex Poiret) showed higher values of GDRI for 60 min (11 to 29 %) and also the retardation action lasted longer (120 min).
CONCLUSION In this research, fiber rich fractions with various chemical compositions and functional properties were obtained from quince (Cydonia oblonga Miller) fruit and red beet (Beta vulgaris L. var. conditiva) by means of different procedures involving dehydration, acid and/or ethanolic extraction. Obtention of these products permit to add value to left over of vegetal tissue industrialization as well as to generate multifunctional ingredients for the food industry.
ACKNOWLEDGMENTS The authors acknowledge the financial support of ANPCyT, CONICET and Buenos Aires University.
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REFERENCES Besombes, S. & Mazeau, K. (2005). The cellulose/lignin assembly: assessed by molecular modeling. Part 2: seeking for evidence of organization of lignin molecules at the interface with cellulose. Plant Physiology and Biochemistry, 43, 277-286. Brett, C. T. & Waldron, K. W. (1996). The Physiology and biochemistry of plant cell walls. 2nd. Ed. London: Chapman and Hall. Cadden, A. M. (1987). Comparative effects of particle size reduction on physical structure and water binding properties of several plant fibers. Journal of Food Science, 52, 6, 1595-1631. Canteri-Schemin, M. H., Ramos Fertonani, H. C., Waszczynskyj, N. & Wosiacki, G. (2005). Extraction of pectin from apple pomace. Brazilian archives of biology and technology, 48, 2, 259-266. Castignani, G. L. & Carte, M. E. (1982). Antioxidant Properties of Lignin. Journal of Food Science, 47, 5, 1745-1748. Chau, C. F., Chen, C. H. & Lin, C. Y. (2004). Insoluble fiber-rich fractions derived from Averrhoa carambola: hypoglycaemic effects determined by in vitro methods. Lebensmittel Wissenschaft und Technologie, 37, 331-335. Chau, Chi F., Wang, Yi T. & Wen, Yu L. (2007). Different micronization methods significantly improve the functionality of carrot insoluble fiber. Food Chemistry, 100, 1402-1408. de Escalada Pla, M. F., Ponce, N. M., Stortz, C. A., Gerschenson, L. N. & Rojas, A. M. (2007). Composition and functional properties of enriched fiber products obtained from pumpkin (Cucurbita moschata Duchesne ex Poiret). Lebensmittel Wissenschaft und Technologie, 40, 1176-1185. Dikeman, C. L. & Fahey, G. C. (2006). Viscosity as related to dietary fiber: a review. Critical reviews in food science and nutrition, 46, 649-663. FDA. Department of Health and Human Services. Health Claims: fruits and vegetables and cancer . In: FDA. Code of Federal Regulations. Rockville, MD. Vol. 2, Title 21. Chapter I. Subchapter B. Part 101. Subpart E. Section 101.78; 1993. Femenia, A., Lefebvre, A. C., Thebaudin, J. Y., Robertson, J. A. & Bourgeois, C. M. (1997). Physical and sensory properties of model foods supplemented with cauliflower fiber. Journal of Food Science, 62, 4, 635-639. Fissore, E. N., Ponce, N. M., Stortz, C. A., Rojas, A. M. & Gerschenson, L. N. (2007). Characterisation of fiber obtained from pumpkin (Cucumis moschata Duch) mesocarp through enzymatic treatment. Food Science and Technology International, 13, 141-151. Forni, E., Penc,i M. & Polesello, A. (1994). A preliminary characterization of some pectins from quince fruit (Cydonia oblonga Mill) and pickly pear (Opuntia ficus indica) peel. Carbohydrate polymers, 23, 4, 231-295. Gasztonyi, M. N., Daood, H., Takács Hájos, M. & Biacs, P. (2001). Comparison of red beet (Beta vulgaris var conditiva) varieties on the basis of their pigment components. Journal of the science of Food and Agriculture, 81, 9, 932-933. Gerschenson, L. N., Bartholomai, G. B. & Chirife, J. (1981). Structural collapse and volatile retention during heating and rehumidification of freeze dried tomato juice. Journal of Food Science, 46, 5, 1552-1556.
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Gourgue, C. M. P., Champ, M. M. J., Lozano, Y. & Delort-Laval, J. (1992). Dietary fiber from mango byproducts: Characterization and hypoglycemic effects determined by in vitro methods. Journal of Agricultural and Food Chemistry, 40, 10, 1992, 18641868. Goycoolea, F. M. & Cárdenas, A. (2003). Pectins from Opuntia spp.: A short review. Journal of the Professional Association for Cactus Development, 5, 17-29. Grigelmo-Miguel, N. & Martin-Belloso, O. (1999). Comparison of dietary fiber from byproducts of processing fruits and greens and from cereals. Lebensmittel Wissenschaft und Technologie, 32, 503-508. Guillon, F. & Champ, M. (2000). Structural and physical properties of dietary fibers, and consequences of processing on human physiology. Food Research International, 33, 233245. Guillon, F., Auffret, A., Robertson, J. A., Thibault, J. F. & Barry, J. L. (1998). Relationships between physical characteristics of sugar-beet fiber and its fermentability by human faecal flora. Carbohydrate Polymers, 37, 185-197. Iglesias, M. T. & Lozano, J. E. (2004). Extraction and characterization of sunflower pectin. Journal of Food Engineering, 62, 215-223. Jun, H. I., Lee, C. H., Song, G. S. & Kimet, Y. S. (2006). Characterization of the pectic polysaccharides from pumpkin peel. Lebensmittel Wissenschaft und Technologie, 39, 5, 554-561. Kalapathy, U. & Proctor, A. (2001). Effect of acid extraction and alcohol precipitation conditions on the yield and purity of soy hull pectin. Food Chemistry, 73, 4, 393-396. Kasapis, S. (2008). Recent Advances and Future Challenges in the Explanation and Exploitation of the Network Glass Transition of High Sugar/Biopolymer Mixtures. Critical Reviews in Food Science and Nutrition, 48, 2, 185-203. Kim, D. H., Kim, D. G., Lee, D. Y., Kim, K. E. & Kim, C. W. (2000). Physicochemical characterization of pectin extracted from Cheju mandarin (Citrus unshiu) peels with citric acid. Food Science and Biotechnology, 9, 95-98. Larrauri, J. A. (1999). New approaches in the preparation of high dietary fiber powders from fruit by-products. Trends in Food Science and Technology, 10, 3-8. Legentil, A., Guichard, I., Piffaut, B. & Haluk, J. P. (1995). Characterization of strawberry pectin extracted by chemical means. Lebensmittel Wissenschaft und Technologie, 28, 569-576. Levigne, S., Ralet, M. C. & Thibault, J. F. (2002). Characterisation of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydrate Polymers, 49, 2, 145-153. May, C. D. (1990). Industrial pectins: sources, production, and applications. Carbohydrate polymers, 12, 1, 79-99. Mollea, C., Chiampo, F. & Conti, R. (2008). Extraction and characterization of pectins from cocoa husks: A preliminary study. Food Chemistry, 107, 3, 1353-1356. Pilosof, A. M. R., Boquet, R. & Bartholomai, G. B. (1985). Kinetics of water uptake by food powders. Journal of Food Science, 50, 1, 278-279, 282. Prakongpan, T., Nitithamyong, A. & Luangpituksa, P. (2002). Extraction and application of dietary fiber and cellulose from pineapple cores. Journal of Food Science, 67, 4, 13081313.
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Prato, A. M., Mendes, E. C, Barros, S. T. D. & Costa, S. C. (2005). Extraction and characterisation of acerola pectin. In: Proceedings of the ENPROMER 2nd Mercosur Congress on Chemical Engineering. Rio de Janeiro, Brasil. Rhagavendra, S. N., Rastogi, N. K., Raghavarao, K. S. M. S. & Tharanathan R. N. (2004). Dietary fiber from coconut residue: effects of different treatments and particle size on the hydration properties. European Food Research and Technology, 218, 563-567. Rieger, M. (2009). Available from : http://www.uga.edu/fruit/quince.html. Accessed April 30, 2009. Robertson, J. A., de Monredon, F. D., Dysseler, P., Guillon, F., Amadò, R. & Thibault, J. F. (2000). Hydration properties of dietary fiber and resistant starch: a European collaborative study. Lebensmittel Wissenschaft und Technologie, 33, 72-79. Schieber, A., Stintzing, F. C. & Carle, R. (2001). By-products of plant food processing as a source of functional compounds-recent developments. Trends in Food Science and Technology, 12, 401-413. Silva, B. M., Andrade, P. B., Martins, R. C., Seabra, R. M. & Ferreira, M. A. (2006). Principal component analysis as tool af characterization of quince (Cydonia oblonga Miller) jam. Food Chemistry, 94, 504-512. Silva, B. M., Andrade, P. B., Martins, R. C., Valentão, P., Ferreres, F., Seabra, R. M. & Ferreira, M. A. (2005). Quince (Cydonia oblonga Miller ) fruit characterization using principal component analysis. Journal of Agricultural and Food Chemistry, 53, 111-122. Stevens, B. J. H & Selvendran, R. R. (1980). The isolation and analysis of cell wall material from the alcohol-insoluble residue of cabbage (Brassica oleracea var. Capitata). Journal of the Science of Food and Agriculture, 31, 1257-1267. Strasser, G. R. & Amadó, R. (2002). Pectic substances from red beet (Beta vulgaris L. var. conditiva). Part II. Structural characterisation of rhamnogalacturonan II. Carbohydrate polymers, 48 3, 263-269. Thomas, M. & Thibault, J. F. (2002). Cell-wall polysaccharides in the fruits of Japanese quince (Chaenomeles japonica): extraction and preliminary characterization. Carbohydrate Polymers, 49, 345-355. Vinardell, M. P., Ugartondo, V. & Mitjans M. (2008). Potential applications of antioxidant lignins from different sources. Industrial Crops and Products, 27, 220-223. Vincken, J. P., Schols, H. A., Oomen, R. J. F. J., Beldman, G., Visser, R. G. F. & Voragen, A. G. J. (2003). Pectin-the hairy thing. In: A. G. J. Voragen, H. Schols, & R. Visser (Eds.), Advances in Pectin and Pectinase Research. Dordrecht, Germany: Kluwer Academic Publishers; 47-59.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2010 Nova Science Publishers, Inc.
Chapter 47
RECENT DEVELOPMENTS IN THE PREPARATION AND APPLICATIONS OF CHITOSAN-BASED MICROSPHERES Dongfeng Wang, Haiyan Li, Binjie Liu and Xingyu Wang College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China.
ABSTRACT In recent years, interesting works have been focused on the amino polysaccharide chitosan-based microspheres. The chitosan microspheres have been applied in diversified fields, such as food processing, water treatment and biological fields. To bring new or improved properties, the chemical modification of chitosan-based microspheres is of rapidly growing interest. After chemical modification, chitosan-based microspheres can be used as functional materials with modified properties, such as adsorption resins, magnetic resins and nanocomposites, for specific end use in food industry. This paper presents a review of the developments in the preparation and applications of chitosanbased microspheres. The main objective of this review is to provide recent information and to show the development trend about chitosan-based microspheres.
1. INTRODUCTION Chitosan is an amino polysaccharide derived from of chitin by the N-deacetylation procedure, the second most abundant biopolymer in nature [1]. Chitosan is characterized by its high contents of amino hydroxyl groups, which enable chitosan to adsorb various organic compounds, including organic acids, proteins and dyes [2-4]. To bring new or improved properties, several chemical modifications such as oligomerization, alkylation, acylation, quternization, hydroxyalkylation, carboxyalkylation, thiolation, sulfation, phosphorylation, enzymatic modifications and graft copolymerization along with many assorted modifications
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have been carried out [5]. In addition, chitosan can chelated several metal ions, including transitional metal ions and lanthanide metal ions due to amino groups. [6]. Chitosan has many useful features like biocompatibility, biodegradability and hydrophilicity. Chitosan has been utilized in many fields, such as biomedical applications, wastewater treatment and juice clarification, etc [7-9]. Chitosan can dissolute in acidic solutions, so it is hard to recycle from acidic solution. It is thus necessary to stabilize it chemically for the recycling in acidic solutions. Several processes have been developed to stabilize chitosan, such as suspending cross-linked technique, emulsification technique and spray drying technique [10]. Through stabilization, chitosan can be prepared as microspheres, easily separated from solutions. To get new or improved properties, chitosan-based microspheres have been researched recently. In addition, to expend the range of chitosan applications, chitosan-based microspheres can be used as functional materials with modified properties, such as adsorption resins, magnetic resins and nanocomposites, etc. The preparation and application of different kinds of chitosan-based microspheres are reviewed in this paper.
