Advances in Flow Injection Analysis and Related Techniques, Volume 54 (Comprehensive Analytical Chemistry) (Comprehensive Analytical Chemistry)

COMPREHENSIVE ANALYTICAL CHEMISTRY VOLUME 54 Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Li...

Author: Spas D. Kolev | Ian D. McKelvie

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COMPREHENSIVE ANALYTICAL CHEMISTRY VOLUME

54

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK

First edition 2008 Copyright r 2008 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http:// www.elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing in Publication Data A catalog record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalogue record for this book is available from the Library of Congress ISBN: 978-0-444-53094-3 ISSN: 0166-526X

For information on all Elsevier publications visit our website at books.elsevier.com

Printed and bound in Hungary 08 09 10 11 12 10 9 8 7 6 5 4 3 2 1

ADVISORY BOARD Joseph A. Caruso University of Cincinnati, Cincinnati, OH, USA Hendrik Emons Joint Research Centre, Geel, Belgium Gary Hieftje Indiana University, Bloomington, IN, USA Kiyokatsu Jinno Toyohashi University of Technology, Toyohashi, Japan Uwe Karst University of Mu¨nster, Mu¨nster, Germany Gyo¨rgy Marko-Varga AstraZeneca, Lund, Sweden Janusz Pawliszyn University of Waterloo, Waterloo, Ont., Canada Susan Richardson US Environmental Protection Agency, Athens, GA, USA

Wilson & Wilson’s

COMPREHENSIVE ANALYTICAL CHEMISTRY

Edited by ´ D. BARCELO Research Professor Department of Environmental Chemistry IIQAB-CSIC Jordi Girona 18-26 08034 Barcelona Spain

Wilson & Wilson’s

COMPREHENSIVE ANALYTICAL CHEMISTRY ADVANCES IN FLOW INJECTION ANALYSIS AND RELATED TECHNIQUES

VOLUME

54 Edited by SPAS D. KOLEV School of Chemistry, The University of Melbourne, Victoria 3010, Australia IAN D. MCKELVIE School of Chemistry, Monash University, Victoria 3800, Australia

Amsterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

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CONTENT S

Contributors to Volume 54 Volumes in the Series Preface Series Editor’s Preface Foreword

xv xix xxiii xxv xxvii

PART I: INTRODUCTION TO FLOW ANALYSIS

1. Flow Injection Analysis: Its Origins and Progress

3

Elo Harald Hansen 1. The Conception of FIA 2. The Infancy of FIA 3. Placing FIA into Context 4. The Early Years of FIA 5. The Dissemination of FIA: The Human Factor 6. Miniaturisation of FIA 7. Concluding Remarks References

3 7 9 11 15 15 18 21

2. From Beaker to Programmable Microfluidics

23

Jaromir Ru˚zˇicˇka 1. Introduction 2. Sequential Injection and Programmable Flow 3. Miniaturization 4. Mixing and Dispersion 5. Mixing by Diffusion and Reynolds Number 6. mSI and Lab-on-Valve Design 7. Methods 8. Conclusions Acknowledgment References

23 26 28 28 35 36 37 43 44 44

vii

viii

Contents

3. Theoretical Basis of Flow Injection Analysis

47

Spas D. Kolev 1. Introduction 2. Mass Transfer in FIA Systems 3. Chemical Kinetic Phenomena 4. Sensing Mechanism Abbreviations and Nomenclature References

4. Principles of Flow Injection Analysis

47 48 66 71 74 75

81

Ian D. McKelvie 1. Introduction 2. Sample Dispersion in Flow Injection and Sequential Injection Analysis Systems 3. Components of Flow Injection and Sequential Injection Analysis Systems 4. Operational Modes of FIA and Related Techniques 5. Conclusion Abbreviations References

5. Bibliometrics

81 84 87 98 105 105 106

111

Stuart Chalk 1. Introduction 2. Analytes 3. Application Areas 4. Detection Techniques 5. Other Interesting Statistics 6. Future Trends Abbreviations References

111 112 114 118 121 121 124 125

PART II: ON-LINE SAMPLE MANIPULATION

6. On-Line Sample Pretreatment: Dissolution and Digestion

129

V. Cerda` and J.M. Estela 1. Introduction 2. Dissolution 3. Digestion Abbreviations and Definitions References

129 130 135 154 155

Contents

7. On-Line Sample Pretreatment: Extraction and Preconcentration

ix

159

Shoji Motomizu and Tadao Sakai 1. Introduction 2. Liquid–Liquid Extraction (Solvent Extraction, SE) without Membrane 3. Liquid–Solid Extraction (Solid Phase Extraction, SPE) of Organic and Inorganic Substances 4. Gas–Liquid Extraction Based on Mass Transfer 5. On-Line Pretreatment System, Including Computer-Controlled Automated Systems Abbreviations References

8. Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation

159 160 171 188 196 198 199

203

Maria Dolores Luque de Castro 1. Introduction 2. The General Membrane-Based Separation Module 3. The Continuous Manifold 4. Detectors 5. Chemical Reactions Involved 6. Dialysis 7. Microdialysis 8. Gas Diffusion 9. Analytical Pervaporation Abbreviations References

9. Membrane-Based Separation Techniques: Liquid–Liquid Extraction and Filtration

204 204 207 211 211 212 218 223 226 231 232

235

M.D. Luque de Castro and B. A´lvarez-Sańchez 1. Introduction 2. Membrane-Based Continuous Liquid–Liquid Extraction 3. Continuous Filtration Abbreviations References

10. Chromatographic Separations

235 236 254 261 262

265

Petr Solich 1. Introduction 2. Separation Columns Used in Flow Analysis 3. Pharmaceutical Applications of Sequential Injection Chromatography

265 267 277

x

Contents

4. Comparison of Sequential Injection Chromatography and High Performance Liquid Chromatography 5. Other Chromatographic Approaches 6. Future Trends Abbreviations References

11. Flow Injection Analysis–Capillary Electrophoresis

278 280 281 284 284

287

Pavel Kubańˇ and Peter C. Hauser 1. Introduction 2. Fundamentals of Capillary Electrophoresis 3. On-Line Coupling of FIA and CE 4. Electrokinetically Pumped Flow Analysis 5. Conclusions Abbreviations Acknowledgments References

287 288 291 302 305 305 306 306

PART III: DETECTION 12. Photometry

311

Wolfgang Frenzel and Ian D. McKelvie 1. Introduction 2. Fundamentals of Spectrophotometric Measurements 3. Instrumental Aspects of Flow-Through Spectrophotometry 4. Background Absorbance Correction 5. Refractive Index (Schlieren) Effects 6. Conclusions and Outlook Abbreviations References

13. Luminescence

311 314 317 332 334 338 340 340

343

Paul S. Francis and Conor F. Hogan 1. Introduction 2. Photoluminescence 3. Chemiluminescence 4. Electrochemiluminescence 5. Future Directions Abbreviations References

14. Atomic Spectroscopic Detection

343 344 349 358 367 369 370

375

Elo Harald Hansen and Manuel Miro´ 1. Introduction 2. Interfacing the Flow Network with Detection Devices 3. Flow Systems as Front-End Vehicles for On-Line Processing of Aqueous Samples

375 378 379

Contents

4. Flow Systems as Front-End Vehicles for On-Line Processing of Solid Samples 5. Hyphenation with Atomic Spectroscopic Detectors Abbreviations References

15. Vibrational Spectrometry

xi

397 401 402 403

407

Sergio Armenta, Salvador Garrigues and Miguel de la Guardia 1. A Short Note on the Evolution of Flow Injection Analysis in Recent Years 2. Vibrational Techniques as Detectors in Flow Injection Analysis 3. Scientometric Evolution of Vibrational Spectrometry in Flow Injection Analysis 4. Objectives 5. Infrared Spectrometry 6. Raman Spectrometry 7. Concluding Remarks and Outlook Abbreviations Acknowledgments References

16. Electrochemical Detection

407 408 408 409 410 430 435 435 436 436

441

Ari Ivaska 1. Introduction 2. Detector Design 3. Conductometric Measurements 4. Potentiometric Measurements 5. Voltammetric and Amperometric Measurements 6. Coulometric Measurements 7. Conclusions Acknowledgments References

17. Miscellaneous Detection Systems

441 444 444 447 450 455 457 457 458

461

Kate Grudpan and Jaroon Jakmunee 1. Introduction 2. Conductometric Detectors 3. Miscellaneous Non-Spectrophotometric, Optical Detection Systems 4. Radiometric Detection 5. Thermometric and Enthalpimetric Detection 6. Dynamic Surface Tension Detector 7. Mass Spectrometry 8. Nuclear Magnetic Resonance (NMR) 9. Piezoelectric Detection 10. X-Ray Fluorescence 11. Conclusion Abbreviations References

461 463 464 474 476 477 479 480 481 485 505 505 506

xii

Contents

PART IV: APPLICATIONS OF FLOW INJECTION ANALYSIS

18. Food, Beverages and Agricultural Applications

513

Ildiko´ V. To´th, Marcela A. Segundo and Antońio O.S.S. Rangel 1. Introduction 2. Applications: Beverages 3. Applications: Plants and Vegetables 4. Applications: Milk and Dairy Products 5. Applications: Meat and Fish Products 6. Miscellaneous Food Products Abbreviations References

19. Life Sciences Applications

513 514 545 546 547 548 548 549

559

Jianhua Wang and Xuwei Chen 1. Introduction 2. Deoxyribonucleic Acid (DNA) Assays 3. Assays of Proteins, Peptides and Amino Acids 4. Immunoassays 5. Enzymatic Assays 6. Cellular Analysis 7. Perspectives Abbreviations References

20. Pharmaceutical Applications

559 560 566 575 581 585 586 587 588

591

Miroslav Pola´sˇek 1. Introduction 2. Automated Analytical Flow Methods in Pharmaceutical Research 3. Automated Analytical Flow Methods in Pharmaceutical Production and Drug Quality Control Abbreviations References

21. Industrial and Process Analysis Applications

591 594 599 613 613

617

Celio Pasquini and Ma´rcio V. Rebouc- as 1. Introduction 2. The Advantages and Weakness of Flow Analysis Applied to Industry 3. Process Analysers Based on Flow Systems 4. Selected Applications of Flow Analysis to Industrial and Process Analysis 5. Conclusion Abbreviations References

617 619 627 629 635 636 637

Contents

22. Environmental Applications: Atmospheric Trace Gas Analyses

xiii

639

Kei Toda and Purnendu K. Dasgupta 1. Introduction 2. Collection of Trace Gases 3. Integration of a Gas Collector into a Flow Analysis System 4. Flow System Miniaturization for Atmospheric Analysis 5. Illustrative Examples 6. Applications to Breath Analysis 7. Ancillary Systems for Field Monitoring 8. Conclusions Acknowledgments References

640 640 650 656 661 673 675 680 680 681

23. Environmental Applications: Waters, Sediments and Soils

685

Paul J. Worsfold, Ian D. McKelvie and Grady Hanrahan 1. Challenges of Environmental Analysis 2. Instrumentation and Modes of Application 3. Range of Sample Types 4. Applications 5. Future Trends Abbreviations and Definitions References

Subject Index See Color Plate Section at the End of This Book

686 692 698 705 751 752 754

761


CONTRIBUTORS TO VOLUME 54

´ lvarez-Sańchez B. A Department of Analytical Chemistry, Faculty of Sciences, University of Co´rdoba, Spain Sergio Armenta Departamento de Quı´mica Analı´tica, Universidad de Valencia, Edificio Jeroni Munõz, Avenida Dr. Moliner 50, 46100 Burjassot, Valencia, Spain V. Cerda` Department of Chemistry, Universitat de les Illes Balears, E-07122 Palma de Mallorca, Spain Stuart Chalk Department of Chemistry and Physics, University of North Florida, 1 UNF Drive, Jacksonville, FL, 32224, USA Xuwei Chen Research Center for Analytical Sciences, Northeastern University, Box 332, Shenyang, 110004, China Purnendu K. Dasgupta Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019-0065, USA Miguel de la Guardia Departamento de Quı´mica Analı´tica, Universidad de Valencia, Edificio Jeroni Munõz, Avenida Dr. Moliner 50, 46100 Burjassot, Valencia, Spain Maria Dolores Luque de Castro Department of Analytical Chemistry, Faculty of Sciences, University of Co´rdoba, Spain J.M. Estela Department of Chemistry, Universitat de les Illes Balears, E-07122 Palma de Mallorca, Spain

xv

xvi

Contributors to Volume 54

Paul S. Francis School of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3217, Australia Wolfgang Frenzel Fachgebiet Umweltverfahrenstechnik, Technische Universita¨t Berlin, StraXe des 17. Juni 152, 10623 Berlin, Germany Salvador Garrigues Departamento de Quı´mica Analı´tica, Universidad de Valencia, Edificio Jeroni Munõz, Avenida Dr. Moliner 50, 46100 Burjassot, Valencia, Spain Kate Grudpan Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Grady Hanrahan Department of Chemistry, California Lutheran University, 60 W. Olsen Rd. #3700 Thousand Oaks, CA 91360, USA Elo Harald Hansen Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, DK-2800 Kgs. Lyngby, Denmark Peter C. Hauser Department of Chemistry, University of Basel, Spitalstrasse 51, 4004 Basel, Switzerland Conor F. Hogan Department of Chemistry, La Trobe University, Victoria 3086, Australia Ari Ivaska ˚ bo Akademi University, Laboratory of Analytical Chemistry Process, Chemistry Centre, A ˚ FI-20500, Turku/Abo, Finland Jaroon Jakmunee Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand Spas D. Kolev School of Chemistry, The University of Melbourne, Victoria 3010, Australia Pavel Kubań Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverˇ´ı 97, 61142 Brno, Czech Republic


xvii

Ian D. McKelvie School of Chemistry, PO Box 23, Monash University, Victoria 3800, Australia Manuel Miro´ Department of Chemistry, Faculty of Sciences, University of the Balearic Islands, Carretera de Valldemossa, km. 7.5, E-07122-Palma de Mallorca, Illes Balears, Spain Shoji Motomizu Department of Chemistry, Okayama University, Okayama 700-8530, Japan Celio Pasquini Instituto de Quı´mica, Universidade Estadual de Campinas, C.P. 6154, 13083-970 Campinas-SP, Brazil Miroslav Pola´sˇek Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, 500 05 Hradec Kra´love´, Czech Republic Antońio O.S.S. Rangel Escola Superior de Biotecnologia, Universidade Cato´lica Portuguesa, Rua Dr. Antońio Bernardino de Almeida, 4200-072 Porto, Portugal Ma´rcio V. Rebouc- as Braskem S.A., Unidade de Insumos Ba´sicos, Laborato´rio, Rua Eteno 1561, Complexo Petroquı´mico de Camacari, 42810-000 Camacari/Bahia, Brazil Jaromir Ru˚zˇicˇka Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA Tadao Sakai Department of Applied Chemistry, Aichi Institute of Technology, 1247 Yachigusa, Yakusa-cho, Toyota-shi, Aichi 470-0392, Japan Marcela A. Segundo REQUIMTE, Servic- o de Quı´mica-Fı´sica, Faculdade de Farmaćia, Universidade do Porto, Rua Anı´bal Cunha, 164, 4050-047 Porto, Portugal Petr Solich Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, 500 05 Hradec Kra´love´, Czech Republic

xviii


Kei Toda Department of Chemistry, Kumamoto University, Kumamoto 860-8555, Japan Ildiko´ V. To´th Escola Superior de Biotecnologia, Universidade Cato´lica Portuguesa, Rua Dr. Antońio Bernardino de Almeida, 4200-072 Porto, Portugal Jianhua Wang Research Center for Analytical Sciences, Northeastern University, Box 332, Shenyang, 110004, China Paul J. Worsfold School of Earth, Ocean and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, United Kingdom

VOLUMES IN THE SERIES

Vol. 1A

Vol. 1B Vol. 1C Vol. 2A

Vol. 2B

Vol. 2C

Vol. 2D Vol. 3

Vol. 4

Vol. 5

Vol. 6 Vol. 7 Vol. 8

Vol. 9

Analytical Processes Gas Analysis Inorganic Qualitative Analysis Organic Qualitative Analysis Inorganic Gravimetric Analysis Inorganic Titrimetric Analysis Organic Quantitative Analysis Analytical Chemistry of the Elements Electrochemical Analysis Electrodeposition Potentiometric Titrations Conductometric Titrations High-Frequency Titrations Liquid Chromatography in Columns Gas Chromatography Ion Exchangers Distillation Paper and Thin Layer Chromatography Radiochemical Methods Nuclear Magnetic Resonance and Electron Spin Resonance Methods X-ray Spectrometry Couiometric Analysis Elemental Analysis with Minute Sample Standards and Standardization Separation by Liquid Amalgams Vacuum Fusion Analysis of Gases in Metals Electroanalysis in Molten Salts Instrumentation for Spectroscopy Atomic Absorption and Fluorescence Spectroscopy Diffuse Reflectane Spectroscopy Emission Spectroscopy Analytical Microwave Spectroscopy Analytical Applications of Electron Microscopy Analytical Infrared Spectroscopy Thermal Methods in Analytical Chemistry Substoichiometric Analytical Methods Enzyme Electrodes in Analytical Chemistry Molecular Fluorescence Spectroscopy Photometric Titrations Analytical Applications of Interferometry Ultraviolet Photoelectron and Photoion Spectroscopy Auger Electron Spectroscopy Plasma Excitation in Spectrochemical Analysis

xix

xx

Volumes in the Series

Vol. 10 Vol. 11 Vol. 12

Vol. 13

Vol. 14 Vol. 15 Vol. 16 Vol. 17 Vol. 18 Vol. Vol. Vol. Vol. Vol.

19 20 21 22 23

Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.

24 25 26 27 28 29 30 31 32 33 34

Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.

35 36 37 38 39 40 41 42 43

Vol. 44 Vol. 45 Vol. 46

Organic Spot Tests Analysis The History of Analytical Chemistry The Application of Mathematical Statistics in Analytical Chemistry Mass Spectrometry Ion Selective Electrodes Thermal Analysis Part A. Simultaneous Thermoanalytical Examination by Means of the Derivatograph Part B. Biochemical and Clinical Application of Thermometric and Thermal Analysis Part C. Emanation Thermal Analysis and other Radiometric Emanation Methods Part D. Thermophysical Properties of Solids Part E. Pulse Method of Measuring Thermophysical Parameters Analysis of Complex Hydrocarbons Part A. Separation Methods Part B. Group Analysis and Detailed Analysis Ion-Exchangers in Analytical Chemistry Methods of Organic Analysis Chemical Microscopy Thermomicroscopy of Organic Compounds Gas and Liquid Analysers Kinetic Methods in Chemical Analysis Application of Computers in Analytical Chemistry Analytical Visible and Ultra-violet Spectrometry Photometric Methods in Inorganic Trace Analysis New Developments in Conductometric and Oscillometric Analysis Titrimetric Analysis in Organic Solvents Analytical and Biomedical Applications of Ion-Selective Field-Effect Transistors Energy Dispersive X-ray Fluorescence Analysis Preconcentration of Trace Elements Radionuclide X-ray Fluorecence Analysis Voltammetry Analysis of Substances in the Gaseous Phase Chemiluminescence Immunoassay Spectrochemical Trace Analysis for Metals and Metalloids Surfactants in Analytical Chemistry Environmental Analytical Chemistry Elemental Speciation – New Approaches for Trace Element Analysis Discrete Sample Introduction Techniques for Inductively Coupled Plasma Mass Spectrometry Modern Fourier Transform Infrared Spectroscopy Chemical Test Methods of Analysis Sampling and Sample Preparation for Field and Laboratory Countercurrent Chromatography: The Support-Free Liquid Stationary Phase Integrated Analytical Systems Analysis and Fate of Surfactants in the Aquatic Environment Sample Preparation for Trace Element Analysis Non-destructive Microanalysis of Cultural Heritage Materials Chromatographic-mass Spectrometric Food Analysis for Trace Determination of Pesticide Residues Biosensors and Modern Biospecific Analytical Techniques Analysis and Detection by Capillary Electrophoresis Proteomics and Peptidomics: New Technology Platforms Elucidating Biology

Volumes in the Series

Vol. Vol. Vol. Vol. Vol. Vol. Vol.

47 48 49 50 51 52 53

Modern Instrumental Analysis Passive Sampling Techniques in Environmental Monitoring Electrochemical (Bio) Sensor Analysis Analysis, Fate and Removal of Pharmaceuticals in the Water Cycle Food Contaminants and Residue Analysis Protein Mass Spectrometry Molecular Characterization and Analysis of Polymers

xxi


PREFACE

What is it about flow injection analysis (FIA) that has captured the enthusiasm of two generations of analytical researchers? For those whose early experience of analytical chemistry was the tedium of repetitive batch wet chemical analysis using conventional laboratory apparatus. Assembling an FIA system from a few simple components, and seeing it used to perform analyses with high reproducibility and great rapidity was certainly a satisfying experience. Add to that the potential for enhancement of analytical selectivity by kinetic control or by on-line separations, along with the ability to otherwise modify the sample on-line, with minimal reagent use and waste production, and it is hardly surprising that many analytical chemists have become enamoured by the flow injection approach. FIA is where it all began. Even though subsequent generations of its development have spawned a plethora of acronyms, many of which are used in this book (e.g., FI, SIA, SI, MSFIA, FI-LOV, BIA, etc.), we have tended to use for consistency, albeit not exclusively, the term FIA, and by corollary, SIA, although the others have equal currency. The name Flow Injection Analysis is perhaps the most inclusive, generic nomenclature that can be applied to all of these flow techniques. The power and scope of FIA is already widely appreciated by the research community, as reflected by nearly 20,000 publications in this area. We hope that with this book we will also stimulate instrument manufacturers and industry to fully embrace these flow analysis techniques for more widespread routine applications in the laboratory and in remote and automated field monitoring. This book is the product of a team of leading international experts in different areas of FIA, all of whom graciously agreed to contribute. We acknowledge with appreciation their dedicated efforts, and trust that the final product meets their approval, and that of a wider readership. We gratefully acknowledge the referees’ input in further enhancing the scientific quality of the manuscript. We thank Professor Damia Barcelo, CAC Series Editor, for his invitation to undertake this task, and the support of our managing editor, Derek Coleman and all other Elsevier production staff.

xxiii

xxiv

Preface

We hope that the material in this book provides an accurate picture of the state of the flow injection art, that it adequately encapsulates the innovative and intriguing developments in the field, and that it communicates to the next generation of researchers and practitioners, the excitement and satisfaction that the present generations have experienced in working with FIA. Spas D. Kolev Ian D. McKelvie

S ER I E S E D I TOR ’ S P R E F A CE

Flow injection analysis has already an extensive history in the field of analytical chemistry. One of my duties as editor of the Comprehensive Analytical Chemistry series is to cover as much as possible the whole field of analytical chemistry. Although there are many books on flow injection analysis available in the market, we have not had a specific book on this topic in the series. It is my pleasure to introduce this new book covering one of the most prolific areas in analytical chemistry today. This volume on Flow Injection Analysis (FIA), edited by Spas D. Kolev and Ian D. McKelvie, is designed to give the reader not only an understanding of the basics of each technique but also to provide ideas on how to apply FIA in different fields. The book is divided into four main parts: Introduction to flow analysis, On-line sample manipulation Detection, and Applications. Each part contains several chapters and in total the book comprises 23 chapters including principles, bibliometrics, membrane based separation techniques, chromatographic separation, luminescence, photometric and electrochemical detection among other topics. The chapters on applications show a great variety of fields where the technique has been successfully applied—from food and beverages to life sciences, industrial and environmental applications. All contributions are by recognized experts in the field, which makes the book a unique and useful resource for the analytical chemistry community worldwide. The book is suitable for a wide audience, from students at the graduate level to experienced researchers and laboratory personnel in academia, industry and government. As well as a good introduction to the topic, it is state of the art and fits perfectly in the Comprehensive Analytical Chemistry series. Finally, I would like to extend special thanks to the two editors and all the contributing authors of this book for their time and efforts in preparing this excellent and useful book. Prof. D. Barcelo´ Dept. of Environmental Chemistry, IIQAB-CSIC Barcelona, Spain

xxv


FOREWORD

Flow injection analysis is more than an analytical technique. Essentially it is a technology that provides a platform for the use of most analytical techniques and methods and presents a modus operandi that promotes reproducibility, speed, flexibility and ready automation whilst being simple in operation and relatively inexpensive. These features have endeared the concept of flow injection analysis to researchers worldwide over the last thirty years, and are fully demonstrated in this book. Some dozen books on various aspects of flow injection analysis have been published since the first edition of Ruzicka and Hansen’s Flow Injection Analysis in 1981. These include books, handbooks and practical guides on principles, instrumentation and applications, and on applications for pharmaceuticals, enzymes and antibodies, preconcentration, atomic spectrometry, and agriculture and environmental science. So one must ask, is another book needed at this stage? The answer is definitely Yes! The last book appeared in 2000, and most of the others over a decade ago. Much has happened during the intervening years, and the FIA and related literature has exploded in the past decade. New developments have advanced the state-of-the-art of flow injection-based techniques, in instrumentation, in new modes, in sample handling, in detection, and in expanded applications. The editors have done a remarkable job in assembling world leaders in key areas of flow injection and related techniques, to provide comprehensive indepth, up-to-date coverage of all aspects, beginning with the history, theory and principles of flow injection analysis, and newer generations of FIA. Different and new modes of flow injection techniques are covered by pioneers in areas including membrane separation, capillary electrophoresis, chromatography, and online sample preparation, extraction and preconcentration. Different modes of detection are then explored by leaders in each area. The most popular, of course, is photometric detection, but luminescence, vibrational spectrometry, atomic spectrometry, and electrochemistry and others are increasingly valuable for different applications. Important applications are described by leading experts for food and agriculture, life sciences, bioprocess and pharmaceutical analysis, industrial and process analysis, and environmental analysis. The literature of flow injection and related techniques now exceeds 17,000 publications, and Chapter 5 provides valuable information on databases for extracting literature of interest.

xxvii

xxviii

Foreword

Editors McKelvie and Kolev are to be commended for envisioning this book, for its topics and organization, and for involving and prodding many leaders in the field to contribute. This will be essential reading for all researchers and practitioners in the field, for beginners to seasoned users. Gary D. Christian University of Washington Alan Townshend University of Hull

PART I Introduction to Flow Analysis


CHAPT ER

1 Flow Injection Analysis: Its Origins and Progress Elo Harald Hansen

Contents

1. The Conception of FIA 2. The Infancy of FIA 3. Placing FIA into Context 4. The Early Years of FIA 5. The Dissemination of FIA: The Human Factor 6. Miniaturisation of FIA 7. Concluding Remarks References

3 7 9 11 15 15 18 21

1. THE CONCEPTION OF FIA It was spring 1974, more precisely in late April. My former colleague Jaromir (‘‘Jarda’’) Ru˚zˇicˇka and I were — following a number of years where we had been deeply engaged in research and development of ion-selective electrodes — conducting a series of experiments for on-line measurements with a recently developed novel gas sensor. Named the air-gap sensor [1], it consisted of a pH-electrode which, furnished with a thin film of electrolyte solution in contact with a reference electrode, was situated in an enclosed chamber above the sample solution, that is, the sensor was aimed at measuring the partial pressure of protolytic gases, in casu ammonia. The rationale for construction of this device was simply, that since all problems with the then existing gas sensors were ascribed to the gas-permeable membranes generally used, the solution to get rid of these problems would be to eliminate the membrane and replace it by an air-gap. The system that we used comprised two streams propelled by a peristaltic pump. One stream consisted of ammonium chloride, alternately of different Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00601-6

r 2008 Elsevier B.V. All rights reserved.

3

4

Elo Harald Hansen

Figure 1 Photograph of the system in which the very first FIA experiments were performed. The system was originally designed to monitor ammonium concentrations in effluents via their conversion to ammonia which was then determined potentiometrically by means of an air-gap sensor (the white cylinder at the left of the picture). The ammonia was generated by mixing intermittently ammonium solutions, delivered from the reservoirs shown at the rear, with a solution of NaOH, both solutions being propagated by the peristaltic pump situated at the front of the box. The FIA experiments were executed by injected defined volumes of ammonium solutions directly into the alkaline carrier stream by means of a syringe with a hypodermic needle.

concentrations, and the other one was sodium hydroxide, the two streams being merged immediately prior to the measuring cell. The whole setup is depicted in Figure 1, where the white cylinder at the left is the air-gap sensor. However, to our dismay the sensor appeared to respond very slowly to changing concentrations. One day we lost patience and therefore decided to take a different approach, that is, merely to pump the sodium hydroxide stream and then to inject — by puncturing the wall of the base carrying tube, using a syringe with a needle — identical and precisely metered volumes of different concentrations of ammonium chloride. And to our exhilaration/surprise the sensor responded to our injections with discrete signals, which on the attached recorder yielded sharp, reproducible peaks, the heights of which were proportional to the concentrations of the injected solutions (Figure 2). Of course, it was essential to fix all other experimental parameters, notably the position of the syringe injection, and therefore we had contraptions made by our workshop that would ensure that this prerequisite was duly and precisely fulfilled (one of the very early injection ports is shown in the manifold depicted in Figure 4). Without fully appreciating the real significance of our experiments, we nevertheless wrote our first flow injection analysis- (FIA) patent application, which was filed in September 1974, and followed by our first scientific FIA-publication in

Flow Injection Analysis: Its Origins and Progress

5

ELECTRODE RESPONSE (pH4)

1.5

1.0

0.5

0

6

4 2 TIME (MIN)

0

Figure 2 Readouts for the very first FIA experiments, as obtained by injecting three different solutions of ammonium concentrations (1.0, 5.0 and 10.0 mM) using the system depicted in Figure 1 with the potentiometric air-gap detector. Reproduced from Ref. 2 by permission of Elsevier.

early 1975 [2]. In this communication the term ‘‘flow injection analysis’’ was coined for the concept, and its applications for potentiometric as well as optical detection were demonstrated (Figures 2 and 3). Actually the completion of this first FIA paper was logistically complicated. As it happens, in July 1974 Jarda took up a planned one-year appointment with the International Atomic Energy Agency (IAEA) in order to establish an analytical facility at Centro de Energia na Agricultura (CENA) in Piracicaba, Brazil. Having drafted the manuscript before Jarda left Denmark, and the only means of communication being letters (very slow), telephone (virtually prohibitive because of prices) and telex (only short messages) — conditions unknown to the young generation of today — we nevertheless managed to write the manuscript and submit it to the Editor of Analytica Chimica Acta, Dr. Alison McDonald. Many years later, at the International Conference on Flow Injection Analysis in Prague in 1999 (ICFIA 99) we had the immense pride to learn, via a letter from Alison, that ‘‘In my 28 years of editing Analytica Chimica Acta, I had the pleasure of reading many interesting papers, but perhaps the most

6

Elo Harald Hansen

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0

5 10 TIME (min)

Figure 3 Calibration runs for the spectrophotometric determinations of phosphate by the molybdenum blue method in the concentration range 2.5–25 mg P l1. Reproduced from Ref. 2 by permission of Elsevier.

fascinating was the first paper on flow injection analysis from Jaromir Ru˚zˇicˇka and Elo Hansen, sent from Brazil in 1975’’. During his tenure at CENA, Jarda was confronted with the immense number of analyses which a routine laboratory is faced with in its daily work, and spurred by this, he and the local scientists, headed by Professor Henrique Bergamin Fo, pursued the idea of FIA. This resulted eventually in four more FIA papers (on determination of nitrate-N, phosphate and chloride). Inspired by their children’s fascination with Lego, they adapted these building blocks very ingeniously to arrange the tubes and manifold components in a neat manner (Figure 4). Along with the continued research in Denmark our conception of FIA matured and added to the practical design of the individual components of the systems. Notably, the sample introduction by means of a syringe with a hypodermic needle was replaced by the use of different types of dedicated rotary or sliding valves. Yet the term ‘‘injection’’ was (and is) affixed for sampling in FIA.


7

Figure 4 Manifold employed for the determination of phosphate by the molybdenum blue method as used by Jarda Ruzicka in Brazil in 1974, where Lego building blocks for the first time were used to arrange and affix the various components. The sample solution was injected into the molybdate stream (entering at top, right), which was subsequently merged with a stream of reducing agent (ascorbic acid, top, left), and then via the reaction coil at the bottom left guided to the detector. (See Color Plate Section at the end of this book.)

Although the injection of the sample into an unsegmented carrier stream is vital to FIA, we realized, as we verbalised it in our first paper, that it was equally well founded on two additional cornerstones, namely the controlled dispersion — and our ability to manipulate it to suit our analytical purposes — and on reproducible timing which allowed us to abandon the concept of steady-state measurements, which not only meant, that we could perform the individual analysis faster than the traditionally known air-segmented continuous-flow systems, but resulted in much smaller consumption of sample and hence reagents.

2. THE INFANCY OF FIA As is apparent from the early FIA publication, we (and others) always derived the analytical readout from the peak maximum. One may, however, ask (in retrospect — everything appears so simple when you look at it in the rear view mirror): why did we go through all the gymnastics of creating a well-defined concentration gradient of the injected sample zone within the reagent stream — corresponding to, and in reality containing, a virtually infinite number of

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elements of different concentration ratios of sample and reagent — and then only exploit a single one of them? The reason was that it was easy to identify and quantify on the recorder since better electronic aids were non-existent at that time. However, we need to recall that the then popular air-segmented systems conscientiously, and indeed conceptually, were mimicking the batch analysis by homogeneously mixing sample with reagent and by attaining, if at all possible, chemical equilibrium. Therefore, the early AutoAnalyzer (AA) systems marketed by Technicon, had reaction coils almost 10 m long in order to attain reaction times up to 5 min. In fact, generations of analytical chemists have been indoctrinated to believe that not only was it essential, but also the ‘‘Gospel Truth’’, that in order to do chemical analysis, whether manually or by automated procedures, a complete physical homogenization of sample and reagent must be attained, preferably followed by establishment of chemical equilibrium, so that the readout can be taken at the steady-state level. Actually, this preoccupation with imposing the ‘‘batch-concept’’ to continuous-flow systems is also the basic philosophy in those procedures where peak areas are measured (re. chromatography), where all information is condensed into merely ‘‘a single’’ assigned value for each recorded response. This is why the full potentials of FIA were only gradually unravelled as we worked. In 1977 we took the first steps towards gradient techniques with the development of the FIA titration procedure [3], which was soon followed by stopped-flow techniques [4]. Later a number of FIA-gradient techniques were added, notably gradient dilution and calibration, selectivity evaluation methods and gradient scanning — which truly revealed the vast potential of FIA [5,6]. In fact, we came to believe that it was, more than anything else, the gradient techniques, which inherently distinguished FIA from all other continuous flow techniques. This might readily be seen from our definition of FIA, which in the first edition of our book [7] was defined as ‘‘A method based on injection of a liquid sample into a moving unsegmented continuous stream of suitable liquid. The injected sample forms a zone, which is then transported towards a detector that continuously records the absorbance, electrode potential, or any other physical parameter, as it continuously changes as a result of the passage of sample material through the flow cell’’. Yet, in the second edition [8] this was rephrased to read as ‘‘Information-gathering from a concentration gradient formed from an injected, well-defined zone of fluid, dispersed into a continuous unsegmented stream of carrier’’. Conforming perfectly well to the original description, this more concise definition does, however, more clearly spell out that FIA represents a source of dynamic and kinetic information, the concentration gradient formed carrying the dynamic or physical information, and the occurring reactions embodying the kinetic or chemical information. In the gradient techniques one or several of these elements along the concentration gradient, controlled in space and time, are selected and used for analytical purposes. In assays that rely on controlled kinetic processes, like reaction rates, catalysis, diffusion of ions or gases through membranes or packed reactors, rate of adsorption and desorption, as well as on response characteristics of diffusion controlled detectors, advantage is


9

similarly taken by the precise mixing and rigorous timing of all operations and events. As the FIA-literature has subsequently shown, and as demonstrated by many examples in this book, numerous applications of exploiting these features have appeared, ranging from utilizing bio- and chemiluminescence, over kinetic discrimination schemes to enzymatic assays.