2. PREPARATION OF CHITOSAN-BASED MICROSPHERES There are numerous studies in literature on the preparation of chitosan-based microspheres. The methods of preparing chitosan-based microspheres can be generally classified in the following ways.
2.1. Inverse Crosslinking-Emulsion This method is the well-known method. The acetic acid solution of chitosan was mixed with liquid paraffin which worked as the dispersant agent, Span 80 as an emulsifier was added into the mixture. The mixture was emulsified to form the W/O emulsification. Then crosslinking agent was added drop by drop, the pH value was adjusted by means of NaOH solution, after a few hours of the crosslinking reaction, the microspheres were filtered and rinsed with distilled water. Chitosan can be crosslinked by a reaction with the hydroxyl or amino groups of the chains with the crosslinking agents to form water-insoluble cross-linked resins [11].The crosslinking step is the important reaction, and a relatively easy method to prepare chitosan resins. The crosslinking can be accomplished with different crosslinking agents, such as glutaraldehyde, epichlorhydrin or chloromethyl thiirane. The aldehyde ends of glutaraldehyde and amine moities of chitosan form imine functions through a Schiff’s base reaction [12]. Epichlorhydrin or chloromethyl thiirane can react with the amino groups of chitosan to form the macromolecule polymer with net structure. Li optimized the preparation conditions of chitosan microspheres [13].The preparation of chitosan microspheres has many influence factors, including temperature, stirring speed and cross-linking agent. Stirring speed is extremely important for the formation and diameter of chitosan. With controlling the influence factors, this method can prepare various kinds of microspheres, such as macroporous resins and nano-grade chitosan microspheres [14, 15].
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Hu et al. prepared uniform size chitosan microspheres by a novel T-shaped microchannel [16].To form the uniform size chitosan emulsion, the W/O emulsion of chitosan flowed through the microchannel with the diameter of 350μm. Then the crosslinking agent was added into the emulsion. After two hours, the uniform size chitosan microspheres were formed.
2.2 Coacervation The acid solution of chitosan can form gels when added into alkaline solutions in drops, such as NaOH and KOH [17, 18]. Shanmuganathan et al. improved this method [18]. Chitosan microspheres (CSM) were prepared by the W/O emulsification process along with an ionic coacervation technique. The KOH solution in n-octanol was added into the w/o emulsion of chitosan drop by drop. Then the ionic gelation was initiated. After the reaction, the chitosan microspheres were removed and rinsed in demineralized water baths. The whole process was carried out at room temperature. The method has advantages of simple operation, mild reaction conditions and easy division. So it is usually applied to prepare sustained release drugs or immobilized bioactive substances.
2.3 Spray Drying The acidic solution of chitosan was injected into the spray drying apparatus (Figure 1) through a peristaltic pump [19]. The atomization occurred through air compression force. The solvent was evaporated rapidly with a temperature above its ebullition temperature. Then the product in the form of dry microspheres was collected. Weerakody et al. prepared chitosan microspheres for encapsulation of α-lipoic acid (LA) using the spray drying technique [20]. LA-loaded microspheres had smooth surface morphology. Particle size distribution of LA-loaded microspheres indicated that 50% of the particles were less than 7.89μm diameter. The antioxidant activity of encapsulated LA demonstrated a significant level (75%) of retention of activity when compared to free LA.
Figure 1. High pressure spray jets. A: the nozzle shown in the diagram is an atomizer with internal mixing of gas and liquid.
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Figure 2. Schematic representation of molecular imprinting.
2.4. Solvent Evaporation The acidic solution of chitosan was added into the solvent (methylbenzene). The mixture formed the W/O emulsion through ultrasonic treatment. Then the crosslinking agent was added into the emulsion. After crosslinking, the solvent was evaporated. To improve this method, the solvent was evaporated vacuum evaporation [21].
3. CHITOSAN-BASED MICROSPHERES In different application fields, chitosan microspheres have been prepared and modified as different materials, including molecular imprinting microspheres, chitosan gels, chitosan nanoparticles, multifunctional resins, affinity resins, chitosan magnetic microspheres and adsorption resins.
3.1. Molecular Imprinting Microspheres Molecular imprinting is a newly developed methodology which based on molecular interaction. Molecular imprinted polymers can provide molecular assemblies of desired structures and properties. So the molecular imprinting technique can be applied to different kinds of target molecules, such as metal ions, pesticides, proteins and whole cells [22-25]. The principle of molecular imprinting is the assembly of a cross-linked polymer matrix around a template; when the template is removed. The principle of molecular imprinting is shown in Figure 2 [26]. The template molecule assembles with a cross-linked polymer matrix via non-covalent or covalent interactions. Then the template molecule is removed from the polymers. The processed polymers possess stable capacity of specific recognition and selectivity to the template molecule. Cross-linked chitosan microspheres are low-cost, stable and easily fabricated materials. Chitosan microspheres have already been used as the supporting matrix. Molecular imprinting polymers have been applied in water treatment. There are several reports about the metal ions imprinted chitosan polymers. The metal ions mainly include Ni2+, Co2+, Pb2 +, Cu2+ and Zn2 +, etc [27-31]. The metal ions solutions mix with the chitosan acidic solution to form the complexes, then the complexes mixtures crosslink with crosslinking agents. After removing the metal ions, the metal ions imprinted chitosan polymers are obtained. This method is one of the
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popular methods. He et al. prepared the Ni2+ template chitosan chelated resins [27]. The adsorption capacities of the Ni2+ template chitosan chelated resins to Ni2+, Cu2+, Zn2+ were two times as those of general chitosan resins, but no difference for Cr6+. To separate Cu (II) ions from multi-element coexistence solution, Sun et al. synthesized a new adsorbent material [32]. N-Succinyl chitosan (NSC) synthesized via introduction of succinyl groups at the N-position of the glucosamine units of chitosan. NSC was added into the copper acetate solution to form the Cu (II) template NSC. After crosslinking, the crosslinked NSC resins with Cu (II) as template ions were obtained. The results of the adsorption experiments showed that the crosslinked NSC template can selectively adsorbed Cu (II) ions from the solution of Cu (II), Zn (II), Co (II), and Ni (II) ions coexistence. The crosslinked NSC template also showed a good reusability. After reuse for eight times, the adsorption capacity of the crosslinked NSC template for Cu (II) ions decreased slightly from 2.11 to 1.88 mmol/g. Birlik et al. prepared double-imprinted chitosan beads which can be used for the selective removal of Cu (II) ions from synthetic waters [33]. Chitosan-succinate was mixed with Cu (II) ions and then reacted with 3-(2-aminoethylamino) propyltrimethoxysilane (AAPTS). Both Cu (II) and AAPTS were used as templates. After crosslinked with tetraethoxysilane, the Cu (II) ions were removed using HNO3 solution. Then the double-imprinted beads were formed. The double-imprinted beads were used in the adsorption–desorption of Cu (II) ions from metal solutions. The maximum adsorption capacity for Cu (II) ions was 47.63 mg/g. To increase metal loading capacities and selectivity, the double-imprinted beads were superior to the Cu (II)-imprinted chitosan-succinate. In addition, molecular imprinting technique is also used in anion recognition. Ozkutuk et al. reported the preparation and adsorption ability of the phosphate-imprinted chitosansuccinate beads [34]. Chitosan was modified with succinic anhydrides firstly. Second the mixture of chitosan-succinate and Fe (III) ions stirred continuously at room temperature. And Na3PO4 was added to Fe (III)-chitosan-succinate mixture. This mixture was slowly dropped into NaOH solution to form beads. Afterwards, beads were crosslinked with epichlorohydrin and the template (phosphate ions) was removed using 1M KOH solution. Selective cavity for the phosphate ion was obtained in the phosphate imprinted metal-chelate polymer. The phosphate-imprinted metal-chelate polymer was used in the adsorption–desorption process. The adsorption process was fast and equilibrium was reached around 30 min. The adsorption behaviour of this system was described approximately by the Langmuir equation. Molecular imprinted technique can be applied in separation and purification of proteins. Recently, there are several reports about chitosan as the supporting matrix in proteins imprinted technique. Guo et al. [35] prepared free hemoglobin-imprinted chitosan beads. Feng et al. preparated a bovine serum albumin (BSA) imprinted chitosan microspheres for protein recognition [36]. The functional biopolymer chitosan (CS) microspheres were formed by inverse crosslinkingemulsion method. The template BSA was immobilized on the surface of chitosan microspheres. They also prepared the free-template imprinted chitosan microspheres. Compared with the free-template imprinted composite microspheres, the surface-imprinted chitosan microspheres possessed higher adsorption capacity towards template protein. The surface-imprinted chitosan microspheres characterized by easy preparation and high affinity can be broadly applied in biotechnology for down-stream processing and biosensor.
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3.2. Chitosan Gels Chitosan gels have been also used in several fields. Dambies et al. prepared modified chitosan gel beads by the molybdate adsorption and the coagulation technique [37]. The modified chitosan gel beads were tested for As (III) and As (V) removal from dilute solutions (in the range 5-20mg As L-1). The results of adsorption experiments indicated: In acidic solutions, the sorption capacity of As (V) was significantly higher than As (III) uptake. So the modified chitosan gel beads can be applied in the treatment of industrial effluents or as a preconcentration process from acidic solutions. In order to improve the metal ion adsorption capacity of the chitosan beads and their mechanical stability, Chen et al. prepared the chitosan gel beads chemically modified by polyethyleneimine (PEI) using the coacervation technique [38]. The adsorption properties of the modified beads and the density of the NH2 groups were significantly increased. To improve the bead stability, the modified beads were encapsulated with polysulfone (PSF) by the emulsion phase inversion method. Even after coating with PSF, the absorption capacity and adsorption kinetics of the PSF-PEI-CTS were still higher than the unmodified beads. So the modifications improve the mass transfer performance of the chitosan beads as well as the bead stability. In addition, chitosan can be modified physically. For example, Hasan et al. [39] prepared chitosan-coated perlite beads to remove Cd2+ from the aqueous solutions. The acid-washed perlite was mixed with chitosan flakes in oxalic acid. The mixture was dropwise added into a NaOH precipitation bath. Then the chitosan-coated perlite beads were obtained. Except for the adsorption of metal ions, chitosan gels can be applied in the adsorption of organic substances. Novel gels from phenylboronate derivatives of chitosan were prepared for the adsorption of saccharides by Matsumoto et al. [40]. The adsorption characteristics of Dglucose and 1-methyl-α-D-glucoside on the gels were examined and compared with a commercial gel. The resulted showed that the chitosan gels were inferior to the commercial gel in their adsorption capacity. Though the preparation of chitosan gels has advantages of simple operation and mild reaction conditions, the disadvantage of friability has impeded the development of chitosan gels.
3.3. Chitosan Nanoparticles Nanoparticles carriers could be used to increase the availability of drug for oral uptake, targeted release drugs, controlled or sustained release drugs, across the physical barriers and so on. Chitosan nanoparticles as biodegradable carriers have been widely used in the development of drug control releasing systems. Chitosan nanoparticles have been used as a carrier for insulin, vaccine, and proteins [41-43]. Calvo et al. first prepared the chitosan-poly ethylene oxide nanoparticles as protein carriers using a tripolyphosphate (TPP) cross-linking method [44]. In addition, poly (ethylene glycol) (PEG)-modified chitosan nanopartilces used as insulin carrier have been produced [45]. To improve hydrophilicity of chitosan, Kwon et al. [46] prepared hydrophobically modified glycol chitosans (HGCs) by covalent attachment of 5β-cholanic acid to glycol chitosan through amide formation. The mean size of selfaggregates was controllable by changing the DS of 5β-cholanic acid.
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Recently, the immobilization of enzyme onto the nanoparticle has drawn considerable interest. For instance, Chen et al. prepared nanosized chitosan particles immobilized trypsin [47]. Trypsin was immobilized on linolenic acid modified chitosan using glutaraldehyde (GA) as crosslinker. The results showed that the activity of trypsin immobilized onto linolenic acid modified chitosan nanoparticles increased with increasing concentration of GA up to 0.07% (v/v) and then decreased with increasing amount of GA. The particle size increased (from 523 to 1372 nm) with the increasing concentration of GA (from 0.03 to 0.1% v/v). In addition, the thermal stability and optimum temperature of trypsin immobilized on nanoparticles were improved. Furthermore, Chitosan nanocomposites have been applied in other fields, such as water treatment and bacterial inhibition [48, 49].
3.4. Multifunctional Resins Except for the adsorption properties, chitosan-based microspheres also have other functions by chemically modifying. Chitosan is a natural biopolymer, it is hydrophilic, and it has the ability to form complexes with metals. Wang et al. [50] prepared resins of chitosan-Ce4+ microspheres (RCCM) using the suspending cross-linked method in the adverse phase. Chitosan was solubilized in acetic acid solution. Ce(NH4)2(NO3)6, Ethyl acetate, paraffin, cross-linked agents were added to the above solution with heating and stirring. In the end, RCCM were obtained by washing with petroleum ether, acetone, ethanol and water. RCCM are characterized as the multifunctional polysaccharide polymers. Yu et al. reported RCCM could be employed in the apple juice production [51]. The contents of methamidophos and parathion-methyl in the juice treated by RCCM decreased 58.76% and 71.92%. Furthermore, RCCM was used to clarification for beer by the ways of a column [52]. RCCM could hydrolyze sensitive protein and increase contents of free amino acid in the beer. Therefore, the beer treated by RCCM could stay oxidation and aging procedure due to some metal ions being absorbed by RCCM. Both batch experiment and column technique was used to investigate the effect of RCCM on bittering of citrus juice [53]. The result of experiment showed that the quantity of naringin and limonin were reduced by 54.86% and 43.20% in juice treated by RCCM column, respectively. In addition, RCCM can also be used in tea beverage to improve its quality. RCCM showed considerable adsorption properties for citric acid [54]. Static adsorption experiments were carried out to study the adsorption kinetics and thermodynamics of citric acid on RCCM. The results indicated that the adsorption of RCCM on citric acid followed the Langmuir isotherm. The adsorption followed second-order kinetic equation. The adsorption rates of citric acid on RCCM were controlled by the intraparticle diffusion. With the hydrolysis activities and the adsorption capacity, RCCM is considered as a novel functional resin with its promising applications in several fields, such as food processing, water treatment and biological purification.
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3.5. Affinity Resins Research revealed crosslinked chitosan microspheres to be a good carrier of immobilized enzymes. The covalent attachment of protease inhibitors to chitosan can be achieved via the primary amino groups or the hydroxyl groups of chitosan. Several researches have reported chitosan microspheres as the support to immobilize enzymes [55, 56]. For instance, Wan et al. used chitosan microspheres as the support to immobilize laccase [57]. Chitosan microspheres were added to the laccase solution in phosphate buffer. After 24 h under stirring, the laccase immobilized chitosan microspheres were obtained. Compared with the free enzyme, immobilized laccase of chitosan microspheres showed a lower specific activity but had a similar substrate affinity with improved stability which made it more attractive in the application aspect. Furthermore, they also investigated the reuse of the immobilized laccase. The results showed: After being reused fifteen times, the immobilized laccase maintained at least 80% of its initial activity. Zhang et al. prepared the trypsin immobilized chitosan resin to separate soybean trypsin inhibitor [58]. The chitosan resin was activated by epichlorohydrin and coupled with trypsin. The determination results of immobilization trypsin activity showed that the immobilized trypsin chitosan resin had the adsorption affinity to soybean trypsin inhibitor. The scavenging rate for soybean trypsin inhibitor was 33.3%. So the immobilization trypsin chitosan can be a new cheap affinity carrier material.
3.6. Chitosan Magnetic Microspheres Chitosan-based microspheres have been extensively applied in several fields, including food processing, water treatment and biotechnology. But it is difficult to effectively separate from aqueous solutions. So it is necessary to find a simpler and effective method to solve this problem. Chitosan magnetic microspheres can be separated from the medium by a simple magnetic process after the adsorption. So in recent years, magnetic adsorbent technology has received considerable attention. The preparation process of chitosan magnetic microspheres can be divided into two steps. First, the Fe3O4 particles were prepared by coprecipitating Fe2+ and Fe3+ ions by ammonia solution and treating under hydrothermal conditions. Second, the Fe3O4 particles and chitosan acidic solutions were mixed to form the microspheres. Several researchers have prepared chitosan-based magnetic microspheres and applied in many fields, such as wastewater treatment, immobility of enzymes, drug release and so on [59-61]. In wastewater treatment, several investigations have reported about chitosan magnetic microspheres. Rorrer et al. prepared the porous-magnetic chitosan beads to remove of cadmium ions from waste water [62]. These beads can effectively remove Cd2+ from waste water. The adsorption efficiency was influenced by the beads size. To improve uptake properties of metal ions, Zhou et al. prepared magnetic chitosan microspheres chemically modified with thiourea (TMCS). Compared to the unmodified ones, the uptake properties of metal ions such as Hg2+, Cu2+, and Ni2+ ions was improved significantly. The adsorption kinetics followed the pseudo-second-order equation [63]. To improve the selectivity of metal ions, magnetic chitosan resin modified by Schiff’s base derived from thiourea and
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glutaraldehyde was prepared to separate Hg (II) from aqueous solution by Ahmed et al [64]. It was found that Hg (II) could be selectively separated from Cu (II), Pb (II), Cd (II), Zn (II), Ca (II) and Mg (II) at pH 1. Furthermore, chitosan magnetic microspheres have been used to remove dyes and fluoride from wastewater [65-67]. Chitosan magnetic microspheres have also applied in other application fields. For instance, Zong et al. immobilized lipase in magnetic chitosan microspheres [60]. Zhou et al. [61] prepared the chitosan magnetic microspheres containing aspirin for targeting drug release.
3.7. Adsorption Resins Chitosan have good sorption properties. With a large number of functional groups, chitosan-based microspheres have been applied in water treatment to remove metal ions or dyes. Recently, much attention has been paid to the adsorption of metal ions on chitosan, which is proved to be an extremely promising material due to its high chelating ability, higher hydrophilicity, environmentally safe, and abundant base material. Chitosan-based microspheres are well known as an excellent biosorbent for metal cation removal, such as Hg2+ [63], Ni2+, Cu2+, Zn2+,[68], Pb2+ [69]and Cd2+[70], etc. Taking mercury removal for example, chitosan-based microspheres proposed as adsorbents for metal removal is reviewed. Shafaei et al. [71] studied the Hg (II) adsorption capacity of chitosan. The results showed that the sorption isotherm presented a good fit to Langmuir isotherm. They also studied the effect of pH and chitosan particle size on the Hg (II) adsorption capacity. The higher adsorption capacity obtained was 11.7 mg g-1 at pH 6.0 and chitosan particle size 0.177 mm. To improve the adsorption capacity, several methods have been used to modify chitosan physically or chemically [72, 73]. Grafting of different functional groups in the crosslinked chitosan can enhance the adsorption capacity and the selectivity [74, 75]. Zhou et al. [76] prepared poly (ethyleneimine) grafted chitosan microspheres and studied the adsorption capacity of Hg (II). The Hg (II) maximum adsorptive capacity was 441.3 mg·g-1 and selective separation of Hg 2+ and UO2 2+ was performed at pH < 3. They also studied the adsorption of Hg (II) by ethylenediamine modified chitosan magnetic microspheres [77]. For the high adsorption properties towards Hg (II) ions of mercapto and amino groups, chitosan resins containing function groups of S and N have been synthesized. Chloromethyl thiirane (CT) modified Chitosan was synthesized by C. Ni et al. The Hg (II) adsorbing capacity of chitosanCT was 922.8 mg g-1 significantly higher than chitosan (676.0 mg g-1) [78]. To improve the capability and sensitivity for trace metals adsorption, Hakim et al. synthesized a new cross-linked chitosan (CCTS) modified with tris(2-aminoethyl)amine (TAA) moiety to Hg(II) [79]. A cross-linked structure of chitosan was synthesized with ethyleneglycoldiglycidylether (EGDE) as the cross-linker. EGDE cross-linked chitosan was reacted with epibromohydrin. The CCTS-epibromohydrin was then mixed with TAA in dioxane, and the mixture was refluxed for 3h. The synthesized resin showed good adsorption toward mercury in a wide pH range, and the adsorbed mercury can be easily eluted. Compared to Hg (II) adsorption on CCTS, the adsorbed Hg on CCTS-TAA can be easily eluted by using HNO3 without any addition of complexing agent. The synthesized resin has a strong application potential in Hg (II) determination of environmental samples. They also synthesized the cross-linked chitosan-threonine (CCTS-Thr) resin and applied to adsorb the
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trace amounts of Mo, V and Cu in environmental water samples [80]. The adsorbed elements can be easily eluted using nitric acid, and their recoveries were found to be 90–100%. In addition, chitosan-based microspheres also have been applied in the adsorption of dyes, proteins, organic acids, etc [81, 82].
4. CONCLUSION As a biopolymer, chitosan is easily processible into microspheres by many methods. Chitosan-based microspheres have been applied in such fields as biological engineering, medicines, wastewater treatment and food processing. With the unique molecular architecture of amino groups, Chitosan has a high potential for providing novel functions for special utilization. Progress in chitosan modification is quite rapid as evidenced by a sharp increase in the number of papers and patents in the past decade. However, there are also disadvantages which impeded the development of chitosan-based microspheres, such as friability, single function and tedious process of modification. So in the future, our work should focus on two main points: On the one hand, the preparation process should be optimized and improved to suit to industrial production and application. On the other hand, the modification should be easy and feasible. Moreover, it is necessary to expand the application field further through controlled modification reactions.
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[43] Xu, YM ; Du, YM ; Huang, RH. Preparation and modification of N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride nanoparticle as a protein carrier. Biomaterials, 2003, 24, 5015-5022. [44] Calvo, P; Lope, CR; Vila-Jato, JL; Alonso, M.J. Novel hydrophilic chitosan-poly ethylene oxide nanoparticles as protein carriers. J. Appl. Polym. Sci., 1997, 63, 125132. [45] Ohya, Y ; Cai, R ; Nishizawa, H ; et al. Preparation of PEG-grafted chitosan nanoparticle for peptide drug carrier.Proc. Intl. Symp. Controlled Release Bioact. Mater., 1999, 26,655-656. [46] Kwon, S; Park, JH; Chung, H. Physicochemical characteristics of self-assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid. Langmuir, 2003, 19, 10188-10193. [47] Liu, CG; Desai, KGH; Chen, XG. Preparation and characterization of nanoparticles containing trypsin based on hydrophobically modified chitosan. J. Agric. Food Chem., 2005, 53, 1728-1733. [48] Namdeo, M; Bajpai, SK. Chitosan–magnetite nanocomposites (CMNs) as magnetic carrier particles for removal of Fe (III) from aqueous solutions. Colloids and Surfaces A: Physicochem. Eng. Aspects, 2008, 320, 161-168. [49] Shi, ZL ; Neoha, KG ; Kang, ET. Antibacterial and mechanical properties of bone cement impregnated with chitosan nanoparticles. Biomaterials, 2006, 27, 2440-2449. [50] Wang, DF; Luo, Y; Du, DH; et al. Organophosphorous pesticide degradation by cerium complexes. Periodical of Ocean University of China, 2004, 34(4), 577-581. [51] Su, L; Wang, DF; Yu, LN; et al. Application ofchitosan-Ce4+ particles in the production of pellucid fruit juice. Food and Fermentation Industries, 2006, 32(1), 141-143. [52] Yu, LN; Wang, DF; Li, HY. Hydrolysis Activities of Resins of Complexes Made from Polysaccharides and Ce4 +. Journal of Rare Earths, 2006, 24, 125-129. [53] Li, HY; Wang, DF; Yu, LN. Effects of resins of chitosan-Ce4+ microspheres on bittering and quality of orange juice. Chinese Rare Earths, 2008, 29(4), 21-25. [54] Li, HY; Wang, DF; Yu, LN. Adsorption behavior of citric acid on resin of chitosan microspheres. The Chinese Journal of Process Engineering, 2009, 9(1), 86-91. [55] Ren, GZ; Li, ZH; He, BL. The immobilization of urease on magnetic chitosan microspheres I. synthesis and characterization of magnetic chitosan microspheres. Ion Exchange and Adsorption, 2000, 16(4), 304 -310. [56] Li, H; Wang, WJ; Xu, FC. Studies on preparation of chitosan microspheres and immobilization of papain on them. Journal of South China Agricultural University, 2000, 21(2), 49-53. [57] Wan, YY; Du, YM; Shi, XW. Immobilization and characterization of laccase from Chinese Rhus vernicifera on modified chitosan. Process Biochemistry, 2006, 41, 13781382. [58] Zhang, B; Wang, DF; Fan, Y; et al. Affinity purification of trypsin inhibitor with antiaspergillus flavus activity from cultivated and wild soybean. Mycopathologia, 2009, 167, 163-171. [59] Rorrer, GL; Hsien, TY. Synthesis of porous-magnetic chitosan beads for removal of cadmium ions from waste water. Ind Eng Chem Res, 1993, 32, 2170-2178. [60] Liu, ZW; Zhao, YF; Guang, Y. Investigation of immobilization of lipase in magnetic chitosan microspheres. Food Machinry, 2008, 24(1),13-15.