3. PLACING FIA INTO CONTEXT It is true that we were neither the first to employ unsegmented streams, nor to inject a defined volume of sample into a continuously moving stream. In fact, Nagy et al. in 1970 published a paper [9] in which they injected a discrete volume of sample into an unsegmented stream. Yet, still being entrapped by the concept of attaining equilibrium conditions for all processes (physical as well as chemical) they incorporated into their system a mixing chamber, which obviously obviated all attempts to create a concentration gradient of the injected samples and hence to exploit it analytically. Actually, in their initial experiments, which were centred on voltammetric studies and did not involve any chemistry per se, they noted that ‘‘reproducible results were obtained only if rapid and total homogenization of the solution to be analyzed and the supporting electrolyte was ensured’’. Likewise, Beecher et al. [10], who were on the same track, conscientiously included long mixing coils (of the order of 8–10 m) to ensure that all participating chemical reactions were to proceed to completion (exactly as in the then very well-known Technicon AA system relying on air-segmentation). But what FIA was demonstrating was that all this mimicking of manual operations was becoming unnecessary (or, indeed, obsolete) and that an intelligent combination of the three cornerstones of FIA would and could accomplish what was essential in order to obtain a definite and reliable analytical readout. Not everybody agreed with this conclusion. In a discussion in Analytical Chemistry in 1977 [11,12] with Dr. Marvin Margoshes, one of the ranking scientists of Technicon, we wrote [11] ‘‘that unsegmented flow systems have been known for many years’’. But ‘‘what has kept them from coming into general use has been the limitation of our knowledge as to how to employ the dispersion patterns for analytical purposes’’. His answer [12] was that, yes ‘‘unsegmented flow systems have been known for many years y [but] the limitations of unsegmented flow have kept these systems from coming into general use’’. However, what Dr. Margoshes chose to overlook was that we had, de facto, demonstrated exactly how to overcome these limitations. In this context it might furthermore be of interest to note that in a previous personal contact with Dr. Margoshes in 1976 he had dismissed FIA with the comment that dialysis was totally impossible to implement (Technicon’s AA instruments were primarily aimed at the clinical area where dialysis of the biological samples was essential). This made us so hotheaded that we became determined to prove him wrong. And we did. In fact, the sixth paper in our first series of ten FIA papers dealt with the determination of chloride and phosphate in serum as effected via dialysis [13]. That is exactly the

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reason why that paper in the acknowledgment mentions that thanks are due to ‘‘one of the leading scientist at Technicon for having drawn our attention to this area of research’’. At Flow Analysis I in Amsterdam in 1979, where Dr. Snyder of Technicon gave a presentation entitled ‘‘Continuous-Flow Analysis: Present and Future’’ [14], in which he compared air-segmented continuous flow analysis (CFA) with FIA, the tone of argumentation had somewhat mellowed, but still it was maintained that ‘‘FIA is limited to very simple situations, and to dedicated as opposed to flexible or multi-channel analysers’’. As a conciliatory note Dr. Snyder did, however, add that ‘‘there is a place for both FIA and CFA in automated chemical analysis’’. Time has proved him more than right, or rather prophetic. The concept of airsegmentation was at its origin truly a breakthrough in automated analytical chemistry, but in spite of its many advantages it did not as such add any novel or conceptual dimensions to performing chemical assays. Rather it facilitated the mechanistic handling of samples in areas where large quantities were required, particularly in the clinical field — which in its own merit is commendable — but it did not, unlike FIA, open new and unique avenues for executing analytical chemical procedures per se. Looking back at the air-segmented flow Technicon systems versus FIA, it is, through the spectacles of time, interesting to recall our own encounter with that, in the 1970s in the field of automated analysis, very dominant company. As it happens Jarda and I were actually invited by Technicon to present and demonstrate our invention in the spring of 1976 at their headquarters in Tarrytown, NY. When we parted after our American venture — Jarda for Brazil to fulfil his obligations there, and I for Denmark — we were quite optimistic about having found a commercial partner, but in August of the same year we received a couple of letters which dimmed our expectations. Thus, Morris H. Shamos, Senior Vice President & Chief Scientific Officer at Technicon on August 4 of that year wrote ‘‘I see no point in spending money to obtain worthless patents, or to support a research that cannot lead us to a proprietary position’’. And the day afterwards we received a letter from Dr. Margoshes which stated ‘‘When you were here I had some reservations about the ability of your method to extend to smaller sample and reagents flows — as would be needed for applications in clinical chemistry — without excessive pumping pressure. I have done some calculations that show, in a rather straightforward way, that simply scaling your procedure down to use 1/10 the volume of sample and reagent would cause the pressure to increase 10,000 fold’’. This was supplemented by a letter the same year from the chairman of Technicon that said, ‘‘I understand that the problem was one of patentability. Our people evidently felt that your Danish patent application was somewhat deficient, in that a search showed considerable prior art. Thus, our patent attorney did not feel that a truly protective patent could be issued in the United States and other countries. To confirm or deny this feeling, we hired both a Swedish and a Dutch patent attorney, whose opinions verified our own experts’ opinions. It was for this reason that the matter was dropped here at Tarrytown’’. Thus, it was clear that they did not have any confidence in our idea.


11

Fortunately, time has proven them wrong. Not only did we in the years following our encounter with Technicon, obtain more than 10 US patents, but also FIA blossomed with the help of many scientists all over the world. In contrast, today there are not many who recall the name of Technicon, its AA having gone into oblivion. As Jarda fittingly has phrased it (and I agree): ‘‘There are two lessons in this story: (1) Lack of vision is a fatal flaw of the R&D division of any company; and (2) beware of patents and patent attorneys’’.

4. THE EARLY YEARS OF FIA There are several ways to assess the development and/or the impact of an analytical concept or technique. Thus, one may look at how it is accepted and used by one’s fellow scientists, which, in turn, is reflected in the number of scientific publications it generates, or how it fares commercially. If we look at the latter point first, we did not fare well at the outset, as mentioned above with our encounter with Technicon. However, in June 1976 Jarda and I attended the Analytical Days in Lund, Sweden, where we met Rune Lundin, who was employed by a Swedish Chemical Sales Company and Dr Bo Karlberg, who then worked at Astra. Already knowing Rune Lundin, I told him to attend Jarda’s lecture, where he, for the first time at an international conference, would talk about FIA, because I thought it would attract Rune Lundin’s interest. Being in fine company with Sir Alan Walsh, the father of Atomic Absorption Spectrometry (AAS), who that year received the prestigious Torben Bergman Medal from The Swedish Chemical Society, Jarda delivered his lecture, and supported by Dr Karlberg’s lecture, which was focused on the use of FIA for pharmaceutical applications, Rune Lundin was afterwards absolutely enthusiastic. He was simply wild about FIA. He wanted to commercialise it. So, he decided to quit his safe, regular job, to mortgage his house above the chimney to get capital and persuaded Bo Karlberg, who at Astra had become an FIA aficionado, to wave goodbye to Astra and join him at his small company, Bifok. And together they started to produce FIA equipment, and engaged themselves in travelling around the world at FIA-conferences to promote their apparatuses. In fact, Rune Lundin did so well that within a few years he aroused the interest of one of the biggest chemical companies in Sweden, Perstorp, which bought him out. Some years later, it became a separate division of the Perstorp Corporation, called Tecator, which, in turn, some years ago was bought by the Danish company Foss Electric, and was renamed Foss-Tecator. Thanks to the vigour of Ju¨rgen Mo¨ller, Tecator negotiated with Perkin–Elmer an agreement to allow them to exploit FIA in conjunction with AAS. This turned out to be highly beneficial for both parties, not the least for procedures based on hydride generation, as many probably are aware of, based on personal experience. In all honesty, it should be added that the commercial exploitation never became the success that we expected, probably because people decided to build their own equipment. However a number of companies around the world are happily producing a variety of FIA systems, now that the various patents have expired.

Elo Harald Hansen

14000

Cumulative no. FIA publ.

12000 10000 8000

1200 1000

No. FlA publications

12

6000

800 600 400 200 0 1974 1979 1984 1989 1994 1999 2004

4000 2000 0 1974

1979

1984

1989 Year

1994

1999

2004

Figure 5 Growth of the publications of FIA from 1975 and on. Prepared for presentation at Flow Analysis IX in Australia 2003, it depicts in a semilogatithmic diagram the cumulative number of FIA publications as a function of time. For comparison the number of publications as of April 2007 was 16800 (See also Figure 1, Chapter 5).

Fortunately for us, the academic community welcomed FIA with open arms. This is amply reflected in the exponentionally growing number of scientific articles that emerged in the years following the publication of our first paper. Thus, depicting the cumulative number of publications as a function of time in a semi-logarithmic diagram, the doubling time, TD (i.e., the time span required for the literature to double in size) can be read out as the slope of the curve [15]. Figure 5 shows such a diagram for FIA publications, which I prepared for Flow Analysis IX in Australia in 2003. Initially, this doubling time for FIA was less than one year, later it tapered off to 1–2 years, increased in the 1990s to 3–4 years, and in recent years it has been of the order of around 7–8 years. Nevertheless, this is still quite spectacular, if one, as we have ambitions to do, wishes to track the current FIA publications and maintain an up-to-date FIA bibliography [16]. Besides pointing out that there is a scientific journal, Journal of Flow Injection Analysis, published by the Japanese Society of Flow Injection Analysis, exclusively aimed at FIA and related concepts, one might in this context also look at how frequently FIA-related front covers have emerged on major analytical chemical journals. Thus, this has happened four times on both Analytical Chemistry and The Analyst. If we go into the scientiometric literature in order to evaluate the criteria for the success of FIA, we might, as I have earlier reported [17], however find other, and in some instances, strange explanations. In 1984 Braun and Lyon wrote a paper [18], entitled ‘‘the Epidemiology of Research on Flow-Injection Analysis: An unconventional Approach’’ in which they tried (indeed very unconventionally) to analyse exactly why FIA had encountered the propagation it had had. Although it is easy to criticise in retrospect, it might, nevertheless, be of interest, and indeed entertaining, to recount the reasons which these two authors judged to be the major ones. In making their evaluation, they noted that our first FIA paper in Analytica Chimica Acta [2] had been quoted 105


13

times in the period 1975–1982, or 10 times more frequently than papers from that journal statistically were cited annually. Furthermore, in an independent paper [19], comprising the years 1981–85, they ascertained that our papers had a relative citation rate (RCR, defined as the ratio of observed to expected citation rate) of 1.32, which on a global basis made Denmark rank second only to Switzerland and well ahead of countries such as the USA, Japan and the UK. Besides, one of our papers [20] proved in a survey conducted and published in Current Contents, ISI, in 1983 to be the most cited analytical chemical article in the time span 1980–1982 [21]. In trying to find explanations for these phenomena Braun and Lyon [18] discussed several possibilities, but finally arrived at the conclusion that there were only two plausible explanations, or rather concepts, which could be taken into account, that is, the ‘‘Invisible College’’ and the ‘‘Matthew Effect’’. According to Braun and Lyon, the concept of the Invisible College was first used in the eighteenth century ‘‘to refer to the collection of scientists who eventually formed the Royal Society of London. The group represented a collection of intellectuals who had a sense of allegiance to each other and who frequently interacted both professionally and socially. The adjective invisible was used because the membership of the group was not confined to a particular academic setting, and was not obvious to persons who had little knowledge about 17th century science’’. The term was reintroduced and rephrased by de Solla Price in 1963 [22] to describe the existence of such groups in modern sciences, i.e., ‘‘the groups are collections of scientists who live in disparate geographical locations, but who often attend the same conferences, who publish in the same journals, who invite each other to give presentations at their home institutions, and who share preprints of their research endeavours’’. Therefore, according to Braun and Lyon, it is through the political power of such ‘‘colleges’’ that many of the changes in science are made, because only through the formation of reasonably small, homogeneous groups can the individuals break their isolation and protect themselves from the pressures of ‘‘Big Science’’ and the ‘‘publish or perish’’ syndrome. Or said more plainly and directly, in order to make it in science today it is necessary to be a member of a close-knit, or even parochial group. Braun and Lyon were of the conviction that they had found evidence that early on there existed such an invisible college for FIA, and gradually ‘‘this college expanded to include analytical chemists at many institutions in Brazil, UK, USA and elsewhere’’. As a comforting note they did, however, add that ‘‘an important point to note about the concept of invisible colleges is that it is not intended to connote a conspirational or subversive process in which power groups engage in Machiavellian struggles for control of science’’. But nevertheless, they were convinced that it was this invisible college ‘‘who wrote and promoted the Ruzicka-Hansen article [2] during the second half of the 1970s’’. The second, even more bizarre, explanation forwarded by Braun and Lyon is the one associated with the Matthew Effect. For those who are not so ardent in their biblical recollections, I can add that that the Matthew Effect takes its prerogative from the Gospel, Matthew 25:29, which says ‘‘For onto every one that

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hath shall be given, and he shall have abundance; but from him that hath not shall be taken away even that which he hath’’. Hence, one of the manifestations of the Matthew Effect is that most persons who publish journal articles receive very few citations to their papers (they ‘‘hath not’’), while on the other hand there are a minor number of persons who also publish articles in scientific journals, and their papers attract a large number of citations (they ‘‘hath abundance’’). As Braun and Lyon stated in evaluating our efforts, ‘‘such researchers have established reputations [did we have that?], have ample funding [unfortunately, we were not blessed with such privileges], and travel frequently to meetings [tight economic restraints made this out of reach for us]’’. But nevertheless, the authors finally concluded that ‘‘in short, the initial promotion of the Ruzicka-Hansen article by the flow-injection analysis Invisible College was compounded by the Matthew Effect into generating that high number of citations’’. That is, of course, one (or two) way(s) to look at it, but I sincerely believe that we (invisible college or not) were not handed anything on ‘‘a silver platter’’, but worked hard for it. Some might even say that we preached the Gospel (albeit not the Matthew one!) whenever we had the opportunity to do so. And if others have chosen to adopt what we suggested, it is not at the expense of our fellow scientists. But primarily, I do not think that we have, or are blessed with, the powers nor bestowed with the supernatural capacities, to exert manipulation of our fellow scientists or others, despite the potentials that Braun and Lyon concoct us to possess (however flattering it might be). Secondly, I am more than convinced that the number of FIA publications from all over the world (and in most instances from authors that we, in fact, do not know personally, nor do have any contacts with) amply reflect that the reason lies more in the fact that FIA proved itself a viable and practical concept in its own merit — and possibly at the time of its appearance filled a need in analytical chemistry and offered itself as an attractive alternative of choice. In this context I also take comfort in the fact that our paper presented at the Flow Analysis I conference in Amsterdam in 1979, and later published in the special issue of Analytical Chimica Acta (Vol. 114) [20], became the most cited analytical article in the period 1980–1982 [21]. Since most of the papers of that special issue were devoted to FIA, it appears odd that our paper should have had any particular prerogative over the others. Or that we should have been able to spellbind the participants of the conference or, indeed, manipulate the readers of that journal. Finally, it could be added that if Braun and Lyon arrived at these conclusions at the time of writing their epidemiological essay in the early 1980s, where they based their evaluation on the development of FIA on a generation of 300–400 papers, it would be interesting to learn what they would call the ensuing growth in the following three decades to the now more than 16,800 publications (Spring, 2007), and if they still would maintain their original prerogatives concerning the presence of our manipulating hands behind the scene. Personally, I think that they would retract their original speculations, not the least because Braun et al. in 2001 [23] acknowledged that the second edition of our FIAmonograph within the time frame 1980–1999 had been the third most cited monograph within the field of Analytical Chemistry.


15

5. THE DISSEMINATION OF FIA: THE HUMAN FACTOR Apart from what was said above, I personally think that a very important factor in the dissemination of an idea rests in the human contact and collaboration. At the onset of developing FIA, Jarda spent, as mentioned earlier, a year in Brazil, where there was a tremendous quest for making analysis of an array of various species. As the literature reveals, some of the earliest FIA publications originated from Brazil, entailing that the staff there became familiar with using this analytical concept. Several members of the team headed by Professor Henrique Bergamin Fo, including Francisco (‘‘Chico’’) Krug and Elias Zagatto, each spent one year studying in Denmark in the late 1970s and early 1980s as a joint collaboration between our two countries and financed by the Danish International Development Agency (DANIDA). It is therefore not surprising that some of the early innovations of FIA were made in Brazil. But people all over the world caught up with using FIA. Thus, just to mention a few, Bo Karlberg in Sweden was the first one to demonstrate and utilize on-line extraction, Dr. Paul Worsfold from England was the first one to combine FIA and chemiluminescence, Professors Miguel Valcarcel and M. D. (‘‘Lola’’) Luque de Castro in Spain not only made a number of original contributions as well but also wrote a FIA-monograph, and Professor Zhaolun Fang proved the unique combination of FIA with AAS. Jarda Ruzicka has within the last years published a CD-ROM about FIA [24], and in the latest third version there is an extensive photo gallery of many who have contributed to the development of FIA and more detailed description of their specific contributions, and I recommend readers to consult this source.

6. MINIATURISATION OF FIA A disadvantage of conventional FIA is that once the flow system is started, the continuous pumping of carrier and reagent consumes chemicals and generates waste, even if no samples are injected. And as disposal of chemical waste became increasing costly — often more costly than buying the chemicals — focus was set on miniaturising the flow systems. Going hand in hand with the emergence of computer-controlled operation and sophisticated software, FIA was in 1990, as described by Jarda Ruzicka in Chapter 2, supplemented by sequential injection analysis (SIA), and then later by lab-on-valve (LOV) systems, that focused on reduction of consumption of chemicals and production of waste. In this way flow injection became an important tool of what nowadays is termed ‘‘Green Chemistry’’. Yet, miniaturisation of FIA had already been initiated in the mid-1980s [8,25], spurred by economy of sample and reagent consumption and particularly by suitable integration of the detector within the FIA channel itself, so that the detection could be executed exactly within that section of the channel where the dispersion and other conditions of an individual assay were optimum. The resulting microconduits [25], being the size of a credit card, permitted the

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integration of traditional manifold components such as coils, connectors, injection valve, dialysis and gas diffusion units as well as columns containing ion-exchangers or immobilized enzymes. Not having sophisticated facilities at our disposal for their production, they were simply made by pressuring a thin wire, wound in the shape of the channel pattern, into the surface of a transparent PVC plastic bloc (normally 70 45 10 mm in dimensions), which was afterwards covered by a flat plate with the aid of pressure-sensitive glue or by means of a layer of adhesive material. Introduction of liquids into the channels and their withdrawal were effected through perpendicular holes drilled at appropriate positions and equipped with externally communicating tubes. First used for potentiometric measurement of pH by incorporating into the microconduit a miniaturised PVC-based pH-electrode and a reference electrode, they were also adapted for enzymatic assays with chemiluminescent detection (a microconduit for this is shown in Figure 6 [26]), pH reflectance measurements by means of bifurcated optical fibres interfaced with a layer of immobilized indicator, and, together with Art Janata, for determining potassium and chloride via CHEMFETS [8]. It was also at that juncture that we, in order to implement the exact injection of very small volumes, developed the so-called hydrodynamic flow injection approach [27]. Although our microconduits never ‘‘caught on’’ for practical applications as such (probably due to the cumbersome way of their production, or possibly because we, as Jarda very aptly has phrased it, were almost 20 years ahead of time [24]), I do suspect, in the fear of sounding preposterous, that it was possibly Jarda’s lectures in Switzerland about the FIA microconduits that prompted H. M. Widmer to hire one of our students and be inspired along with A. Manz to develop what they later termed the micro Total Analysis Systems (mTAS) [28], initially running into many of the mistakes that we had already experienced, and where the hydrodynamic injection approach found extensive use. Today mTAS, or as it is also known nowadays, Lab-on-Chip, is a very ‘‘hot’’ research area. While it is characteristic that the three generations of FIA, resulting in manifold miniaturisation, were developed by chemists due to evolving demands, dictated by either practical considerations, or by the chemistries to be executed, mTAS/Lab-on-Chip is predominantly developed at institutions manned by physicists. An example of such a system is shown in Figure 7, left. The channel network, which is made by various sophisticated procedures, such as micro-drilling, etching, photolithography, or laser erasing, is impressively exact and reproducible, allowing different channel profiles to be obtained, and it can in many instances be made using inexpensive materials and mass-produced at low cost, in fact, at much lower expenditures than the LOV. However, the systems are usually dedicated, i.e., they have a fixed architecture for predetermined chemistries, and to this day operate on a continuous flow basis, mimicking the traditional FIA mode. Seen from a chemical point of view it might in this context be of interest to compare the LOV (Figure 7, right) and the mTAS [29]. While the channel dimensions in the mTAS systems are of the order of 10–100 mm, the corresponding channel dimension in the LOV is typically 0.5–1.6 mm. When comparing these


17

Figure 6 (a) Manifold and (b) integrated FIA-microconduit for measurement of chemiluminescence by means of a light detector consisting of two photo diodes (cf. insert) and comprising an injection valve and a reactor containing immobilized enzyme (ER). C, carrier stream; R1, reagent 1 (luminol); R2, reagent 2 (hexacyanoferrate(III)); S, sample; and W, waste. Reproduced in amended form from Ref. 8 by permission of John Wiley & Sons, Ltd.

two devices, one may then ask: what is the crucial difference between the two systems? Intuitively, the response would be to point to the channel dimensions. It is true that they are very different, requiring that the liquids to be injected into the mTAS system must be extremely clean, because even the minute presence of particulate matter would result in blocking of the conduits, while small particles generally would not pose a problem in LOV. Yet, in our opinion the crucial difference is rather associated with the ways and means of moving the liquids within the manifolds, that is, the propelling device. While the movements of liquids in the mTAS systems to a large extent have been based on electroosmotic

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Elo Harald Hansen

Figure 7 Comparison of a mTAS manifold (at left) and an LOV (at right). While the channel diameters in the mTAS generally are of the order of 10–100 mm, the corresponding dimensions in the LOV system are around 0.5–1.6 mm. (See Color Plate Section at the end of this book.)

or electrophoretic forces (which, in turn, set certain requirements on the solutions to be handled), and even on surface tension or diffusion, in LOV they rely on the use of an external syringe pump. This makes a fundamental change, because a syringe pump not only allows propulsion and aspiration to be implemented, but also permits stopping of the flow and all operations for any length of time, completely at will. Thus, we are not being dictated to by the system in order to implement our chemistries, but in LOV we are controlling the parameters in order to adapt the physical movements of the liquids to the chemistries to be implemented. This, very importantly, entails that we can intelligently exploit the interplay between thermodynamics and kinetics. Said in other words, when we are in control of the fluidics, we can adapt them to the chemistry taking place, which, in turn, essentially gives us an extra degree of freedom. This is crucial in executing different chemistries, especially if we are dealing with chemistries that are not fast or instantaneous, or even require stepwise reaction sequences. In this context it is interesting to note that the authors of mTAS articles are always demonstrating their capacity for fast, single-step chemistries (re. the old batch assays), which leaves out a multitude of very interesting and intriguing chemistries. In conclusion, one may also phrase it differently: we are not limited by choosing our chemistries to satisfy the manifolds of the mTAS systems, rather we can design our LOV systems to fulfil exactly the requirements to be met by the chemistries that are necessary to solve the specific problem at hand.

7. CONCLUDING REMARKS With well over 30 years behind it, FIA is now becoming of age. From its timid start, it has grown in scope, applicability and utilization. The maturing of a scientific


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technique is manifested in many ways: by its age, by the number of publications it has generated (and continues to generate), by the array of acronyms and hyphenations it has produced (e.g., rFIA, FI-AAS, FI-HGAAS, SIA, SI, LOV, PSAFIA-AAS, SPEC-FIA-AAS, FIA-ICP, FIA-MS, FIA-ICPAES, FIA-AEF), by its variety of applications, or possibly by its inclusion into textbooks on analytical chemistry. Of these, the growing range of applications is undoubtedly the most significant. Thus, looking back at the history of flow injection, the development shows that this analytical technique, which was originally conceived as a concept to automate serial assays, rapidly revealed itself to entail novel and unique options, and increasingly found its use in a multitude of areas as diverse as oceanography, clinical chemistry, agricultural, pharmaceutical and environmental analysis, for monitoring of industrial processes, and, most recently, in the bioanalytical field [30]. Because of its rapid acceptance during a relatively short period of time, and considering the wealth of literature that FIA and its sequels have generated, one might in retrospect get the impression (or fear) that it has reached not only a level of maturity, but is approaching senility. However, although it is true that FIA has been readily accepted as a tool, it is still far from being fully recognized as an analytical concept. Thus digging through the many thousands of FIA publications, I maintain, as I have phrased it previously [31], that FIA is still far from being fully exploited, ‘‘because the ultimate test for an analytical approach is not that it can do better what can be done by other means, but that it allows us to do something that we cannot do in any other way’’. And indeed FIA does allow unique applications. Just to mention a few, one can point to the stopped-flow techniques [5,6], which in the earlier years were somewhat overlooked, but in recent years have enjoyed widespread use within biotechnology, the exploitation of detection principles relying on transient effects such as bio- and chemiluminescence, performing assays based on metastable/transient constituents, and combining FIA and chemometrics. Amply illustrated by the emergence of the sequels of FIA, i.e., SIA and the LOV, I am therefore confident that despite its maturity, novel developments of FIA are still yet to be encountered. Yet in the chemical laboratory FIA has over the years tantalized the ingenuity of a multitude of researchers, resulting in the development of a continuous stream of cleverly designed analytical approaches, aimed both at fundamental chemical research and at the more mundane tasks of routine applications. Although I secretly have asked myself if this could continue, I keep being surprised and delighted with new developments. In all modesty, we ourselves have contributed to this, most recently via our work on the determination of trace-level concentrations in complex matrices by using the LOV configuration as a front end to implement separation and preconcentration by means of solid-phase extractions and ultimate detection by atomic spectroscopy techniques (described in detail in Chapter 14), and by designing micro-extraction column reactors for use with SIA or LOV in order to conduct sequential extraction/fractionation of solid samples (e.g., soils or sediments) to obtain information on the mobility, bioavalability and the eventual impact of anthropogenic metal species under conditions mimicking those taking place in natura.

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Elo Harald Hansen

All scientific achievements appear, at least in retrospect, to be the result of theoretical rationale, careful planning and meticulous experimental execution. Anyway, that is the impression one gets from reading the scientific literature (probably because this is the picture the researchers would like to convey to their fellow scientists). But in reality that is frequently far from true. Often one stumbles upon a discovery by chance — although the extent of the chance is arguable. Because, naturally, we direct our research along a path guided by our previous experience, supplemented by intuition (hopefully) and imagination (if so blessed). Yet, what ultimate determines the value of a scientific or other achievement lies in how it is accepted by others. And in this respect we must admit that we have been truly fortunate, thanks to the many analytical chemists all over the world who have found it of value and interest to use FIA in their research and development activities. We owe them our sincere thanks and gratitude. Due to the explosive developments in hardware and software, analytical chemistry today is very much different from what it was in the 1970s — and so are we, Jarda and I, as can be realized from Figure 8, which shows a photo taken around 1979/1980. When I, on behalf of Jarda and myself, gave a ‘‘dinner lecture’’ at the conference dinner of Flow Analysis IX in Geelong, Australia, in 2003, the last slide of my presentation showed a quote from the former President of France, George Pompidou, which read as follows: ‘‘There are three roads to ruin: Women, gambling and technology. The most pleasant one is with women. The quickest is with gambling. And the surest is with technology’’. At the end of the slide I had added our own comment, which said: ‘‘Agree, and although they all give you a lot of fun, the latter one additionally gives you many interesting experiences’’.

Figure 8 Photo of Jarda Ruzicka and myself at the end of the 1970s. While Jarda has given up smoking (including pipe) years ago, I am, as many will know, still addicted to that vice/quality of life. Besides these alterations, our hair (and my beard) has in the ensuing years assumed another hue.


21

Thus, while FIA has not been an economic success for us personally, it has given us something even more precious, namely friendships with many scientists all over the world, and the possibilities to attend conferences and visit countries in most corners of our globe, and hereby strengthen the personal relationships.

REFERENCES 1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 69 (1974) 129–141. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 78 (1975) 145–157. J. Ruzicka, E.H. Hansen and H. Mosbæk, Anal. Chim. Acta, 92 (1977) 235–249. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 99 (1978) 37–76. S. Olsen, J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 136 (1982) 101–112. E.H. Hansen, Fresen Z. Anal. Chem., 329 (1988) 656–659. J. Ruzicka and E.H. Hansen, Flow Injection Analysis, Wiley, New York, 1981. J. Ruzicka and E.H. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1988. G. Nagy, Zs. Feher and E. Pungor, Anal. Chim. Acta, 52 (1970) 47–54. G.R. Beecher, K.K. Stewart and P.E. Hare, Proceedings of a symposium entitled Symposium on Chemical and Biological Methods for Protein Quality Determination, Marcel Dekker, New York, 1975, pp. 411–421. J. Ruzicka, E.H. Hansen, H. Mosbæk and F.J. Krug, Anal. Chem., 49 (1977) 1858–1861. M. Margoshes, Anal. Chem., 49 (1977) 1861–1862. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 87 (1976) 353–363. L.R. Snyder, Anal. Chim. Acta, 114 (1980) 3–18. T. Braun, W.S. Lyon and E. Bujdoso´, Anal. Chem., 49 (1977) 682A–688A. Hansen’s FIA-bibliography, http://www.fialab.com/ E.H. Hansen, Anal. Chim. Acta, 308 (1995) 3–13. T. Braun and W.S. Lyon, Fresen Z. Anal. Chem., 319 (1984) 74–77. T. Braun, W. Gla¨nsel and A. Schubert, Trends Anal. Chem., 8 (1989) 281–284. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 114 (1980) 19–44. E. Garfield, Curr. Cont., ISI Press, 35 (1983) 5–8. D. de Solla Price, Little Science. Big Science, Columbia University Press, New York, 1963. T. Braun, A. Schubert and G. Schubert, Anal. Chem., 73 (2001) 667A–669A. J. Ruzicka, Flow Injection Analysis: CD-ROM Tutorial, 3rd ed., 2005 (available free of charge from www.flowinjection.com). J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 161 (1984) 1–25. B.O. Petersson, E.H. Hansen and J. Ruzicka, Anal. Lett., 19 (1986) 649–665. J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 145 (1983) 1–15. A. Manz, N. Graber and H.M. Widmer, Sens. Actuators B, 1 (1990) 244–248. M. Miro´ and E.H. Hansen, Anal. Chim. Acta, 600 (2007). E.H. Hansen and M. Miro´, TrAC — Trends Anal. Chem., 26 (2007) 18–26. E.H. Hansen, Quim. Anal., 8 (1989) 139–150.


CHAPT ER

2 From Beaker to Programmable Microfluidics Jaromir Ru˚ˇzicˇka

Contents

1. 2. 3. 4. 5. 6. 7.

Introduction Sequential Injection and Programmable Flow Miniaturization Mixing and Dispersion Mixing by Diffusion and Reynolds Number mSI and Lab-on-Valve Design Methods 7.1 Reagent-based assays 7.2 Bead injection 7.3 mSI affinity chromatography 7.4 Sequential injection chromatography 7.5 mSI titrations 7.6 Sample pretreatment 8. Conclusions Acknowledgment References

23 26 28 28 35 36 37 37 39 40 41 41 43 43 44 44

1. INTRODUCTION A vast majority of (bio)chemical assays rely on precise and reproducible solution handling, since samples and reagents have to be metered, mixed, incubated, heated, separated, and monitored by spectroscopy, electrochemistry, or other means for quantification of target analytes. This chapter follows the development of solution-handling techniques from manual to mechanized, and into a microfluidic format. It focuses on microsequential injection (mSI) techniques, not because of their novelty, but for their well-documented versatility, that opens

Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00602-8

r 2008, Published by Elsevier B.V.

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Jaromir Ru˚zˇicˇka

yet unexplored avenues for further research in this dynamic field of analytical chemistry. Indeed, compared to traditional flow injection analysis (FIA) that operates on continuous forward flow, sequential injection (SI), bead injection (BI), and sequential injection chromatography (SIC) utilize flow programming, to enhance their usefulness and to reduce consumption of reagents. It will be shown how flow programming in mSI format, can, due to its unprecedented flexibility, accommodate all the above-mentioned analytical techniques in the same instrument. Analytical chemistry is the oldest branch of chemistry [1], since prior to the development of quantitative analysis, chemical experimentation remained within the realm of alchemy. It is in Lavoisier’s book [2], where we find the first description of solution-handling tools, including volumetric glassware. By 1806 volumetric analysis was perfected, and has remained in its form almost unchanged to this day [3]. Thus originated the solution-handling method, often referred to as ‘‘beaker chemistry’’. As time went by, this approach became refined, miniaturized, and mechanized, ultimately evolving into current microwell plate formats that have been designed to meet the needs of high throughput pharmaceutical assays. The characteristic feature of this approach, known as ‘‘batch analysis’’ is that each sample solution is processed within a container (test tube, beaker, microwell), where it is homogenously mixed with auxiliary reagents and the readout (end point, absorbance, etc.) is being taken after equilibrium has been reached. When mechanized for serial assays, the individual containers are moved around, through stations, where samples are pipetted, reagents are added, solutions are mixed, etc., as required by assay protocol. While precise, reproducible, and well suited for parallel processing of slow, and end-point-based assays, batch analysis is labor intensive and it becomes less reliable when microminiaturized down to microlitre volumes, where it suffers from the adverse effects of evaporation, differences in solution viscosities, and inability to carry out separations. For certain applications, such as routine clinical assays, or drug screening, discrete analysers dominate the field, since they offer ‘‘black box with prepacked chemistry’’ approach, albeit at high cost capital investment, and expensive reagent cost and maintenance. It was Tsvett, a botanist, who unwittingly became the father of continuous-flow analysis. In 1906, he published his pioneering work on the separation of components of chlorophyll using column of calcium carbonate, eluted continuously by mobile phase (petroleum ether) [4]. For almost 50 years, chromatography, which Tsvett discovered and named, was the only analytical technique where samples were analysed while being carried by a flow-through tubing towards a detector. This all changed, in 1957, when Skeggs, a clinical chemist, designed an air segmented, continuous-flow analyser (Figure 1), where sample solutions were drawn into a system of flow channels by a peristaltic pump, metered and mixed with reagents on the way to detector, while being heated, filtered, extracted, etc [5,6]. The essential feature of Skegg’s design was air segmentation, which divided the moving carrier stream into many separate segments using numerous air bubbles that prevented intermingling of adjacent

From Beaker to Programmable Microfluidics

25

Figure 1 Skegg’s continuous-flow analyser. Top: samples were drawn from the autosampler carousel (S) by a peristaltic pump that also aspirated air and reagent (R). Air segmented stream was pumped through a reaction coil, air bubbles were removed and reacted mixture was pumped and into a flow through cell, where absorbance was measured. The readout was obtained at a ‘‘steady state’’ flat portion of a peak. Below: air bubbles separated individual aqueous segments, where the circular movement of liquid promoted mixing. Adapted from Ref. [30] with authors permission.

samples. Also, friction of liquid with walls of the tubing facilitated homogenous mixing of samples with regents, by promoting solute circulation, within each forward moving liquid segment. Skeggs’ invention became an undisputed success, largely in the field of clinical assays, as almost all clinical laboratories in technically advanced countries used the Technicon Autoanalysers for serial assays of multiple analytes. Interestingly, academic research, textbooks, and university teachings, mostly ignored this revolutionary approach to solution handling, which dominated the field of real-life assays for the almost 20 years. It was in 1974, that unexpected, yet in hindsight almost trivial, discovery was made. During investigation of response rate of an electrode, it was realized, that air segmentation was not necessary for performing reagent-based assays. The subsequent study of dispersion of solutes in tubular conduits within nonsegmented, continuous flow, led to the development of a new technique [7], termed FIA. The method was based on combination of three principles: sample injection, controlled dispersion, and reproducible timing [8,9,20]. Sample injection defined the volume of analyte and its initial geometry in the tubular channel, while the controlled dispersion was achieved by holding the flow rate of the carrier stream constant and by maintaining a fixed geometry of the flow path. The precise timing of the start of the assay cycle and of the residence time of the analyte zone in the system was controlled by flow rate and by the volume of the flow path. To begin with, FIA was not well received, since the idea of using controlled dispersion rather than homogenous mixing of sample with reagents was entirely at odds with the accepted concept of reagent-based assays [9]. It took several years

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before experimental evidence finally prevailed, documenting that the dispersion process and timing of all events could be controlled with such repeatability, confirming that FIA assays, based on monitoring of concentration gradients, and incomplete chemical equilibria, can be as precise and reproducible, as those obtained in the traditional batch format, where sample and reagent solutions are homogenously mixed and chemical equilibrium is attained. The scope of this chapter documents that FIA and its related techniques are now widely used both for routine as well as research work, in laboratories worldwide. After 30 years of existence with over 16,000 papers published on FIA alone, novel, ingenious modifications and applications of flow injection techniques are still being discovered [9]. One of them, termed SI [10], benefits from many offerings of computerization and from advances in software and laboratory hardware. Improved pumps, valves, and miniaturized solid state spectrophotometers and light sources (LED) allowed the initial design of SI methodology to be gradually transformed from proof of concept, to a powerful, miniaturized tool for research and routine applications. The result, mSI is far more versatile than the original, continuous flow-based techniques.

2. SEQUENTIAL INJECTION AND PROGRAMMABLE FLOW It takes a long time for a novel method to mature and to be accepted. This is because initially, it is difficult to visualize the full potential of a new approach, and to anticipate its future applications. The proof of concept is often hampered by lack of suitable hardware components, that not yet been developed, or, by the use of less suitable components, that were cannibalized from available instrumentation, or had to be improvised due to lack of funds. The first step towards present form of mSI was made in 1990 [10], when the principle of sequential injection analysis (SI) was proposed and experimentally verified. From the outset it was clear that SI belongs to the family of FIA techniques, being based on the same principles: sample (and reagent) injection, controlled dispersion, and reproducible timing. Therefore, the success of SI critically depends on strict control of the two kinetic processes that occur simultaneously, while the injected sample moves through the channel, namely the physical process of dispersion of the injected sample and reagent zones, and the chemical process of formation of chemical species to be detected downstream. The difference between FIA and SI is in the way, by which solutions are being manipulated. In SI mode, sample and reagent zones are sequentially stacked upstream from the injection valve (Figure 2, top) and subsequently mixed by reversing the flow. Dispersion is controlled by means of flow programming. Flow reversal and sudden accelerations are used to promote mixing and to speed up flushing of the system, while stopping the flow is used to control reaction time. Also, in SI mode, solutions are pumped only when a sample is being processed and, for that reason, smaller volumes of reagents are used and smaller volumes of waste are produced, as compared to continuousflow based FIA. The research community rapidly recognized these advantages,


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Figure 2 Top (A–E). Principle of sequential injection analysis. (A) Metered volume of sample solution is injected via multiposition valve upstream. (B) Sequential injection of a metered volume of reagent forms a well-defined zone. (C) Injection of carrier solution (or of a second reagent) pushes the stack further upstream. (D) Flow reversal carries reaction mixture downstream and towards detector. (E) Reaction product reaches detector where it is being monitored. Below (a–e). Principle of bead injection. (a) Metered volume of bead suspension is injected into the system and captured within the flow channel. (b) Solution of target analyte is injected and transported towards microcolumn of captured beads. (c) Analyte molecules react with ligands immobilized on bead surfaces. (d) Reaction product is either detected on bead surfaced or eluted to be detected downstream. (e) Beads are discarded. Adapted from Ref. [20] with author’s permission. (See Color Plate Section at the end of this book.)

and therefore SI has been soon applied to a variety of assays, using spectroscopic, electrochemical, radiochemical, turbidimetric, and chemiluminescent detection. An excellent review published by Lenehan et al. [11] summarized the development of SI in 300 references up to 2002. Since then, SI has expanded into separation techniques, of which SIC (see below) is an innovative technique with potential for many practical applications.

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3. MINIATURIZATION Being initially designed for automated monitoring of industrial processes [10,11] SI instrumentation and reagent consumption was of little or no importance. Therefore SI systems designed for laboratory use, were assembled in the same fashion as FIA instruments, by connecting the pump, valve, and detector with tubing that, when coiled, served as a reactor. What prompted the development of mSI technique was the impact of microTAS techniques [12–14] where the research was focused on dramatic downsizing of solution-handling systems, designed as ‘‘lab-on-chip’’. The obvious advantages of microminiaturization: low sample and reagent consumption, minimized production of chemical waste, small instrument size and portability, inspired downscaling of SI instrumentation, albeit with two important differences. First, mSI systems unlike ‘‘lab-on-chip’’ devices operate on programmable flow and not continuous flow. Second, decrease of channel volume in mSI systems channel is not achieved by decreasing channel diameter, as done in ‘‘lab-on-chip’’ designs, but by decreasing channel length. The reason for and advantages of this design are discussed in next sections (4 and 5). By integrating the flow cell, sample introduction channels, access to reagent reservoirs, microcolumns and jet mixers, within a mezzo-fabricated monolithic structure, mounted atop a multiposition valve [15] (Figure 3, top) a novel solution-handling platform was designed, and named in jest ‘‘lab-on-valve’’ (LOV). Within next 5 years, rather unexpectedly, LOV become a vehicle for new techniques such as BI [16] and SI affinity chromatography [17] in microformat. In addition, mSI has been adopted as a ‘‘front end’’ solution-processing system for mass spectrometry [18], capillary electrophoresis [19], and found application for biomolecular assays [20]. For trace analysis by atomic spectroscopy, mSI in the LOV format was used as a sample processing ‘‘front end’’ to atomic absorption and inductively coupled plasma (ICP) [21–28]. Indeed, virtually all reagent-based assays can be automated in the mSI–LOV mode (Table 1), either in solution, or through interaction of solution and solid surfaces, in BI format [29].

4. MIXING AND DISPERSION The key to any successful flow-based technique is the design of the way, in which sample and reagents are mixed, and their further processing is controlled while chemical reaction(s) are taking place. For real-life application it is the combination of convection and diffusion that brings reactants together, a process that is accomplished rapidly, easily, and transparently in batch systems, where homogenous mixing is the goal. Flow-based system are more complex, since the mixing has to be considered in three dimensions, accomplished in two directions by radial and axial dispersion. It is well known, and well described, how injected bolus of a homogenous liquid (visualized as square wave input), disperses due to initial acceleration into a ‘‘hollow bullet’’ shape, being ultimately transformed into a ‘‘rolling ball’’ of material while traveling down the flow channel. This process is easily observed


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Figure 3 Top. Experimental setup for mSI–LOV system. A multiposition valve integrates a flow cell, associated with port #2, flow through port (#5) that allows access to sample to be analysed and reagent ports. A high precision stepper motor driven syringe pump provides bidirectional solution metering and transport within the LOV channels. The holding coil is used to insulate working channels from the pump, in order to prevent sample and reagent solution entering into the barrel of the pump. Typical pump volume is 1 mL, injected volumes are on microlitre scale and working channel diameter 0.8 mm. Bottom. Tools for breaking laminar flow: (A) Tubing (0.8 mm I.D., 1.6 mm O.D. inserted into LOV channel (I.D. 1.6 mm) can be recessed in order to disrupt laminar flow. (B) Flow channel through stator and rotor of a multiposition valve has been designed to have sharp bends and wider (stator) and narrow (rotor) sections. (C) A narrow tube, inserted into LOV channel at a corner efficiently breaks laminar flow pattern due to ‘‘jet and wall’’ effect. (D) Narrow channel section effectively breaks the laminar flow pattern during flow reversals (see Section 6). Adapted from Ref. [20] with author’s permission. (See Color Plate Section at the end of this book.)

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Table 1 1.

2.

3. 4.

5.

6.

7.

8. 9.

10.

11.

12.

13.

14. 15. 16.

Development and applications of microSI–LOV technique 2006–2000

Yang W, Wang JH, An octadecyl immobilized silica beads packed microcolumn versus polytetrafluoroethylene knotted reactor for Cd(OH)(2) precipitate collection coupled to electrothermal atomic absorption spectrometry for cadmium screening, CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 34: 1078–1082 (2006) Long XB, Miro´ M, Hansen EH, et al. Hyphenating multisyringe flow injection lab-onvalve analysis with atomic fluorescence spectrometry for on-line bead injection preconcentration and determination of trace levels of hydride-forming elements in environmental samples, ANALYTICAL CHEMISTRY 78: 8290–8298 (2006) Chailapakul O, Ngamukot P, Yoosamran A, et al. Recent electrochemical and optical sensors in flow-based analysis, SENSORS 6: 1383–1410 (2006) Ohno S. Studies on spectrophotometric analysis for ultratrace amounts of metal ions coupled with catalytic reactions and flow-based techniques, BUNSEKI KAGAKU 55: 809–810 (2006) Leelasattarathkul T, Liawruangrath S, Rayanakorn M, et al. The development of sequential injection analysis coupled with lab-on-valve for copper determination, TALANTA 70: 656–660 (2006) Long XB, Miro M, Jensen R, et al. Highly selective micro-sequential injection lab-on-valve (mu SI-LOV) method for the determination of ultra-trace concentrations of nickel in saline matrices using detection by electrothermal atomic absorption spectrometry, ANALYTICAL AND BIOANALYTICAL CHEMISTRY 386: 739–748 (2006) Yang M, Xu Y, Wang JH. Lab-on-valve system integrating a chemiluminescent entity and in situ generation of nascent bromine as oxidant for chemiluminescent determination of tetracycline, ANALYTICAL CHEMISTRY 78: 5900–5905 (2006) Ruzicka J, Carroll AD, Lahdesmaki I. Immobilization of proteins on agarose beads, monitored in real time by bead injection spectroscopy, ANALYST 131: 799–808 (2006) Gutzman Y, Carroll AD, Ruzicka J. Bead injection for biomolecular assays: affinity chromatography enhanced by bead injection spectroscopy, ANALYST 131: 809–815 (2006) Hansen EH, Miro M, Long XB, et al. Recent developments in automated determinations of trace level concentrations of elements and on-line fractionation schemes exploting the micro-sequential injection-Lab-on-valve approach, ANALYTICAL LETTERS 39: 1243–1259 (2006) Wang Y, Chen ML, Wang JH. Sequential/bead injection lab-on-valve incorporating a renewable microcolumn for co-precipitate preconcentration of cadmium coupled to hydride generation atomic fluorescence spectrometry, JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 21: 535–538 (2006) Miro M, Hansen EH, Buanuam D. The potentials of the third generation of flow injection analysis for nutrient monitoring and fractionation analysis, ENVIRONMENTAL CHEMISTRY 3: 26–30 (2006) Quintana JB, Miro M, Estela JM, et al. Automated on-line renewable solid-phase extraction-liquid chromatography exploiting multisyringe flow injection-bead injection lab-on-valve analysis, ANALYTICAL CHEMISTRY 78: 2832–2840 (2006) Yuan SG, DeGrandpre M. Comparison between two detection systems for fiber-optic chemical sensor applications, APPLIED SPECTROSCOPY 60: 465–470 (2006) Miro M, Hansen EH. Solid reactors in sequential injection analysis: recent trends in the environmental field, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 25: 267–281 (2006) Edwards KA, Baeumner AJ. Sequential injection analysis system for the sandwich hybridization-based detection of nucleic acids, ANALYTICAL CHEMISTRY 78: 1958– 1966 (2006)


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Table 1 (Continued ) 17. Ohno S, Teshima N, Sakai T, et al. Sequential injection lab-on-valve simultaneous spectrophotometric determination of trace amounts of copper and iron, TALANTA 68: 527–534 (2006) 18. Pimenta AM, Montenegro MCBSM, Araujo AN, et al. Application of sequential injection analysis to pharmaceutical analysis, JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS 40: 16–34 (2006) 19. Chen Y, Carroll AD, Scampavia L, et al. Automated method, based on micro-sequential injection, for the study of enzyme kinetics and inhibition, ANALYTICAL SCIENCES 22: 9–14 (2006) 20. Burakham R, Jakmunee J, Grudpan K. Development of sequential injection-lab-at-valve (SI-LAV) micro-extraction instrumentation for the spectrophotometric determination of an anionic surfactant, ANALYTICAL SCIENCES 22: 137–140 (2006) 21. Economou A. Sequential-injection analysis (SIA): a useful tool for on-line samplehandling and pre-treatment, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 24: 416–425 (2005) 22. Ruzicka J. From beaker chemistry to programmable microfluidics, COLLECTION OF CZECHOSLOVAK CHEMICAL COMMUNICATIONS 70: 1737–1755 (2005) 23. Long XB, Miro M, Hansen EH. On-line dynamic extraction and automated determination of readily bioavailable hexavalent chromium in solid substrates using micro-sequential injection bead-injection lab-on-valve hyphenated with electrothermal atomic absorption spectrometry, ANALYST 131: 132–140 (2006) 24. Burakham R, Lapanantnoppakhun S, Jakmunee J, et al. Exploiting sequential injection analysis with lab-at-valve (LAV) approach for on-line liquid-liquid micro-extraction spectrophotometry, TALANTA 68: 416–421 (2005) 25. Long XB, Miro M, Hansen EH. An automatic micro-sequential injection bead injection Lab-on-Valve (mu SI-BI-LOV) assembly for speciation analysis of ultra trace levels of Cr(III) and Cr(VI) incorporating on-line chemical reduction and employing detection by electrothermal atomic absorption spectrometry (ETAAS), JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 20: 1203–1211 (2005) 26. Long XB, Miro M, Hansen EH. Universal approach for selective trace metal determinations via sequential injection-bead injection-lab-on-valve using renewable hydrophobic bead surfaces as reagent carriers, ANALYTICAL CHEMISTRY 77: 6032–6040 (2005) 27. Erxleben H, Ruzicka J. Atomic absorption spectroscopy for mercury, automated by sequential injection and miniaturized in lab-on-valve system, ANALYTICAL CHEMISTRY 77: 5124–5128 (2005) 28. Wang Y, Wang JH, Fang ZL. Octadecyl immobilized surface for precipitate collection with a renewable microcolumn in a lab-on-valve coupled to an electrothermal atomic absorption spectrometer for ultratrace cadmium determination, ANALYTICAL CHEMISTRY 77: 5396–5401 (2005) 29. Chen XW, Wang WX, Wang JH. A DNA assay protocol in a lab-on-valve meso-fluidic system with detection by laser-induced fluorescence, ANALYST 130: 1240–1244 (2005) 30. Chen XW, Wang JH, Fang ZL. A spectrophotometric procedure for DNA assay with a microsequential injection lab-on-valve meso-fluidic system, TALANTA 67: 227–232 (2005) 31. Long XB, Hansen EH, Miro M. Determination of trace metal ions via on-line separation and preconcentration by means of chelating Sepharose beads in a sequential injection lab-on-valve (SI-LOV) system coupled to electrothermal atomic absorption spectrometric detection, TALANTA 66: 1326–1332 (2005)

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Table 1 (Continued ) 32. Hansen EH. Use of flow injection and sequential injection analysis schemes for the determination of trace-level concentrations of metals in complex matrices by ETAAS and ICPMS, JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH PART A-TOXIC/HAZARDOUS SUBSTANCES & ENVIRONMENTAL ENGINEERING 40: 1507–1524 (2005) 33. Miro M, Estela JM, Cerda V. Potentials of multisyringe flow injection analysis for chemiluminescence detection, ANALYTICA CHIMICA ACTA 541: 57–68 (2005) 34. Estela JM, Cerda V. Flow analysis techniques for phosphorus: an overview, TALANTA 66: 307–331 (2005) 35. Hartwell SK, Srisawang B, Kongtawelert P, et al. Sequential injection-ELISA based system for online determination of hyaluronan, TALANTA 66: 521–527 (2005) 36. Erxleben H, Ruzicka J. Sequential affinity chromatography miniaturized within a ‘‘lab-on-valve’’ system, ANALYST 130: 469–471 (2005) 37. Wang JH. Lab-on-valve mesofluidic analytical system and its perspectives as a ‘‘worldto-chip’’ front-end, ANALYTICAL AND BIOANALYTICAL CHEMISTRY 381: 809– 811 (2005) 38. Jakmunee J, Pathimapornlert L, Hartwell SK, et al. Novel approach for monosegmented flow micro-titration with sequential injection using a lab-on-valve system: a model study for the assay of acidity in fruit juices, ANALYST 130: 299–303 (2005) 39. Liu KL. Review of atomic spectroscopy, SPECTROSCOPY AND SPECTRAL ANALYSIS 25: 95–103 (2005) 40. Wang JH, Hansen EH. Trends and perspectives of flow injection/sequential injection on-line sample-pretreatment schemes coupled to ETAAS, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 24: 1–8 (2005) 41. Jakmuneea J, Patimapornlert L, Suteerapataranon S, et al. Sequential injection with labat-valve (LAV) approach for potentiometric determination of chloride, TALANTA 65: 789–793 (2005) 42. Wang JH, Fang ZL, The third generation of flow injection analysis; Current situation and perspectives of Lab-on-Valve scheme, CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 32: 1401–1406 (2004) 43. Hansen E. The impact of flow injection on modem chemical analysis: has it fulfilled our expectations? And where are we going? TALANTA 64: 1076–1083 (2004) 44. Grudpan K. Some recent developments on cost-effective flow-based analysis, TALANTA 64: 1084–1090 (2004) 45. Hartwell SK, Christian GD, Grudpan K. Bead injection with a simple flow-injection system: an economical alternative for trace analysis, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 23: 619–623 (2004) 46. Long XB, Chomchoei R, Gala P, et al. Evaluation of a novel PTFE material for use as a means for separation and preconcentration of trace levels of metal ions in sequential injection (SI) and sequential injection lab-on-valve (SI-LOV) systems-Determination of cadmium(II) with detection by electrothermal atomic absorption spectrometry (ETAAS), ANALYTICA CHIMICA ACTA 523: 279–286 (2004) 47. Miro M, Frenzel W. A critical examination of sorbent extraction pre-concentration with spectrophotometric sensing in flowing systems, TALANTA 64: 290–301 (2004) 48. Zhu HL, Chen HW, Zhou YL. Determination of immunoglobulin G in human serum by sequential injection renewable surface fluorescence immunoassay system with a chipbased flow-through cell, CHINESE JOURNAL OF ANALYTICAL CHEMISTRY 32: 841–846 (2004)


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Table 1 (Continued ) 49. Ogata Y, Scampavia L, Carter TL, et al. Automated affinity chromatography measurements of compound mixtures using a lab-on-valve apparatus coupled to electrospray ionization mass spectrometry, ANALYTICAL BIOCHEMISTRY 331: 161–168 (2004) 50. Hindson BJ, Brown SB, Marshall GD, et al. Development of an automated sample preparation module for environmental monitoring of biowarfare agents, ANALYTICAL CHEMISTRY 76: 3492–3497 (2004) 51. Chen Y, Ruzicka J. Accelerated micro-sequential injection in lab-on-valve format, applied to enzymatic assays, ANALYST 129: 597–601 (2004) 52. Toda K. Development of miniature key devices for flow analysis and their applications, BUNSEKI KAGAKU 53: 207–219 (2004) 53. Hansen EH, Wang JH. The three generations of flow injection analysis, ANALYTICAL LETTERS 37: 345–359 (2004) 54. Nishihama S, Yoshizuka K, Scampavia L, et al. Optimization of micro sequential injection analysis, BUNSEKI KAGAKU 52: 1187–1192 (2003) 55. Karthikeyan S, Hashigaya S, Kajiya T, et al. Determination of trace amounts of phosphate by flow-injection photometry, ANALYTICAL AND BIOANALYTICAL CHEMISTRY 378: 1842–1846 (2004) 56. Solich P, Polasek M, Klimundova J, et al. Sequential injection technique applied to pharmaceutical analysis, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 23: 116–126 (2004) 57. Erxleben HA, Manion MK, Hockenbery DM, et al. A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve system, ANALYST 129: 205–212 (2004) 58. Miro M, Frenzel W. Flow-through sorptive preconcentration with direct optosensing at solid surfaces for trace-ion analysis, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 23: 11–20 (2004) 59. Wang JH, Hansen EH. On-line sample-pre-treatment schemes for trace-level determinations of metals by coupling flow injection or sequential injection with ICP-MS, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 22: 836–846 (2003) 60. Wang JH, Hansen EH, Miro M. Sequential injection-bead injection-lab-on-valve schemes for on-line solid phase extraction and preconcentration of ultra-trace levels of heavy metals with determination by electrothermal atomic absorption spectrometry and inductively coupled plasma mass spectrometry, ANALYTICA CHIMICA ACTA 499: 139–147 (2003) 61. Ampan P, Ruzicka J, Atallah R, et al. Exploiting sequential injection analysis with bead injection and lab-on-valve for determination of lead using electrothermal atomic absorption spectrometry, ANALYTICA CHIMICA ACTA 499: 167–172 (2003) 62. Wu CH, Scampavia L, Ruzicka J. Micro sequential injection:automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system, ANALYST 128: 1123–1130 (2003) 63. Carroll AD, Scampavia L, Luo D, et al. Bead injection ELISA for the determination of antibodies implicated in type 1 diabetes mellitus, ANALYST 128: 1157–1162 (2003) 64. Wang JH, Hansen EH. Sequential injection lab-on-valve: the third generation of flow injection analysis, TrAC-TRENDS IN ANALYTICAL CHEMISTRY 22: 225–231 (2003) 65. Miro M, Jonczyk S, Wang JH, et al. Exploiting the bead-injection approach in the integrated sequential injection lab-on-valve format using hydrophobic packing

34


Table 1 (Continued )

66.

67.

68.

69.

70.

71.

72. 73. 74.

75.

76.

77.

78.

materials for on-line matrix removal and preconcentration of trace levels of cadmium in environmental and biological samples via formation of non-charged chelates prior to ETAAS detection, JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 18: 89–98 (2003) Grudpan K, Ampan P, Udnan Y, et al. Stopped-flow injection simultaneous determination of phosphate and silicate using molybdenum blue, TALANTA 58: 1319–1326 (2002) Schulz CM, Scampavia L, Ruzicka J. Real-time monitoring of lactate extrusion and glucose consumption of cultured cells using a lab-on-valve system, ANALYST 127: 1583–1588 (2002) Ogata Y, Scampavia L, Ruzicka J, et al. Automated affinity capture-release of biotincontaining conjugates using a lab-on-valve apparatus coupled to UV/visible and electrospray ionization mass Spectrometry, ANALYTICAL CHEMISTRY 74: 4702–4708 (2002) Schulz CM, Ruzicka J. Real-time determination of glucose consumption by live cells using a lab-on-valve system with an integrated microbioreactor ANALYST 127: 1293–1298 (2002) Hansen EH, Wang JH. Implementation of suitable flow injection/sequential injectionsample separation/preconcentration schemes for determination of trace metal concentrations using detection by electrothermal atomic absorption spectrometry and inductively coupled plasma mass spectrometry, ANALYTICA CHIMICA ACTA 467: 3–12 (2002) Carroll AD, Scampavia L, Ruzicka J. Label dilution method: a novel tool for bioligand interaction studies using bead injection in the lab-on-valve format, ANALYST 127: 1228–1232 (2002) Lenehan CE, Barnett NW, Lewis S. Sequential injection analysis, ANALYST 127: 997–1020 (2002) Wu CH, Scampavia L, Ruzicka J. Microsequential injection: anion separations using ‘Lab-on-Valve’ coupled with capillary electrophoresis, ANALYST 127: 898–905 (2002) Wang JH, Hansen EH. Interfacing sequential injection on-line preconcentration using a renewable micro-column incorporated in a ‘lab-on-valve’ system with direct injection nebulization inductively coupled plasma mass spectrometry, JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 16: 1349–1355 (2001) Wu CH, Ruzicka J. Micro sequential injection: environmental monitoring of nitrogen and phosphate in water using a ‘‘Lab-on-Valve’’ system furnished with a microcolumn, ANALYST 126: 1947–1952 (2001) Wang JH, Hansen EH. On-line ion exchange preconcentration in a sequential injection lab-on-valve microsystem incorporating a renewable column with ETAAS for the trace level determination bismuth in urine river sediment, ATOMIC SPECTROSCOPY 22: 312–318 (2001) Wu CH, Scampavia L, Ruzicka J, et al. Micro sequential injection: fermentation monitoring of ammonia, glycerol, glucose, and free iron using the novel lab-on-valve system, ANALYST 126: 291–297 (2001) Ruzicka J. Lab-on-valve: universal microflow analyser based on sequential and bead injection, ANALYST 125: 1053–1060 (2000)

Note: This table summarizes manuscripts published using the lab-on-valve USA, Denmark, Spain, Thailand, China, Japan, Germany, Czech Republic, and Poland.


35

by injection a bolus of dye, and by recording peak changes from asymmetrical to Gaussian. The point is, that the key to successful design of any flow system is to promote radial dispersion as much as possible and to control axial dispersion to suit the analytical protocol at hand [20,30]. Obviously, for chromatographic separation axial dispersion ought to be minimized, while for SI-based techniques it should be initially maximized, in order to promote mutual penetration of sample and reagent zones, that have been sequentially stacked into a tubular conduit (Figure 2). Next, radial mixing has to be promoted, in order to bring reactants together. This of course, is impossible to achieve rapidly at conditions of laminar flow in tubes that have large enough diameters for practical applications, and where diffusion is too slow process to provide adequate mixing in radial direction.

5. MIXING BY DIFFUSION AND REYNOLDS NUMBER Osborne Reynolds studied motion of water in straight smooth tubes by determining the critical flow velocity at which the laminar parabolic profile was disturbed by ‘‘the birth of eddies’’. The result of his immaculate research [31], known as Reynolds number, the combination of conduit diameter, liquid velocity and its properties, is often quoted as a starting point for design of microfluidic analytical systems. Yet in Reynolds’ apparatus (Figure 4.), the dimensions of the pipes, (which were kept absolutely straight and ranged from 6 to 500 mm I.D., with lengths from 3 to 5 m), as well as his means for generating and controlling the flow, were far different from conditions encountered in microflow analytical systems. This is why comparison of flow conditions in an FIA system (Re 100–150) with the Re value at which the laminar pattern is broken (Re W 2,000), is grossly misleading, if interpreted as an absolute threshold under which laminar flow (and inadequate mixing in radial direction) will persist. Such an unfortunate conclusion leads to a ‘‘blind alley’’ of designing FIA conduits with I.D. of 100 mm or less, where the job of mixing in the radial direction relies on diffusion alone. The limited success of this approach is well documented in the literature on ‘‘lab-on-chip’’ flow through systems. Yet reading Reynolds’ work, reveals numerous careful precautions, he made in order to preserve laminar flow, until the critical flow velocity has ultimately been reached. The flow was kept absolutely pulse-free, being driven by gravity, the reservoir being open ‘‘several hours after it has been filled in order to avoid any fluid disturbances’’. Any obstruction, like a spiral made from copper wire, inserted into the tube (Figures 16 and 17 in Ref. [31]), reduced the critical velocity (related to Re) by half. This is not to say that laminar (or quasi-laminar flow) is not to be observed in microfluidic systems, but to point out that reading of Reynolds’ work offers insight into lengths that he went to, in order to work at conditions of stabilized flow in long straight internally polished circular pipes. Therefore, his finding that laminar flow is broken at Re W 2,000 should not be viewed as a limitation for design of microfluidic devices, where, consequently, mixing could only be achieved by diffusion alone.

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Figure 4 Reynolds’ apparatus for study of laminar and turbulent flow used gravity to propel the liquids and flow meter (on his left) to measure flow rates. The dye container (right) was connected with tubing to a funnel-shaped orifice that could be exchanged allowing different tube I.D. and materials to be investigated. Reproduced from Ref. [31] with permission of Royal Society.

Indeed in order to optimize mixing in microfluidic devices, all one has to do is to destabilize the flow. There are two tools available to accomplish this goal; geometry of flow conduit and flow programming. Classical FIA systems have been designed with coiled (or knitted) reaction coils, placed downstream from confluence points in order to promote mixing. Sample injection destabilizes the flow, and sudden acceleration of the injected bolus further promotes radial and axial mixing [30]. Flow generated by peristaltic pumps is never pulse-free. By combination of luck, accident and intent, FIA systems were designed with these artifacts and devices in place, which disrupted laminar flow (at Re computed to be 100 or less), and provided sufficient mixing to make FIA systems operational.

6. lSI AND LAB-ON-VALVE DESIGN The concept of mSI–LOV exploits the means for disrupting the laminar flow pattern, by intentionally designing flow geometry of sample/reagent path to be nonlinear and nonuniform in diameter [15,20] (Figure 3). It uses programmable flow to design a flow protocol that involves flow reversals, flow acceleration, stopped flow, and bursts of high flow velocity. These tools promote fast radial


37

mixing, while axial mixing is controlled by choice of injected volumes, and by the length of solute traveled in forward and reversed direction. Since reaction (incubation) times are promoted by stopping the flow (either in holding coil or better yet in a the flow cell), the length of the conduit through which the sample will travel can be minimized. At the same time, by avoiding narrow flow channels (i.e., smaller than 0.5 mm I.D.), the surface volume ratio is minimized, thus minimizing adsorption of target analytes on conduit walls. In addition, the low flow resistance of short and wide flow conduits, allows precise flow manipulation, and the use of ultra fast bursts of flow that wash out channels rapidly and easily, remove stray air bubbles or clogging particles. The overall experimental setup of system (Figure 3) and geometry of its conduits, exemplify some of many conceivable system designs, the part of which is also a multipurpose flow cell. Absorbance, fluorescence and their combination can be carried out with the universal flow cell that is integrated within LOV module (Figure 5). The key feature of this design is the use of quartz optical fibres sheeted within a tubing that has slightly smaller diameter than LOV channel, and can be fixed in position by standard Upchurch nuts and ferrules. The distance between the fiber ends defines the optical path length, and can be varied from 1 (Figure 5A) to 10 mm (Figure 5B) and up to 200 mm (Garth’s cell, Figure 5C). Fluorescence measurement (Figure 5D) is carried with fibres inserted at right angles, while combination of fluorescence and absorbance measurement requires use of three optical fibres (Figure 5E). Note that the same LOV module is used for BI technique as well as for microaffinity chromatography (Figure 6).

7. METHODS Publications on mSIA–LOV and its applications, summarized in Table 1, are an excellent source of information on various formats and uses of this technique, as adopted in laboratories worldwide. Since additional details on system configuration, its components and uses, are also available in the 3rd edition of Tutorial on Flow Injection Analysis [20], the methods which mSI–LOV can accommodate are outlined only in brief.

7.1 Reagent-based assays In mSI format, reagent-based assays fall into two categories [20]: flow-through measurement, monitored as the sample zone passes through a detector, while the readout has form of a peak, or reaction rate measurement, monitored while the sample zone is arrested within a detector, where the readout is a reaction rate curve. For reaction rate measurement, the activity of catalyst and auxiliary reagent concentrations are selected in such a way that pseudo-zero-order reaction conditions are maintained, assuring that the concentration of the analyte, cA, is proportional to the slope dA/dt of the reaction rate curve. There are two advantages of reaction rate measurement in a stopped-flow mode. First, since the

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Figure 5 Configurations of a flow cell integrated into an LOV module. (A) Absorbance (1 mm light path). (B) Absorbance (10 mm light path). (C) Absorbance (Garth’s Extended Light Path cell; 100 mm long). (D) Fluorescence. (E) Combined fluorescence and absorbance. The vertical arrow indicates the length of light path (see also Figure 6). (See Color Plate Section at the end of this book.)

readout is based on the slope of the reaction rate curve any background signal caused by initial absorbance of sample and/or reagents is eliminated. This is an asset when samples to be analysed vary in colour due to matrix components. Also if sequentially injected reagents absorb at the monitored wavelength, or if the refractive index between sequentially injected zones varies, the stopped-flow technique eliminates resulting artefacts. Next, the stopped-flow technique offers doubling of sampling frequency. This is achieved by injecting stacked reagent/ sample zone into the holding coil, while the reaction rate of the previously injected sample is being monitored in the stopped-flow mode in the flow cell [32]. While so far mainly enzymatic assays have been successfully automated in mSI


39

Figure 6 (A) Experimental setup for bead injection spectroscopy and microaffinity chromatography (for details see text). (B) Configuration of a flow cell for capturing and monitoring of beads by UV–VIS spectrophotometry. (C) Configuration of a flow cell for microaffinity chromatography. The beads are retained as a microcolumn by sheath of optical fibre, designed to allow mobile phase to pass freely into the flow cell (light path 10 mm). For details see text and Ref. [20]. Reproduced from Ref. [20] with author’s permission. (See Color Plate Section at the end of this book.)

format, equally promising is automation of kinetic reagent-based assays that allow detection limit of spectrophotometric methods of target analytes to be extended by several orders of magnitude, compared to absorbance measurements with regent/analyte complex formation.

7.2 Bead injection BI [16–29] can be viewed as a reagent-based assay carried out in mSI mode, whereby the reagent is immobilized on the surface of a solid phase, which is in the form of micropspheres (typical bead size: 30–200 mm in diameter). The mSI–LOV apparatus has been modified to meter and to retain beads (Figure 6A).