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[61] Guo, ZY; Dong, YY; Guo, XM. Preparation and evaluation of magnetic targeting drug release chitosan microspheres containing aspirin. Chinese Journal of Applied Chemistry, 2002, 19(2), 8711-2811. [62] Rorrer, GL; Hsien, TY. Synthesis of porous-magnetic chitosan beads for removal of cadmium ions from waste water. Ind Eng Chem Res, 1993, 32, 2170-2178. [63] Zhou, LM; Wang, YP; Liu, ZR. Characteristics of equilibrium, kinetics studies for adsorption of Hg (II), Cu (II), and Ni (II) ions by thiourea-modified magnetic chitosan microspheres. Journal of Hazardous Materials, 2009, 161, 995-1002. [64] Ahmed, MD; Asem, AA; Khalid, ZE. Selective separation of mercury (II) using magnetic chitosan resin modified with Schiff’s base derived from thiourea and glutaraldehyde. Journal of Hazardous Materials, 2008, 151, 372-379. [65] Safarik, I. Removal of organic polycyclic compounds from water solutions with a magnetic chitosan based sorbent bearing copper phthalocyanine dye. Wat Res, 1995, 29(1), 101-105. [66] Chang, YC; Chen, DH. Adsorption kinetics and thermodynamics of acid dyes on a carboxymethylated chitosan-conjugated magnetic nano-adsorbent.Macromol. Biosci, 2005, 5, 254-261. [67] Ma, W ; Ya, FQ ; Han, M; et al. Characteristics of equilibrium, kinetics studies for adsorption of fluoride on magnetic-chitosan particle. J Hazard Mat, 2007, 143, 296-302. [68] Juang, RS; Shao, HJ. A simplified equilibrium model for sorption of heavy metal ions from aqueous solutions on chitosan. Water Res, 2002, 36, 2999-3008. [69] Ruiz, M; Sastre, AM; Guibal, E. Palladium sorption on glutaraldehyde-crosslinked chitosan. React Funct Polym, 2000, 45, 155-173. [70] Kim, BS; Lim, ST. Removal of heavy metal ions from water by cross-linked carboxymethyl corn starch. Carbohydr Polym, 1999, 39, 217-23. [71] Shafaei, A; Ashtiani, FZ; Kaghazchi, T. Equilibrium studies of the sorption of Hg (II) ions onto chitosan. Chem. Eng. J., 2007, 133, 311-316. [72] Baba, Y ; Matsumura, N ; Shiomori, K; et al. Selective adsorption of mercury (II)on chitosan derivatives from hydrochloric acid. Anal. Sci., 1998, 14, 687-690. [73] Jeon, C; Holl, WHH. Chemical modification of chitosan and equilibrium study for mercury Ions Removal. Water Res., 2003, 37, 4770-4780. [74] Fujiwara, K; Ramesh, A; Maki, T. Adsorption of platinum (IV), palladium (II) and gold (III) from aqueous solutions onto l-lysine modified crosslinked chitosan resin. Journal of Hazardous Materials, 2007, 146, 39-50. [75] Ramesh, A; Hasegawa, H; Sugimoto, W. Adsorption of gold (III), platinum (IV) and palladium (II) onto glycine modified crosslinked chitosan resin. Bioresource Technology, 2008, 99, 3801-3809. [76] Zhou, LM; Liu, SR; Huang, QW. Adsorption of mercury and uranyl onto poly(ethyleneimine) grafted chitosan microspheres. Mod. Chem. Ind., 2007, 27,175177+179. [77] Zhou, LM; Wang, YP; Huang, QW. Adsorption of Hg 2+ and UO22+ by ethylenediamine modified chitosan magnetic microspheres. J. Nucl. Radiochem., 2007, 2911, 184-188. [78] Ni, C; Xu,Y. Studies on syntheses and properties of chelating resins based on chitosan. J. Appl. Polym. Sci., 1996, 59, 499-504. [79] Hakim, L; Sabarudin, A. Oshita K. Synthesis of chitosan-based resins modified with tris(2-aminoethyl)amine moiety and its application to collection/concentration and
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determination of trace mercury by inductively coupled plasma atomic emission spectrometry. Talanta, 2008, 76, 1256-1260. [80] Hakim, L; Sabarudin, A; Oshita, K. Synthesis of cross-linked chitosan functionalized with threonine moiety and its application to on-line collection/concentration and determination of Mo, V and Cu. Talanta, 2008, 74, 977-985. [81] Wang, YJ; Sun, Y. Preparation of crosslinked chitosan resins suitable for protein adsorption. Journal of Tianjin University, 2001, 34(6),819-822. [82] Xia, CF; Xing, DY; Chen, BR. Study of chloroethanoic and trichloroethanoic acid thermodynamics with cross-linked chitosan resin. Ion Exchang and Aasorption, 2004, 20(2), 125-130.
In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 48
CHEMOENZYMATIC SYNTHESIS OF AMYLOSEGRAFTED POLYMERS Yoshiro Kaneko and Jun-ichi Kadokawa* Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan.
ABSTRACT In this chapter, we review the synthesis of amylose-grafted polymeric materials with well-defined structures by chemoenzymatic method, which is the combination of following chemical with enzymatic reactions. As a chemical reaction for the present chemoenzymatic method, introduction of a maltooligosaccharide as a primer, which is an initiating point for the amylose-forming polymerization, to polymer backbones or polymerizable groups is carried out to produce maltooligosaccharide-grafted polymeric materials or macromonomers having maltooligosaccharide components. As an enzymatic reaction, formation of amylose is performed by phosphorylase-catalyzed enzymatic polymerization using α-D-glucose-1-phosphate as a monomer. This enzymatic polymerization is initiated from a meltooligosaccharide as a primer and the propagation proceeds by the reversible reaction to produce amylose. The main-chain backbones of the amylose-grafted polymeric materials describing in this chapter are polystyrene and polyacetylene as organic synthetic polymers, polydimethylsiloxane and silica gel as inorganic materials, and chitin, chitosan, cellulose, and polypeptide as biopolymers.
1. INTRODUCTION Amylose, a natural linear polysaccharide linked through (1J4)-α-glycosidic linkages (Figure 1), is one component of starch, which has been studied for many years in the materials research field because of its low cost, biodegradability, and renewability [1]. *
Corresponding author: Tel: +81-99-285-7743, Fax: +81-99-285-3253, E-mail:
[email protected] 1362
Yoshiro Kaneko and Jun-ichi Kadokawa
Amylose can be prepared by phosphorylase-catalyzed enzymatic polymerization using α-Dglucose 1-phosphate (G-1-P) as a monomer from a meltooligosaccharide as a primer, which is hereinafter described in detail [2]. Because of the natural polysaccharides such as amylose are recycled carbon resources and considered to be eco-friendly substances [3], it is expected that use of the polysaccharides as one component of hybrid polymers will lead to the production of environmentally benign materials. Graft copolymer is one of the hybrid polymers widely applied in the industrial fields [4], and has the structure of polymer main-chain as a backbone covalently linked to graft-chains as the branches. Generally, graft copolymers can be prepared by three synthetic methods; “grafting onto,” “grafting through,” and “grafting from” methods (Scheme 1) [5]. In the “grafting onto method,” the polymer main-chain and the graft-chain are individually prepared by polymerization. The main-chain has functional groups distributed along the chain that can react with the other polymers as graft-chains (Scheme 1a). On mixing the main-chain and the graft-chains in the desired proportion and under the appropriate experimental conditions, a coupling reaction takes place resulting in the graft copolymers. In the “grafting through method,” previously prepared macromonomer is copolymerized with another monomer to produce the graft copolymer (Scheme 1b). The macromonomer has the structure of a polymeric chain having a polymerizable end group. In this case, the macromonomer comprises the graft-chain in the copolymer and the main-chain is formed in situ. In the “grafting from method,” after the preparation of the polymer main-chain, active sites are produced on the main-chain that are able to initiate the polymerization of the second monomers (Scheme 1c). Polymerization of the second monomer results in the formation of graft-chain and the final graft copolymer. Graft copolymers composed of synthetic polymers alone can be easily prepared by above-mentioned preparation methods, because numerous methods for polymerizations to form well-defined synthetic polymers and coupling reactions to link a polymer to another polymer have been reported so far. On the other hand, graft copolymers containing polysaccharides have only been obtained in limited cases. As examples of such materials, a type of graft copolymers composed of polysaccharide main-chains and synthetic polymer graft-chains is common [6]. However, little has been reported regarding preparation of graft copolymers containing polysaccharide graft-chains. Because the solubility of polysaccharides is generally poor in any organic solvents, direct reaction of polysaccharides with polymeric main-chains (grafting onto method) is quite difficult. In addition, it is not easy way to construct the polysaccharide-grafted polymeric materials by grafting through and grafting from methods, because the polymerization methods to form well-defined polysaccharides have hardly been known, except the enzymatic method that is hereinafter described. OH HO HO
O OH OH
O
OH
O HO
O
OH n
Figure 1. Structure of amylose.
O HO OH
OH
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Scheme 1. Synthetic methods for graft copolymers.
Scheme 2. Formation of (1J4)-α-glucan (amylose) catalyzed by phosphorylase.
The enzymatic polymerization is a useful tool for the regio- and stereocontrolled preparation of polysaccharides [7]. For example, phosphorylase-catalyzed enzymatic polymerization using G-1-P proceeds with the regio- and stereoselective construction of an αglycosidic bond under mild conditions, leading to the direct formation of amylose in aqueous media. This polymerization is initiated from a maltooligosaccharide primer. Then, the
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propagation proceeds through the following reversible reaction to produce a (1J4)-α-glucan chain, that is amylose (Scheme 2). In the reaction, a glucose unit is transferred from G-1-P to the nonreducing 4-OH terminus of a (1J4)-α-glucan chain, resulting in inorganic phosphate (P) [2]. By means of the combination of the above-mentioned enzymatic polymerization forming amylose with a chemical reaction, i.e., a chemoenzymatic approach, several research groups have developed polysaccharide-grafted polymeric materials with well-defined structures. For example, the maltooligosaccharide primer is firstly introduced to the polymer backbones to obtain primer-grafted polymers, and then the phosphorylase-catalyzed enzymatic polymerization of G-1-P from the primer-grafted polymers is performed to produce amylosegrafted polymers. In the chemoenzymatic method, the length of amylose graft-chain can be controlled by changing the feed ratio of G-1-P to the primer on the enzymatic polymerization, whereas the number of amylose graft chain can be controlled by changing the feed ratio of the macromonomer containing amylose-chain to the comonomer (grafting through method) or the number of the primers on the main-chain (grafting from method). The present chapter reviews research developments in the area for the preparation of amylose-grafted polymeric materials by the chemoenzymatic method. In the first part, we describe the general methods to link maltooligosaccharides as the primers to polymer backbones or polymerizable groups by the chemical reaction. In the second part, we introduce the preparation of amylose-grafted polymers using organic synthetic polymers as main-chains such as polystyrene and polyacetylene (Figure 2a,b). In the third part, the preparation of amylose-grafted inorganic materials such as polydimethylsiloxane and silica gel is described (Figure 2c,d). In the last part, we describe the preparation of amylose-grafted biopolymers such as chitin, chitosan, cellulose, and poly(L-glutamic acid) (Figure 2e-g), assuming production of the eco-friendly materials because they were composed of only biopolymers as recycled carbon resources.
amylose
a) polystyrene b) polyacetylene c) polydimethylsiloxane d) silica gel e) chitin and chitosan f) cellulose g) poly(L-glutamic acid) Figure 2. Various amylose-grafted polymers.