40


The assay protocol comprises five steps: (a) injection of precisely metered volume of bead suspension into a flow channel, where beads are captured in a welldefined geometry; (b) injection of a metered volume of sample; (c) perfusion of the bead column by the sample zone, while analyte molecules are retained or a reaction occurs at the surface of the beads; (d) monitoring of reaction product; (e) discarding beads from the conduit. BI assays can be carried out in numerous, not yet fully exploited ways that fall into three broad categories. The first, bead injection spectroscopy (BIS) is based on monitoring analytes as they are captured on the beads trapped within a flow cell and monitored by UV–VIS spectrophotometry [16,29] (Figure 6B). The second approach involves monitoring analytes eluted from the captured beads. While the first approach can be viewed as ‘‘on-column spectroscopy’’, the second can be viewed as miniaturized chromatography carried out on a renewable microcolumn [17]. The third approach is based on flushing the beads from the column into a graphite furnace, where beads are dried, incinerated, and target elements are vaporized and atomized for detection by AAS. Enhancement of the performance of atomic spectroscopies by BI was proposed by Hansen and Wang and documented in a series of innovative research articles [21–28]. The second approach was based on elution of the target element, followed by atomization of the eluate. In both cases the microcolumn, comprising Sephadexs C-25 cation-exchange beads, was automatically renewed for each assay by BI technique carried out in an LOV manifold. The central issue of all BI techniques is the manipulation of a bead suspension, as the beads need to be precisely metered and transported through a system of channels and into a flow cell, where solution/surface reactions are taking place. Bead transport and bead metering is accomplished by precise flow control. Beads are transported and packed at moderate flow rates (10 mL s1), perfused by analytes at 1–2 mL s1 and discarded by means of flow acceleration, at flow rates up to 200 mL s1. The critical issue of BI spectroscopy is bead monitoring, since the same amount of beads has to be captured within the flow cell and the beads be uniformly packed. A detailed description, including movie clips of functioning a BI system is given in Ref. [20].

7.3 mSI affinity chromatography Immunoaffinity chromatography, used for separation of immunoglobulins, is based on molecular recognition between a site fixed on a stationary phase and a target immunoglobulin that is being captured, while unwanted matrix components, such as salts and proteins, are washed out by the mobile phase. Conventional affinity chromatography is carried out in a continuous-flow format on a stationary phase of Sephadexs or Sepharoses beads, furnished with Protein A or G as a selective bioligand. Protein A has multiple antibody-binding domains that are recognized by the Fc portion of IgG molecules. At pH of 6 or higher, histidine residues become charged and selectively capture mammalian IgG, while at pH lower than 3, captured IgG is released. In mSIC format [17,33], the


41

bead column is formed within the LOV module, in a conduit situated between the central port of the multiposition valve and the flow cell (Figure 6C). The bead column volume is 10 mL, the flow cell volume 8 mL, and the dead volume between the flow cell and the end of the column is 0.1 mL. By using a 5 mL pulse of 0.1 N HCl, almost all IgG molecules retained on the column are eluted into the flow cell, where they are detected spectrophotometrically at 280 nm. The use of programmable flow is tailored to perform IgG on-column adsorption at a flow rate of 5 mL s1, and IgG is eluted from the column at 2 mL s1. The column is loaded at 40 mL s1, and is discarded at 400 mL s1. The entire assay is completed within 120 s, using only 2 mL of mobile phase [17,33]. Compared to BI spectroscopy, chromatography on a renewable bead column is more robust, since the volume of the beads captured within a renewable column is far less critical and also because the target analyte is eluted and monitored in solution and not on bead surfaces [33]. By using a short pulse of eluent, the front elution allows almost all target molecules to be in the flow cell at the same time, yielding excellent limits of detection [17,33].

7.4 Sequential injection chromatography SIC was the first chromatographic technique that benefited from introduction of programmable flow. It was conceived by Sˇatıńsky´ et al. [34], and demonstrated by the assay of pharmaceutical compounds separated on monolithic reversed phase columns. In contrast to HPLC, which employs conventional, particle-based columns and must use high-pressure pumps to force mobile phase through the column, work on SIC uses porous silica rods termed ‘‘monoliths’’. These columns, fabricated by sol-gel process, exhibit very low flow resistance and have a sufficient number of theoretical plates to provide separation efficiency suitable for resolution of closely related compounds [34,35]. Programming the flow of mobile phase allows injected volumes of sample and/or eluent to be conveniently varied. As the injected solvent disperses into a carrier stream, it forms a concentration gradient, used to elute analytes sequentially from the stationary phase. The assay protocol comprises six stages: (1) column conditioning, (2) sample injection, (3) retention of analytes and elution of matrix materials, (4) gradient formation, (5) analyte elution and detection, and (6) column purification. The advantage of the SIC format is that flow rates can be individually tailored to accommodate the needs of each stage of an assay protocol. Thus steps 1 and 6 can be carried out at high flow rates, while steps 3 and 4 are carried at low flow rates [34,35].

7.5 mSI titrations mSI titrations with spectrophotometric detection [36] are based on measuring residual alkalinity within sequentially stacked zones of sodium hydroxide, indigo carmine indicator, and acid sample. The reaction mixture is bracketed by two air bubbles that prevent the stacked zones from dispersing into the carrier

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Figure 7 Separation and sample pretreatment by peripherals adjacent to LOV module. (The primary syringe pump has been omitted from the graphic design as compared to Figures 3 and 6). (A) External low-pressure chromatographic column as used for affinity chromatography. (B) Sample pretreatment by dialysis using stopped-flow acceptor stream. (C) Sample cleanup achieved by capture of matrix components on external disposable column. (D) Sample preconcentration and cleanup by external column designed to capture target analyte. The captured analyte is eluted from the column by eluent supplied via the LOV module in a stop flow period and subsequently aspirated into the LOV by flow reversal for further processing. (See Color Plate Section at the end of this book.)

stream during mixing, which was accomplished by a single flow reversal. To obtain a smooth response, air segments were discarded prior to absorbance measurement (at 609 nm). This innovative approach was recently reviewed and expanded [37].


43

7.6 Sample pretreatment This is carried by peripheral devices that are clustered adjacent to the LOV module (Figure 7A–D). While this task needs an additional drive, such as a syringe (Figure 7B) or a peristaltic pump (Figure 7D), it enables the LOV system to accomplish sample pretreatment by capture of species on a microcolumn, or to eliminate matrix components by dialysis. Use of programmed flow for these operation offers various advantages that are not realized when continuous flow is used for the same sample cleanup technique. On-line sample pretreatment using a wide variety of microcolumns has been pioneered by Hansen et al. with detection of traces of elements by atomic spectroscopies [39,40].

8. CONCLUSIONS This chapter documents the versatility of the flow injection concept, by showing an astonishing variety of flow system configurations, designed to accommodate numerous analytical techniques that rely on automated sample/reagent handling. And yet, there is, without doubt, room for further novel designs, since two basic components of any flow system (pump and valve), can be supplemented by additional components. One, however, should keep in mind that the ultimate goal of research in analytical instrumentation is the development of a tool that is not only novel, but also practical. If a method fails to fulfill this goal, it will fail to become useful. Also, if not versatile and amenable to continuous improvements, the technique, however ingenious, will be replaced by another approach, better suited for real-life assays. Complex systems, with network of pumps and/or valves, are not practical, since if one component fails, it is difficult to identify it. The example of such poor design are, of course, ‘‘black boxes’’ such as the automated analysers with prepacked reagents, that are supposed to carry out automated assays, when a button is pressed, while the flow path and its components remain obscure to the user. The goal, therefore, should be the design of microfluidic analytical systems that are visually transparent, so that their function can be observed and understood, allowing any malfunction to be identified by sight. The challenge is to design the simplest possible system configuration, comprising ideally, only one pump and one valve. While well-written software allows addition of multiple pumps and valves with ease, an effort should be made to minimize the number of additional mechanical components. All SI techniques, including BI, SIC, and especially mSI–LOV, have been designed accordingly. It is hoped that this chapter will inspire many, by showing the combined advantages of flow and software programming to pursue this line of research. Well-designed software provides a powerful research and development tool, as it opens the door to techniques that cannot be designed for batch or continuous-flow system. In other words, the software should offer, in addition to trivial pump and valve control, integration of all system functions including the detector. Only when the baseline can be automatically readjusted (as in BI, or for stopped-flow reaction rate

44


measurement), can data be collected when the detector reads the presence of sample in the flow cell, and washout cycles will be programmed in an expedient way. In addition, optimization should be carried out by writing software ‘‘loops’’ that systematically adjust selected crucial parameters stepwise (injected volume, flow rates, flow reversals, bead volumes, etc.), until the investigated parameter is optimized. Also, detector readout, available in real time, should be superimposed on previous experimental runs, to provide instant identification of the changing of system response, caused by a change of a parameter investigated during method development. Purity and stability of reagents, blank values, and system drift are examples of parameters that can be identified and corrected more easily than by a manual, time-consuming approach. Software-assisted development will ease the transformation of traditional batch-based assay protocols, that rely on ‘‘end-point’’ format, into a kinetic-based reaction rate mode. The result will be improved reagent economy, and often a higher selectivity, as the ‘‘kinetic advantage’’ of flow injection technique can be conveniently exploited in the mSI–LOV stopped-flow format. As to the trends in the design of instruments for automated solution handling, further miniaturization beyond microlitre level should be carefully considered, and attempted only when the amount of available sample is limited, such as in research on the biochemistry of single cells. What will be rewarding to explore, is the design of systems that combine the advantages of batch processing (long incubation time, parallel automated processing of many samples) with flow-based processing, which offer the advantages emphasized in this chapter. Design of systems that can be deployed, for oceanography, hydrology, environmental and process monitoring, may seem to be ‘‘only’’ a technological challenge, yet the usefulness of such enterprises is without any doubt. We have not yet reached the end of the road leading from beaker to microfluidics. It has been a challenging journey that continues to offer many interesting views and unexplored opportunities [38].

ACKNOWLEDGMENT The author wishes to express his gratitude to Garth Klein, whose expertise in software development has made programmable flow a reality.

REFERENCES 1 F. Szabadvary, History of Analytical Chemistry, Pergamon Press, Oxford, 1966. 2 A. L. Lavoisier, Traite elementaire de chimie, Paris, 1789, p. 101. 3 E. R. Madsen, The Development of Titrimetric Analysis till 1806, G.E.C Gad Publishers, Copenhagen, 1958. 4 M. Tsvett, Ber. Deut. Bot. Ges., 24 (1906) 316–323. 5 T.L. Skeggs, J. Am. J. Clin. Pathol., 28 (1957) 311–322. 6 T.L. Skeggs, Anal. Chem., 38 (1966) 31A–44A. 7 J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 78 (1975) 145–147. 8 J. Ruzicka and E.H. Hansen, Anal. Chim. Acta, 99 (1978) 37–76. 9 E.H. Hansen, Talanta, 64 (2004) 1076–1083.


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10 J. Ruzicka and G.D. Marshall, Anal. Chim. Acta, 237 (1990) 329–343. 11 C.E. Lenehan, N.W. Barnett and S.W. Lewis, Analyst, 127 (2002) 997–1020. 12 Micro Total Analysis Systems, Proceedings of mTAS ‘94 Workshop, University Twente, van den Berg A. and Bergveld P. (Eds.), Kluwer Academic Publishers, London, 1995. 13 Micro Total Analysis Systems, ’98 Proceedings of mTAS ‘98 Workshop, Banff, Canada, Harrison D.J. and van den Berg A (Eds.), Kluwer Academic Publishers, London, 1998. 14 Micro Total Analysis Systems 2000, Proceedings of mTAS ‘2000 Symposium, Enschede, Netherlands, A. van den Berg and P. Bergveld (Eds.), Kluwer Academic Publishers, London, 2000. 15 J. Ruzicka, Analyst, 125 (2000) 1053–1060. 16 J. Ruzicka and A. Ivaska, Anal. Chem., 69 (1997) 5024–5030. 17 H. Erxleben and J. Ruzicka, Analyst, 130 (2005) 469–471. 18 Y. Ogata, L. Scampavia, J. Ruzicka, C.R. Scott, M.H. Gelb and F. Turecek, Anal. Chem., 74 (2002) 4702–4708. 19 Wu Chao-Hsiang, L. Scampavia and J. Ruzicka, Analyst, 128 (2003) 1123–1130. 20 J. Ruzicka, Flow Injection Analysis, 3rd ed., CD-ROM Tutorial, Published by FIAlab Instruments, Inc., 2004. Available free of charge at www.flowinjection.com 21 J. Wang and E.H. Hansen, Anal. Chim. Acta, 424 (2000) 223–232. 22 J. Wang and E.H. Hansen, Anal. Chim. Acta, 435 (2001) 331–342. 23 J. Wang and E.H. Hansen, Atom. Spectrosc., 22 (2001) 312–318. 24 J. Wang and E.H. Hansen, J. Anal. At. Spectrom., 16 (2001) 1349–1355. 25 E.H. Hansen, J. Flow Injection Anal., 19 (2002) 1. 26 J. Wang and E.H. Hansen, Trends Anal. Chem., 22 (2003) 225–231. 27 M. Miro´, S. Jonćzyk, J. Wang and E.H. Hansen, J. Anal. At. Spectrom., 18 (2003) 89–98. 28 J. Wang, E.H. Hansen and M. Miro´, Anal. Chim. Acta, 499 (2003) 139–147. 29 J. Ruzicka and L. Scampavia, Anal. Chem., 71 (1999) 257A–263A. 30 J. Ruzicka and E.H. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1988 (Z-L. Fang, Trans.), Beijing University Press, Beijing, 1991 (in Chinese). 31 O. Reynolds, Phil. Trans. Roy. Soc. Lond., 174 (1883) 935–982. 32 Y. Chen and J. Ruzicka, Analyst, 129 (2004) 597–601. 33 Y. Gutzman, A.D. Carroll and J. Ruzicka, Analyst, 131 (2006) 809–815. 34 D. Satinsky, P. Solich, P. Chocholous and R. Karlicek, Anal. Chim. Acta, 499 (2003) 205–214. 35 D. Satinsky, J. Huclova, P. Solich and R. Karlicek, J. Chromatogr. A, 1015 (2003) 239–244. 36 J. Jakmunee, L. Pathimapornlet, S.K. Hartwell and K. Grudpan, Analyst, 130 (2005) 299–303. 37 K. Grudpan, S. Khonyoung, S.K. Hartwell, S. Lapanantnoppakhun and J. Jakmunee, J. Flow Injection Anal., 23 (2006) 94–101. 38 J. Ruzicka, Flow Injection Analysis, 4th ed, CD-ROM Tutorial, FIAlab Instruments, Inc., 2007. 39 J. Wang and E.H. Hansen, Trends Anal. Chem., 24 (2005) 1–8. 40 M. Miro´ and E.H. Hansen, Trends Anal. Chem., 25 (2006) 267–281.


CHAPT ER

3 Theoretical Basis of Flow Injection Analysis Spas D. Kolev

Contents

1. Introduction 2. Mass Transfer in FIA Systems 2.1 Straight open tubes 2.2 Helically coiled open tubes 2.3 Parallel-plate sections 2.4 Mathematical modelling of the flow pattern in FIA systems 3. Chemical Kinetic Phenomena 4. Sensing Mechanism Abbreviations and Nomenclature References

47 48 49 55 57 58 66 71 74 75

1. INTRODUCTION The principles on which flow injection analysis (FIA) is based are relatively simple, well understood, and successfully implemented in analytical practice; however, the development of its theoretical basis is far from completed. The main reason for this deficiency, as pointed out in books [1–4] and journal reviews [5–15] on this topic, is the complexity of the physicochemical phenomena (e.g., diffusion, convection, chemical kinetics) taking place in even the simplest of FIA systems. From a process analysis point of view, an FIA system can be considered as an assembly of subsystems, tied up together by common flows of materials [16]. The performance of such a system is determined not only by the characteristics of the individual subsystems, but also by their interactions and interrelations. In general, these subsystems do not correspond to any physical element of the system studied. A comprehensive theoretical understanding of the subsystems of a system and the relationships between them is required for the development of a Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00603-X


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formalized representation of this system, often referred to as its physical model. The mathematical model of a system consists of the mathematical description of the corresponding physical model. Once validated, the mathematical model provides not only an in-depth understanding of how the system functions, but it also allows the optimization of the system parameters through numerical simulation. Such models are often referred to as analytical-experimental models [15]. The name of these models originates from the fact that they may incorporate both fundamental physical constants and empirical parameters that can only be determined experimentally. Depending on the scale on which the physical and chemical processes in the system studied are considered, the analyticalexperimental models can be divided into probabilistic and deterministic models. The former models view processes at molecular level where movements of particles are stochastic in nature. The deterministic models such as the dispersion model and tanks-in-series model (TSM) operate at macro level and are much more frequently used in the mathematical description of FIA systems than their probabilistic counterparts. Unlike the TSMs, which view the system as homogenous, dispersion models take into account detailed variations in behaviour from point to point throughout the system they describe. For this reason dispersion models, such as the convective-diffusion equation and the axially dispersed plug flow model (ADPFM), dominate the scientific literature on the theory of FIA [15]. In contrast to the analytical-experimental models, ‘blackbox’ models describe a system on the basis of its input–output relationships without taking into consideration the real processes responsible for the transformation of the input into output [15]. Unlike analytical-experimental models, the applicability of ‘black-box’ models is usually confined within the region of experimental data used for their development. Though the main types of ‘black-box’ models used in describing FIA systems will be briefly outlined later in this chapter, the theoretical basis of FIA will be discussed mainly with the help of the dispersion models mentioned above. In this approach the main subsystems of a typical FIA system describe: (a) mass transfer phenomena, (b) chemical kinetic phenomena, and (c) the sensing mechanism involved in producing the analytical signal. All three subsystems act in unison and thus are equally important for the functioning of any FIA system. However, the mass transfer subsystem is arguably the one which mainly differentiates FIA from other flowthrough and batch analytical techniques. For this reason the current chapter will focus on the mass transfer processes in FIA and their effect on the generation of the analytical signal.

2. MASS TRANSFER IN FIA SYSTEMS The mass transfer subsystem is primarily responsible for defining the Residence Time Distribution (RTD) function or curve of an FIA system. This is a probability function describing the distribution of residence times of fluid elements in the FIA system between the injection and detection points. The RTD function can be related in a straightforward manner to the analytical signal (i.e., FIA peak) in

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Theoretical Basis of Flow Injection Analysis

single-line FIA systems without a chemical reaction. The RTD curve will be identical to the corresponding FIA peak if the detector signal is numerically equal to the average bulk concentration of the analyte in the cross-section of the flow where sensing takes place. The two mass transfer phenomena primarily responsible for the transportation of samples through FIA systems are convection and diffusion. They both affect the broadening of the sample zone, which is referred to as sample dispersion in analogy with both chromatography and chemical reaction engineering. Most FIA systems are made up of predominantly tubular (straight or coiled open tubes) and parallel-plate flow-through sections. The mass transfer in these sections will be discussed in more detail in this chapter. Flow-through sections with other geometries such as mixing chambers, packed-bed or singlebead string reactors are only occasionally used in FIA systems. The analysis of the physicochemical phenomena taking place there, and subsequent mathematical modelling, is outside the scope of this chapter and can be found elsewhere [1–15].

2.1 Straight open tubes A cylindrical coordinate system is most suitable for describing the physicochemical processes taking place in a straight open tube. In this system the position of each point is determined by its radial (r) and axial (x) coordinate (distance) (Figure 1a) and therefore, mass transfer can be viewed as consisting of a radial and axial component. Convection is mass transfer by the bulk motion of a fluid. The convective transport in an FIA system is mainly due to the pressure difference between its inlet and outlet. This is created by the use of a propelling device, which is in most cases a peristaltic pump. It can also be caused by density or temperature differences between the sample and the carrier solution. However, such effects Streamlines

Velocity profile

a

Laminar flow

r/a

x 1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1 0

0.5

1

1.5

2

u/u

b

Turbulent flow

Figure 1 Streamlines and velocity profiles in laminar (a) and turbulent (b) tubular flow.

50

Spas D. Kolev

are usually insignificant in typical FIA applications and will be neglected in the subsequent considerations. In a straight open tube with radius (a), depending on ¯ dynamic viscosity (m), and density (r) of the the average linear flow velocity (u), liquid, the flow can be either laminar or turbulent in nature. Under laminar flow conditions all fluid elements follow streamlines parallel to the main direction of the flow and the velocity profile, described by Equation (1), has a parabolic shape (Figure 1a). This flow pattern is also referred to as Poisseulie flow. The linear flow velocity (u) at the tube walls is zero and it reaches its maximum value, which is twice the average linear velocity, at the centre of the tube (r ¼ 0). Both pressure and linear velocity are independent of time and viscous forces are dominant. r 2 u ¼ 2u¯ 1 (1) a The symbols used throughout this chapter and their definitions are given in Abbreviations and Nomenclature. When instead of the viscous forces, the inertial forces are dominant, the flow is characterized by random vortices and eddies which result in a velocity profile resembling plug flow (Figure 1b). This transition from a flow dominated by viscous forces (i.e., laminar flow) to one dominated by inertial forces (i.e., turbulent flow) occurs when the so-called Reynolds number (Equation (2)) exceeds 2,100 [17]. ¯ 2aur (2) m The Reynolds number is one of the numerous dimensionless groups used in the description of multi-parameter flow systems. The mathematical modelling of such complex systems can be substantially simplified by reducing the number of system parameters. This can be achieved by converting the original equations, comprising the corresponding mathematical model, into dimensionless form. This results in replacing the dimensional parameters of the system (e.g., tube radius, average linear flow velocity) by dimensionless groups (e.g., Reynolds number) which are considered as the ‘natural’ groups of the system [16]. The use of dimensionless groups, as will be demonstrated later in this chapter, allows also generalization of the results obtained for a concrete system and making them applicable to other systems described by the same equations. The flow rates and tube radii used typically in FIA, i.e., less than 5 mL min1 and between 0.3 and 0.8 mm, respectively, define the convective tubular transport as laminar in nature (Reo150). Assuming that the sample is initially present as a sample plug and the only mass transfer mechanism is laminar convection, one would expect that the analyte will remain evenly distributed among the streamlines stretching between the tube walls and the tube centre (Figure 2a). This suggests that the analyte at the tube walls (r ¼ a, Figure 1), where the linear velocity is zero (Equation (1)), will remain stationery, thus producing ‘infinite’ dispersion. The RTD curve will be highly asymmetrical with a pronounced tailing (Figure 2a). Under the assumption made above, there will be no mass exchange between parallel Re ¼


a

b

51

c

t

τ = 1.0

τ = 0.3

τ = 0.1

c

r x

Figure 2 RTD distribution curves and schematic presentation of the sample zone in the case of: (a) dispersion dominated by convection (t ¼ 0.1); (b) dispersion under mixed convectiondiffusion control (t ¼ 0.3); and (c) dispersion under diffusion control (t ¼ 1.0). The arrows (m k) represent radial diffusion.

streamlines. Therefore, chemical reactions between the analyte and reagents present in the carrier stream could take place only at the parabolic sample zone/ carrier stream interface (Figure 2a). Under such conditions FIA would have never developed into a viable analytical flow-through technique. However, the mass transfer subsystem depicted in Figure 2a is characterized by substantial concentration gradients between the sample zone and the adjacent carrier solution. With time, these gradients will promote diffusion mass transport of the analyte from the sample zone towards the adjacent carrier solution. At the same time, reagents in the carrier solution will diffuse into the sample zone and react with the analyte. The diffusion mass flux in both radial ( Jr ) and axial ( Jx ) direction can be described by Fick’s first law (Equation (3)). J x ¼ D

@c @x

and

J r ¼ D

@c @r

(3)

where D and c are the diffusion coefficient and the concentration of the diffusing chemical species. Due to the low value of D (B1091010 m2s1) this diffusion flux is much smaller than a typical convective mass flux ( ¼ ucx). It can be expected that in most cases the mass transfer in axial direction will be dominated completely by laminar convection. At the same time radial diffusion is the only mass transfer mechanism responsible for transporting analyte between streamlines of different linear velocity. As a result of this process, analyte in the trailing section of the sample zone (Figure 2a), close to the tube walls, is transported to the centre of the tube where the velocity is much higher. At the same time analyte from the leading section of the sample zone is transported towards to tube walls. By transporting analyte in radial direction, radial diffusion gradually transforms the parabolic sample zone (Figure 2a), formed immediately after the injection of the

52

Spas D. Kolev

sample, into a sample plug with diffused boundaries (Figure 2b and c). This process leads to a more uniform concentration distribution of the analyte in radial direction, thus reducing sample dispersion and producing more symmetrical RTD curves. It can be expected that the effect of radial diffusion on sample dispersion will be more pronounced in narrower tubes (i.e., small a) and for analytes with higher diffusion coefficients (D). The effect of radial diffusion will be more prominent at a longer mean residence time of the sample ¯ where L is the characteristic length). This is because in the FIA system (tm ¼ L=u, the longer radial diffusion acts, the higher the concentration uniformity in radial direction is. The mass transfer parameters mentioned above (i.e., a, D, and tm) control the extent to which convective and diffusion mass transfer affect sample dispersion. These parameters are linked through the general mathematical model of a fully developed laminar tubular flow (Equation (4)). This model is based on first principles and is also known as the convective-diffusion equation [18]. 2 @c @ c @2 c 1 @c r2 @c ¼D (4) þ þ 2 u¯ 1 2 @t @x2 @r2 r @r @x a where t is time. This equation will be applicable to FIA systems with steady-state flow pattern. However, the flow is briefly stopped prior to sample introduction by an injection valve or by hydrodynamic injection. This is followed be a period of unsteady motion. During this time, the flow is accelerated from the condition of rest to condition of steady motion. Theoretical calculations and experimental investigations conducted by Kolev and Pungor have shown that the period of unsteady motion is much shorter than the mean residence time of the analyte (tm) and thus can be neglected [19]. Dispersion analysis in fully developed laminar tubular flow can be simplified and generalized if Equation (4) is transformed into an equation incorporating dimensionless quantities and variables. Various closely related semidimensionless or dimensionless forms of Equation (4) have been reported in the literature (e.g., [20–22]). Among them Equation (5) appears to be more suitable for the subsequent discussions and it will be used in the remainder of this chapter. 2 @C 1 @2 C @ C 1 @C @C ¼ (5) þt þ 2ð1 R2 Þ 2 @y PeL @X2 R @R @X @R where X ¼ x/L and R ¼ r/a are the dimensionless axial and radial distances, respectively; y ¼ t/tm is the dimensionless time; C ¼ c/c0 is the dimensionless concentration; and c0 is the characteristic dimensional concentration. The two dimensionless numbers characterizing this mass transfer system are the Fourier number (Equation (6)) and the Pećlet number (Equation (7)). t¼

tm D a2

(6)


PeL ¼

¯ uL D

53

(7)

The Fourier number is the mean residence time reduced to molecular diffusion scale and represents the ratio between the mean residence time and the time required by the system to reach steady-state. The Pećlet number, defined by Equation (7), expresses the ratio between mass transfer by convection and mass transfer by diffusion. This number can incorporate axial or radial dispersion ¯ instead of the coefficients instead of D, the maximum linear flow velocity (2u) ¯ and the tube radius (a) as the characteristic average linear flow velocity (u), distance parameter instead of L. The axial dispersion form of the Pećlet number will be introduced later in this chapter. In most dimensionless forms of Equation ¯ (4) the Pećlet number incorporates a and 2u. Due to the mathematical complexity, a general analytical solution of the convective-diffusion equation, which can be used in a straightforward manner, has not yet been derived. Various numerical techniques have been successfully applied for solving this equation. These include finite-difference methods [22–31], finite-element methods [32], Laplace transforms [33], and orthogonal collocations [34]. Simple relationships which allow substantial reduction in computational time have been proposed by Kolev [35]. Numerical simulations of the convective-diffusion equation have revealed that dispersion under typical FIA conditions is unaffected by PeL (Equation (7)) [22–26]. This result indicates that diffusion mass transfer in axial direction, represented by ð1=PeL Þ @2 C=@X2 in Equation (5), is negligible compared to the convective mass transfer, i.e., 2ð1 R2 Þð@C=@XÞ. Therefore, sample dispersion in straight open tubes of FIA systems can be characterized solely by the Fourier number (Equation (6)). Depending on its value, the following three stages in the development of sample dispersion can be distinguished: Dispersion dominated by convection (to0.1). Immediately after the injection of the sample into the carrier stream (t o 0.01), its dispersion is due to convective mass transfer only. The sample zone is parabolic in shape and the RTD is an asymmetrical peak with a substantial tailing (Figure 2a). With time, the radial diffusion contribution increases and this results in the formation of doublehumped RTD curves [23,24,26,29,30,32,36]. The leading and trailing humps represent the convective and diffusion mass transfer processes, respectively. The leading hump corresponds to the remnants of the front of the initial parabolic sample zone (Figure 2a). The trailing hump is the result of radial diffusion which transports analyte from the front of the initial parabolic sample zone to streamlines of lower linear velocity closer to the tube walls. This process takes place in parallel with the diffusion of analyte from streamlines of lower velocity to the centre of the tube. Dispersion under mixed convection-diffusion control (0.1 o t o 0.8). At higher Fourier numbers, sample dispersion is influenced by both convection and diffusion mass transfer. The parabolic shape of the sample zone gradually changes into a plug shape which is accompanied by increasing symmetry of the corresponding single-humped RTD curves (Figure 2b).

54

Spas D. Kolev

Dispersion dominated by diffusion (t W 0.8). If the mean residence time is sufficiently long, the concentration distribution in the radial direction becomes highly uniform, though the concentration non-uniformity in the axial direction remains (Figure 2c). The sample zone acquires a plug shape and the corresponding RTD curve (Figure 2c) approaches a symmetrical Gaussian shape. The position of the peak maximum (ymax ¼ tmax/tm) on the dimensionless time axis becomes close to unity. The reproducibility of FIA measurements is generally higher in this t range because the dispersion due to diffusion is less sensitive to random perturbations in the flow pattern. Several asymptotic or series analytical solutions of Equation (4) and its dimensionless forms have been proposed. Only those valid at high values of PeL (Equation (7)), which are relevant to dispersion under typical FIA conditions, will be discussed here. Asymptotic solutions give satisfactory results at either low (i.e., less than 0.1) or high (i.e., greater than 0.6) Fourier numbers. Vrentas and Vrentas [37], Lighthill [38], and Hunt [39] have proposed asymptotic solutions for convection-dominated mass transfer with insignificant axial diffusion contribution. The latter restriction has been relaxed in the solution derived by Chatwin [40]. Taylor [20,41] solved analytically Equation (4) for high Pećlet and Fourier numbers. Under these conditions the dispersion process is diffusion controlled, the axial diffusion mass transfer is negligible, and Equation (4) is reduced to a much simpler from mathematical point of view partial differential equation (Equation (8)). @c @2 c @c ¼ DL 2 u¯ @t @x @x

(8)

The parameter DL, often referred to as the axial dispersion coefficient, is defined by Equation (9) [20]. DL ¼

a2 u¯ 2 48D

(9)

It should be noted that Ruzicka and Hansen [1] have introduced a dimensionless parameter called also dispersion coefficient which is very different in nature from the diffusion-like dispersion coefficients used in chemical reaction engineering [42] (e.g., DL, Equation (9)). Ruzicka and Hansen’s dispersion coefficient, referred to by Valcarcel and Luque de Castro [2] as ‘practical dispersion coefficient’, has been defined as ‘the ratio of concentrations of sample material before and after the dispersion process has taken place in that element of fluid that yields the analytical readout’ [1]. Equation (8) is identical to the mathematical description of the axially dispersed plug flow model (ADPFM), which is one of the most popular hydraulic models used in the mathematical description of the flow pattern in chemical reactors [42]. It has been also used extensively in mathematical modelling of FIA systems and these applications will be discussed later in this chapter.


55

Aris [21] has extended Taylor’s theory to the case of diffusion-controlled dispersion where both radial and axial diffusion are taken into account. The axial dispersion coefficient in this case can be calculated by Equation (10). However, as mentioned earlier, under typical FIA conditions (e.g., high PeL) the axial diffusion does not affect dispersion and Equation (10) reduces to Equation (9). DL ¼ D þ

a2 u¯ 2 48D

(10)

Series solutions of the various forms of the convective-diffusion equation have been also proposed and they can be viewed as exact analytical solutions when the number of terms in the series approaches infinity. However, the practical benefits of using such solutions are severely restricted by the necessity of calculating a large number of series terms. Building up on earlier research involving the use of series solutions of the convective-diffusion equation [29,43,44], Gill and Sankarasubramanian [45] have proposed a generalized ADPFM with an infinite series of time-dependent axial dispersion coefficients. After an appropriate truncation of this series, the generalized model describes accurately either convection or diffusion-dominated laminar dispersion. However, deviations from numerical results for dispersion involving both convective and diffusion mass transfer have been observed. Tseng and Besant have solved the convective-diffusion equation using a combination of eigenvalues and eigenvectors and a Bessel function series [46,47]. Yu [48–50] has employed a similar approach based on the radial expansion of the local solute concentration in a series of Bessel functions. Shankar and Lenhoff [51] have proposed a modification of Yu’s method with a higher computational efficiency.

2.2 Helically coiled open tubes The centrifugal forces in a helically coiled tubular flow of an incompressible viscous liquid cause the superimposition of a double vortex on the flow in the longitudinal direction (Figure 3) [1,52]. This secondary flow enhances radial mixing, thus reducing sample dispersion and narrowing the corresponding RTD curve [53,54]. These effects are governed to a great extent by the so-called aspect ratio (j), which is the ratio of the tube radius to the coil radius. Transition from laminar to turbulent flow has been found experimentally to take place [52] at Reynolds numbers calculated by Equation (11): Re ¼ 2100ð1 þ 12j1=2 Þ

(11)

The effect of the aspect ratio on the velocity profile in the absence of diffusion mass transfer has been studied first by Dean [55,56]. He has solved analytically the Navier–Stokes equations for very small values of j, thus demonstrating that a fully developed helically coiled tubular flow is governed by a dimensionless group known as the Dean number (Equation (12)). De ¼ Rej1=2

(12)

56

Spas D. Kolev

Figure 3 Secondary flow in coiled tubes at low and high values of the Dean number. (Reproduced from Ref. [1] with permission of John Wiley & Sons, Inc.)

Topakoglu [57] has extended the solution of Dean for a wider range of j values. Both the solutions of Dean [55,56] and Topakoglu [57] are valid for Dean numbers lower than 36 [58]. Truesdell and Adler [58] have numerically calculated the axial and secondary velocities for closely wrapped helices of circular and elliptical cross-sections for De up to 280. McConalogue and Srivastava [59] and Austin and Seader [60] have proposed numerical solutions valid for even higher values of the Dean number (e.g., 1,000 [60]). Søeberg [61] has solved the equations for fluid motion and continuity of a fully developed laminar flow in a helically coiled tube by a technique based on the symmetry of the secondary-flow field. His solution covers the entire laminar range. In addition to the Dean number (De), mass transfer in helically coiled tubes depends also on the so-called Schmidt number (Sc), which relates inertial to molecular diffusion forces. It is defined as: Sc ¼ n=D

(13)

where n is the kinematic viscosity ( ¼ m/r) of the flowing liquid. Assuming that axial diffusion is negligible and using Dean’s solution for the velocity distribution, Janssen has demonstrated that the mass transfer process in helically coiled tubes can be characterized by De2Sc [62]. Depending on the value of this dimensionless parameter, three regions of dispersion can be distinguished [52]. At very low flow velocities (De2Sco10) the centrifugal forces are too small to affect the flow pattern. For De2ScW10 the secondary flow gradually develops and it is fully established at De2ScW104 where the flow is divided into two equal parallel halves as shown in Figure 3a. A further increase in De2Sc leads to the establishment of a linear velocity profile (Figure 3b), which causes a dramatic decrease in dispersion (e.g., at De2Sc ¼ 109 the dispersion is 100 lower than that in straight


57

tubes). Though under typical FIA conditions (DB1010 m2 s1, n B106 m2 s1, Reo150, and j B0.1) secondary flow is fully established in mixing coils, the De2Sc is far lower than the value required for the development of a linear velocity profile. The mathematical complexity of the equations describing mass transfer in helically coiled tubular flow has resulted in the derivation of analytical solutions with severely restricted validity. These solutions have very limited applicability to FIA systems. McConalogue [63] and Ruthven [64] have examined the case of dispersion completely dominated by convection which is typical for very short tubes. A theoretically derived RTD function has been proposed by Ruthven [64]. However, cases where dispersion is affected by both convection and diffusion mass transfer are of much greater relevance to FIA. Following the approach applied earlier by Taylor [20,41], both Erdogan and Chatwin [65] and Nunge et al. [66] have derived theoretical expressions for the axial dispersion coefficient in helically coiled tubes based on the velocity distribution obtained by Dean [55,56] and Topakoglu [57], respectively. The expression of Nunge et al. [66] is valid for a wider range of aspect ratios. However, both expressions predict negative axial dispersion coefficients for higher values of De2Sc (W700) [62] while, as mentioned earlier, typical De2Sc values in FIA are substantially higher. Another theoretical expression for the axial dispersion coefficient for small values of De2Sc has been derived by Golay [67] based on convoluting a Gaussian concentration distribution and a Poiseuille curved tubular flow. As with straight open tubes, the Fourier number determines the effect of diffusion on the overall dispersion process and the suitability of the ADPFM for its description. Trivedi and Vasudeva [68] have proposed an empirical relationship (Equation (14)) for the minimal value of the Fourier number at which the ADPFM is still valid. t Re1

(14)

Numerical solutions have produced results for dispersion in helically coiled tubes for higher De2Sc values, which are more relevant to FIA conditions. Janssen [62] has presented detailed numerical results for calculating DL for De2Sc values of up to 5,000. Using Monte Carlo and numerical techniques, Johnson and Kamm [69] have obtained results qualitatively similar to those of Janssen [62]. Daskopoulos and Lenhoff [70] have utilized the method of Horn [71] which is a modification of the method of moments of Aris [21]. The agreement at low Schmidt numbers (Sc) has been satisfactory, however, substantial discrepancies have been observed for Sc values of relevance to FIA (i.e., greater than 1,000). The results of Daskopoulos and Lenhoff [70] show that the region of applicability of previously computed dispersion coefficients [15] is limited and in most cases irrelevant to mass transfer in FIA mixing coils. For this reason a number of empirical or semi-empirical equations for calculating DL in helically coiled tubes have been proposed (e.g., [72–77]).

2.3 Parallel-plate sections In addition to industrial heat- and mass-exchangers and medical haemodialysers, parallel-plate laminar flow is often encountered in dialysis or gas-diffusion cells

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used in FIA systems. Sample dispersion in such a flow can be described by a convective-diffusion equation (Equation (15)), similar to the one for laminar tubular flow (Equation (4)), i.e., 2 @c @ c @2 c 3 z2 @c ¯ ¼D þ u 1 @t @x2 @z2 2 a2 @x

(15)

where a is half the channel height and z is the transverse distance. For this reason, approaches similar to those used in the solution of Equation (4) have been applied equally successfully to Equation (15). Adopting the Taylor– Aris approach for laminar tubular flow, Wooding [78] has derived an equation for DL similar to Equation (10). DL ¼ D þ

2 a2 u¯ 2 105 D

(16)

Kolev and van der Linden [79] have used an approach similar to that of Gill and Sankarasubramanian [45] to derive a general time-dependent expression for DL which reduces to Equation (16) for Fourier numbers greater than 1, i.e., tmWa2/DL. They have used the convective-diffusion equation (Equation (15)) to describe the transient laminar mass transfer in a single stream parallel-plate laminar flow system with one impermeable wall and a constant concentration on the other [80]. Relationships for predicting the mass transfer coefficient and the Pećlet number in this system have been derived. These relationships have been found to be valid for co-current parallel-plate dialysers as well [80]. By simulations of the model, conclusions with regard to optimum design and operation have been drawn and possibilities for simplifying the model have been proposed [81].

2.4 Mathematical modelling of the flow pattern in FIA systems Due to the complexity of the mass transfer phenomena outlined above it is no wonder that the convective-diffusion equation has been rarely used in the mathematical modelling of even single-line FIA systems. This complexity has prompted the widespread use of a variety of ‘black-box’ and analyticalexperimental models [15].

2.4.1 Probabilistic models of the flow pattern in FIA systems Most probabilistic models of FIA systems are based on the random walk simulation approach [82–85]. This views the sample zone as a discrete number of individual molecules and the time interval of observation as consisting of a number of time increments (Dt) of equal duration. During each increment, each molecule is transported downstream by convection (e.g., laminar flow) and then takes a random step (DL) in x, y, or z direction, i.e., DL ¼

pffiffiffiffiffiffiffiffiffiffiffi 2DDt

(17)


59

Random walk models are based on first principles since they only use fundamental physical constants (e.g., diffusion coefficients, temperature, viscosity, kinetic constants). Unlike other deterministic models based on first principles, such as the convective-diffusion equation, the mathematics involved with the random walk models is less complex. However, it should be noted that an explicit relationship describing the velocity profile is still required and this will drastically increase the mathematical complexity in the case of flow patterns other than laminar flow in straight open tubes. The ease with which the results can be visualized is another advantage offered by the random walk models. However, the statistical noise in the simulation results and difficulties in fitting experimental data have prevented these models from becoming an attractive alternative to dispersion models. The drawbacks mentioned above can be reduced to some extent by decreasing the size of the random step and by refining the wall approximations [85]. However, such an approach will involve a considerable increase in computation time.

2.4.2 Axially dispersed plug flow model From mathematical point of view the ADPFM (Equation (8)) is much simpler than the convective-diffusion equation and this explains its widespread use in the modelling of process and analytical flow-through systems (e.g., FIA and chromatographic columns). This model is most appropriate for patterns of flow where the radial variation in composition is relatively small. The most frequently used dimensionless form of this model is expressed by the following equation: @C 1 @2 C @C ¼ þS @y PeL @X2 @X

(18)

where S is the dimensionless source or sink term (e.g., chemical reaction). In the mathematical description of FIA systems by the ADPFM, the flow system has been usually assumed as infinitely long (i.e., ‘open vessel’ assumption) and in most cases the introduction of the sample has been approximated by an ideal delta-function input, Equation (19) [15]. 1 for y ¼ 0 dðyÞ ¼ (19) 0 for y40 The solution of Equation (18) under these conditions is: 1 PeL 1=2 PeL ðX yÞ2 C¼ exp 2 py 4y

(20)

The Pećlet number can be related directly to the plate height (H) in chromatography by Equation (21) [15]. PeL ¼ 2L=H

(21)

The sample injection can only be approximated by a delta-function input if the sample volume is much smaller than the volume of the FIA system [86,87].

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This condition is rarely valid in FIA systems and the finite volume of the sample must be taken into account. This is usually done by approximating the sample injection with a rectangular function. The analytical solution of Equation (18) in this case is Equation (22) [35]. ( " # " #) 1 y1 1þay erf C¼ þ erf (22) 2 2ðy=PeÞ1=2 2ðy=PeÞ1=2 where a is the reduced sample volume ( ¼ Vs/V). The ADPFM outlined above assumes that an FIA system can be reduced to a uniform tube (i.e., ‘open vessel’ assumption). This is an oversimplification which does not take into account that in reality even the simplest FIA system consists of various flow-through sections with different geometrical and dispersion properties [88]. Usually the sections upstream of the injection device (fore-section) and downstream of the measuring cell (after-section) are bent empty tubes or a series of tubes with diameters which differ from the diameters used in the other sections of the FIA system. Kolev and Pungor have conducted a theoretical study [89], based on the ADPFM, which has revealed that the mass transfer phenomena taking place in short fore- and after-sections may affect the overall dispersion in the flow system. These effects are often referred to as ‘end effects’ [89]. Kolev and Pungor [90] have developed and experimentally verified a general mathematical model of a single-line FIA system with bulk detection (i.e., detector sensitive to the average analyte concentration in the cross-section of the flow). This model views the flow system as consisting of several tubular sections connected in series, each having a different Pećlet number. Each section is described by a modified form of Equation (18), i.e., @Ci c @2 C i @Ci i Si ¼ 0 þ ci 2 @y PeL;i @X @X

(23)

where ci are coefficients, derived earlier by the same authors [88], which make possible the use of the ADPFM for the description of flow systems comprised of tubular sections with various diameters. The boundary conditions introduced by Wehner and Wilhelm [91] have been used in the mathematical model based on Equation (23). Three different types of sample introduction techniques, i.e., syringe, hydrodynamic [92], and valve injection, have been described mathematically [90]. Kolev and Pungor have conducted numerical simulations to study the influence of the main design, and operational parameters, of a single-line FIA system on its sensitivity and sampling rate [86,87]. Kolev et al. have further expanded the applicability of their modelling approach to FIA systems with surface detectors (e.g., ion-selective [93] or enzyme [94,95] flow-through electrodes). The modelling of the sensing mechanism of these electrodes will be discussed later in this chapter. The FIA models with surface detection have been experimentally verified by the same authors [96,97]. In most cases it is difficult if possible at all to derive a time domain analytical solution of Equations (23). However, the derivation of an analytical solution in


61

the Laplace domain is often possible. Such a solution is usually a cumbersome expression and its inverse transformation into the time domain is possible only by numerical methods. Kolev and Pungor [98] have compared existing methods for numerical inverse Laplace transformation. They have established that best results, with respect to precision and consumption of computation time, are offered by the methods employing expansion of the Laplace domain function into Chebyshov polynomials of the first kind or into Fourier sine series. In a subsequent study [99], Kolev and van der Linden have developed a fast numerical technique for solving more complex mass transfer mathematical models where an analytical solution in the Laplace domain is not possible. This technique is based on numerical integration of the model equations in the Laplace domain, followed by a numerical inverse transformation of the Laplace domain solution. The ADPFM has been also used for the mathematical description of twochannel FIA systems. A mathematical model of an FIA system with a membrane separation module has been developed by Kolev and van der Linden [100]. It takes into account the geometrical dimensions and the dispersion properties of the main sections of the FIA manifold, the mass transfer in the channels of the separation module, and the characteristics of the membrane (thickness and diffusion coefficient). Relationships for calculating the mass transfer coefficients and Pećlet numbers in both channels, derived earlier by the same authors [80,81], have been used in the model simulations. These numerical simulations have provided an in-depth understanding of the online separation processes taking place in the modelled parallel-plate dialyser and have allowed its optimization. In addition, this model can be applied for characterizing the transport properties of membranes used in other separation systems. The absolute limits of mass transfer across the membrane in a parallel-plate dialyser set by the flow pattern in both channels have been determined on the basis of the model mentioned above [101].

2.4.3 Ideally mixed tanks models TSMs view a flow system as composed of a finite number of ideally mixed tanks connected in series. The model with equally sized tanks (Figure 4a) has been extensively used in chromatography where it is known as the ‘plate model’ [102]. In the case of only one tank, the TSM reduces to the mathematically simpler ideally mixed tank (IMT) model. This could be the main reason for the widespread use of this model in the mathematical description of the dispersion process in FIA systems [103–150]. The IMT model is described by the following equations in dimensional or dimensionless quantities and variables [16,18]: dc dC ¼ nðcin cout Þ or ¼ Cin Cout (24) dt dy where Vt is the volume of the tank, n is the volumetric flow rate, and subscripts in and out refer to influent and the effluent concentrations. This model is best suited to the description of FIA systems incorporating a mixing (gradient) chamber. The IMT model has been used to describe FIA Vt

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Figure 4 Schematic of the tanks-in-series and parallel-tanks [122] models.

systems with and without chemical reactions [103–122]. Stewart and Rosenfeld [138] have reported on the experimental verification of a number of theoretical calibration expressions [105,106,135], derived on the basis of the IMT model. The models involving chemical reactions will be discussed in more detail later in this chapter. The IMT model has been also applied for the description of FIA systems without a gradient chamber. Ramsing et al. [136] and Olsen et al. [137] have shown experimentally that the descending part of the concentration curve in an FIA system, with a short mixing coil and a sample volume smaller than the volume of the mixing coil, can be described by the IMT model. The volume of the tank in this case is equal to the volume of the mixing coil and half of the sample volume. Similarly, Tyson et al. [116–118] have modelled an FIA system with atomic absorption detection, with and without a mixing chamber, as one hypothetical IMT with volume which can only be determined experimentally. In a subsequent article [119], Tyson has outlined the mathematical basis of a calibration method based on measuring peak width. He has derived a linear relationship between peak width and the logarithm of (c0/c1), where c is the concentration at which the peak width is measured. This model has been further refined [120] to take into account experimentally observed deviations from this linear relationship [121]. Lucy and Cantwell [142] have used the IMT model to describe the mixing between each organic segment and the liquid in the wetting film adjacent to the aqueous segment in an FIA system with liquid–liquid extraction. Powell and Fogg [143] have shown that a resistance–capacitor circuit can be used as an analog simulator of the IMT model. In studying the hydrodynamically limited precision in FIA manifolds with a gradient chamber, Gisin et al. [144] have convoluted the transfer function of an IMT with two


63

different sample input functions, i.e., a rectangular and an exponential wash-out function. The model has been validated with experimental data produced in FIA systems with and without a mixing chamber. Appleton and Tyson [122], and later Stone and Tyson [123] have demonstrated that models based on the tanks-in-parallel configuration (Figure 4b) can also successfully approximate the flow pattern in FIA systems. The mathematical description of the TSM in the case of N ideally mixed tanks consists of N equations similar to Equation (24). In these equations the influent concentration of each tank is in fact the effluent concentration of the preceding tank. In the case of a delta-function input (Equation (19)) the RTD function in the last of the N ideally mixed tanks is described by the following equation [18].

C ¼ NðNyÞN1 =ðN 1Þ! expðNyÞ

(25)

The sample injection can be approximated by a delta-function only if the sample volume is much smaller than the volume of the FIA system. This condition is rarely valid in FIA systems and the finite volume of the sample must be taken into account. Reijn et al. [145] have obtained an analytical solution of the TSM for the more realistic case of slug injection which is expressed by two Chi-squared distribution functions. Gisin et al. [144] have proposed an approximate solution of the TSM for slug injection by multiplying the delta-function solution (Equation (25)) by the reduced sample volume ( ¼ Vs/V). Van der Linden [146] has based the mathematical description of the mass transfer processes in a gas-diffusion FIA cell on the TSM. Satisfactory agreement with experimental results has been reported. Quintella et al. [151] have quantified the effect of gravity on dispersion using the TSM. One ideally mixed tank has been sufficient to describe dispersion in upward and horizontal flow, while two tanks have been required for the description of dispersion in downward flow. The TSM has been also applied for the description of FIA systems with chemical reactions [147–149] and the sensing mechanism in atomic absorption spectrometry [150].

2.4.4 ‘Black-box’ models and impulse-response functions The most mathematically simple types of ‘black-box’ models used to describe the performance of FIA systems are the so-called regression models. These models consist of equations (e.g., polynomials, power, or exponential functions) relating a response variable and several predictor variables. In most cases the response variables are various FIA peak characteristics (e.g., Ruzicka and Hansen’s practical dispersion coefficient [152–154]; peak height [155,156]; travel time [23,90,154]; baseline-to-baseline time [23,87,121,154,157], time at peak maximum [1,154]; and dispersion volume [155]), while the predictor variables are geometrical or operational parameters of the FIA system studied (e.g., flow rate, sample volume, length and diameter of tubing, analyte diffusion coefficient, and temperature). In some cases regression equations for calculating the axial and radial dispersion coefficients have been proposed [158–162]. The data used in

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constructing the relationships mentioned above are usually obtained experimentally, though, in some cases numerical simulations have been used as well [87,157]. The ‘black-box’ models discussed above view an FIA system as one indivisible module, thus neglecting the influence of its individual sections on the overall performance. This limits severely the applicability of the results to other, even similar, flow configurations. In a series of papers van der Linden and co-workers [163–165] have shown that it is much more advantageous to apply the ‘black-box’ principle to the main flow-through sections of an FIA manifold, than to the system as a whole. This has been achieved by calculating the impulseresponse function of the corresponding flow-through section (e.g., coiled and knitted tubes, mixing T-pieces [163,164], and various measuring cells [165]) by deconvoluting the RTD curves of the FIA system with and without the section concerned. The impulse-response function obtained in this way is in fact the response of the flow-through section to an idealized delta-function input (Equation (19)). The response function of any FIA manifold can be predicted by simply convoluting the impulse-response functions of its sections. This approach has been experimentally validated by the same authors [163–165]. The numerical convolution and deconvolution has been carried out using the Fast Fourier Transform (FFT) algorithm [163]. The experimental noise in the Fourier domain has been successfully reduced with a Hunt filter [164].

2.4.5 Statistical moments and special functions The RTD curve and its statistical moments can provide useful information about the prevailing flow pattern in a flow-through system [16,18]. The two statistical moments, most frequently used in this analysis, are the first moment about the origin (m1, Equation (26)), called the mean, which defines the centre of gravity of the RTD curve and the second moment about the mean, (s2, Equation (27)), often referred to as the variance, which characterizes the RTD width [18]. Z 1 m1 ¼ t cðtÞ dt=m0 (26) 0

s2 ¼

Z

1 0

ðm1 tÞ2 cðtÞdt=m0 ¼ m2 m21

(27)

The quantity m0 is the so-called zeroth moment of the RTD curve (Equation (28)). In a single-line FIA system this statistical moment multiplied by the volumetric flow rate is equal to the amount of analyte injected in the absence of sources and sinks. Z 1 m0 ¼ cðtÞ dt (28) 0

The overall variance of an FIA system ðs2peak Þ without a chemical reaction can be calculated as the sum of the peak variance contributions from the injection device ðs2injection Þ, the detector ðs2detector Þ, the sections connecting the injection


65

device with the detector ðs2transport Þ, and the electronics which are usually well designed and can be neglected [145,166–171]. s2peak ¼ s2injection þ s2transport þ s2detector

(29)

The additivity of the total variance allows the dispersion contribution of various flow system components to be studied by simply inserting them between the injection device and the measuring cell, or by replacing one component by another (e.g., a straight tube by a coiled tube, an empty injection loop by a packed injection loop) [166–169]. An additional term taking into account the effect of a chemical reaction ðs2reaction Þ has been introduced on the right-hand side of Equation (29) [166], but this seems to lack any theoretical justification. The variance can be directly related to sample throughput and sample dilution [1,72,170], and as such it can be used as the function which must be minimized in the optimization of FIA manifolds [168,169,172]. The statistical moments about the origin can be related directly to the Laplace domain solutions of the analytical-experimental models outlined earlier in this chapter by the relationship proposed by van der Laan [173]. j d c¯ mj ¼ ð1Þ j (30) dp j p!0 where c¯ is the Laplace transform of the RTD function while p is the Laplace complex variable. Equation (30) can be used for determining the unknown parameters in analytical-experimental models (e.g., number of ideally mixed tanks in the TSM, axial dispersion coefficient in the ADPFM) [72,90,136]. The statistical moments of experimentally measured FIA peaks can be calculated directly by numerical integration. However, such calculations often result in substantial errors because they weigh heavily the tailing portion of the peak, where the signal to noise ratio is at its lowest [15,90,169,174]. These errors can be minimized by using relationships between the moments and other directly measurable peak characteristics (e.g., area, maximum height, or width at various heights) assuming a known RTD. In the case of a Gaussian RTD (G(t), Equation (31)), often used to describe chromatographic peaks, s2 can be calculated in a straightforward on the basis of peak area (A) and peak maximum pffiffiffiffiffiway ffi (s ¼ A Gmax 2p where Gmax ¼ G(tm)), or peak width (e.g., at G(t) ¼ 1/2Gmax the peak width is equal to 2.355s). " # A t tm 2 GðtÞ ¼ pffiffiffiffiffiffi exp pffiffiffiffiffiffi (31) s 2p 2s As mentioned earlier, the RTD in a typical FIA system is asymmetric and its approximation with Gaussian distribution, though used occasionally (e.g., [136,175]), has its limitations. It has been shown that FIA peaks can be described more accurately by an exponentially modified Gaussian (EMG) function [166,169]. This function (Equation (33)) is asymmetric and can be obtained by

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the convolution of a Gaussian function (Equation (31)) and an exponentially decay function (E(t), Equation (32)) with a time constant z [176]. 1 t EðtÞ ¼ exp (32) z z A HðtÞ ¼ GðtÞ EðtÞ ¼ pffiffiffiffiffiffi zs 2p

Z

t 0

" # t0 tm 2 t t0 exp pffiffiffiffiffiffi exp dt0 z 2s

(33)

where tu is a dummy variable of integration. The EMG function (Equation (33)) contains an integral form that is difficult to estimate numerically. A simple method for generating an acceptable approximation has been proposed by Berthod [176] who has found that in some cases FIA peaks can be better approximated by an exponentially modified square function than an EMG function. Foley [177] has derived empirical equations for calculating the area of EMG peaks at several peak height fractions (0.10, 0.25, 0.50, and 0.75). These equations together with the well-known Gaussian RTD relationships between peak area, peak maximum, and peak width outlined above, have been used in deciding if an FIA peak is Gaussian or EMG in nature [166]. This identification of the peak character is necessary for choosing the appropriate equations for calculating its statistical moments. The concept of using EMG functions for peak description has been further extended by generating EMG functions of second and third order, i.e., with two and three time constants, respectively [178]. Other special functions that have been used in the description of FIA peaks are Gamma-distribution function [150], Lorentzian function [179], and orthogonal polynomials [180].

3. CHEMICAL KINETIC PHENOMENA Chemical reactions involving the analyte often take place in the conduits of FIA systems and therefore play a crucial role in the generation of the analytical signal. Not only do chemical reactions affect the magnitude of FIA peaks but they can also induce double-humped peaks. These types of peaks would not normally appear if the monitored species undergoes only physically driven dispersion [25]. When the sample is injected into a carrier containing the reagent(s), the chemical reaction does not proceed uniformly throughout the entire sample zone. It starts at the edges of the sample zone and progresses towards its centre as the sample zone undergoes dispersion and mixes with the carrier/reagent solution. In the case of a large sample volume, laminar dispersion often cannot provide sufficient mixing between the carrier and the central zone of the sample. This leads to the formation of reaction induced double-humped peaks. Such undesirable effects can be circumvented by enhancing the mixing between the sample and the reagent(s). One way of achieving this is by merging the sample zone with a


67

reagent(s) stream at a triple connector (T-piece), with subsequent further mixing in a mixing coil. The superposition of the mass transfer and chemical kinetic phenomena further complicates the mathematical description of the corresponding physical models, especially in the case of analytical-experimental models. This explains the limited number of attempts to model FIA systems with a chemical reaction. If a solution of the equations describing the mass transfer subsystem exists, it is possible to derive a solution in the case of a first or pseudo-first-order homogeneous reaction. This can be achieved by establishing a relationship between either the solutions of the mass transfer and kinetic subsystems [181–183], or between the desired solution and the solution to the Green’s function problem in the absence of a chemical reaction [184–186]. A fully developed tubular laminar flow with an irreversible first or a pseudo first-order reaction is a special case of the more general problem mentioned above. This case is occasionally encountered in FIA and the corresponding system is described by the following equation: 2 @c @ c @2 c 1 @c r2 @c ¯ ¼D kc (34) þ þ 2 u 1 @t @x2 @r2 r @r a2 @x where k is a first-order kinetic rate constant. If the solution for the transient concentration (c) in the corresponding mass transfer subsystem in the absence of a chemical reaction (Equation (4)) is known, the solution of Equation (34) will be given by Equation (35) [187]: c ¼ cn ekt

(35)

This equation can be used for the determination of (pseudo) first-order reaction rate constants [187] by comparing the FIA peaks recorded in the presence and absence of the reaction. In the case of a first-order reaction the RTD, not the exact flow pattern in the system, determines the degree of conversion of the analyte into product. However, in the case of a pseudo first-order reaction, the actual distribution of the analyte and the reagent in the cross-section of the flow must be known. This is because Equation (35) is valid only if the reagent concentration remains constant and well in excess of the analyte concentration for the entire reaction time. It should be noted that in the case of a chemical reaction, it is the product not the analyte concentration that is sensed by the detector. This requires the calculation of the transient product concentration in the measuring cell of the FIA system by taking into account the relevant initial and boundary conditions. If the diffusion coefficients of both the analyte and the product are equal or very close in value, the transient product concentration can be related to the transient analyte concentration by Equation (36). This then simplifies substantially the mathematical description of this mass transfer-kinetic system. cp ¼ cn ð1 ekt Þ

(36)

Otherwise, it is necessary to solve the convective-diffusion equations for both the analyte and the product.

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Andreev and Khidekel [188,189] have employed the method of moments, used earlier by Aris [21] in solving Equation (34), to derive analytical expressions for the zeroth (Equation 28) and first (Equation (26)) moments about the mean, as well as for the variance (Equation (27)) of the analyte and product RTD curves. The product RTD curve, which is sensed by the detector, has been calculated by convoluting the fundamental solution in the form of a Gaussian function with a rectangular initial sample distribution [189]. The influence of the main system parameters on the sensitivity has been investigated by numerical simulations. In a subsequent study [190], Andreev and Kondratieva have derived an asymptotic solution of the convective-diffusion equations for the analyte and product in the case of a fast second-order reaction. An acceptable agreement with experimental data reported by Wada et al. [26] has been obtained. Finite-difference solutions of the mathematical description of FIA systems based on the convective-diffusion equation (Equation (4)) with first or secondorder chemical kinetics have been presented by Painton and Mottola [25] and Wada et al. [26], respectively. Painton and Mottola [25] have used an oscillating first-order kinetic constant to compensate for the non-ideal mixing that occurs when the sample is injected as a slug in the flowing reagent stream. Numerical simulations of their model have revealed that the higher the rate constant, the shorter the time at which the peak maximum appears. This result suggests that the interactions between the mass transfer and chemical kinetic subsystems create an effect of faster transport of the sample zone [25]. The simulations carried out by Wada et al. [26] have predicted the appearance of double-humped peaks at relatively short mean residence times due to insufficient mixing between the sample zone and the carrier, as discussed above. These theoretical predictions have been also experimentally confirmed by the same authors. The mathematical complexity involved in the analytical solution of models, based on the convective-diffusion equation, has prompted the use of the alternative approaches to describe the mass transfer subsystem outlined earlier in this chapter. Betteridge et al. [82] have used the random walk approach to describe the product RTD curves in the case of second (Analyte + Reagent#Product) and third (Analyte + 2 Reagent#Product) order reactions. It has been assumed that the sample plug is introduced into a tubular laminar stream of the reagent solution. The order of the reaction can be expected to have an effect since the third-order reaction requires twice as much of the reagent as the second-order reaction to generate the same concentration of the product. The random walk simulations have revealed that under the typical non-equilibrium FIA conditions the maximum of the product RTD curve for a third-order reaction is lower and lags behind the maximum for a second-order reaction. The same approach has been applied by Crowe et al. [83] to the modelling of a merging-zone FIA system where the carrier stream with the sample plug merges with the reagent stream. Numerical simulations of this model have allowed to study the influence of the main design and operational parameters of the FIA system on its performance. Satisfactory agreement between the random walk model and experimental


69

results involving the reaction of Ca(II) with o-cresolphthalein complexone has been reported. The ADPFM (Equation (8)) has also been used in the description of FIA systems with a chemical reaction. Kolev et al. [191] have applied this model with a chemical kinetic term (S ¼ kc) to an open–closed FIA system in the case of a pseudo first-order reaction: @c @2 c @c ¼ DL 2 u¯ þ S @t @x @x

(37)

In the case of second and higher-order reactions, Equation (37) becomes nonlinear and a numerical solution will be required. Montesinos et al. [192,193] have modelled an FIA system consisting of an infinitely long tubular reactor with a sample plug inserted between the same or two different carrier solutions. In this case, the same or two different second-order reactions have been assumed to take place at both edges of the sample zone. Satisfactory agreement with experimental results, including double-humped peaks for large sample volumes, has been obtained. Parab et al. [194] have refined the model of Montesinos et al. [192] for the determination of glucose by using a more accurate kinetic equation for the glucose oxidase — peroxidase cascade and the correlation to predict axial dispersion proposed by Kolev and Pungor [86]. Temperature effects have also been taken into account, and theoretical predictions regarding the optimal temperature for this analysis have been made. Among the lump parameter models, both the IMT model and TSM have been utilized in the description of FIA systems with chemical reactions [103–115,120,124,125,128–132,147–149]. The chemical reactions included in all FIA models based on the IMT approximation, except for the one described by Echols and Tyson [129], have been assumed to be very fast. For this reason reaction kinetic effects on the analytical signal have not been taken into account, therefore further simplifying the mathematics involved. Ruzicka et al. [103] and Ruzicka and Hansen [104] have used the IMT model to describe the RTD curves in two FIA systems for continuous flow titration. In one of these systems, discrete samples are passed through the gradient chamber where they disperse before being mixed with a continuously flowing stream of the titrant stream. In the other system, the titrant is present in the carrier stream and the titration reaction takes place in the gradient chamber which is positioned between the injection valve and the measuring cell. Pardue and co-workers [105–112] have reported a comprehensive theoretical and experimental treatment of FIA systems with gradient chambers and chemical reactions with complex stoichiometry. The associated IMT models have been based on the assumption that both dispersion and chemical reaction effects take place only in the gradient chamber. Mottola and co-workers [113–115] have studied flow-through systems with measuring cells equipped with a mechanical stirring device. The samples have been directly injected into the measuring cell and the carrier has been continuously recirculated through the system. The chemical reaction and mass transfer processes have been described by first-order kinetic equations. There is a strong similarity between these equations and the solution of the IMT model. Tyson [120] has

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studied the combined effect of dispersion and a fast chemical reaction in a singleline FIA system in the case of chemically induced double-humped peaks. This system has been without a gradient chamber. Using the IMT model he has derived an expression (Equation (38)) for calculating the time distance between the two humps (Dteq), which has been found to be directly proportional to the logarithm of the analyte concentration in the original sample (c0). Vt V expðVs =Vt Þ 1 Dteq ¼ ln c0 þ ln (38) n cr n where cr is the reagent concentration and Vs is the volume of the sample. The concentrations of the analyte and the reagent at each of the humps are chemically equivalent, and therefore correspond to the equivalence point in traditional titration. The model has been also successfully applied to the description of an FIA system incorporating an actual gradient chamber [124]. Similar flow systems and models have been subsequently used for the determination of stability constants [125] and for comparison of various mixing devices [128]. The alternating helical reactor has been identified as the best alternative to a gradient chamber when high degree of mixing between the sample and the carrier is required [128]. Echols and Tyson [130] have also utilized a two-channel FIA system with a gradient chamber to implement a previously developed batch method [195] for the determination of stability constants. The same authors have derived equations for the RTD curves of reactants and products in the same flow system in the case of a first-order reaction [129]. This model allows the experimental determination of first-order rate constants in a relatively simple fashion. Using the IMT model Berthod et al. [131] have described mathematically a gradient chamber FIA system for the determination of the critical micelle concentration of a surfactant. The model has been developed earlier by the same group [132]. Reijn et al. [147] have solved the TSM for a (pseudo) first-order chemical reaction in the cases of both delta-function and slug injection (i.e., rectangular input function), and have derived expressions for calculating the statistical moments of the RTD curve of the product. The model has been validated by experiments performed with an FIA system incorporating a single-bead string reactor. Van Veen et al. [148] have extended the model of Reijn et al. [147] to two consecutive (pseudo) first-order reactions (i.e., Analyte-Intermediate-Product), and have derived equations for the maxima of the RTD curves of the intermediate and the product. Rules for choosing the optimal reactor length have been formulated and experimentally confirmed using an FIA manifold with a single-bead string reactor. Hungerford and Christian [149] have generalized the TSM approach by substituting the flow rate with an empirical mass transfer rate coefficient. Their model includes equations describing the dispersion due to mass transfer, and to an overall second-order reaction of all the participants in the chemical interaction (i.e., the analyte, the reagent, and the product). Model solution in the case of a slug injection has been obtained using Laplace transforms. Good agreement with experimental results for a slow chemical reaction has been reported.