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Scheme 3. Representative reaction methods to link the maltooligosaccharide to polymer backbones or polymerizable groups.
2. GENERAL REACTION METHODS TO LINK MALTOOLIGOSACCHARIDE TO POLYMER BACKBONES OR POLYMERIZABLE GROUPS In this section, representative three reaction methods to link the maltooligosaccharides to polymer backbones or polymerizable groups are described (Scheme 3).
2.1. Reaction of Maltooligosaccharide having Lactone Group at Reducing Terminus with Amino Group The reducing terminus of maltooligosaccharide can be easily converted to the corresponding aldonic acid by oxidation, which leads to aldonolactone by drying process. Generally, the aldonolactone selectively reacts with the amino group to obtain the corresponding aldonamide without protection of hydroxy groups on the maltooligosaccharide (Scheme 3a) [8]. Use of amine-functionalized polymer backbones or polymerizable groups on this reaction can provide maltooligosaccharide-grafted polymer backbones or macromonomers having maltooligosaccharide components.
1366
Yoshiro Kaneko and Jun-ichi Kadokawa
2.2. Reductive Amination of Maltooligosaccharide with Amino Group Reductive amination is the process that amine reacts with aldehyde or ketone to form imine, which is subsequently reduced to amine (Scheme 3b) [9]. Reductive amination can be utilized to link amine-functionalized molecules to maltooligosaccharides because the reducing terminus of maltooligosaccharide has a hemiacetal structure, which is in equilibrium with an aldehyde group. Sodium cyanoborohydride (NaBH3CN) and sodium triacetoxyborohydride (NaBH(OCOCH3)3) are generally employed as reducing agents for the reductive amination of the reducing terminuses in oligosaccharides under mild conditions.
2.3. Reaction of Maltooligosaccharide Having Amino Group at Reducing Terminus with Carboxyl Group Introduction of amino group to the reducing terminus of maltooligosaccharide can be achieved by treatment with aqueous ammonium hydrogen carbonate [10]. The resulting maltooligosaccharide having glycosyl amine terminus can be condensed with the carboxyl group via the formation of an amide linkage (Scheme 3c).
3. AMYLOSE-GRAFTED ORGANIC SYNTHETIC POLYMERS 3.1. Amylose-grafted Polystyrene In the research field of the synthetic polymer chemistry, polystyrene and its derivatives are one of most representative synthetic polymers because of some unique properties that make them useful in a wide range of products [11]. The commercial success of polystyrene is due to transparency, ease of fabrication, thermal stability, relative high modulus, and low cost. Hybridization between polystyrene and amylose is a promising research topic from the viewpoints not only of preparation of new hybrid materials, but also of fusion of two symbolic polymers in both synthetic and natural polymer chemistries. However, it may be difficult to hybridize the polystyrene and amylose by blend method of these two polymers, because of immiscibility of these polymeric chains caused by quite different polarities. In this section, therefore, as one of the methods to solve this problem, we describe the preparation of amylose-grafted polystyrene by the chemoenzymatic method according to the following reaction manners [12]. Kobayashi et al. investigated two different synthetic approaches for the preparation of these graft copolymers (Scheme 4). Route I is the phosphorylase-catalyzed enzymatic polymerization of G-1-P from vinylbenzyl maltopentaose amide (1), followed by radical polymerization of the resulting vinylbenzyl amylose amide macromonomer (2) to prepare amylose-grafted polystyrene (4) (grafting through method) (Scheme 4). Every repeating unit has a hydrophilic amylose graft-chain which is connected with a hydrophobic styrene component via an amido group. Route II is the radical polymerization of 1 at first and then the enzymatic polymerization of G-1-P from the resulting maltooligosaccharide-grafted polystyrene (3) to obtain 4 (grafting from method) (Scheme 4). In the grafting from method, amylose-chain was probably elongated from a part of maltooligosaccharide sites on 3,
Chemoenzymatic Synthesis of Amylose-Grafted Polymers
1367
because of steric hindrance of crowded graft-chain on 3. Therefore, amylose-chains on 4 probably possess rather uniform chain length, and sterically hindered primers would be left.
3.2. Amylose-grafted Polyacetylene Researches concerning conjugated polymers, such as polyacetylene, polypyrrole, and polyphenylene, have attracted much attention in terms of the various practical applications of their interesting electrical and optical properties [13]. Moreover, interests and applications of the conjugated polymers have been extended to the biological fields. In order to endow the conjugated polymers with the biological functions derived from sugar residues, for example, the sugar-substituted conjugated polymers were synthesized by using the conjugated mainchain structures of polyaniline [14], polyisocyanide [15], polythiophene [16], polyphenylene [17], poly(p-phenylene ethynylene) [18], and poly(p-phenylene vinylene) [19]. In the series of these studies, we reported the synthesis of poly(N-propargylamide) with galactose residues [20]. It contained a cis-polyacetylene main-chain and was obtained by the Rh-catalyzed polymerization of a corresponding N-propargylamide monomer. On the basis of the above study, the polyacetylene having graft saccharide chains with higher degree of polymerization (DP), e.g., natural polysaccharides, would be functional polymers with hybrid properties of polyacetylene and polysaccharide. In this section, therefore, we describe the synthesis of the amylose-grafted polyacetylene by the chemoenzymatic method as an effective preparation method for the polyacetylene-polysaccharide hybrid. The maltooligosaccharidegrafted polyacetylenes as key materials for the amylose-grafted polyacetylenes were prepared according to the synthetic manners of “macromonomer (grafting through) method [21]” and “polymer reaction (grafting onto) method [22]” as shown in Scheme 5 and 6, respectively.
Scheme 4. Synthetic approaches for amylose-grafted polystyrene (4).
1368
Yoshiro Kaneko and Jun-ichi Kadokawa (a)
OH
HO HO
OH
CH O OH OH
O O HO
O
C [Rh(cod)2]BF4HO HO CH2 HN water HO
OH
O
OH
5
5
HN
O HO
O
O
n
OH OH
HO O
OH
5
6
OH
H
O
OH cod = 1,5-cyclooctadiene
CH2
OH OH
OH OH G-1-P, phosphorylase
O
HO HO
n
OH O
OH
acetate beffer (pH = 6.2)
O HO
CH2
HN HO
H
O
OH O
OH
m
7
OH OH
(b) 5
OH
CH +
O
C [Rh(cod)2]BF4 HO HO CH2 HN water
OH OH
O O HO
O
HN
OH
HO
H
HN
O
O
CH2
H
n
O
OH
5
8
CH2
9
OH OH
OH G-1-P, phosphorylase acetate beffer (pH = 6.2)
HO HO
O OH OH
O O HO
HN
OH
HO O
m
10
CH2 O
H
HN
CH2
H
n
O
OH OH
OH
Scheme 5. Chemoenzymatic synthesis of amylose-grafted polyacetylenes (7) and (10) by macromonomer method.
First, we describe the macromonomer method to prepare the amylose-grafted polyacetylenes. A N-propargylamide macromonomer (5) having a maltooligosaccharide substituent was firstly prepared by the reaction of maltoheptaonolactone with propargylamine without protection of sugar hydroxy groups. Then, Rh-catalyzed polymerization of 5 was carried out to give the polyacetylene (6) with pendant maltooligosaccharide chains (Scheme 5a). Furthermore, the copolymerizability of 5 was investigated by the copolymerization with the other N-propargylamide monomer (8). The reaction was performed in the various feed ratios of 5 to 8 under the similar conditions as those of the homopolymerization of 5 as described above to produce copolymer (9) (Scheme 5b). The enzymatic polymerization of G-1-P using 6 and 9 as primers was performed to produce the two types of amylose-grafted polyacetylenes (7) and (10) (Schemes 5a and b). The complex formation with iodine is a well-known characteristic property of amylose [23]. The colorless solutions of 7 and 10 in DMSO turned to violet after the addition of a standard iodine-iodide solution to the polymer solution, as the same color change in the complex formation of amylose with iodine. Values of the λmax of UV-Vis spectra of the iodine
Chemoenzymatic Synthesis of Amylose-Grafted Polymers
1369
complexes with 7, 10, and amylose were 577, 586.5, and 586.5 nm. These data indicated that the average DP of 10 was probably comparable to that of amylose, whereas the average DP of 7 might be lower in comparison with amylose. On the basis of the above data, we assume the difference in the enzymatic polymerization using 6 and 9. The enzymatic polymerization was hardly initiated from all the potential sites of the primers on 6 due to steric hindrance, resulting in the lower average DP. On the other hand, the less hindered orientation of the primers on 9 due to existence of units 8 in the main-chain was probably more suitable for initiation from the most of the potential sites for the chain elongation reaction. Therefore, the average DP of 10 became same as that of amylose. To obtain the amylose-grafted polyacetylene with the higher DP of main-chain, we designed the approach according to the polymer reaction method (Scheme 6) [22]. We first prepared the amine-functionalized polyacetylene (11) with the high DP (ca. 72-112) according to Scheme 7, which had the ability to react with maltoheptaose lactone, giving the maltooligosaccharide-grafted polyacetylene (12). Then, the phosphorylase-catalyzed polymerization from the maltooligosaccharide chain on 12 produced the desired amylosegrafted polyacetylene (13). OH + n
NH
O
HO HO
OH O
OH
NH3Cl
O HO
O
OH O
OH
11
5O
HO
O
maltoheptaose lactone
OH
O NH OH Et3N ethylene glycol
HO HO
O OH
n
O
OH O HO
NH HO
NH3Cl
O
OH 5O
OH
12 OH OH
O NH OH G-1-P, phosphorylase acetate beffer (pH = 6.2)
HO HO
O OH
n
O
OH O HO
NH HO
O
OH mO
NH3Cl
OH
13 OH OH
Scheme 6. Chemoenzymatic synthesis of amylose-grafted polyacetylene (13) by polymer reaction method.
1370
Yoshiro Kaneko and Jun-ichi Kadokawa O N H 14 +
n
[(nbd)Rh+B–(C6H5)4]
NH
THF
O
O
HCl aq. / MeOH
11
NH O O
N H 15
O nbd = norbornadiene
16
Scheme 7. Synthesis of amine-functionalized polyacetylene (11) by copolymerization of Npropargylethanamide (14) with tert-butyl propargylcarbamate (15), followed by deprotection.
OAc CH3 O Si CH3
CH3 O Si
O
AcO AcO
+
OAc O
OAc OAcO
n
OAc O
OAc 5O
17
AcO
18
NH2
O OAc
CH3 O Si CH3
OAc AcO AcO
O
CH3 O Si
OAc
AcO
O
5O
O
n
HN
O HO
O
OAc
CH3 O Si
OH OH
HN
OAcO
CH3 O Si CH3
OH
HO HO
n
OAc O
deacetylation
HO
O
OH 5O
OAc
OH
20
19
OH
OAc OH
OAc
Scheme 8. Synthetic approach (II) for maltooligosaccharide-grafted PDMS (20). CH3 O Si CH3
OAc
18
AcO NH2 AcO allylamine
O OAc O
OAc
HN
OAcO
AcO 5O
n
22 O
OAc
CH3 O Si H
deacetylation 19
20
hydrosilation
OAc
21 OAc OAc
Scheme 9. Synthetic approach (III) for maltooligosaccharide-grafted PDMS (20).