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4. SENSING MECHANISM The detectors incorporated in FIA systems are of a surface or bulk type. Surface detectors respond to changes in the concentration (activity) of the analyte at the sensor/solution interface. Bulk detector sense the average concentration in a cross-section or volume of flow. Most FIA models incorporating a sensing mechanism subsystem involve surface detection (e.g., potentiometry, voltammetry, and sensing based on the use of membrane based optodes). Bulk detectors (e.g., conductometric, spectrophotometric) are generally assumed to respond in a linear fashion to the RTD function of the detected chemical species. Thus, there is no need of a separate subsystem to describe this linear transformation. Exceptions to this rule are usually models of FIA systems with atomic absorption detection. Most models which take into account a surface sensing mechanism are distributed parameter analytical-experimental models [93–97,135,196,198–203]. Meschi et al. have developed a model of an FIA system with a tubular amperometric electrode [196]. The model is based on the equations for the limiting steady-state current (Iss) in a tubular electrode of length W (Equation (39)) [197], and Taylor’s radial distribution in a fully developed tubular laminar flow [41] (Equation (40)). I ss ¼ 5:43 n FW 2=3 n1=3 D2=3 c a2 u¯ 1 r 2 1 r 4 dcav ðx; tÞ þ cðx; r; tÞ ¼ cav ðx; tÞ þ 3 a 2 a dx 4D

(39) (40)

where n is the number of electrons exchanged in the corresponding electrode halfreaction, F is the Faraday constant, and cav is the average concentration in the cross-section of the flow. Equation (22) in dimensional quantities and variables has been used for calculating cav. This equation is the solution of the convective-diffusion equation (Equation (5)) in the case of dispersion dominated by diffusion (Equation (9)). The model predictions agree well with experimental results obtained at lower flow rates, when the condition mentioned above is valid. In a subsequent study, the same authors have integrated the solution of their amperometric model to describe the analytical signal in an FIA system with coulometric detection [198]. Model simulations have revealed that the coulometric analytical signal is independent of sample dispersion. Assuming a Gaussian concentration distribution for cav and Taylor’s radial distribution (Equation (40)), Evans et al. [199] have developed a model to predict the refractive index effects in high-sensitive absorbance detectors. These are used in both liquid chromatography and FIA. This is one of the examples of FIA models incorporating the sensing mechanism of bulk detection. Analytical-experimental models of the sensing mechanism of FIA detectors, not yet coupled to the mass transfer subsystem, have also been developed. Fosdick and Anderson [200,201] have calculated the steady-state current and absorbance of a thin-layer spectroelectrochemical FIA cell, in which one of the

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walls acts as the working electrode. The concentration in the cross-section of the flow has been calculated using either a Nernst diffusion-layer approximation or by solving numerically Equation (15) under steady-state conditions (qc/qt ¼ 0). This modelling work has not covered transient analytical signals, which are typical in FIA. Wilke and Picht [202] have derived expressions for the impulseresponse functions of potentiometric and amperometric (both direct current and alternating current) flow-through detectors with membrane-stabilised liquid– liquid interfaces. All these models assume Fickian diffusion of the chemical species of interest. Hisamoto et al. [203] have described the response of an ionselective optode flow-through cell packed with beads and operating in reflection mode. The beads have been coated with a liquid layer containing a neutral ionophore and a dye. The mass transfer within this layer has been reduced to one-dimensional Fickian diffusion. The Kubelka–Munk theory of reflectance has been also applied [203]. This model has been solved for a step-function change in the concentration of the analyte in the flow cell, and therefore it can only describe the rising section of the corresponding FIA peaks. Kolev et al. [93] have developed a model of an FIA system incorporating an iodide ion-selective electrode. The mass transfer of the analyte to the measuring cell in this model is described by the ADPFM (Equation (8)). Two sensing mechanisms have been initially considered. In the first one, the rate-determining step is the diffusion of the iodide ion through a stagnant liquid layer separating the flowing carrier solution from the electrode membrane. The ratedetermining step in the second mechanism has been assumed to be a reversible first-order adsorption process. The comparison of the experimental and theoretically predicted RTD curves has shown clearly that neither the diffusion nor the kinetic sensing mechanism alone is suitable to describe the electrode response [96]. A ‘multi-electrode’ sensing mechanism successfully describing the transient electrode response has been proposed. It is based on the assumption that a fraction of the electrode surface, which is free of defects, responds instantaneously to changes in the analyte concentration. The response of the remaining electrode surface is exponential in character and can be described by the IMT model. This study has demonstrated the suitability of FIA for studying the dynamic behaviour of chemical sensors. The model developed can be viewed as an example of a mixed lump/distributed parameter analytical-experimental model. Kolev et al. [94] have also developed a pure distributed parameter model of an FIA system with a single-layer enzyme electrode. The model has been experimentally verified [97]. Numerical simulations have been conducted for studying the influence of the main system parameters on sensitivity and sampling rate [95]. The mass transfer in this model is based on the ADPFM. The sensing mechanism assumes Fickian diffusion within the electrode membrane and a biocatalytic process obeying the Michaelis–Menten kinetics. A similar approach regarding the mass transfer subsystem has been used in the modelling of copper(II) detection in an optode-based FIA system [204]. The sensing element in this optode is a Nafion membrane incorporating the colorimetric reagent 1-(2upyridylazo)-2-naphthol (PAN). The model takes into account the diffusion of copper(II) across the stagnant liquid layer separating the membrane from the


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carrier stream, the ion-exchange equilibrium at the membrane/solution interface and the protolytic and complexation equilibria involving PAN and copper(II) within the membrane. Because of its mathematical complexity, this model has been solved by a numerical technique employing a finite-difference scheme. Excellent agreement has been obtained between the experimental results and the theoretical predictions of the model. Lumped parameter models based on the IMT model and the TSM have also been utilized for the description of the sensing mechanism in FIA. Pratt and Johnson [139] have developed a model for the response of a vibrating wire amperometric electrode in an ideally mixed FIA measuring cell. They have neglected any sample dispersion upstream of the measuring cell and have derived an expression for calculating the peak area. Pungor and co-workers [133–135] have described mathematically the potentiometric and voltammetric signal in a flow system with a gradient chamber by the IMT model, assuming a rectangular analyte input. They have shown that a more precise description of the analytical signal can be achieved by taking into account not only the mixing in the gradient chamber, but also the axial dispersion of the analyte in the tubing connecting it to the injector [135]. The corresponding model combines the solution of the ADPFM in the case of delta-function injection (Equation (20)) and the IMT model. This is another example of a mixed lump/distributed parameter analytical-experimental model. Appleton and Tyson [122] have described the nebuliser performance in FIA atomic absorption spectrometry by a parallel-tanks model (Figure 4b). This assumes that only a fraction of the influent analyte is dispersed whilst the remainder flows directly to the waste. Stone and Tyson [126] have observed that both this model, in the case of two tanks, and the simpler IMT model describe successfully FIA manifolds with flame atomic absorption or spectrophotometric detection. Turner et al. [140,141] have modelled an FIA system with an integrated gradient chamber and potentiometric detector for a step-function input. Fick’s second law has been used to describe the diffusion of the hydrogen ions through the hydrated glass layers of a glass electrode, located in the gradient chamber [141]. Another FIA model combining the IMT concept with Fickian diffusion across a membrane is that of Tsai et al. [205]. They have described the probe dynamics in flow injection/membrane introduction mass spectrometry. Analytical solutions have been provided for step and rectangular function input of the sample directly into the probe, which consists of a gradient chamber with a nonporous pervaporation membrane separating it from the mass spectrometer. Smit and Scheeren [150] have described the transport process between the nebuliser and the torch in an inductively coupled plasma optical emission spectrometric detector by the TSM convoluted with a time delay function. The solution of the model for delta-function input has produced a Gamma density function. The Laplace domain solutions of the model in the cases of a rectangular input and input which can be described also by the TSM, but with a different number of tanks, have been presented and equations for calculating their statistical moments using Equation (30) have been derived.

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ABBREVIATIONS AND NOMENCLATURE ADPFM EMG FIA IMT PAN RTD TSM a A c c c¯ C D DL De F G(t) Gmax H(t) Iss J k L n p PeL r R Re S Sc t t’ tm tmax u u¯ V Vs n

Axially dispersed plug flow model Exponentially modified Gaussian Flow injection analysis Ideally mixed tank 1-(2’-Pyridiylazo)-2-naphthol Residence time distribution Tanks-in-series model Radius (tubular flow) or half of the channel height (parallel-plate flow) [m] Peak area Concentration [mol m3] Transient concentration distribution in the absence of chemical reaction [mol m3] Laplace transform of the RTD function Dimensionless concentration ( ¼ c/c0 or ¼ (V c)/(Vs c0)) Diffusion coefficient [m2 s1] Axial dispersion coefficient (Equation (9)) [m2s1] Dean number (Equation (12)) Faraday constant ( ¼ 96,486 C mole1) Gaussian RTD [mol m3] (Equation (31)) Maximum value of G(t) Exponentially modified Gaussian (EMG) function [mol m3] (Equation (33)) Limiting steady-state current [A] (Equation (39)) Flux [mol m2 s1] Kinetic rate constant Characteristic length [m] Number of electrons exchanged in an electrode half-reaction Laplace complex variable Pećlet number (Equation (7)) Radial distance [m] Dimensionless radial distance ( ¼ r/a) Reynolds number (Equation (2)) Dimensionless source or sink term Schmidt number (Equation (13)) Time [s] Dummy variable of integration [s] (Equation (33)) ¯ Mean residence time [s] (¼ L=u) Time at peak maximum [s] Linear flow rate [m s1] Average linear flow rate [m s1] Volume of the FIA system [m3] Sample volume [m3] Volumetric flow rate [m3 s1]


Vt W x X z

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Volume of an ideally mixed tank (gradient chamber) [m3] Length of a tubular electrode [m] Axial distance [m] Dimensionless axial distance ( ¼ x/L) Transverse distance [m]

Greek symbols a Reduced sample volume ( ¼ Vs/V) Dt Time increment [s] Dteq Time interval between the two peaks in a double-humped peak [s] DL Random step [m] z Time constant [s] (Equations (32) and (33)) y Dimensionless time ( ¼ t/tm) ymax Dimensionless time at peak maximum ( ¼ tmax/tm) m Dynamic viscosity [Pa s] m0 Zeroth moment [mol m3 s] (Equation (28)) m1 First moment about the mean [s] (Equation (26)) n Kinematic viscosity [m2 s1] ( ¼ m/r) r Density [kg m3] 2 s Second moment about the mean (variance) [s2] (Equation (27)) t Fourier number (Equation (6)) j Aspect ratio c Coefficients (Equation (23)) Note: Subscripts av, 0, and r refer to the average concentration in the cross section of a tubular laminar flow, characteristic concentration or initial sample concentration, and reagent concentration, respectively. Subscripts in and out refer to influent and effluent concentrations in the tanks-in-series model, respectively. Subscripts injection, transport, reaction and detector refer to the contribution of these processes to the overall variance of the corresponding FIA system whose subscript is named peak (Equation (29)). Subscript max refers to the maximum value (e.g., peak maximum).

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CHAPT ER

4 Principles of Flow Injection Analysis Ian D. McKelvie

Contents

1. Introduction 2. Sample Dispersion in Flow Injection and Sequential Injection Analysis Systems 2.1 Injection volume 2.2 Manifold variables 3. Components of Flow Injection and Sequential Injection Analysis Systems 3.1 Propulsion 3.2 Injection 3.3 Conduits manifolds and mixing 3.4 Detection 3.5 Description and performance of a flow injection or related analysis system 4. Operational Modes of FIA and Related Techniques 4.1 FIA and SIA manifolds for analyte detection with or without sample reaction 4.2 FIA and SIA determinations based on kinetic measurements 4.3 Flow manifolds involving phase separations 4.4 Determinations based on solid-phase reactors 4.5 Determinations requiring sample modification 5. Conclusion Abbreviations References

81 84 85 85 87 87 91 95 96 97 98 98 99 100 102 105 105 105 106

1. INTRODUCTION The publication of the first paper on flow injection analysis in 1975 ushered in a new paradigm in analytical chemistry. In this paper, Ruzicka and Hansen [1] introduced the term ‘‘flow injection analysis’’ (FIA) for the first time, and initiated Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00604-1


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a field of research that would, over the next three decades, involve thousands of researchers, and which to date has resulted in more than 16,500 publications in the scientific literature (cf. Chapter 1) [2]. From this first generation of FIA systems, subsequent generations of sequential injection analysis (SIA) and labon-a-valve systems have developed (cf. Chapter 2), along with other microfluidic devices involving the unsegmented transport of liquids in narrow channels. While it may seem odd to devote a chapter to the basics of FIA in a book whose objective is to emphasize the more recent advances in this now mature technique, the intention of this chapter is to outline the principles that underpin non-segmented flow analysis for newcomers to the technique, as well as for existing users who may not be versed in the wider aspects of flow analysis. No matter how sophisticated the control software, how ergonomic or glossy the instrument, how fast the sample throughput or how turn-key the operation might be, it is essential that researchers and users alike have a clear understanding of the principles of this versatile and labour-saving analytical approach. This understanding is necessary to ensure that the best possible performance is obtained from a flow analysis system, to enable informed troubleshooting when things do go wrong, and to be able to develop new, and refine existing, FIA methods. Accordingly, this chapter gives an outline of the rudiments of flow and SIA, describes the components of classical flow injection analysers, discusses more recent developments in fluid delivery and detection, and summarizes some of the principal modes of operation. A number of these topics are explored in greater depth in subsequent chapters that focus on various methods of detection, and on applications in a range of different sample matrices. In FIA (Figure 1) a small, fixed volume of liquid sample is injected as a discrete zone using an injection device, into an unsegmented, liquid carrier flowing through a narrow bore tube or conduit. The sample zone is progressively dispersed into the carrier by convection and by axial and radial diffusion as it is transported through the conduit under laminar flow conditions. Reagents may be added at various confluence points and these mix with the sample zone under the influence of radial dispersion, to produce reactive or detectable species that can be sensed by any one of a variety of flow-through detection devices. The height or area of the peak-shaped signal thus obtained can be used to quantify the analyte using calibration data from known concentrations of the analyte. The whole process of sample injection, transport, reagent addition, reaction and detection, can be accomplished rapidly (seconds to 10 s of seconds), using minimum amounts of sample and reagents, with a high degree of precision. Figure 1 shows some of the common components that are used for the various unit operations in the flow-based analytical process, i.e. propulsion, injection, reaction/mixing/modification, detection and data analysis. The assemblage of pumps valves, flow tubes and detector is often referred to collectively as the manifold. The second clearly identifiable generation of FIA to emerge was SIA [3]. In its simplest form (Figure 2), SIA involves only a single reversible pump, usually of the syringe type, and a multifunction selection valve. These permit a defined sequence of sample and reagent intercalation, and zone merging and mixing

S

Time

P MC

IV

D

C

W

R

PROPULSION

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Signal

Principles of Flow Injection Analysis

INJECTION

MIXING/ SEPARATION/ MODIFICATION

DETECTION

Photometry

Peristaltic pump

Rotary valve

Proportional injector

Coiled reactor

Potentiometry

Knotted reactor Atomic spectrometry Single bead string reactor

Δ

Gas pressure

hv Piston or syringe pump

Hydrodynamic injection

Digestor/ photo-reactor/ separation device

Voltammetry

Figure 1 Schematic diagram of a simple flow injection analysis (FIA) system, showing various options for sample and reagent delivery, injection, mixing and detection.

Figure 2 Schematic diagram of a sequential injection analysis (SIA) system.

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before switching the valve and transporting the modified sample or reaction product, to the sensing section of the SIA manifold. The arguable advantage of SIA compared with FIA, is that only a single pump is required, and that the pump can be isolated from potentially damaging reagents or samples by a buffer zone of unreactive liquid, e.g. deionized water. Sample treatment and conditioning steps, such as digestion or enzymatic hydrolysis, etc., can be performed in a series of holding or reaction coils attached to the multifunction valve, but in an off-line, discontinuous mode. Ideally, such an arrangement is more easily reconfigured for different detection reactions using the same manifold simply by software control. Again, because of the flexibility that SIA systems offer, they should inherently be more suitable for multiple parameter determination than is FIA. FIA and other related techniques, e.g. SIA and lab-on-valve, are founded on three important principles, viz. injection of sample or reagents, controlled dispersion of the injected zone and reproducible timing of processes in the flowing stream. Hence the injected zone does not undergo homogeneous mixing, nor is there a need for the reaction to reach equilibrium (i.e. steady-state conditions) before detection is performed, in order for quantification to be achieved.

2. SAMPLE DISPERSION IN FLOW INJECTION AND SEQUENTIAL INJECTION ANALYSIS SYSTEMS In FIA, the tubing used is of small internal diameter (typically 0.3–1.0 mm) and laminar flow conditions apply, with Reynolds number, Re, of {2,000. Consequently, a zone of sample or reagent injected into a flowing carrier/reagent stream will rapidly adopt a parabolic velocity profile, and the major dispersive influence at this stage will be axial convection. Sample near the walls radially diffuses into the flowing carrier stream, and the sample concentration profile changes successively downstream from the point of injection as radial and axial diffusion occurs. Radial mixing can be further promoted by inducing secondary flow, i.e. by using coiled rather than straight tubes. In a single-line flow injection manifold, the extent of dispersion and mixing and hence the dilution, of the injected sample zone that occurs as it is transported through the conduit is expressed in terms of the dispersion coefficient, D, which is defined by Co (1) Cmax where C1 is the original concentration of the injected sample, Cmax the maximum concentration of the sample zone after it has undergone all the dispersive processes and is passing through the detector [4]. Except when preconcentration is performed, the dispersion value must always be greater than unity. Flow injection systems with 1 o D o 3 are classified as having limited dispersion, and are used in conjunction with detection systems such as ion-selective electrodes and atomic spectroscopy, where minimal sample dilution is desirable. Flow D¼


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systems with 3oDo10 are classed as medium dispersion, and are most commonly used where significant sample and reagent mixing is necessary, as is the case for methods involving spectrophotometric or fluorometric detection. Large dispersion systems (DW10) are used where extensive dilution of sample and reagent is required. An FIA system may be designed to fall into one of these three categories, depending on the purpose of the system. For example, very lowdispersion manifolds may be used in the evaluation of potentiometric flow cells, where maintaining maximum sample zone integrity is necessary in order to avoid errors associated with dilution of the sample, and the flow system is used only as a means of conveying the sample to the detector [5]. However, for most FIA determinations involving spectrophotometric detection, where sample and reagent mixing is necessary to promote the colour-forming reaction, manifolds with medium dispersion are used. Thus, the manipulation of dispersion in a flow system can be readily accomplished by varying a number of manifold parameters.

2.1 Injection volume The effect of increasing injected sample volume, Sv , is to decrease dispersion. As Sv increases, the detector response is observed to increase, until it eventually reaches maximum value corresponding to the minimum dispersion case. The relationship between dispersion and injection volume can be expressed by 1 ¼ 1 ekSv D

(2)

where k ¼ constant. If S1/2 is the volume of sample required for 50% dilution of the original sample zone concentration, i.e. D ¼ 2, it can be shown that S1=2 ¼

ln 2 k

(3)

Since steady-state (D ¼ 1) conditions are not a prerequisite for successful FIA measurements, injection volumes in excess of 2S1/2 are seldom required. Selection of injection volume is a convenient and powerful means of selecting the dispersion of a flow injection manifold.

2.2 Manifold variables Dispersion is proportional to the radius of the conduit through which the injected zone travels [6]. For a given injection volume, the length of tubing occupied by the injected zone increases as the tube diameter is decreased, and thus the contact area between the sample and carrier decreases with a commensurate reduction in mixing and dispersion processes [4]. An important practical implication is that zero dead-volume connectors should be used between different manifold components, and that changes in manifold tube diameter be avoided, if unnecessary sample dispersion is to be avoided [7].

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Dispersion is also influenced markedly by the geometry of the manifold tubing. If reaction tubes are coiled, fluid passing though the tube will be subjected to secondary flow. This is caused by the centripetal force exerted on elements of the fluid, and results in radial circulation of the fluid as it flows through the coiled section of the tube. At low flow rates the velocity profile remains essentially parabolic, but at higher flow rates the flow differs markedly from laminar flow conditions, with much higher radial velocities occurring at the walls. This secondary flow effect becomes more pronounced the smaller the diameter of the coil, and for this reason, coiled reactors are preferred in FIA. Even more efficient reagent and sample mixing may be achieved with knotted, knitted or serpentine [8] reactors, in which the knots acts as very tight radius coils, or by the use of single-bead string reactors (SBSR). An SBSR consists of a length of tubing packed with small glass beads only marginally smaller than the internal diameter of the tube (Figure 1). The other factor that critically affects dispersion is the length of tubing used, and for a straight tube, this is given by the relation [4] pffiffiffi D¼k L (4) where L is the length of tube through which the sample passes, whereas in coiled tubes, D will also be affected by secondary flow [7]. The effect of flow rate, Q, on of dispersion is less well defined. Ruzicka and Hansen reported that dispersion decreased as flow rate decreased [4], whereas Valcarcel and Luque de Castro in their monograph [6] suggested that D decreased as Q was reduced from ca. 4–2 mL min1, but then increased at lower flow rates (i.e. 0.5oQo2 mL min1). Subsequently Li and Ma [9] have shown that D increases as Q increases to a maximum value at ca. 4 mL min1 (in accord with Ruzicka and Hansen [4]), but thereafter in the range Q ¼ 4–8 mL min1, D decreases. The value of Q at the inflection point was independent of tube diameter, injection volume, mixing coil radius and injection volume, and depended only on the diffusion coefficient of the injected analyte species. Within the commonly used range of flow rates (1.6–4.0 mL min1) used in FIA, Karlberg and Pacey have reported that Q has little effect on dispersion if other manifold conditions are held constant [10]. In multiple line flow injection systems, sample zone dispersion will also be affected by the diluting effect of each of the contributing reagent streams, R1, R2, y RN, according to Equation (5): Q þ QR1 þ QR2 . . . þ QRN Dilution factor ¼ C (5) QC For a two-line system consisting of a carrier, C, and reagent, R, which have equal flow rates, dispersion due to merging of R with C will approximately double, i.e.: (QC+QR)/QC ¼ 2 and hence the overall value of dispersion could never be o2, regardless of the injection volume selected. For multiple line FIA systems, involving the addition of several reagents, the dilution factor is an important consideration, especially if high sensitivity is required. Therefore it is preferable to choose lower flow rates of more concentrated reagents, and thus


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minimize the dilution effect, than to maintain similar flow rates for carrier and multiple reagent streams, and to increase the dispersion many fold. This approach also has the practical advantage of avoiding the production of large volumes of dilute waste solutions. Thus the extent of sample zone dispersion, and hence the sensitivity of the analytical determination, can be selected by suitable manipulation of manifold parameters such as flow rate, internal diameter and length of tubing used. Of these, the two most influential factors are the injection volume and the dilution factor (Equation (5)). However, the preceding discussion applies only to the physical dispersion that occurs when an injected zone is transported in a non-reactive carrier, and is subsequently diluted by the confluence with additional streams. The more realistic situation, where the sample zone also undergoes a series of chemical reactions to form a detectable product, which is also subject to dispersive processes, is described in Chapter 3.

3. COMPONENTS OF FLOW INJECTION AND SEQUENTIAL INJECTION ANALYSIS SYSTEMS 3.1 Propulsion Figure 1 shows some of the propulsion devices that have been used in flow injection systems for delivery of carrier and reagent solutions, where the flow requirements are typically 0.2–4 mL min1. Historically peristaltic pumps have been favoured, perhaps because of their ready availability and multichannel capacity. However, the limitations of peristaltic pumps are that they produce slight pulsation of the flow, the flow delivered varies with time as the peristalsis tubes wear, and it is necessary to replace tubes regularly. Furthermore, stopping, restarting and reversing the flow may not be instantaneous, because of the momentum of the motor, gearbox and pump head, and this can be problematic in stop-flow measurements, or in sequential injection methods. However, peristaltic pumps are still the most popular choice of multichannel propulsion device used in FIA. Some early FIA systems and their predecessors utilized constant-head reservoirs as a means of reagent delivery [11], and this has more latterly been used in capillary batch injection [12] and micro flow analysis systems [13]. Similarly gas-pressurized reservoirs can be used for liquid propulsion in FIA systems, and both approaches provide a simple, inexpensive means of achieving pulseless flows. A limitation of this approach is that it is difficult in multiline systems to reproducibly regulate the individual flows [6]. However, LyddyMeaney et al. [14] have described a field-portable water-monitoring system that used compressed gas for reagent delivery in a reverse FIA (multicommutation) mode that was not susceptible to these problems. Helium was stored at high pressure (6,900 kPa) in a miniature compressed gas cylinder (internal volume 50 cm3) and after regulating to a constant pressure of between 50 and 100 kPa,

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was used to pressurize a series of reagents held in a machined Perspexs reagent block consisting of a number of wells of ca. 10 mL volume. Each well was connected to a two-way solenoid valve that was used to reproducibly inject microlitre volume of reagent into a continuously flowing stream of filtered sample. This approach has several advantages in that only the potential energy of the compressed gas is used to propel the reagents once the system is pressurized, and thus no gas is consumed or lost by the system during the course of several thousand injections, provided that all connections are gas tight. Furthermore, the use of multiple reagent injections of microlitre amounts means that the reagent reservoir holds sufficient reagent for ca. 1,000 analyses, and the use of an inert propulsion gas such as He or Ar reagents may prolong the operational lifetime of reagents that are not stable in air. However, the disadvantages include the need to have access to a source of compressed gas, which may not be readily available in remote areas, and the restrictions that apply to the transport of devices containing compressed gases by commercial aircraft. Suction has been used for fluid transport in FIA systems, and is integral to the operation of an SIA system which typically utilizes a syringe pump to draw sample and reagents into the flow manifold through a multiport selection valve, before pushing them out through the detection line. Use of suction has the advantage that a single-channel pumping device can be used to draw sample and reagents from different reservoirs through a selection or injection valve, the manifold and the detector. For example, Grudpan et al. used a simple diaphragm aquarium pump in the suction mode, as a means of promoting liquid transport in an FIA system [15], and this approach was also applied by Wang et al. using a peristaltic pump (Figure 3) [16]. However, an important disadvantage of the use of reduced pressure to induce flow is that it favours degassing of the reagents and sample, unless these are first rigorously sonicated, vacuum filtered or sparged with helium to remove dissolved gases. While this is routinely done in laboratory applications, it is not a practicable option for flow analysis systems used in a field environment. Another type of pump that has been used is the self-priming solenoid actuated diaphragm or membrane pump, which is capable of flow rates of up to 15 mL min1 [17]. While these pumps can be individually controlled, a feature

Figure 3 Use of suction for liquid delivery in an FIA system for determination of phosphate. M, acidic molybdate reagent stream; A, ascorbic acid reagent stream; I, injection valve; MC, mixing coil; D, 660 nm detector; R, recorder; P, aquarium air pump and waste bottle, aspiration rate, 0.6 mL min1. Modified from Ref. [15]. Copyright (1993), with permission from Springer.


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that enhances both the versatility of manifold design and economy of reagent use, a potential disadvantage is that they do produce a pulsed flow (typically 120–250 strokes per minute). However, this can be partially overcome by use of a pulse-dampening device [17], or by background correction at a non-absorbing wavelength when photometric detection is employed [18]. Lo´pez-Garcıá et al. recently described their use for sample introduction in flame AAS [19]. Reciprocating piston pumps that are commonly used for liquid chromatography can also be used for FIA. These are designed to provide a continuous liquid flow at high pressure, but are expensive, and a separate pump is required for each reagent stream. A related device is the screw-driven syringe pump, which provides a pulse-free flow. However flow from a syringe pump is discontinuous because the syringe must be periodically filled by valve switching and reversing the drive direction. While this does not pose a problem in SIA, which by definition involves discontinuous flow, these pumps are generally not suitable for conventional FIA. A recent innovation is the milliGATt1 pump, which consists of four, spring-loaded reciprocating sapphire pistons in cylinders machined in a stepper-motor driven rotor. As the rotor turns, the bottoms of the pistons follow a circular track over a series of raised lobes that displace them vertically. As one piston fills through a port in the cylinder head, the opposite piston discharges through a second port, while the two adjacent pistons are either part filling or emptying, and a pulseless flow is achieved. These pumps are reversible and can deliver flows ranging from a few nanolitre per minute to several millilitre per minute. However, they are restricted to pumping a single stream, and hence are more suitable for SIA than FIA. There is increasing emphasis on miniaturization of analytical devices, driven perhaps by the demand for automated, rapid, portable, in situ or even in vivo measurement capacity, and the desire to minimize reagent consumption and waste production. A generalization is that flow analysis research has tended to focus either on the downscaling of functional FIA/SIA systems and methods and the development of new generation systems such as lab-on-valve (described in Chapter 2) and other permutations of so-called zone fluidics, or on the development of microfluidic systems such as mTAS or lab-on-a-chip but with a greater emphasis on device development rather than applications. However, research in both areas has highlighted the need for liquid propulsion systems that are compatible with small volume, microchannel manifolds. Micropump devices investigated to date for these applications fall into two major categories: (a) dynamic or continuous micropumps, and (b) displacement micropumps [20,21]. The use of electroosmotic flow (EOF) in FIA is an example of dynamic micropumping. An EOF micropump FIA system has been described by Dasgupta et al. who used 400 mm, 75 mm i.d. fused silica capillaries connected to a buffer/ reagent selection valve and an injection valve for sample injection, to perform separate spectrophotometric determinations of chloride and Fe(III) [22,23].

1

The milliGATt is a patented design of Global FIA, and manufactured by VICI Valco.

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Pu and Liu reported a miniaturized chip-SIA system that used EOF for fluid delivery and applied this to the determination of enzyme activity (Figure 4) using a confocal microscope for fluorescence detection [24]. An interesting feature of this EOF pumping system were the 32 parallel pumping channels etched into a microfluidic chip that were used to overcome backpressure and flow rate limitations. A Nafions ion-exchange membrane was also used as an electric field decoupler (EFD) in order to achieve high stability flows and to avoid sample solution contact with the etched pump channels. EOF-induced liquid transport originates at the wall surface of the capillary, and hence there is not the same physical dispersion that occurs due to convection in pressure-driven systems. However, the currents required to induce EOF may be sufficient to cause significant Joule heating which can result in de facto sample zone dispersion. Other types of dynamic micropumps include electrohydrodynamic (EHD), magnetohydrodynamic (MHD) and ultrasonic pumps. The former are restricted to pumping dielectric liquids, whereas MHD pumps, like EOF pumps, are capable of propelling a wide range of liquids, including deionized water and buffers, albeit at microlitre per minute flows [21].

Figure 4 A miniaturized SIA system that employs electroosmotic flow as the means of fluid transport. Reprinted from Ref. [24]. Copyright (2004), with permission from Elsevier B.V.


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Displacement micropumps are generally of the reciprocating displacement type. These comprise a chamber with entrance and exit check valves, and a deformable plate or diaphragm. Displacement of the fluid within the chamber is achieved by deforming the diaphragm by means of piezoelectric disks, or thermopneumatic [25], piezoelectric, electromagnetic or electrostatic actuation [21]. The so-called microperistaltic pump comprises three such chambers in series and is made from an etched silicon wafer bonded between two glass plates, and uses piezoelectric disks as actuators [26]. By adjusting the phase of each pump so that an element of fluid is propelled from one pump chamber to the next, in a manner analogous to the action of peristaltic pump rollers, the assemblage can be made to produce a much less pulsed flow than that obtained using a single chamber pump. Rainelli et al. compared the performance of electrokinetic, piezoelectric and syringe pumps for delivery of reagents in mechanically engraved polymethylmethacrylate manifold blocks for FIA determinations of iron, chloride, cyanide, nitrite and some heavy metals [27]. They concluded that the first two pumping methods were less than ideal for photometric FIA because of reagent incompatibilities with the conditions necessary to induce EOF low flows and the pulsed nature of flows from piezoelectric pumps, and elected instead to use syringe pumps. However developments in the in situ manufacture of silica frits has enabled the production of electroosmotic pumps capable of delivering higher pressure flows [28]. For example, Chen et al. described the successful application of a lab-on-chip electroosmotic pump formed from a monolithic silica matrix that was capable of a maximum pressure of 0.4 MPa with flow rate of 0.4 mL min1 [29]. Improvements in piezoelectric pump technology has resulted in the availability of pumps capable of flows of up to 4–9 mL min1 and a pulse frequency of 0.5–200 Hz, and Ribeiro et al. have successfully applied these in an FIA system for the spectrophotometric determination of garbapentin in pharmaceuticals (Figure 5) [30]. While the literature abounds with reports of the use of many of these types of micropumps in microfluidic devices such as lab-onchip or micro-PCR systems, their application in flow injection systems has been limited. Now that they are more readily available and with better performance, it is to be expected that will gain much wider use in flow injection systems.

3.2 Injection FIA and its progeny rely on the reproducible injection of sample or reagent with volumes of 10 s–100 s of microlitres. The earliest FIA systems used injection through a septum with a syringe, and it was from this practice that the term ‘‘FIA’’ originated. The requirement for high-precision automated injections saw rotary injection valve rapidly adopted (Figure 6a) and stepper-motor driven valves are still widely used in FIA, especially in fully automated commercial instruments for laboratory use. An early injection valve developed by Bergamin et al. was the proportional valve [31], shown in Figure 6b in its simplest form. The drilled, moving block containing the by-pass channel and the volume of sample for injection was

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Solution in

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Figure 5 (a) Diagrammatic representation of a piezoelectric activated pump. A, piezoelectric actuator; B, diaphragm; C, pump chamber; D, inlet check valve; E, outlet check valve. (b) Flow injection manifolds for determination of gabapentin. (A) Aspiration mode, (B) propulsion mode. S, sample; V, three-way solenoid valve; R, reagent; D, detector (480 nm); W, waste; P, P1, P2, piezoelectric micro pumps. Reprinted from Ref. [30]. Copyright (2007), with permission from Elsevier B.V.

actuated using a solenoid, a process that was described at ‘‘commutation’’. Another approach to sample introduction is that of hydrodynamic injection (Figure 7) in which a known length of conduit is used to define the volume of sample that is delivered into the manifold by redirecting the sample and carrier flow paths by either switching the pumps, or three-way solenoid valves that are used to connect the sample and carrier flow streams. This approach is intrinsically more reliable than the use of rotary or proportional injectors because there is not the same potential for mechanical wear of valve rotors or commutators. The injection reproducibility is also comparable with that of the rotary or proportional valves [32]. Time-based injection is an alternative to the volumetric injection techniques described above. Time-based injection relies on pumping or bleeding sample into the conduit through a manifold connection at a known flow rate for a defined period. A three-way solenoid valve provides a convenient means of performing these injections, which should be made while the carrier flow is halted in order to avoid undue pressure pulsation. The strength of this approach is that the injection volume can be easily varied by altering the time that the dosing solenoid is opened. However, the implicit weaknesses are that the reproducibility will be adversely affected by any fluctuations in flow rate and that unless a different valving arrangement is assumed [33], the sample dosing line must be purged through the manifold, which will slow sample throughput. However timed injection is ideal for reverse or reagent-injection FIA in which sample is pumped continuously and reagent is injected into it to produce some analyte-related detector

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Figure 6 Common injection methods employed in FIA: (a) rotary injection valve, (b) solenoidactuated proportional injector.

response [34]. Because reagents do not have to be purged through the manifold, minimal reagent is consumed and the major consideration is whether there is sufficient sample; such an approach is ideally suited to on-line process or environmental monitoring where sample volume is usually not limited [35]. A variation on the reagent injection technique was the use of microporous membranes to either pulse [36] or diffuse [37,38] reagent into a flowing sample stream. The Holy Grail of any automated serial assay system is the ability to perform fast determinations of multiple analytes. The limitations of single zone injections of sample or reagents were recognized in this regard early in the evolution of flow injection techniques, and considerable research has been focussed on the development of alternative injection systems. FIA monographs from the later 1980s [4,6] contain numerous descriptions of how rotary or proportional valves can be plumbed to achieve injection of multiple adjacent zones of sample and reagents to achieve what were synonymously termed sandwich [39,40], chasing zone [41], multiple reagent-injection [14,42], time division multiplex [16] or multicommutation [43–45] techniques. The latter term derives from the proportional, or commutator valve, which if equipped with several injection ports and connecting tubes could be configured to perform multiple injections, nested injections, zone trapping, on-valve ion-exchange and the like [33].

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ON

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Figure 7 Comparison of (a) hydrodynamic and (b) time-based injection modes. In (a) the injection volume is defined by the volume of the conduit contained between the two T-pieces or three-way solenoid valves (represented by broken circles), whereas in (b) the injection volume is dependant on the injection switching time of the single three-way valve and the flow rate at which sample is propelled into the carrier.

While the advantages of multicommutation were clearly recognized, the mechanics and fluidics of execution could be complex, and this provided the impetus for the development of the SIA approach (Figure 2) [3,46]. SIA provides the means of performing multicommutation using a relatively simple instrumental configuration that can be modified to meet the requirements of different detection chemistries largely through software, as described in Section 4.1. Because multicommutation by SIA is discontinuous and involves flow reversal/s, sample throughput may not be as great compared with that of a continuous flow FIA system, and matrix matching and associated refractive index problems can be more difficult (see Chapter 12). However these potential limitations can be addressed by careful programming and use of multiwavelength detection, and are far outweighed by the versatility, multiparameter capability and reagent economy of SIA. Other approaches variously termed multicommutated flow injection analysis (MCFIA), multisyringe flow injection analysis (MSFIA) [47], multipump flow injection analysis (MPFIA) [48] and all injection analysis (AIA) [49] have been described using different hardware configurations for performing multicommutation (Figure 8). It is a matter of semantics whether these variants should be considered as different flow techniques that justify their own name, or whether they are recognized simply as different fluidic arrangements used to perform multiple reagent injection or multicommutation.


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Figure 8 Representation of several different approaches used to perform multicommutation of sample S, with reagents R1, R2 and R3 and carrier, C, as illustrated by the different reagent and sample combinations in the flow conduit. SIA: sequential injection analysis, which uses a syringe pump, SP, to draw sample and reagent solutions in sequence through the multiport selection valve into the holding coil, HC, before expelling them into the reaction coil, RC, and then to the detector, D. MPFIA: multipump FIA, involving the use of two or more membrane pumps (P1, P2) for sample, S, and reagent, R, and controlled by a flow controller, FC. MCFIA: multicommutated FIA, which uses solenoid three-way valves to inject zones of sample into a common carrier with suction from a single pump. MSFIA: multisyringe FIA, which is a variant of SIA in which the multifunction valve is replaced by multiple syringe pumps, SP. AIA: all injection analysis, which use nested injection loops of several rotary injection valves to achieve stacking of different sample zones and sample.

3.3 Conduits manifolds and mixing FIA and SIA manifolds are generally comprised of flow tubing or conduits with an internal diameter of 0.3–1.0 mm to maintain laminar flow conditions and controlled dispersion of injected sample or reagent zones. Tubing connectors and junctions should have negligible dead volume. Any tubing that is chemically unreactive such as PTFE (Teflons) is commonly used because it is chemically inert, but PVC tubing (such as Tygons) may also be suitable for a wide range of reagents. Microconduit manifolds consisting of polymeric blocks with etched, engraved or micromachined channels have also been used [50–55], but a disadvantage is that they are generally dedicated to particular detection chemistries, and for this reason the versatility of SIA or lab-on-valve is preferred [56–58]. Mixing device/s are used to promote radial mixing, and hence reaction between injected zones of sample and reagent/s. These usually consist of coiled

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Figure 9 Mixing devices commonly used in FIA and related techniques: (a) a PTFE coiled tubing reactor, (b) a knitted PTFE tubing reactor and (c) a serpentine PTFE tubing reactor.

tubes (Figure 9a), and if a sufficiently small coil radius is used, secondary flow is induced which enhances the radial mixing. For this reason, knotted or knitted coils [59] (Figure 9b) or serpentine reactors [8] (Figure 9c) are employed because they provide superior radial mixing with minimal dispersion compared with coiled tubular reactors. Other devices such as SBSR, packed bed reactors or a coaxial jet mixer [60] have been described, but their use is less common. Mixing between flowing streams of sample and reagents is also dependent on the characteristics of the confluence point. Clark et al. have shown that the best mixing is achieved when a T-junction is used that has connections converging at 301 [59]. Efficient mixing may also be achieved by the use of multicommutation if the injected adjacent reagent and sample segments are sufficiently small, and are allowed to disperse into each other as flow proceeds. Flow reversal assists this process [61] and is a key to the successful operation of SIA.

3.4 Detection A flow analysis system requires one or more flow-through detection devices that are used to sense changes in absorbance, fluorescence, chemiluminescence, atomic emission or absorption, infrared absorption, pH, electrode potential, diffusion current, electrical conductivity, turbidity, mass and so on. Photometric detection is one of the most favoured modes of detection [62], although Chalk (Chapter 5) argues that electrochemical techniques in toto are even more widely used. Detection devices should have a minimal swept volume (ca. 6–30 mL) in order to avoid undue increases in sample zone dispersion, long flushing times, and be made so as to avoid the entrapment of bubbles or adsorption of coloured reaction products. Detectors and sensors for FIA and related techniques should ideally give rapid and reproducible signals with no hysteresis effects, have a linear response over a wide concentration range, and be resistant to mechanical wear or chemical degradation and interference when exposed to large numbers of samples with complex and aggressive matrices. The use of FIA/SIA techniques confers a number of advantages in this regard, since the detector/ sensor need only be exposed to samples for brief periods thus minimizing the potential for damage or contamination. Furthermore, the use of a flow system readily facilitates flushing or conditioning of the detector and periodic calibration and running of quality assurance checks [63–65].


Table 1

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Descriptions of flow analysis detection systems and their applications

Detection approach

Techniques and coverage

Chapter

Photometry

Absorbance and reflectance: principles, 12 photometry, spectrophotometry, diode array and charge-couple detectors, LEDs and fibre optics, optrodes, capillary flow cells, liquid core waveguide cells, multireflection cells, sandwich cells, background and schlieren correction methods

Photo-luminescence

Fluorescence, chemiluminescence and electroluminescence: principles of photoluminescence and detection in FIA, chemiluminescence, bioluminescence, electrochemiluminescence

13

Atomic spectroscopic detection

AAS, AFS, ICP-AES, ICP-MS: interfacing atomic spectrometry with flow systems, flow systems for front-end sample processing of liquid and solid samples

14

Vibrational spectrometry

FTIR, Raman, NIR: principles of vibrational spectroscopic detection in flow analysis, applications of vibrational spectroscopic techniques

15

Electrochemical detection

Conductometry, potentiometry, voltammetry and amperometry coulometry: detector design, principles of measurement and applications

16

Miscellaneous detection systems

Conductometry, non-spectrophotometric optical 17 detectors, radiometric detection, thermometric detection, dynamic surface tension measurement, mass spectrometry, NMR, piezoelectric detectors and X-ray fluorescence

It is beyond the scope of this chapter to cover the detection methods that have been used in FIA, and readers are referred to other chapters of this book where more detail is provided (Table 1).

3.5 Description and performance of a flow injection or related analysis system The parameters necessary to unambiguously specify the operation of an FIA or SIA system should include the flow rates of carrier and reagent streams,

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injection volumes or multicommutation sequences for sample and reagents, tubing diameter and length of mixing coils. While classical FIA involves a continuous flow of carrier and reagents, other modes of operation such as SIA can involve stopped-flow, flow reversal, intermittent, pulsed and gradient flows and the time and flow sequences for each of these should be clearly specified, preferably in table form [66]. Similarly, the composition of reagents and carrier should be defined. The detector used should be reported with specification of the flow cell volume, and the aperture of the entrance window, lmax reported for photometric measurements, or lex and lem for fluorescence measurements. It is recommended that the performance of a flow-based analysis system should be described in terms of the following analytical figures of merit, viz. accuracy, precision, sensitivity, detection limit, selectivity and linear dynamic range [67]. With respect to sample throughput, it is useful to report the number of injections per hour which is a measure of the fluidic and kinetic characteristics of the flow manifold, rather than the so-called sample throughput which is also a function of the number of replicates (often undefined) as well as the flushing and filling time required to change from one sample to the next. Reported methods should also be properly validated against one or more standard method/s, and involve the use of certified reference materials.

4. OPERATIONAL MODES OF FIA AND RELATED TECHNIQUES It is not possible to summarize all of the modes of application of flow and SIA systems that have been described over the last 30 + years. However, in this section a number of important modes of application are briefly described for both FIA and SIA. It is evident from the following discussion that in many cases SIA is more versatile than an equivalent FIA systems with respect to ease of tuning and reconfiguration. However, FIA systems still do have their place because in some instances, the fluidics are simpler and they may exhibit higher sensitivity and faster injection rates than the equivalent SIA manifold.

4.1 FIA and SIA manifolds for analyte detection with or without sample reaction The most rudimentary FIA and SIA systems shown in Figure 10a and b can be used as a means of transporting the sample to a flow-through detector such as an ion selective electrode or an atomic spectroscopic detector, in which case minimal dispersion is desirable. Alternately, manifolds such as these are used where some simple chemical reaction or derivatization is required for detection. In the latter case, direct injection of sample into a reagent stream may be unsatisfactory (Figure 10a I), especially for trace analysis involving photometric detection, because of schlieren effects that occur due to refractive index differences between the sample and the carrier (cf. Chapters 12 and 17). This can be overcome in many cases by matching the matrices of the sample and the carrier in FIA (Figure 10a II),


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Figure 10 (a) Simple FIA systems for transport and analyte derivatization. (I) Injection into reagent stream, R, with possible refractive index effects. (II) Sample injected into matrixmatched carrier, C. (b) SIA system with and without background correction using dual wavelength detection. S, sample; P, pump; D, detector; W, waste.

but matrix matching is inherently more difficult in SIA because of the mode of reagent and sample zoning. An effective matrix correction approach in SIA is the use of multiwavelength correction, using either a dual-light emitting diode (LED) or a charge coupled device (CCD) detector. Reagent can also be injected into a continuously flowing stream of sample, and this is the so-called reverse, or reagent-injection mode. It is of considerable benefit in process and environmental monitoring where sample is in abundant supply. When more than one reagent is injected, the process is sometimes referred to as multicommutation, as described in Section 4.3.2. In photometric detection this has the advantage that the background absorbance of the sample and any other confluent reagents are measured continuously, and that the signal peak obtained over the baseline is due only to the analyte and the injected reagent blank. Another advantage of the reagent injection approach is that by continuous on-line matching of the refractive indices of injected reagents and carrier prior to merging with continuously pumped sample, schlieren effect errors can be overcome [68].

4.2 FIA and SIA determinations based on kinetic measurements In the stop-flow mode (Figure 11), the carrier flow is stopped for a certain period at a predetermined time after injection to allow further reaction between sample and reagents to occur. If the analytical reaction is slow, then the resultant peak consists of a steadily increasing peak, the gradient of which is a measure of the reaction rate, superimposed on the peak obtained before the stop period was initiated. This approach may be used to (i) enhance the sensitivity of analyses based on reactions with slow kinetics, (ii) discriminate analyte signal from background signal in samples having a large blank, e.g. interfering colour or (iii) obtain kinetic data for the reaction of interest by use of the gradient of the peak

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Figure 11 (a) An FIA system employing stop-flow for the purpose of background correction or measurement of kinetics. T is an injection and pump controller. (b) An SIA system for stopflow kinetic measurements. S, sample; P, pump; C, carrier; R, reactant; D, detector; W, waste.

Figure 12 (a) An FIA system for liquid–liquid extraction. (b) An SIA liquid–liquid extraction system. S, sample; P, pump; C, aqueous carrier; OP, organic phase; SE, segmentor; EC, extraction coil; BE, back-extraction reagent; WS, wash solvent; D, detector; W, waste.

obtained during the stop period, e.g. determination of enzyme activity [69–71], or to measure different chemical species or suppress interference by the use of kinetic discrimination FIA [72–76].

4.3 Flow manifolds involving phase separations 4.3.1 Liquid–liquid extraction Solvent extraction can be performed using flow injection, and a typical manifold is illustrated in Figure 12a. Aqueous-phase analyte is injected into an aqueous carrier and a continuous stream of organic phase added at the organic-phase segmentor. The flowing stream of organic–aqueous segments that is produced passes through an extraction coil, EC, where mass transfer from aqueous to organic phase occurs. The segmented stream is then separated into pure organic phase that usually contains the analyte, and a mixture of organic–aqueous phase by the phase separator (e.g. a T-piece with a PTFE thread in one arm, a planar [77] or tubular [78] hydrophobic membrane separator, or gravity separation [79])


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prior to detection. This technique has been widely used for solvent extraction of samples prior to atomic absorption spectrometry [80–82], and in the analysis of pharmaceutical products [83] and beverages [84]. Liquid–liquid extraction may also be performed by extracting across a hydrophobic membrane without the need for solvent–aqueous segmentation [85], or by the use of a chromatomembrane [86]. SIA solvent extraction need not involve segmentation because extraction can be performed by a film of solvent adhering to the PTFE conduit walls (Figure 12b), which is referred to as wetting film extraction. Analyte can then be back-extracted from the wetting film with a suitable reagent or solvent [87–89] (see also Chapters 7 and 9).

4.3.2 Membrane-phase separation techniques: gas diffusion, dialysis and tangential flow filtration Membrane techniques are described in detail in Chapters 8 (gas diffusion, pervaporation, dialysis) and 9 (filtration). Simple gas-diffusion FIA systems can be used for the determination of gaseous species such as CO2 [90], H2S [91,92], HCN [93], SO2 [94,95] or NH3 [96,97] or other volatile species, e.g. arsine [98] that form due to reaction with acid, base or some derivatizing reagent. The gaseous species thus formed diffuse through the membrane into an acceptor stream and are detected by means such as electrical conductivity, photometry fluorescence, etc. If the acceptor contains an acid–base indicator, acidic or basic gases can be detected by absorbance change (Figure 13a). In-valve, static preconcentration of gaseous species can be achieved by incorporating the acceptor side of the gas diffusion block in the injection loop of the detector, and allowing diffusion of analyte gas from a continuously pumped donor stream prior to injection. For example, sensitive ammonia determination using FIA can be performed using the simple manifold shown in Figure 13a [99]. FIA operates on a quasi-continuous flow basis, i.e. the indicator reagent is pumped continuously, and only the sample is introduced periodically. In contrast, the corresponding SIA system, which involves discontinuous pumping, uses much less sample and reagent, but is somewhat more complex. To function effectively, it requires either a selection

Figure 13 Typical gas-diffusion (a) FIA and (b) SIA systems for determination of acidic or basic gases. R, acid or base; In, acid–base indicator acceptor reagent; S, sample; P, pump; C, carrier; GD, gas-diffusion assembly containing a hydrophobic porous membrane; D, detector; W, waste.

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valve and an injection valve (shown in Figure 13b [100] or two selection valves and two syringe pumps [101] in order to perform the determination of ammonia. The complexity of SIA systems for other membrane separation techniques such as tangential flow filtration (TFF) and dialysis is also quite high. In part, this is because the donor flow is separated from the flow of analyte to the detector by the membrane, and the manifold connections required to operate an SIA system using a single pump and a single selection valve, are necessarily more complex than in FIA, and while feasible, would make the operation of the system quite slow. For this reason, multiple pumps and valves are often used in this context, even though it is counter to the general SIA philosophy of developing a generic analyser. Dialysis involves the separation of high molecular mass species, e.g. biomolecules, from smaller molecular mass species such as anions and cations, using a membrane with a certain molecular mass cut-off. Dialysis can be performed in either static mode, or dynamically, and it is the latter that is used in flow analytical techniques. Sample is pumped continuously across the membrane and the small molecules and ions diffuse through the membrane into an acceptor stream of deionized water. Dialysis, like other dynamic membrane techniques, does not operate with quantitative efficiency, and only a small fraction of the analyte may actually diffuse into the acceptor. However, process is highly reproducible, and on this basis it can be used for quantitative analysis. Similarly, on-line filtration is most effectively performed using tangential or cross flow filtration (TFF), in which turbulent flow is induced on the donor side of the membrane to minimize the formation of a filter cake layer or growth of biofilms. These TFF units may have a large membrane area, and be operated as a separate sampling system with a high flow sample feed [14,18], or be miniaturized, and be incorporated in the FIA/SIA system. Membranes with pore size r0.2 mm are usually preferred to avoid the ingress of bacteria into the flow manifold [102]. The manifold arrangement for dialysis and filtration are similar, and typical configurations are shown in Figure 14 for FIA [103] and SIA [104] applications. Other filtration modes that have been utilized included the use of ultrasonic particle separation, [105] which involves no membrane, and shows promise for long-term unattended applications.

4.4 Determinations based on solid-phase reactors In-line packed reactors may be utilized in FIA and SIA (a) for solid-phase microextraction (SPME) of analytes as a means of preconcentration and sample clean-up [106], or (b) for chromatographic separation. In FIA in-valve preconcentration can be achieved by passing a known volume of sample or standard through a small chromatographic column which replaces the injection loop in the injection valve (Figure 15a). In addition to the advantages of preconcentration and separation of interferences, this approach allows time-based calibration using different volumes of a single calibration standard. Rapid separations for multianalyte determinations, analyte preconcentration and enhancement of


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Figure 14 Typical applications of dialysis and tangential flow filtration (TFF) techniques in (a) FIA. (I) Schematic diagram of a generic FIA dialysis system where the dialysis unit, DU, forms part of the manifold. The volume of sample that can be dialysed is restricted by the size of the injection loop. (II) An in-valve system that enables a large volume of sample to be dialysed before the sample is injected into the carrier. (III) A TFF system where sample is pumped continuously through the feed side of the assembly, preferably at high velocity, while the filtrate fills the loop of the injection valve. (b) SIA systems incorporating (I) dialysis and (II) TFF.

Figure 15 (a) Schematic diagrams of flow injection systems incorporating packed bed reactors, solid-phase extraction or chromatographic media: (I) chromatographic column (e.g. ion exchange) in-line for separation or preconcentration. Loading and elution is achieved by alternate injection of sample, S, and eluent, E. (II) In-valve preconcentration with a packed column of adsorbent or stationary phase. A carrier that will act as an eluent is selected in this instance. (b) Representation of a simple sequential injection chromatography system. Samples S1, S2, S3, are introduced through the selection valve, and eluted with mobile phase, E, through a monolithic chromatographic column.

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selectivity may also be achieved by the use of small (10 s–100 s of mL) chromatographic columns. The development of SIA-lab-on-valve [56], described in Chapter 2, allows the manipulation of ion-exchange or reverse-phase bead suspensions such that these can be injected into the manifold, used for SPME and either regenerated on-line, automatically replaced or even injected into electrothermal atomic absorption [57] or ICPMS systems [107]. The advent of monolithic chromatographic columns with very low backpressure has enabled the development of SIA-chromatography that embodies all of the advantages of liquid chromatography with the pre- and post-separation sample processing power of a sequential injection system [108–110] (cf. Figure 15b and Chapter 10). Solid matrices may also be used for immobilization of enzymes, cells or antibodies. The advantages of the use of enzymes in an immobilized form include minimal enzyme consumption, and potentially improved activity, reduced inhibition and greater longevity [111]. In some cases multiple enzymes can be immobilized on the same support [112].

Figure 16 Flow and sequential injection systems for on-line sample pretreatment: (a) (I) A simplified diagram of a flow injection system that includes a flow-through reactor (FR) typically used for on-line microwave or thermal digestion or photo-oxidation. (II) An FIA system used for the determination of total organic carbon by UV photo-oxidation in the presence of an oxidant such as peroxydisulfate. The oxidized product (carbonate/ hydrogencarbonate) is acidified and the carbon dioxide produced is determined by diffusion through a gas diffusion membrane (GD) that causes a change in the absorbance of the weakly buffered indicator acceptor stream (In). Inorganic carbon must be removed off-line beforehand in a separate step. (b) An SIA system for the determination of inorganic (IC) and organic (OC) carbon, that uses the same UV photo-oxidation procedure shown in (a). IC and OC were determined sequentially by first acidifying the sample and measuring the liberated carbon dioxide, followed by photo-oxidation of a fresh sample that measures the sum of OC and IC. A cresol red (CR) indicator was used in the photometric detection of the diffused CO2.


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4.5 Determinations requiring sample modification FIA and SIA provide a powerful means of performing sample pretreatment prior to analyte detection. Detailed information on FIA/SIA sample pretreatment pertinent to the determination of metals, and the analysis of food and beverages, industrial and environmental samples is given in Chapters 6, 14, 21 and 23. On-line sample modification can be performed using flow-through thermal or microwave digestion [113], UV photo-oxidation or electrochemical dissolution. On-line microwave acid digestion has proved to be a very effective means of preparing waters and biological samples for metals analysis by atomic spectroscopy [114]. Similarly UV photo-oxidation can be used to rapidly oxidize organic matter in the determination of metal species [115], or in the measurement of total or organic phosphorus [116], carbon [117] or nitrogen [118] in waters and wastes (Figure 16). While most of these methods have focussed on the pretreatment of liquid samples, a metal dissolution technique has been reported that involves on-line electrolytic leaching of solid metal alloy samples prior to either photometric or ICP-AES detection [119]. Another recent development has been the use of SIA as a means of performing sequential extraction procedures for metals and nutrients on solid soil samples [120,121]. In addition to providing accurate information under very controlled leaching conditions about the metal or nutrient speciation in the soils, this approach also provides additional information on the kinetics of release that would not be available if batch techniques were performed.

5. CONCLUSION The material in this chapter illustrates the principles on which flow and SIA and their derivative techniques are founded. The history of FIA and related techniques has been a rich one, and has given rise to thousands of interesting publications on a multitude of different analytes in an extensive range of different sample matrices. The fluidics, detection and automation of even simple systems were very real challenges when FIA was in its infancy 30 years ago, but despite that, many ingenious systems were contrived and fascinating chemistry explored. Future developments in flow analysis techniques will be even more intriguing, with the availability of scarcely imaginable miniaturized fluidics and detector elements, and with the ability to collect and process data at speeds and with such power as never before. However, it is important not to forget that the same FIA principles of injection of sample or reagent, controllable partial dispersion and reproducible timing have, and will continue to underpin the development of new microanalytical flow systems.

ABBREVIATIONS AIA BIA

All injection analysis Batch injection analysis

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C1 CCD CE Cmax D EFD EHD EOF L LED MCFIA MHD MPFIA MSFIA Q Re S1/2 SBSR SPME Sv TFF

Original concentration of the injected sample Charge coupled device Capillary electrophoresis Maximum concentration of the sample zone when it reaches the detector Hydrodynamic dispersion Electric field decoupler Electrohydrodynamic Electroosmotic flow Length of manifold tubing Light emitting diode Multicommutated FIA Magnetohydrodynamic Multipumped FIA Multisyringe FIA Volumetric flow rate Reynolds number The volume of injected sample corresponding to dispersion of D ¼ 2. Single-bead string reactors Solid-phase microextraction Injection volume Tangential flow filtration

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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CHAPT ER

5 Bibliometrics Stuart Chalk

Contents

1. Introduction 2. Analytes 3. Application Areas 4. Detection Techniques 5. Other Interesting Statistics 6. Future Trends Abbreviations References

111 112 114 118 121 121 124 125

1. INTRODUCTION This chapter briefly summarizes statistics (bibliometrics) on the literature in flow analysis. It has been put together based on the data available in the Flow Analysis Database [1] collected over the past 15 years by this author. The Flow Analysis Database was originally based on the scanning and optical character recognition of references in the FIAStar Database (published in the 1980s by Tecator) and the ‘‘List of FIA References’’ at the back of second edition of Jarda Ruzicka and Elo Hansen’s book on Flow Injection Analysis (FIA) [2]. Indeed, the author would like to thank Elo Hansen for generation of the bibliography in that book as it was the inspiration to start collecting citations in the first place. The Flow Analysis Database now contains over 17,000 citations to work on flow analysis (as of June 2007). The database contains papers on FIA, sequential injection analysis (SIA), lab on valve (LOV) and zone fluidics (ZF). In addition, there are papers on post-column derivatization in liquid chromatography and a small number of papers on microfluidics. While the database is ‘‘comprehensive’’, it is estimated that it currently represents 95% of the literature. And of course the literature is dynamic, so the statistics in this chapter have been reported as percentages (overall and by decade) because hard numbers are not Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00605-3


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112

Stuart Chalk

18000 16000 14000 12000 10000 8000 6000 4000 2000 0 1975

1980

1985

1990

1995

2000

2005

2010

Year

Figure 1 Journal papers published on flow analysis by year.

useful to the reader. Please consult the Flow Analysis Database website for current totals. The growth of flow analysis techniques (shown in Figure 1) is a testament to the revolutionary approach the technologies embody. Flow processing, FIA, SIA, LOV, ZF and microfluidics have been integrated with almost all analytical detection technologies (see Section 3). The area has also reinvigorated analytical reagent chemistry by removing the limitations of reagent stability and reaction kinetics, and given birth to the important area of automated cold vapor/hydride generation.

2. ANALYTES Since the determination of phosphate in the first flow injection paper in 1975, flow analysis has been applied to over 2,000 different analytes, inorganic and organic, molecular and elemental. Table 1 shows the top 25 analytes. It can be seen that the majority of analyses have been applied to transition metals and inorganic anions. Of particular interest is the wealth of papers on the analysis of mercury and arsenic, especially since 2000. This can be attributed to the advantages of using flow analysis in the generation of volatiles combined with the need to speciate these metals. Speciation of arsenic has become a major focus of the analytical community worldwide due to the toxic nature of arsenic(III) in the environment and propensity of organic arsenic to bioaccumulate (Table 2).

Bibliometrics

Table 1

113

Analytes determined by flow analysis techniques

Analytes

Decade (%) 1970

Arsenic Mercury Iron Copper Glucose Hydrogen peroxide Chloride Lead Selenium Ammonia Cadmium Cobalt Nitrite Amino acids Phosphate Nitrate Aluminum Calcium Ascorbic acid Zinc Ethanol Manganese Urea Nickel Magnesium Potassium

0.65 0.00 5.84 0.65 2.60 0.65 0.65 0.00 0.00 1.30 0.00 0.00 0.65 0.65 1.30 3.25 0.65 3.90 0.65 0.00 0.00 0.65 0.65 0.00 0.00 0.65

1980

1.08 0.83 2.86 2.46 2.58 1.38 1.51 1.17 0.31 2.03 0.46 0.95 1.48 0.89 1.44 1.51 0.92 1.54 0.49 0.55 0.77 0.68 0.49 0.52 0.43 0.25

1990

2.69 2.39 2.84 2.89 3.06 2.03 1.42 1.49 1.13 1.39 1.12 1.08 1.05 1.28 0.90 0.93 1.07 1.16 0.78 0.63 0.69 0.39 0.57 0.39 0.41 0.50

Overall (%) 2000

6.57 5.49 2.90 2.67 2.09 1.69 1.66 1.69 2.37 0.69 1.74 1.38 1.22 0.99 0.96 0.78 0.86 0.36 1.14 0.96 0.64 0.50 0.33 0.41 0.14 0.13

3.76 3.20 2.87 2.69 2.59 1.76 1.51 1.48 1.42 1.24 1.21 1.15 1.18 1.09 1.02 1.00 0.95 0.95 0.85 0.73 0.68 0.49 0.46 0.42 0.31 0.31

The trend is the same for mercury analysis (Table 3). Mercury speciation is extremely important around the world due to the bioaccumulation of methylmercury in fish, the extreme toxicity of dimethylmercury and the general usage of the metal (e.g. thermometers). It should be noted that in surveying the literature, there are many additional papers not included in these statistics that use flow analysis as the analytical tool specifically for mercury in environmental and health monitoring (these are papers where the focus is the analytical method itself). The initial focus on speciation using flow techniques was the discrimination of iron(II) and iron(III) (Table 1 (values given in italics)). Scientists continue to focus on this analysis as there is significant research showing that the ratio of iron(II)/iron(III) is important to the health of the oceans and consequentially to CO2 levels in the atmosphere.

114

Table 2

Stuart Chalk

Arsenic species

Arsenic species

Decade (%)

Overall (%)

1970

1980

1990

2000

Arsenic(total) Arsenite/As(III) Arsenate/As(V) Arsenobetaine Arsenocholine Monomethylarsenic Dimethylarsenic Trimethylarsenic Arsinic acid

0.65 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.80 0.06 0.18 0.03 0.00 0.00 0.00 0.00 0.00

1.94 0.14 0.19 0.20 0.14 0.04 0.04 0.00 0.00

3.67 0.94 0.96 0.53 0.17 0.11 0.14 0.03 0.02

2.34 0.42 0.47 0.29 0.12 0.06 0.07 0.01 0.01

Total

0.65

1.08

2.69

6.57

3.76

Table 3

Mercury species

Mercury species

Decade (%)

Overall (%)

1970

1980

1990

2000

Mercury (total) Mercury (II) Monomethylmercury Dimethylmercury Ethylmercury Phenylmercury

0.00 0.00 0.00 0.00 0.00 0.00

0.61 0.09 0.09 0.03 0.00 0.00

1.62 0.22 0.39 0.01 0.07 0.08

3.40 0.47 1.18 0.06 0.19 0.19

2.06 0.28 0.62 0.03 0.10 0.10

Total

0.00

0.83

2.39

5.49

3.20

Inorganic nutrients such as phosphate and nitrate are also high the list of analytes and have resulted in standard US Environmental Protection Agency methods of analysis. The clinical origins of flow analysis, as a development from segmented continuous flow analysis (CFA) are also seen from the large literature on glucose and amino analysis. This is supplemented by a significant number of papers on the analysis of drugs and enzymes (Table 4).

3. APPLICATION AREAS Over 1,000 different types of sample have been analyzed using flow analysis technology with the majority of these, not surprisingly, in the areas of environmental and clinical analysis. Table 5 shows the top seven sample type

Bibliometrics

Table 4

115

Enzyme and drug analyses

Enzymes

Overall (%)

Drugs

Overall (%)

0.110 0.104 0.069 0.069 0.058 0.046 0.046 0.040 0.040 0.035 0.035 0.035 0.029 0.029 0.029 0.023 0.023 0.023 0.023 0.017 0.017

Acetaminophen Dopamine Isoniazid Tetracycline Atrazine Captopril Morphine Codeine Promethazine Sulfonamides Theophylline Nitroprusside Penicillamine Reserpine Warfarin Digoxin Ranitidine Dopa Isoprenaline Perphenazine Promazine

0.283 0.208 0.144 0.133 0.110 0.098 0.081 0.069 0.058 0.052 0.052 0.046 0.046 0.046 0.040 0.035 0.035 0.029 0.029 0.029 0.029

Enzyme, peroxidase Enzyme, subtilisin Enzyme, triacylglycerol lipase

0.017 0.017 0.017

Total

1.652

Total

0.953

Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme, Enzyme,

Table 5

alpha-amylase lactate dehydrogenase alkaline phosphatase cholinesterase glucose oxidase acetylcholinesterase protease galactosidase urease amyloglucosidase horseradish peroxidase lipase aspartate aminotransferase chymotrypsin creatine kinase acid phosphatase catalase cellulase malate dehydrogenase atpase guanase

Application areas of analysis

Matrix

Overall (%)

Environmental Biological fluid Food and drink Pharmaceutical Agricultural Reference material Drinking water Geological

19.48 15.03 7.57 6.76 5.01 4.40 2.36 2.33

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Stuart Chalk

areas and drinking water (the highest single matrix) that account for over 70% of the papers in the literature. Other areas with significant focus are alloys, vegetables, plants, fermentation broth and industrial and commercial products. The analysis of solid samples is accomplished via injection of either hotplate or microwave digests/extracts, or via slurries. Breaking down these areas further we see some interesting trends. In environmental matrixes (Table 6) the dominant type is waters particularly river and seawater. This is likely due to the ability of flow analysis techniques to preprocess samples of variable salinity prior to detection-and still provide accurate analyses. Also, automation and miniaturization of these analyses makes them easily applied in the field or aboard research vessels trawling the oceans. The analysis of digestates or leachates of soils and sediments is also becoming an important analytical application area. Clinical analysis has been very important to the success of flow techniques in general and Table 7 shows it is still important today. The major biological fluids analyzed are blood and urine with a significant increase in the report of urine analyses in recent years. This is an indicator that more attention is being paid to the metabolism of toxic species, which can be estimated from ingestion/excretion studies. Analysis of other biological samples such as hair, teeth, muscle, kidney and heart tissue have been reported in the literature, but with much lower frequency than for blood and urine. Table 6

Environmental sample types

Environmental

Decade (%)

Overall (%)

1970

1980

1990

2000

Water Estuarine water Ground water Lake water Pond water Rain River Seawater Snow Spring/Mineral water Surface water Well water Other Soil Air Sediment Other

3.90 0.00 0.00 0.65 0.00 1.95 0.00 0.00 0.00 0.00 0.00 0.00 1.30 3.25 0.00 0.00

7.13 0.03 0.18 0.18 0.12 0.98 1.87 2.00 0.15 0.22 0.40 0.25 0.74 1.94 0.55 0.40

11.33 0.41 0.50 0.54 0.19 0.82 2.54 3.70 0.15 0.45 0.51 0.18 1.35 2.58 0.73 0.84

18.01 0.63 0.82 0.82 0.19 0.63 3.39 5.77 0.16 0.85 1.54 0.30 2.93 3.83 1.90 2.04

12.85 0.41 0.55 0.57 0.17 0.79 2.69 4.08 0.15 0.54 0.86 0.23 1.81 3.17 1.11 1.18 1.16

Total

7.14

10.02

15.47

25.77

19.48

Bibliometrics

Table 7

Biological fluid sample types

Biological fluid

Decade (%)

Overall (%)

1970

1980

1990

2000

Blood Serum Plasma Whole Urine Saliva Other

17.53 14.94 1.95 0.65 3.25 0.00

11.92 8.33 2.37 1.23 3.53 0.25

8.79 5.25 2.47 1.07 3.58 0.23

10.13 4.66 3.40 2.07 5.55 0.30

9.89 5.66 2.77 1.46 4.27 0.25 0.62

Total

20.78

15.70

12.60

15.98

15.03

Table 8

117

Food and drink sample types

Food and drink

Decade (%)

Overall (%)

1970

1980

1990

2000

Beverages Wine Beer Soda Other Food

0.00 0.00 0.00 0.00 0.00 1.30

2.80 1.04 1.20 0.03 0.52 1.66

4.59 1.98 1.70 0.09 0.81 2.77

4.49 1.99 1.13 0.06 1.30 5.21

4.14 1.78 1.37 0.07 0.92 3.42

Total

1.30

4.46

7.36

9.69

7.57

Foods and drinks (Table 8) are also major sample types analyzed via flow analysis system. Beer (for a-amylase) and wine (for sulfite/sulfur dioxide and nitrite) make up the majority of the analysis. Milk and milk powder, vinegar, honey, cheese and baby formula make up the majority of the foodstuffs analyzed. The analysis of drugs in pharmaceuticals (Table 9) is another already large and growing area, primarily in the area of dissolution testing of tablets. Agricultural samples (excluding plants or vegetables) like dairy and crops continue to be important (Table 10). An important finding from the database is the increase in the use of standard reference materials (SRMs) to validate proposed new methodologies. Table 11 shows the types of reference materials researchers have used in the literature and the agencies from which they obtained these samples. The increased use of SRMs is not limited to the area of flow analysis as it is becoming important in all areas of analytical science to show equivalency of new methods to existing standard protocols. In the future expect the majority of research papers to include the analysis of at least one reference material.

118

Table 9

Stuart Chalk

Pharmaceutical sample types

Pharmaceutical

Decade (%)

Overall (%)

1970

1980

1990

2000

Ampoule Capsule Cream Drop Herbal Injection Lotion Patch Syrup Tablet Vial Other

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.60 0.65 0.65

0.06 0.25 0.03 0.22 0.00 0.00 0.06 0.00 0.09 1.35 0.00 1.01

0.08 0.27 0.18 0.38 0.12 0.08 0.05 0.00 0.24 2.09 0.18 2.42

0.05 0.39 0.36 0.25 0.42 0.03 0.05 0.03 0.13 2.49 0.52 4.91

0.06 0.31 0.21 0.29 0.21 0.05 0.05 0.01 0.17 2.09 0.27 3.04

Total

3.90

3.07

6.09

9.63

6.76

Table 10

Agricultural sample types

Agricultural

Decade (%)

Overall (%)

1970

1980

1990

2000

Crops Dairy Feed Grain Other

0.65 0.65 2.60 0.00 0.00

2.06 0.98 1.17 0.09 0.22

1.93 1.42 1.09 0.19 0.22

1.69 1.99 1.05 0.31 0.52

1.84 1.53 1.10 0.21 0.32

Total

3.90

4.52

4.85

5.57

5.01

The final major area of application of flow analysis is geological. Just as in the analysis of soils and sediments, these analyses are achieved through hotplate or microwave digestion or leaching (Table 12).