4. AMYLOSE-GRAFTED INORGANIC MATERIALS 4.1. Amylose-grafted Polydimethylsiloxane Inorganic polymers such as polydimethylsiloxanes (PDMSs) have various interesting properties, e.g., high oxygen permeability, low toxicity, and biocompatibility, which are advantages as biomaterials [24]. Therefore, saccharide-PDMS hybrid would be expected to have a significant potential for biological applications. In this section, therefore, we describe
Chemoenzymatic Synthesis of Amylose-Grafted Polymers
1371
the preparation of amylose-grafted PDMS by the chemoenzymatic method as an effective hybridization method [25]. Stadler et al. investigated the reaction of maltooligosaccharide with PDMS backbones by following four methods. As a first approach, the reaction of maltoheptaonolactone with aminefunctionalized PDMS (17) was investigated. However, because of difference in solubility between maltoheptaonolactone and 17, this reaction did not proceed. In a second approach, the reaction of peracetylated maltoheptaonolactone (18) with 17 was performed to obtain peracetylated maltoheptaose-grafted PDMS (19), which was converted to maltoheptaose-grafted PDMS (20) by deacetylation (Scheme 8). A third approach was to employ the reactive allylic double bond, which was introduced by the reaction of 18 with allylamine. The resulting derivative (21) was coupled with polydimethylsiloxane (22) containing Si-H groups by hydrosilation to obtain 19, which was converted to 20 by treatment same as that for the second approach (Scheme 9). On the other hand, as a fourth approach, maltoheptaose was peracetylated to obtain 23, which was converted to the β-1-O-allylmaltoheptaoside (24) by reaction with allylalcohol. The allylic double bond was added to 22 by hydrosilation using platinum catalysis. The resulting O-acetylated maltooligosaccharide-grafted PDMS (25) was deprotected to obtain the maltooligosaccharide-grafted PDMS (26) (Scheme 10). The phosphorylase-catalyzed enzymatic polymerization of G-1-P from maltoheptaoside graft-chains of 20 and 26 proceeded in aqueous citrate buffer although these maltooligosaccharide-grafted PDMSs with high molecular weights remain water insolubility. This is because maltoheptaose graft-chains are solvated in an aqueous media and are accessible for phosphorylase to proceed the polymerization.
4.2. Amylose-grafted Silica Gel In this section, we take up a topic of silica gel as the backbone for amylose-grafted materials. Silica gel is a useful media for the separation of various molecules in highperformance liquid chromatography (HPLC). In particular, combination of silica gel with chiral compounds such as polysaccharide derivatives provides enantioseparation for racemic molecules, which is practically used for determining optical purity and for obtaining optical isomers [26]. Phenylcarbamate derivatives of amylose and cellulose are known to show high chiral recognition as chiral stationary phases (CSPs) in HPLC [27]. For example, amylose (27) and cellulose (28) having 3,5-dimethylphenylcarbamate groups have been widely used to separate a broad range of racemates, including drugs (Figure 3). OAc
OAc AcO AcO
O OAc
OH O
OAc OAcO
OAc AcO
O
OAc OAcO
O
OAc
O
AcO
24 CH3 O Si CH3 OAc
OAc
O 5O
OAc
O
OAc
OAc
OAc
OAc AcO hydrosilation AcO
O OAcO
BF3 Et2O
O
5O
22
OAc OAc
allylalcohol
OAc 23
O
AcO AcO
OH
CH3 O Si
deacetylation n
O
OAc
HO HO
OH O
OH O HO
AcO
O
OH
5O
25
CH3 O Si CH3 OH
O
5O
OAc
26
Scheme 10. Synthetic approach (IV) for maltooligosaccharide-grafted PDMS (26).
HO
OH
CH3 O Si n
O
1372
Yoshiro Kaneko and Jun-ichi Kadokawa R
R NH
NH
O
O O
O O
O O HN R
O
R=
O
O
O O
CH3 O
HN R
n
NH
O
O O
n
CH3
NH
R
R
27
28
Figure 3. Structures of amylose (27) and cellulose (28) having 3,5-dimethylphenylcarbamate groups. OH HO HO
OH O O
OH O HO
O
HO 1) I2, KOH / water-methanol HO
OH OH
3
2) Amberlite
O
OH O HO
OH
OH O
OH
(H+)
O HO
OH
3
OH HO HO 3-aminopropyltriethoxysilane
O OH
OH O
Si(OEt)3
O O HO
29
maltopentaose
H2N
OH
OH
OH O
OH O HO
HN
OH
HO
Si(OEt)3
G-1-P phosphorylase
O
HO HO
OH O HO
O
O
O
OH
OH
OH
OH OR
OH HO
silica gel
DMSO-pyridine, 90
HO HO oC
OH
n
31
OH
Si(OEt)3 O
O
OH
3
30
HN HO
OH
O
1) OCN
OH OH
O O HO
HN
OH
HO O
n
32
Si O
O
RO RO
silica gel 2) (CH3)3SiCl
O OR OR
O O RO
OR
O
OH OH
OH
HN RO
n
CSP-I 33
Si
O
silica gel
O OR OR
R=
O H C N
OR
Scheme 11. Synthetic approach (I) for amylose-grafted silica gel (33).
The CSPs are usually prepared by coating and adsorbing the polysaccharide derivatives, e.g., 27 and 28, on macroporous silica gel. Therefore, because such polysaccharide derivatives were dissolved or swelled in the organic solvents such as THF and chloroform, they can not be used as main mobile phases. To overcome this problem, Okamoto et al. prepared CSPs in which the polysaccharide phenylcarbamates were chemically linked to silica gel through hydroxy groups of the polysaccharides [28]. However, these linked CSPs showed lower chiral recognition ability. The introduction of the above-mentioned chemical linkage may disturb the formation of regular higher-ordered structure of the polysaccharides, which is important for high enantioselectivity. In this section, therefore, the preparation of the CSP, in which amylose is chemically linked to silica gel at the reducing terminal residue alone by chemoenzymatic method, is described [29]. Amyloses bearing triethoxysilyl groups at the reducing terminal residues were firstly prepared by the phosphorylase-catalyzed enzymatic polymerization of G-1-P using two kinds of primers derived from maltopentaose. The resulting amylose chains, which had a desired chain length and a narrow molecular weight distribution, were then linked to silica gel to be used as CSPs. Two kinds of synthetic approaches are illustrated in Scheme 11 and 12, respectively. In approach (I), maltopentaonolactone (29) was firstly prepared, followed by reaction with 3aminopropyltriethoxysilane to obtain the maltooligosaccharide (30) bearing triethoxysilyl group. Amylose chains were then extended by the phosphorylase-catalyzed enzymatic
Chemoenzymatic Synthesis of Amylose-Grafted Polymers
1373
polymerization, and the resulting amylose derivative (31) was allowed to react with silica gel to produce amylose-grafted silica gel (32) (Scheme 11). In approach (II), maltopentaose was firstly oxidized to be converted into a potassium gluconate (34) at a reducing terminal residue, and the enzymatic polymerization was then performed. After the lactonization, the amylose was immobilized onto the silica gel to obtain 37 (Scheme 12). Finally, both the amylose-grafted silica gels 32 and 37 were treated with a large excess of 3,5-dimethylphenyl isocyanate to derivatize the remaining hydroxyl groups of amylose to the carbamates (Schemes 11 and 12). These methods have two advantages; one is that the immobilization onto silica gel only at the reducing terminal residue of amylose can be achieved, and the other is that amylose with a desired chain length and a narrow molecular weight distribution can be easily prepared. The chiral recognition abilities of these materials were investigated using racemic molecules, e.g., trans-stilbene oxide (Figure 4) by HPLC. Consequently, the enantiomers eluted at different retention times, indicating that racemic trans-stilbene oxides were completely separated. In addition, the chiral discrimination ability depended on both the DP of amylose and the preparation method. The chiral recognition ability increased with an increase in the DP of amylose, and the CSPs prepared by approach (II) were superior to those by approach (I) if the DPs were similar. Methods I and II differed only in the surface treatment processes. The silica surface in approach (I) has been end-capped with trimethylsilyl chloride, while that in approach (II) has been end-capped with 3,5dimethylphenyl isocyanate.
Figure 4. Structure of trans-stilbene oxide.
OH
OH HO HO
OH O HO
OH OH
1) I2, KOH / water-methanol
O
OH
OH O
HO HO
O
O O HO
OH
OH
O
OH 3
O HO
OK HO O 3
OH
OH
34
maltopentaose
G-1-P phosphorylase
O
HO HO
OH OH
O O HO
O
n
35
OH
HCl (pH = 1) / water
O OH OH
H2N O
O HO
Si
O
silica gel
OH DMSO, 80 oC
O
OH n
O HO
HO HO
O OH O
OH O HO
n
37
2) DMA-pyridine
RO RO
O OR O
OR O RO
HN
OR
RO O
n
CSP-II 38
Si
O
silica gel
O OR OR
R=
O H C N
OR
Scheme 12. Synthetic approach (II) for amylose-grafted silica gel (38).
Si O
OH OH
OH
OR 1) OCN
HO O
O OH
36
HN
OH
O
silica gel
O OH OH
OH
OH
OH
HO O
OH
OH
HO HO
OK
OH
1374
Yoshiro Kaneko and Jun-ichi Kadokawa
5. AMYLOSE-GRAFTED BIOPOLYMERS 5.1. Amylose-grafted Chitin and Chitosan Starch, cellulose, and chitin are the most abundant polysaccharides on earth and have widely been studied for their potential to become substitutes for petroleum-based materials. Because the polysaccharides are naturally recycled carbon resources and are considered to be eco-friendly because of their biodegradability [3], it is expected that the use of polysaccharides will lead to the production of environmentally benign materials. Besides the abundant materials, polysaccharides having branched structures are often found in nature. For example, arabinoxylan, gum arabic, and guaran play important roles in moisture maintenance and protection against bacteria [30]. These materials are composed of two or more different kinds of saccharide components, which contribute to their prominent functions. On the basis of the above, the development of an efficient method for the preparation of branched (grafted) artificial heteropolysaccharides using common polysaccharides is a promising topic in materials research fields. In this section, therefore, we describe the chemoenzymatic synthesis of amylose-grafted chitin [31] and chitosan [31,32] as graft heteropolysaccharides. Synthetic approaches for these materials are shown in Scheme 13. Introduction of maltoheptaose primer to chitosan by reductive amination was firstly carried out using NaBH3CN in a mixed solvent of aqueous acetic acid and methanol to produce maltoheptaosegrafted chitosan (39) [9]. The functionality of maltoheptaose to chitosan in 39 depended on reaction time. Maltoheptaose-grafted chitin (40) was synthesized by N-acetylation of 39 using acetic anhydride in a mixed solvent of aqueous acetic acid and methanol. Phosphorylasecatalyzed enzymatic polymerization of G-1-P from maltoheptaose in 39 and 40 was then performed to obtain amylose-grafted chitosan (41) and chitin (42), respectively. The products 41 and 42 were insoluble in any solvents, e.g., aqueous acetic acid and DMSO, which were good solvents for chitosan and amylose, respectively. The XRD patterns of these materials showed typical A-type crystalline structures due to amyloses. Such crystalline structures are generally attributed to the formation of a double helix of amylose [33]. Therefore, we assume that one of the reasons for the insolubility of these materials is probably caused by molecular aggregation as a result of this crystalline structure. The aggregation in the material would contribute its conversion into film and hydrogel forms. For example, the hydrogel of 41 could be formed by drying the reaction mixture slowly in the vessel at 40-50 ºC.
5.2. Amylose-grafted Cellulose Two representative natural polysaccharides, cellulose and amylose, are composed of the same structural unit, i.e., the glucose unit, but linked through the different (1J4)-β- and (1J4)-α-glucosidic linkages, respectively. The roles of cellulose and amylose in nature are completely different; the former is the structural material and the latter acts as the energy source. In this section, we describe the chemoenzymatic synthesis of the graft heteropolysaccharide composed of these two polysaccharides, i.e., amylose-grafted cellulose [34]. This material has a very interesting and unique structure because that is composed of two polysaccharide chains with the same structural unit but with the different linkages.
Chemoenzymatic Synthesis of Amylose-Grafted Polymers
1375
OH OH
O
HO HO
O
OH
+
O HO
O
OH
OH
O HO
NH2
O
OH
n
chitosan
5
O HO
OH
OH
maltoheptaose NaBH3CN in CH3COOH aq./CH3OH OH
OH
O
O HO
O HO
NH2 O
HO HO
OH
O
OH
O
OH
O
OH
OH
OH
O OH
40
phosphorylase OH O HO
NH
OH
O HO
NH2
OH
O
O HO
NH
O
HO HO
HO
OH
OH
OH O
OH
OH O
m
OH
OH
42
Scheme 13. Synthetic approaches for amylose-grafted chitin (42) and chitosan (41). OH OH O HO
NH2
O O HO
OH
O
HO HO
O
OH O
OH
+
O HO
OH
OH O
OH
n
43 5
O HO
OH
maltoheptaose OH HO HO
O
OH
OH
O
OH
O
OH
OH
HO
5
O OH
HO OH
NaBH3CN in CH3COOH aq./CH3OH
O HO
NH2
O
HN
O
O HO
OH
O
O HO
OH
OH n
44 OH HO HO
O
OH
OH
O
OH
O OH
HO
OH O
m
OH
HO G-1-P phosphorylase in sodium acetate buffer
OH O HO
NH2
O OH
O HO
NH n
CH3 OH
O
O HO
m
41
O
O
OH
OH
O
OH
O
O
HO
HO
n
OH
O
O HO
G-1-P
OH
NH CH3
phosphorylase
O HO
O
5
n
O
NH
G-1-P
OH
NH2
HO HO
OH
O
O HO
O HO
HO
OH
O
39
OH
O
OH
OH
HO
O HO
NH2
OH
O
O
OH
HO
OH
5
HO HO
in CH3COOH aq./CH3OH
NH
OH
O
O HO
n
OH
OH
OH
(CH3CO)2O
O
O OH
HN O HO
O OH n
45
Scheme 14. Synthetic approach for amylose-grafted cellulose (45).
OH
1376
Yoshiro Kaneko and Jun-ichi Kadokawa
OH O HO
CH3
O Cl S CH3 O tosyl chloride
O
O OH O HO
Et3N in LiCl/DMAc
OH n
O
O O HO
OH
OH n
46
cellulose OH
NaN3
N3
O
O HO
(n-Bu)4NI in DMSO
S OO
O HO
OH
NaBH4
O
in DMSO
OH
43
n
47
Scheme 15. Synthesis of amine-functionalized cellulose (43). OH
OH O
HO HO
OH
O O HO
OH O HO
maltopentaose H N
OH OH
OH
O O HO
O
OH 3
O
HO HO NH3HCO3 aq.
OH
OH O
OH
OH
3
O HO
NH2 OH
48
O C n
OH HO O poly(L-glutamic acid)
HO HO
O C
H N
O
O C
H N
n
OH O
OH
OH
O HO
HOBt / BOP in DMSO
O HN
OH 3
O
HO
O
O HO OH 49
OH
G-1-P / phosphorylase
HO HO
O C
H N
O
O C
H N
n
OH OH
O O HO
OH O HN
OH m
O HO
O
HO
O
OH 50
Scheme 16. Synthetic approach of amylose-grafted poly(L-glutamic acid) (50).
For the preparation of desired amylose-grafted cellulose by the chemoenzymatic method as shown in Scheme 14, the amino groups have necessarily been introduced to cellulose because the amino groups efficiently react with maltooligosaccharides by the reductive amination, giving rise to the maltooligosaccharide-grafted cellulose (44). Therefore, we first performed the synthesis of amine-functionalized cellulose (43), which was successfully obtained by the three reaction steps, i.e., the partial tosylation of the OH groups at C-6 positions, the displacement of the tosylates by the azido groups, and the reduction to the amino groups (Scheme 15). The reductive amination of 43 with maltoheptaose was carried out by reaction procedure same as that of chitosan with maltoheptaose as described above. Subsequently, the synthesis of the amylose-grafted cellulose was performed by the phosphorylase-catalyzed enzymatic polymerization of G-1-P from the maltoheptaose graft chains on 44 (Scheme 14).
Chemoenzymatic Synthesis of Amylose-Grafted Polymers
1377
When the reaction mixture obtained by the enzymatic polymerization was kept in a Petri dish at room temperature for around five days, it totally turned into the gel form. The produced gel was washed with water several times to remove some contaminants such as the unreacted G-1-P. This gel has a more tough nature than that formed from the amylose-grafted chitosan as described previous section.
5.3. Amylose-grafted Polypeptide Glycoproteins, peptidoglycans, and proteoglycans are saccharide-polypeptide conjugates which occur in blood, plasma membranes, intercellular matrixes, and connective tissues. These naturally occurring saccharide-polypeptide conjugates play important roles in various kinds of biological processes [35]. Oligosaccharide-polypeptide conjugates have been prepared by many researchers to develop model compounds for molecular recognition [36]. In this section, we describe preparation of polysaccharide-polypeptide conjugate, i.e., amylose-grafted poly(L-glutamic acid), by chemoenzymatic method [37], which can be considered as a new and simple model compound for biological applications. Kobayashi et al. investigated the preparation of such a graft copolymer by following synthetic approach (Scheme 16). Maltopentaose was treated with aqueous ammonium hydrogen carbonate to introduce an amino group to the reducing terminus. The resulting maltopentaosylamine (48) was condensed with the pendant carboxyl group of poly(Lglutamic acid) via the formation of an amide linkage. The condensation step was carried out in the presence of 1-hydroxybenzotriazol (HOBt) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) as the coupling reagents in dimethyl sulfoxide at room temperature to obtain maltopentaose-grafted poly(L-glutamic acid) (49). Maltopentaose graft-chains of 49 were elongated to amylose chains by the phosphorylase-catalyzed enzymatic polymerization of G-1-P. The resulting amylose-grafted poly(L-glutamic acid) (50) was soluble in DMSO and 1 mol/L KOH aqueous solution, but insoluble in water. The CD spectra of 49 depended on pH. A negative strong Cotton effect was observed at 209 and 222 nm at pH below 5.4, and a positive weak Cotton effect was observed at 217 nm at pH above 7.6. This tendency of CD profiles was similar to that of the poly(L-glutamic acid). The above data indicated that the maltopentaose-grafted poly(L-glutamic acid) 49 formed a helical conformation at lower pH and a random coil conformation at higher pH. On the other hand, little α-helix content was observed for 50, indicating that helical formation was disturbed by the amylose chains, probably owing to steric hindrance.
6. CONCLUSIONS We reviewed the development of amylose-grafted polymeric materials by the chemoenzymatic method; the combination of chemical reaction to link the maltooligosaccharide as a primer to polymer backbones or polymerizable groups with the phosphorylase-catalyzed enzymatic polymerization of G-1-P to produce amylose chain. The present works are based on the viewpoint that polysaccharides can form promising new
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hybrid polymeric materials. We conclude this review with the expectation that hybrid materials as described herein will exhibit important applications in biomaterials science in the future.
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[16] Baek, M. G., Stevens, R. C. & Charych, D. H. (2000). Design and synthesis of novel glycopolythiophene assemblies for colorimetric detection of influenza virus and E-coli. Bioconjugate Chem., 11, 777-788. [17] (a) Yamashita, Y., Kaneko, Y. & Kadokawa, J. (2007). Synthesis of poly(p-phenylene)s having alternating sugar and alkyl substituents by Suzuki coupling polymerization and evaluation of their main-chain conformations. Eur. Polym. J., 43, 3795-3806. (b) Yamashita, Y., Kaneko, Y. & Kadokawa, J. (2007). Synthesis of glucose-substituted poly(p-phenylene)s with twisted main-chain in one direction due to induced axial chirality. Polym. Bull., 58, 635-643. [18] Kim, I. B., Erdogan, B., Wilson, J. N. & Bunz, U. H. F. (2004). Sugar-poly(pphenylene ethynylene) conjugates as sensory materials: Efficient quenching by Hg2+ and Pb2+ ions. Chem.-Eur. J., 10, 6247-6254. [19] Takasu, A., Iso, K., Dohmae, T. & Hirabayashi, T. (2006). Synthesis of sugarsubstituted poly(phenylenevinylene)s. Biomacromolecules, 7, 411-414. [20] (a) Suenaga, M., Kaneko, Y., Kadokawa, J., Nishikawa, T., Mori, H. & Tabata, M. (2006). Amphiphilic poly(N-propargylamide) with galactose and lauryloyl groups: Synthesis and properties. Macromol. Biosci., 6, 1009-1018. (b) Kadokawa, J., Tawa, K., Suenaga, M., Kaneko, Y. & Tabata, M. (2006). Polymerization and copolymerization of a new N-propargylamide monomer having a pendant galactose residue to produce sugar-carrying poly(N-propargylamide)s. J. Macromol. Sci. Part A-Pure Appl. Chem., 43, 1179-1187. [21] Kadokawa, J., Nakamura, Y., Sasaki, Y., Kaneko, Y. & Nishikawa, T. (2008). Chemoenzymatic synthesis of amylose-grafted polyacetylenes. Polym. Bull., 60, 57-68. [22] Sasaki, Y., Kaneko, Y. & Kadokawa, J. (2009). Chemoenzymatic synthesis of amylosegrafted polyacetylene by polymer reaction manner and its conversion into organogel with DMSO by cross-linking. Polym. Bull., 62, 291-303. [23] Knutson, C. A. (2000). Evaluation of variations in amylose-iodine absorbance spectra. Carbohydr. Polym., 42, 65-72. [24] (a) Jonas, G. & Stadler, R. (1994). Carbohydrate modified polysiloxanes. 2. Synthesis via hydrosilation of monosaccharide and disaccharide and oligosaccharide allylglycosides. Acta Polym., 45, 14-20. (b) Jonas, G. & Stadler, R. (1991). Polysiloxanes with statistically distributed glucose and galactose units. 1. synthesis and thermal characterization. Makromol. Chem.-Rapid Commmun., 12, 625-632. [25] Braunmühl, V. V., Jonas, G. & Stadler, R. (1995). Enzymatic grafting of amylose from poly(dimethylsiloxanes). Macromolecules, 28, 17-24. [26] (a) Armstrong D. W. (1987). Optical isomer separation by liquid-chromatography. Anal. Chem., 59, A84-A91. (b) Okamoto, Y. (1987). Separate optical isomers by chiral HPLC. Chemtech, 176-181. (c) Allenmark, S. G. (1988). Chromatographic Enantioseparation. Chichester: Ellis Horwood. (d) Pirkle, W. H. & Pochapsky, T. C. (1989). Considerations of chiral recognition relevant to the liquid chromatographic separation of enantiomers. Chem. Rev., 89, 347-362. (e) Ahuja, S. (1991). Chiral Separations by Liquid Chromatography: ACS Symposium Series 471. Washington, DC: American Chemical Society. (f) Taylor, D. R. & Maher, K. (1992). Chiral separations by high-performance liquid-chromatography. J. Chromatogr. Sci., 30, 67-85. (g) Subramanian, G. (1994). A Practical Approach to Chiral Separations by Liquid Chromatography. New York: VCH.
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In: Encyclopedia of Polymer Research Ed: Caroline E. Jones
ISBN 978-1-61761-926-7 © 2011 Nova Science Publishers, Inc.
Chapter 49
POLYSACCHARIDES AND THEIR DERIVATIVES FOR BIOMEDICAL APPLICATIONS Liqun Yang*1,2 and Li-Ming Zhang1 1
Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Xingang West Road, Guangzhou 510275, P. R. China. 2 BME Center, State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-Sen University, Guangzhou 510275, P. R. China.
ABSTRACT Polysaccharides are one of essential biomacromolecules in nature, which play important roles in life activities. Some of them exhibit various biological activities or have special functions. Moreover, polysaccharides contain reactive groups, such as hydroxyl, amino and carboxylic acid groups. Thus, they may react with some compounds to produce polysaccharide derivatives. Amphiphilic polysaccharide derivatives can selfassemble into stable micelles in aqueous solution, promising as a new carrier for the controlled release of drugs. In order to overcome various barriers which hinder drug delivery, the drug molecules may be conjugated with polysaccharide chains through chemical bonds to form prodrugs. The parent drug can be released through an enzymatic or chemical transformation during the metabolizing process. This article reviews the investigations of bioactive and functional polysaccharides, amphiphlic polysaccharide derivatives and polysaccharide-based prodrugs for biomedical applications.
1. INTRODUCTION Polysaccharides, together with proteins and polynucleotides, are essential biomacromolecules in the life activities and play important roles in cell-cell communication,
*
Corresponding author. E-mail address:
[email protected] (L. yang); Tel: +86 020 84110934.
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cell adhesion, and molecular recognition in the immune system [1]. They widely exist in the plants, microorganism (fungi and bacteria), animals, and algae, as listed in Table 1. Polysaccharides consist of multiple sugar residues linked by α- or β- glycosidic bond. Some polysaccharides are linear polymers, such as cellulose, amylose and pullulan. Many polysaccharides are branched polymers, for example, amylopectin and glycogen are well known hyper-branched polysaccharides [2]. Polysaccharides may exhibit different conformations and aggregations in solutions, including random or compact coils, stiff rod-like conformations, single, double or triple helical chains. It is interesting to find that some natural polysaccharides exhibit bioactivities, such as antitumor, immunomodulatory, anticoagulant and antioxidant activities [3-5]. Further studies indicate that the polysaccharides' bioactivities are dependent on their chemical structures, chain conformations and aggregations [6]. For example, the antitumor activities are related to the triple helical conformation of the (1→3)-βD-backbone chain for some polysaccharides, such as lentinan from Letinus edodes [3,7] and schizophyllan from Schizophylium commune [8,9]. On the other hand, polysaccharides contain reactive groups, such as hydroxyl, amino and carboxylic acid groups. As a result, they may react with some compounds to produce polysaccharide derivatives. In recent years, interest has grown in biomedical materials prepared from polysaccharides and their derivatives with good biocompatibility, biodegradability, and low toxicity. This article attempts to review the current developments of bioactive and functional polysaccharides, amphiphilic polysaccharide derivatives, and polysaccharide-based prodrugs applied in the drug delivery system, tissue engineer, and diagnosis and therapy of diseases.
2. BIOACTIVE POLYSACCHARIDES Letinus edodes, as an edible mushroom (Figure 1), play an important role in food and medicine. Maeda et al. [10,11] reported that lentinan, a β-(1→3)-D-glucan isolated from Letinus edodes, showed a stronger antitumor activity against sarcoma 180 implanted subcutaneously in mice. It is believed that the bioactivity is related to the structures of lentinan, such as chemical structure, conformation and molecular weight [7]. As illustrated in Figure 2, lentinan is the β-(1→3)-D-glucan containing glycopyranosyl residues branched in position 6 of glucose unit, and the repeating unit consists of five β-(1→3)-glucose residues and two β-(1→6)-glucose side branches [12]. Zhang et al. [13-16] reported that lentinan existed as a triple-helical chain conformation in aqueous solution, and transformed to single random-coil chains with the changes of solvents and temperatures (Figure 3). It is proved that the (1→3)-β-glycosidic and the triple-helix are two important structural requirements [3,7]. In addition, the triple-helix lentinan with the weight average molecular weight of 1.49×106 exhibited high antitumor activity in vivo, having an inhibition ratio of 49.5%, close to that of fluorouracil [7]. Another β-(1→3)-D-glucan, named schizophyllan as an antitumor polysaccharide, has been obtained from Schizophylium commune [8,9]. Schizophyllan has similar chemical structure and triple-helix as lentinan [17], as shown in Figure 4. Since Sakurai and Shinkai were the first to find that schizophyllan may form a helical complex with single-stranded homo-polynucleotides [18], many works about preparing a complex of schizophyllan and
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DNA or RNA for a non-toxic gene delivery system have been developed [17,19-22]. Figure 5 presents a strategy to deliver oligonucleotides to cells by schizophyllan [21]. Schizophylan has no ability to the plasma membrane to induce endocytosis or other uptake processes, and hardly elicits the immune response, so that schizophyllan may not be recognized by T cell receptors. In order to induce the uptake of oligonucleotides, the side chains of schizophyllan are modified by different functional groups, while the β-(1→3)-D-glucan remains to form a triple helical complex with oligonucleotides. The mechanism for the cellular uptake of oligonucleotides differs from the introduced functional groups. The cellular membrane is negatively charged, so that cationic function groups can bind to the surface with the Coulombic forces. Integrins are natural targets for receptor-mediated cell entry used by a variety of bacteria and viruses. As a peptide which can bind to an intergrin, arginine–glycine– aspartic acid (RGD) is used as a functional group for the increment of the cellular uptake. The arginine-rich peptides corresponding to the human immunodeficiency virus induce a cellular uptake from receptor mediated endocytosis. After entering cells, the complex dissociates to release oligonucleotides. Table 1. Natural polysaccharides. Resources Plants Fungi and bacteria Animals Algae
Polysaccharides Celluose, amylose, amylopectin, pectin, carrageenan, inulin, guar gum Bacterial cellulose, dextran, pullulan, schizophylan, lentinan, xanthan, curdlan, gellan, succinoglycan Chitin, chitosan, heparin, hyaluronic acid, chondroitin sulfate, glycogen Alginate
Figure 1. Photo of Letinus edodes.
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Figure 2. Photo of Letinus edodes.
Figure 3. Schematic representation of the thermally-induced conformation transition of lentinan solution at 137 ºC [15].
Figure 4. (a) chemical structure and (b) triple helical conformation of schizophyllan [17].
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Figure 5. A strategy to deliver oligonucleotides to cells by schizophyllan [21].
3. FUNCTIONAL POLYSACCHARIDES 3.1. Bacterial Cellulose Bacterial cellulose synthesized by Acetobacter xylinun has attracted much attention as a new biodegradable material, because it has unique properties different from those of plant cellulose [23]. For example, the water retention value of the never-dried bacterial cellulose is approximately 1000%, which is superior to the 60% observed in typical plant cellulose like cotton linters [24]. During cultivation, A. xylinun produces cellulose in form of pellicle with a highly swollen fiber network at the air/liquid interface of the culture medium. The cellulose pellicle is quite hydrophilic owing to the presence of pore structures and tunnels [24]. Backdahl et al. [25] reported that the nanofiber network was much denser close to the medium/air transition zone than on the opposite side, and the fine, highly entangled nanofibers with dimensions of approximately 100 nm were clearly observed in their SEM pictures. It is found that bacterial cellulose has high crystallinity, high water absorption capacity, and mechanical strength in the wet state, ultrafine network architecture, and moldability in situ [24]. In addition, bacterial cellulose is biocompatible and biodegradable, thus holding great potential for biomedical applications [26]. Bacterial cellulose has been shown high effectiveness for wound-healing [27]. The bacterial cellulose membranes with different shapes and sizes can be created for covering wound areas of the body [26]. They may improve the healing process of burns and chronic wounds [27]. A bacterial cellulose product named Biofill® has been proved to be successful for healing burns and chronic ulcers, which was shown to be more effective than other wound dressing materials [28]. Biofill®, together with Bioprocess® and Gengiflexm®, are the products of bacterial cellulose that now have wide applications in surgery and dental implants [29].
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Figure 6. Chemical structure of chitosan [33].
Another application approach is to utilize bacterial cellulose as implantable biomaterials entirely integrated into the body. Bacterial cellulose was created as hollow tubes with inner diameter of about 1 mm, named BASYC® (Bacterial Synthesized Cellulose), which could be used as artificial blood vessel interpositions in experimental micronerve surgery [24]. Svensson et al. [30] reported that bacterial cellulose showed significantly higher levels of chondrocyte growth compared with tissue culture plastic and calcium, suggesting the potential for bacterial cellulose as a scaffold for tissue engineering of cartilage.
3.2. Chitosan Chitin is the second most abundant biopolymer commonly existed in shells of crustaceans and insects, and cell walls of fungi. Chitosan, as a deacetylated derivative of chitin, is a linear cationic polysaccharide composed of glucosamine and N-acetyl glucosamine linked in a β(1→4) manner (Figure 6) [31-33]. It is a well-known biomaterial with its outstanding bioactivity, biocompatibility, biodegradability, and non-toxicity [34], thus widely used in wound dressing and healing, drug and gene delivery system, and various tissue engineering applications [33]. Compared with other polysaccharides, chitosan is shown to be more suitable as scaffold materials for tissue engineering applications due to the following properties [31]: (1) The cationic nature of chitosan provides electrostatic interactions with anionic glycosaminoglycans, proteoglycans and other negatively charged molecules, to which a large number of cytokines/growth factors are linked; (2) Chitosan may be biodegraded by lysozyme and other proteolytic enzymes in vivo; (3) Chitosan can be easily molded in various forms; (4) The intrinsic antibacterial activity is responsible for reducing the infection rate. Hydrogels are currently used as potential scaffolds to engineer new tissues [33]. Physical crosslinked chitosan hydrogels can be obtained by increasing the concentration through hydrophobic interactions between residual acetyl groups, by controlling the solution pH, or by increasing the temperature [35]. However, these hydrogels are too weak to support the growth factors and are even more readily biodegraded under physiological conditions. In contrast, chemical cross-linked chitosan hydrogels through chemical cross-linking reactions are far stronger and more stable [36]. Hong et al. [37] reported that the chondrocyte encapsulated in a chemical cross-linked chitosan hydrogel could survive for 12 days in vitro, suggesting the chitosan hydrogel potentially applicable as an injectable scaffold for tissue regeneration. Recently, a new cross-linking agent, genipin, has been reported to bind with chitosan leading to covalent coupling [38]. The cross-linking process was able to undergo in situ and
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thus constitute promising matrices for cells. The solutions of chitosan cross-linked with genipin, under physiological conditions, could form relatively strong elastic hydrogels when compared to those of pure chitosan [38]. Genipin, an effective cross-linking agent for polymers containing amino groups, is biodegradable and much less cytoxic than glutaraldeyde [39], which is mostly used for chitosan cross-linking. Therefore, it might replace glutaraldehyde with the advantages of stability and biocompatibility for cross-linking chitosan in the tissue engineering application.
4. AMPHIPHILIC POLYSACCHARIDE DERIVATIVES Amphiphilic polysaccharide derivatives (APDs) consisting of hydrophilic polysaccharide chains and hydrophobic segments can form micelle structures with hydrophobic inner core and the hydrophilic outer shell in aqueous solutions [40-43]. The critical aggregation concentration (CAC) value, usually determined by pyrene probe fluorescence spectroscopy, is used to evaluate the thermodynamic stability of micelles in aqueous solutions, i.e. the small CAC is, the more stable micelles are [44,45]. Moreover, the formation and properties of the micelles may change with the structures of APDs [46,47]. Various APDs have been synthesized by conjugating different hydrophobic segments with hydroxyl, amino and carboxylic acid groups of hydrophilic polysaccharides, as summarized in Table 2. The hydrophobic segments could be grafted or blocked with polysaccharide chains. The grafted APDs usually tend to form larger micelles than the blocked APDs, owing to the sterical hindrance of the polysaccharide main chains twisting in the process of self-assembly. In recent years, much interest has grown in APDs, since they may be used as drug delivery for poorly water-soluble drugs and protein drugs [55-57]. In most APD micelles, hydrophobic drugs can be entrapped in the hydrophobic microdomains via hydrophobic interactions (Figure 7), while controlled release of the loaded drugs can be achieved through diffusion from the micelles or by biodegradation of APDs. Table 2. Syntheses of amphiphilic polysaccharide derivatives. Hydrophilic polysaccharides Chitosan, dextrin, amylopectin, sodium alginate and hyaluronate Pullulan, carboxymethylcellulsoe, glycol chitosan, sodium alginate Dextran, chitosan, glycol chitosan Pullulan, chitosan, dextran, maltoheptaose Dextran, cellulose diacetate Carboxymethyl--hydroxypropyl guar gum, carboxymethylcellulsoe, dextran
Hydrophobic segments Long chain alkyl residues
References 48-51
Cholesteryl residue
40-41,46,52-56
Bile acid, cholic acid, deoxycholic acid and 5β-cholanic acid residues Poly(ε-caprolactone)
57-61
Poly(lactic acid) N-isopropyl-acrylamide and Nvinylcaprolactam at temperature higher than their lower critical solution temperatures (LCSTs)
66-70 71-73
62-65
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Liqun Yang and Li-Ming Zhang
Figure 7. Drug-loaded micelles prepared through self-assembly of hydrophobic drugs and amphiphilic polysaccharide derivatives.
Cholesterol, with biocompatibility and potential interaction with cholesterol receptors on cell surface, has well-known ability to drive the self assembly of cholesteryl-containing materials [74]. In our work, amphiphilic cholesteryl-bearing carboxymethylcellulse derivatives (CCMCs) have been synthesized at room temperature, which can self-assemble to form polymeric micelles in aqueous solution [52,53]. The AFM phase mode images demonstrate that the sizes of dried micelles are about 50 nm, and there may be multiple hydrophobic microdomains in micelles (Figure 8a-b). Furthermore, the three-dimensional spherical profile of the micelle is clearly displayed in Figure 8c [53]. Indomethaicin (IND) has very low water solubility (