4. DETECTION TECHNIQUES If there is a surprise in the analysis of the literature it is looking at the area of detection techniques. If you were to ask 50 experts of flow analysis about the most widely used detection technology the majority of them would say UV/Vis spectrophotometry. However they would be wrong! Electrochemical analysis

Bibliometrics

Table 11

119

Reference materials

Reference material

Overall (%)

By Standard NRCC CASS Coastal seawater NRCC DORM Dogfish muscle NRCC NASS Open ocean seawater NRCC TORT Lobster hepatopancreas NIST 1643 Trace elements in water NIST 1577 Bovine liver NIST 2670 Toxic metals in freeze dried urine NRCC SLRS Riverine water NIST 1568 Rice flour NIST 1566 Oyster tissue NRCC PACS Harbour sediment NRCC SLEW Estuarine water NIES 1 Pepperbush NIST 1633 Trace elements in coal fly ash NIST 1571 Orchard leaves NIES 2 Pond sediment NIST 1648 Urban particulate NIES 3 Chlorella BCR 144 Domestic sewage sludge NIES 5 Human hair Other

0.27 0.23 0.22 0.20 0.18 0.16 0.14 0.14 0.13 0.11 0.11 0.10 0.07 0.06 0.06 0.04 0.04 0.02 0.02 0.02 2.07

Total By Agency National Research Council Canada (NRCC) National Institute of Standards (NIST), USA Institute for Reference Materials and Measurements National Institute for Environmental Studies (NIES). International Atomic Energy Agency (IAEA), Austria United States Geological Survey (USGS), USA British Chemical Standard (BCS), UK Geological Survey of Japan (GSJ) Other

4.40 1.40 0.89 0.41 0.30 0.13 0.03 0.07 0.03 1.14

Total

4.40

(not even including biosensors) in its different forms is the most prevalent analytical technique. Of course if you break it down by individual technique then UV/Vis is the most commonly used. Table 13 shows the breakdown of detection techniques used in the flow analysis literature and highlights nicely the wide range of detectors that can be using with flow analysis systems. Biosensors, ICPMS, ICP-AES and flame atomic absorption spectrometry (AAS) are being more

120

Table 12

Stuart Chalk

Geological sample types

Geological

Decade (%)

Overall (%)

1970

1980

1990

2000

Minerals Rocks Ores Other

1.30 0.00 0.00 0.00

0.43 0.52 0.34 0.31

0.97 0.35 0.20 0.53

1.55 0.24 0.45 0.86

1.08 0.33 0.32 0.60

Total

1.30

1.60

2.05

3.11

2.33

Table 13

Detection techniques

Detection techniques

Decade(%) 1970

Electrochemical Amperometry Potentiometry Voltammetry Other UV/Vis Spectrophometry Scanning Diode array Atomic Absorption Flame Graphite furnace Cold vapor Other Chemiluminescence Fluorescence Molecular Atomic Biosensor Mass spectrometry ICP-MS Atomic Emission ICP Flame Immunoassay Hydride generation AAS HPLC Ion chromatography

27.27 7.14 11.69 7.14 1.30 21.43 21.43 0.00 3.25 3.25 0.00 0.00 0.00 1.30 3.25 3.25 0.00 0.00 0.00 0.00 1.30 0.65 0.65 0.00 0.00 5.19 0.00

1980

20.93 8.24 4.70 3.93 4.06 17.73 16.72 1.01 7.50 5.13 0.55 0.40 1.41 5.04 6.95 6.88 0.06 0.34 0.65 0.55 2.52 2.12 0.40 1.14 0.52 12.48 1.60

1990

21.03 10.19 4.74 3.98 2.12 19.52 18.46 1.07 9.57 3.06 3.85 1.22 1.44 7.86 8.18 7.80 0.38 6.22 4.68 3.02 3.05 2.61 0.45 2.20 1.73 7.60 2.07

Overall(%) 2000

18.30 9.39 3.87 4.99 0.05 20.56 19.59 0.97 14.70 5.35 4.71 3.81 0.83 13.50 11.50 8.00 3.50 7.95 9.49 5.63 4.02 3.70 0.31 2.27 3.03 7.56 2.32

19.91 9.43 4.44 4.34 1.70 19.45 18.44 1.00 10.94 4.27 3.48 2.00 1.19 9.29 9.07 7.61 1.46 5.65 5.62 3.47 3.27 2.88 0.39 1.99 1.95 8.43 2.04

Bibliometrics

Table 14

121

Hydride generation

Hydride generation

Decade (%)

Overall (%)

1970

1980

1990

2000

HG-AAS HG-AFS HG-ICP HG-ETV HG-Other

0.00 0.00 0.00 0.00 0.00

0.52 0.00 0.06 0.00 0.25

1.73 0.07 0.45 0.34 0.61

3.03 2.24 0.60 0.53 1.51

1.95 0.85 0.42 0.34 0.86

Total

0.00

0.83

3.19

7.90

4.43

widely used this decade, as are both molecular and atomic fluorescence. Shown at the bottom of the table are some chromatographic techniques that had been used to separate analytes before detection using post-column reaction of some kind. An interesting focal point of the detection schemes used in flow analysis is the increasing use of hydride formation. As can be seen from Table 14, hydride generation is now used across the whole range of analytical atomic spectrometry techniques, not just with conventional AAS. New approaches, such as volatile trapping in electrothermal vaporization AAS, and application to a wider array of metals have fueled this growth.

5. OTHER INTERESTING STATISTICS There are over 1,400 different primary (corresponding) authors of the publications in the database, and the top authors (with more than 50 papers) are shown in Table 15a. Many of the authors listed in this table are ‘‘pioneers’’ of flow analysis, and this author would like to acknowledge their significant efforts to the progress of the discipline. Table 15b shows the most productive researchers since 2000 (W20 papers). Research in flow analysis has been published in over 1,000 peer reviewed journals over the last 30 years, and Table 16 shows the top journals (W100 papers) overall. The flow analysis community has stuck with the journal that published the first FIA paper, Analytica Chimica Acta, which has become the prominent journal for this area. It is clear from the list of publications, that flow analysis is published worldwide with significant contributions from China, Japan, South Africa as well as the US and Europe. Thanks to the internet, access to the international flow analysis literature is not a significant problem, as more than 85% of the papers in the database are available online.

6. FUTURE TRENDS Based on where the literature is now, there are a few trends one can delineate looking forward to the next 30 years of flow analysis. The first will be the loss of

122

Table 15

Stuart Chalk

Authors of flow analysis publications

(a) Authors (alltime)

Overall (%)

Valcarcel, M. Ruzicka, J. Van Staden, J.F. Luque De Castro, M.D. Fang, Z.L. Zhang, Z.J. Tyson, J.F. Cerda, V. Wang, J. Martinez Calatayud, J. Trojanowicz, M.A. Worsfold, P.J. Hansen, E.H. Dasgupta, P.K. De La Guardia, M. Townshend, A. Motomizu, S. Perez Ruiz, T. Zagatto, E.A.G. Mottola, H.A. Reis, B.F. Molina Diaz, A. Christian, G.D.

1.83 0.94 0.91 0.78 0.69 0.69 0.69 0.68 0.67 0.59 0.52 0.50 0.45 0.44 0.43 0.42 0.39 0.38 0.37 0.33 0.32 0.29 0.29

(b) Authors (since 2000)

Overall (%)

Zhang, Z.J. Cerda, V. Luque De Castro, M.D. Valcarcel, M. Van Staden, J.F. Martinez Calatayud, J. Molina Diaz, A. Martinez, L.D. Song, Z.H. Yebra, M.C. Reis, B.F. Hansen, E.H. Rangel, A.O.S.S. Lu, J.R. Pingarron, J.M. Yan, X.P. Chen, X.G.

1.29 1.10 0.83 0.74 0.72 0.67 0.67 0.63 0.58 0.58 0.53 0.52 0.50 0.44 0.44 0.44 0.42

Bibliometrics

123

Table 15 (Continued ) (b) Authors (since 2000)

Overall (%)

Themelis, D.G. Zen, J.M. Zagatto, E.A.G. Dasgupta, P.K. Grudpan, K. Lendl, B. Perez Ruiz, T.

0.41 0.38 0.35 0.33 0.33 0.33 0.31

Table 16

Major journals for flow analysis publications

Journal

Overall (%)

Analytica Chimica Acta Talanta Analytical Chemistry The Analyst Analytical and Bioanalytical Chemistry/Fresenius Analytical Letters Analytical Sciences Journal of Analytical Atomic Spectrometry Journal of Chromatography A Electroanalysis Bunseki Kagaku Fenxi Huaxue Journal of Pharmaceutical and Biomedical Analysis Journal of Flow Injection Analysis Spectrochimica Acta B Microchimica Acta Biosensors and Bioelectronics Microchemical Journal Sensors and Actuators B: Chemical Journal of Analytical Chemistry Analytical Proceedings Analytical Biochemistry Trends in Analytical Chemistry Journal of the Association of Official Analytical y Journal of Chromatography B

17.31 6.88 5.62 5.45 4.35 2.78 2.69 2.48 2.19 2.19 2.08 1.83 1.51 1.36 1.28 1.19 1.18 1.13 1.02 0.89 0.81 0.77 0.68 0.65 0.60

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Stuart Chalk

the perception that the word ‘‘flow’’ means that solutions must be continuously pumped. This of course was the original implication when the term ‘‘flow injection’’ was coined back in 1975, but today it only applies to how the operations in the instrument are automated/sequenced. Discontinuous flow, introduced along with the concept of ‘‘sequential injection’’, will allow the flow analysis area to evolve and meet the demands of real world applications, e.g., low reagent consumption, low waste generation, high throughput, etc. In addition, bubbles will continue to make a comeback. The ideas proposed in the conceptualization of ZF [3] take us back to the grandfather of flow analysis, segmented CFA. However, while segmented flow introduced bubbles at regular intervals, future introduction of bubbles will be highly controlled and monitored within the flow system. In the last few years there has been an explosion of research in the area of microfluidics and miniaturization of analytical flow systems. Although this area grew, in part, out of micro total analytical systems (mTAS), the area is significantly different due to the scale of the flow channels involved. Thus, a future area for flow analysis might be called ‘‘semi-microfluidics’’, where the scale of conventional flow analysis systems (e.g. flow-rates and volumes) is decreased by a factor of 10, thus remaining in the laminar flow regime, but significantly decreasing the size of the system. This cannot happen without development of new mixing devices, flow-cells and detection algorithms. Along with the move to a smaller form factor, more emphasis will be placed on integrating flow systems into portable and/or remote monitoring devices. Already the notion of networks of remote monitoring systems has been proposed [4], and it is very likely flow analysis will be a big part of this. Automated collection and processing of samples is attractive in such instruments that will be applied to air-quality monitoring, industrial-waste monitoring and ecosystem tracking. The additional rise in global terrorism also presents opportunities for real-time monitoring and feedback for both military and civilian applications. The existing trend of speciation analysis will continue for many years as researchers more fully understand the environmental and health impacts of arsenic, mercury and other heavy metals. Application in the areas of process monitoring and control will continue to increase as detection technology becomes more sophisticated and can process a wider variety of sample types and compositions. As research progresses there will be a large, but ever evolving need for flow analysis technology. It will be interesting to look back in another 30 years and to analyze the additional 20,000(?) research reports that will have been published by then.

ABBREVIATIONS AAS CFA FIA

Atomic absorption spectrometry Continuous flow analysis Flow injection analysis

Bibliometrics

ICP–AES ICP–MC LOV SIA SRMs ZF mTAS

125

Inductively coupled plasma–atomic emission spectrometry Inductively coupled plasma–mass spectrometry Lab on valve Sequential injection analysis Standard reference materials Zone fluidics Micro total analytical systems

REFERENCES [1] [2] [3] [4]

The Flow Analysis Database, http://www.fia.unf.edu. Accessed 1 June 2007. J. Ruzicka and E. Hansen, Flow Injection Analysis, 2nd ed., Wiley, New York, 1988. G. Marshall, D. Wolcott and D. Olson, Anal. Chim. Acta, 499 (2003) 29–40. Chemical Sciences-Dr. Dermot Diamond, http://www.dcu.ie/chemistry/biographies/dermot_ diamond.shtml. Accessed 1 August 2007.


PART II On-Line Sample Manipulation


CHAPT ER

6 On-Line Sample Pretreatment: Dissolution and Digestion V. Cerda` and J.M. Estela

Contents

1. Introduction 2. Dissolution 2.1 Inorganic samples 2.2 Drugs and pharmaceuticals 3. Digestion 3.1 Analysis of metals 3.2 Organic analysis 3.3 Pharmaceutical analysis Abbreviations and Definitions References

129 130 130 132 135 136 144 153 154 155

1. INTRODUCTION The analytical processing of a sample involves three distinct steps, namely: preliminary operations, measurement and transduction of the analytical signal and acquisition and processing of data. Advances in microelectronics, micromechanics and computer technology have enabled the construction of new instruments and apparatuses to develop efficient, even completely automatic analytical methodologies, which have considerably facilitated the chemical processing of samples. However, not all steps of the analytical process have benefited to the same extent from their availability. Thus, there have been dramatic advances in the measurement and transduction of the analytical signal, and also in the acquisition and processing of data, but not quite so in the preliminary operations of the analytical process. This has no doubt been the result of their constituting the most complex and variable step of the three. In



129

130

V. Cerda` and J.M. Estela

fact, preliminary operations include collecting and pretreating samples, their subjection to the analytical reaction and transfer of the reaction mixture to the detection system, the nature of which can vary widely depending on the particular state of aggregation of the sample and the presence of potential interferents in its matrix. Continuous flow analysis (CFA) systems are excellent tools for handling solutions and hence for implementing wet chemical analysis methods. In fact, CFA systems allow the sample to be isolated from its outer environment in order to avoid contamination or analyte losses. This results in substantial savings in sample and reagents, good reproducibility in both implementing the analytical methodology concerned and acquiring and processing analytical data, and facilitates automation. Usually, analytical methods using CFA systems allow analysts to operate in a clean laboratory atmosphere, avoid the need for inconvenient, hazardous, labour-intensive and/or time-consuming tasks and produce less waste; as a result, they are environmentally benign, non-hazardous and compliant with the basic principles of ‘‘green chemistry’’. However, sample collection and pretreatment (particularly by dissolution or digestion) remain highly difficult to implement in CFA systems, especially if the sample is solid or contains a substantial amount of suspended solids. On-line dissolution is intended to facilitate the use of specific instrumental techniques to analyse certain samples or study the release of active principles in pharmaceuticals. Digestion is used to remove potential interferences with the subsequent instrumental measurement, transform the analyte to a measurable form and/or facilitate the measurement of specific parameters by breaking down the sample into more simple components with the aid of time, heat, reagents and/or catalysts. These operations can be performed by hand which can pose well-known health safety and sluggishness problems, or by using a more or less automated system allowing such problems to be lessened by dissolving or digesting the sample on-line for subsequent detection of the analyte which is also usually done on-line. The following sections describe available approaches and methodologies for on-line dissolution and digestion of samples in various states of aggregation spanning a wide range of matrix types.

2. DISSOLUTION 2.1 Inorganic samples Because most analytical instruments are used to handle liquid samples, most endeavors in this area have focused on improving sample dissolution. Also, sample preparation is becoming the largest single contributor to variance in analytical results and also to analysis times and costs. As can be seen from the available literature on the topic, solid samples are inserted mainly as slurries into CFA systems and dissolved in a reactor accommodated in a microwave oven prior to determining their mineral contents

On-Line Sample Pretreatment: Dissolution and Digestion

131

with an atomic spectroscopic technique. Thus, flow injection analysis (FIA) has been used to determine various metals in silicate rocks. The conventional procedure for dissolving geological materials involves heating the sample in an acid medium for a long time under continuous supervision in order to avoid analyte losses. Very often, samples consist of refractory materials or other compounds resisting acid attack, so they require several hours of heating in an acid solution or alkaline fusion for efficient dissolution. Almeida et al. [1] determined magnesium by flame atomic absorption spectroscopy (AAS) following dissolution of the samples in an HF–HNO3 mixture. In this way, they succeeded in analysing (sample preparation included) about 10 samples per hour. The results for reference rock samples were consistent with their certified values. Quaresma et al. [2] used an on-line automated FIA system with microwave-assisted sample digestion to dissolve silicate rocks in an acid medium (viz. an acid mixture consisting of HF, HCl and HNO3) with a view to determining iron. For this purpose, they assembled a continuous flow system from an automatic FIA manifold that was coupled to a flame atomic absorption spectrometer and additionally connected to a focused microwave oven. The relative standard deviation of the method as applied to the rock-certified materials ranged from 1% to 11%. Some dissolution methods for inorganic samples implemented in flow systems require no microwave assistance. Thus, Sweileh [3] accomplished on-line dissolution with borate melt for the simultaneous matrix isolation, concentration and flame atomic absorption determination of lead in phosphate rock. The flow system was capable of handling slurries and afforded solid sample digestion, simultaneous matrix isolation and analyte concentration. Compared with the direct determination by flame AAS, the ensuing method allowed almost complete elimination of matrix effects and provided a 15-fold increase in detectability. Also, it allowed 10 samples per hour to be processed and analysed for lead with a detection limit of 0.13 mg g1 by using simple equipment and a flame atomic absorption spectrometer. The analytical results compared favourably with those obtained by mixed-acid digestion of the phosphate rock in combination with electrothermal atomic absorption spectroscopy (ET-AAS). One other method allows the on-line dissolution of ZnS to determine sulfide in water samples stabilized with zinc acetate, using spectrophotometry in combination with the Methylene Blue method for detection [4]. The stabilization of S2 as ZnS is a widely used strategy in batch analyses for this ion in water. The sample, containing stabilized S2 as ZnS slurry, is injected into a carrier stream obtained by mixing acidic Fe3+ with N,N-dimethyl-p-phenylenediamine. In the reactor coil, S2 is drifted by the acid used to prepare the solutions and H2S is released to react with the reagents forming Methylene Blue. The solutions are stable, no loss of S2 being observed over a period of at least 7 days. However, use of the method of standard additions is recommended with samples having complex matrices. Monitoring analyses of effluents revealed good agreement between the results obtained each day. The analytical throughput was 60 samples per hour.

132


2.2 Drugs and pharmaceuticals The determination of dissolution profiles for solid dosage forms of pharmaceutical products is required by regulatory agencies, pharmaceutical factories and research institutes. Dissolution testing is performed under precisely specified conditions of temperature, volume and stirring rate, which may mimic processes in the human gastrointestinal tract. Because of differences in the content uniformity of pharmaceutical formulations among samples, parallel dissolutions tests (usually six samples per batch) are often required. Typically, such assays are performed by withdrawing filtered aliquots of sample, for analysis from ultraviolet (UV) spectrophotometric or liquid chromatographic measurements. The manual sampling approach, however, is labour-intensive and timeconsuming, and the resolution of the dissolution profile is usually low owing to the limited number of samples, which can be withdrawn. Various types of automated devices for dissolution testing have to date been developed most of which are of the FIA or SIA type. FIA systems have enabled near real-time monitoring of drug dissolution processes with high throughputs, excellent accuracy and precision and low sample and reagent consumption. The literature abounds with examples of the testing and use of these systems. Thus, Valca´rcel and coworkers [5,6] have illustrated the advantages of FIA dissolution systems with a variety of examples clearly showing their flexibility for adaptation to the problems posed by other automatic and non-automatic alternatives. Georgiou et al. [7] studied the long-term stability of a computer-controlled FIA dissolution system for studying sustained-release formulations of iron(II) using its sensitive reaction with ferrozine. According to these authors, an ideal automated dissolution system for sustained-release formulations should be capable of providing the entire dissolution profile over a prolonged period of time; also, the analyser should remain stable over a very long period, reagent consumption should be low and batch operation possible. While FIA systems meet these requirements quite closely, the use of peristaltic pumps as liquid drivers introduces a problem because pump tubes tend to wear with time, they eventually cause flow-rate oscillations affecting the slope of the calibration curve and the system calls for periodic recalibration as a result. Kouparis and Anagnostopoulou [8] reported an automatic FIA system for the determination of sulfonamides using the Batton–Marshall reaction for clinical analysis, assays and dissolution studies of formulations. The method was used to conduct automated dissolution studies of tablets, and also to determine sulfonamides in control serum and urine samples, feeds and formulations, using the pseudo-titration technique. Lamparter and Lunkenheimer [9] developed a fully automated system for dissolution testing known as AUTO DISSs which allows the on-line determination of active ingredient concentrations with the aid of an integrated autosampler in combination with various measuring instruments. The effectiveness of the system was demonstrated by studying the dissolution of brotizolam from tablets by FIA and that of bepafant from capsules by diode-array spectroscopy.


133

Potentiometry and fluorimetry have also been used for the continuous monitoring of drug dissolution processes in response to the lack of selectivity or sensitivity of UV–Vis spectrophotometry in some situations. Thus, Solich et al. [10] designed a diflunisal ion sensor for assay, content uniformity and dissolution studies of formulations in automated flow-injection systems. No serious interference from common ions or tablet excipients was encountered. Also, the drug was directly determined in coloured samples without the need for a prior separation. The ensuing automated method for dissolution testing provided the entire dissolution profile. The previous authors proposed using fluorimetry to monitor dissolution tests of lisuride tablets [11] and bumetanide in pharmaceuticals [12]. Worth special note here is a system using fluorimetric detection for the simultaneous on-line dissolution monitoring of multi-component solid preparations containing vitamins B1, B2 and B6 by use of a fibre-optic sensor [13]. A new means of in vitro therapeutic drug monitoring was developed by combining the fibre-optic sensor technique with a chemometric method. An artificial neural network method was employed to construct the mathematical model for the simultaneous analysis of the mixture of vitamins B1, B2 and B6 using synchronous spectrofluorimetry. The system provided an advantageous new methodology for simultaneous on-line process monitoring of therapeutic drugs and their metabolites in biological body fluids. The earliest automatic sequential injection analysis (SIA) system for monitoring drug dissolution processes was reported in 1998. The original system allowed the automatic monitoring of the dissolution profiles for ibuprofen tablets, sustained-release capsules and controlled-release matrix tablets [14]. A computer was used to control the movements of a syringe pump and a multi-position valve. Six of the eight ports of the multi-position valve were connected to six vessels of the dissolution tester via six on-line filters and transport lines (Figure 1). After an aliquot of each aspirated solution was delivered into the transferring line connected to the spectrophotometric detector, the residual solution in the holding coil was fed back to the dissolution vessel in order to clear the filter and reduce sample consumption. Every hour, 7 samples run with 6 replicates were processed with a total of 42 measurements. The results provided by this SIA system testify to the operational simplicity of SIA, the ability to computer-control the analytical process, its substantial savings in sample and reagents and its increased robustness and reliability relative to FIA systems, particularly during prolonged operation. The only disadvantage of SIA dissolution relative to an FIA dissolution system is that the throughput is somewhat lower with the former owing to the additional time needed to fill the syringes. For most uses, however, the throughput is still adequate, even for fast dissolution studies performed with six dissolution vessels. This SIA design has also been used for the simultaneous monitoring of aspirin, phenacetin and caffeine in compound aspirin tablets using partial leastsquares calibration [15]. As with FIA systems, potentiometry and fluorimetry have also been used to monitor drug dissolution in SIA. Thus, an automatic method for formulation

134


6

VALVE1

VALVE 2

5

7

S

4 3 2

8

HC

1

9

C

10

W

TC C

D

W Personal Computer PUMP

V1 V2 V3 V4 V5 V6 Dissolution Tester

Figure 1 Schematic diagram of the SI drug-dissolution system. C, carrier; HC, holding coil; TC, transferring conduit; Valve 1, 10-position selection valve (showing position with holding coil connected to detector); Valve 2, two-way valve of the syringe pump; S, standard; W, waste; D, detector; V1–V6, dissolution vessels. Reprinted from Ref. [15]. Copyright (1999), with permission from Elsevier B.V.

assays and dissolution tests exists based on an SIA system including a tubular salicylate-selective electrode as sensing device. The SIA set-up was used to assay acetylsalicylic acid in plain tablets, composed tablets and effervescent tablets, and also for dissolution testing of normal and sustained-release tablets [16]. Legnerova et al. proposed using an automatic SIA system for the fluorimetric determination and dissolution testing of ergotamine tartrate in pharmaceuticals [17] and prazosin hydrochloride in tablets [18]. The fully automated SIA systems used for drug dissolution testing in the previous examples were based on the coupling of an SIA manifold to a conventional dissolution system (i.e. stirred thermostatted vessels). Klimundova and coworkers [19,20] used fully automated SIA systems coupled to a Franz diffusion cell for the in vitro release testing of semi-solid dosage forms. The Franz cell is a device endorsed both by the FDA [21] and OECD [22]. It consists of a donor part and an acceptor part separated by a membrane, which enables skin permeation tests and might be useful as a surrogate for bioavailability. Originally, these authors [19] used their automated SIA system to monitor release profiles for various ointments containing salicylic acid. Thus, they used the fluorescence of salicylic acid for its fluorimetric detection. Subsequently they coupled a Franz diffusion cell to a fully automated sequential injection chromatography (SIC) system furnished with a Chromolith Flash RP-18 monolithic column and a UV detector for the simultaneous release testing of lidocaine and prilocaine in topical pharmaceutical formulations (Figure 2) [20].


135

HOLDING COIL WASTE

DETECTOR S1 CARRIER

Monolithic column Donor compartment

S2

MEMBRANE

Standards S3 50% ethanol

Acceptor compartment Stirrer FRANZ DIFFUSION CELL

Figure 2 Schematic diagram of the SIC system connected to a Franz cell for release test measurements. Reprinted from Ref. [20]. Copyright (2004), with permission from the American Association of Pharmaceutical Scientists.

3. DIGESTION A number of strategies have been devised with a view to accomplishing faster, easier, more automated sample preparation which may include test-tube heating block digestion, fusion with suitable fluxes, sequential closed vessel microwaveassisted digestion, selective leaching of the target analyte and direct aspiration of a sufficiently small particle size slurry. The development of dissolution/digestion techniques using microwave radiation has provided many advantages over classical and non-classical alternative methods including shorter solubilization times, the ability to digest difficult matrices and that to effect dissolution in closed systems, which reduces losses of volatile analytes and atmospheric contamination, instrumental simplicity and low operational costs. As a disadvantage, safely operating with microwaves involves high equipment costs. Microwave radiation has been used to digest biological materials and organic matrices and, much less often, inorganic matrices. The digestion unit can be placed in front of or behind the injection unit in the manifold. In the former arrangement, the sample is introduced into the microwave oven in a continuousor stopped-flow mode and, following digestion, a discrete aliquot is delivered to the detector. In the latter arrangement, the injected sample, together with the reagents to be digested, is propelled to the microwave unit, where it is also cooled and degassed prior to delivery to the detector. Developments in microwave sample preparation have been the subject of several specialized reviews [23–29].

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3.1 Analysis of metals On-line mineralization of samples with microwave assistance in flow systems has been more extensively used than other mineralization methods; in most cases, the nature of the analytes imposed the use of an atomic optical detection technique such as flame atomic absorption spectrometry (FAAS), inductively coupled plasma-atomic emission spectrometry (ICP-AES), hydride generation atomic absorption spectrometry (HG-AAS) or electrothermal-AAS (ET-AAS). In any case, applications can be distinguished in terms of the state or aggregation of the sample (solid or liquid).

3.1.1 Solid samples Coupling the digestion of solid samples to an FIA system is made difficult by the typically long times required for digestion, which detract from throughput, and by the nearly invariable need to filter the insoluble residue left by many sample preparation procedures. Injecting a fixed volume of a solid sample into a flow system entails using a homogeneous suspension. This can be accomplished by aspirating a finely powdered sample suspension in an appropriate liquid containing anti-settling agents under efficient stirring. This measure is indispensable for volumetric sampling and necessary for complete transfer of the slurry to the digestion unit. Particle size distribution and settling continue to be the sources of major problems in these systems, however. Slurry injections with conventional valves cause frequent system blockages; also, they can scratch soft moving parts in valves and lead to leakage and eventual failure. Aspiration by peristaltic pumps suffers from incomplete sample transfer, potential losses on pump tube walls and connectors and carry-over between samples. Moreover, on-line sample digestion causes the formation of gas bubbles and pressure to build up during the dissolution step. A de-bubbling unit must therefore be used prior to a continuous flow spectrophotometric or potentiometric detection step. Obviously, only complete dissolution can prevent the clogging of tubes by insoluble residues. However, complete dissolution may require lengthy multistep digestion and harsh conditions, which do not lend themselves easily to automation. In many cases, partial dissolution suffices to reduce matrix interferences, and save time and reagents. Karanassios et al. [30] developed and effective stopped-flow system for the microwave-assisted wet dissolution and digestion of solid samples where a coiled Teflon PFA tube served as both a sample container and a digestion vessel, and the sample plug, consisting of a water slurry mixed with an acid mixture, was pumped into the coil. After the sample flow was stopped, the coiled tube was sealed and microwave power applied for 2 min in order to digest the sample (powdered botanical and biological reference materials). The resulting digests were analysed by inductively coupled plasma-atomic emission spectrometry. Elemental recoveries were found to be dependant on sample type and digestion time, and comparable with, and occasionally better than, those obtained by hotplate digestion for 3 h. Haswell and Barclay [31] developed another FIA system for on-line microwave digestion of slurried samples with direct elemental (Ca, Fe,


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Mg and Zn) determinations by flame AAS. Organically based elemental reference samples were prepared as slurries in 5% v/v HNO3 and bubble formation during digestion was controlled by post-digestion cooling and pressure regulation. The major source of error was found to be in the dispersion of solids (o180 mm) as slurries in dilute HNO3. The analysis time was 1–2 min per sample. Mineralizing biological samples is a frequent need in analytical procedures and can be made easier by using on-line automated methods. Burguera et al. [32] developed a focused microwave-assisted procedure for the mineralization of adipose tissue and determination of iron and zinc by graphite furnace AAS. Samples were mineralized by using a mixture of sulfuric and nitric acids, and alternately exposing the sampling unit in the microwave-irradiated zone. Additional streams of Triton X-100 and Pd–Mg matrix modifier were inserted into the system in order to avoid the detrimental accumulation of solids on tubing walls and minimize matrix interference effects, respectively. Selected aliquots of mineralized samples were introduced by means of a sampling arm assembly, using air displacement, into the graphite tube atomizer. The main advantage of the ensuing method is it allows iron and zinc in adipose tissue to be determined in a totally closed system with minimal sample manipulation, exposure to the environment and operator intervention. Gluodenis and Tyson [33] developed a double FIA manifold incorporating a resistively heated oven for directly coupling the digestion of solid samples with an analytical spectrometric technique. The potential of the system was illustrated with the dissolution of samples and subsequent determination of copper and iron by flame AAS. Cocoa powder was slurried in 10% nitric acid, injected into the manifold and digested under stopped-flow, high-pressure conditions. Gas–liquid separation was effected by a two-stage de-pressurization system. The procedure provides a clear solution and affords an increased sample throughput while minimizing sample contamination and decreasing sample and reagent consumption. In subsequent work, the previous authors [34] used an FIA system incorporating a stopped-flow microwave-heated reactor to prepare solutions for the subsequent determination of various minor elements in cocoa powder, horse kidney and coal by atomic spectrometry. Slurry samples were injected into the manifold and transferred to a glass reactor mounted inside a microwave oven for dissolution or acid digestion of the samples. After cooling, the reactor was vented and its contents were flushed out into a calibrated flask for dilution to volume. A comparison of the results revealed no significant difference in trace element contents of the previous materials between the FIA method and other sample preparation methods. Also, an analysis of variance showed no indication of sample slurry heterogeneity. Recoveries were low for the coal material, possibly as a result of incomplete dissolution of its silicate constituents. A high-temperature/high-pressure on-line flow digestion system for biological and environmental samples has been proposed for the determination of metals by plasma optical emission spectroscopy. The system includes a novel type of interface intended to remove evolved gases during digestion (CO2 and NOx), which have a strongly adverse impact on ICP stability [35]. The interface consists of a porous polypropylene tube with an inner rod made of PEEK which

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causes the formation of a thin film of liquid at the inner surface of the porous tube, thereby considerably increasing the ratio of gas exchange area to liquid volume and simultaneously decreasing the dead volume. The recoveries thus obtained are quite good for most metals except Sb, Ag and Ga. NBS certified samples of bovine liver, tomato and orchard leaves, pine needles and spinach were injected as slurries into the system in order to determine Al, B, Ba, Ca, Cr, Cd Cu, Fe, Mg, Mn, Mo, Ni, Pb, Sr and Zn. Finally, Sturgeon et al. [36] tested a continuous-flow microwave-assisted digestion technique for the decomposition of environmental samples. A CEM SpectroPrept system was used at moderate powers and pressures to effect the on-line digestion of slurried samples of biological tissues and marine sediment. The efficiency of oxidation of biological matrices, as characterized by the residual carbon content of the solutions, was 64%. Recoveries of trace elements were 9071% and accommodated with the use of suitable internal standards. Accuracy was verified by analysing certified reference materials from the National Research Council of Canada (viz. marine sediment BCSS-1 and lobster hepatopancreas tissue LUTS-1). The precision of measurements, as reflected in the determination of trace metal contents in replicate solutions by using a variety of atomic spectrometric techniques, was better than 1% as relative standard deviation. The determination of metals in sediments is one other analytical area of special interest. A number of methods for on-line digestion of solid samples have been reported. Most are FIA methods where the sample is transferred as an acid/ oxidant slurry for microwave-assisted digestion/dissolution, which is followed by direct insertion into the flame of an atomic absorption spectrophotometer for detection without the need for filtration. Burguera et al. developed several such methods for the determination of lead in sewage sludge [37], and that of Cu and Mn in sewage sludge and diet samples dispersed in a mixture of HNO3 and H2O2 and digested for 2–4 min [38], which afforded a throughput of more than 15 samples per hour. De la Guardia et al. [39] proposed a similar FIA method for determining Cu and Mn in various solid matrices including vegetables, powdered dietary products and sewage sludge. The use of an appropriate interface to digest, cool and degas samples prior to insertion into the nebulizer of a flame atomic absorption spectrometer facilitated full automation of the digestion and measurement steps for the elemental analysis of solids and resulted in a throughput of 180 injections per hour. The ensuing method was also used to determine Pb and Zn in certified sewage sludge samples; the results were accurate for Pb, but underestimated for Zn. Mercury in environmental samples has also been determined by directly injecting them in the form of acid slurries into a hydrogen chloride carrier leading to a microwave oven for digestion and subsequent detection of mercury with an on-line cold vapour (CV) atomic fluorescence spectrometer. Morales et al. [40] reported a fast method (15 samples per hour) for the determination of mercury in BCR-certified sewage sludge homogeneously dispersed in nitric acid. Lamble and Hill [41] used the previous digestion and analysis procedure with the National Research Council Canada certified reference materials DORM-2 and


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PACS-1; however, they injected the slurried samples into a carrier stream of hydrochloric acid that was merged with a solution of potassium bromide– bromate before passing through a PTFE coil accommodated in the microwave cavity of the microwave digester. Following mixing with a solution of hydroxylammonium chloride to remove excess bromine, mercury was determined by its atomic fluorescence. The throughput was similar to that of the previous method. The dissolution/digestion of pharmaceuticals and foods with a view to determining their mineral components is another field that has benefited from the ability to manage solid samples. The procedure is similar to those described above. Thus, samples are carried in the form of slurries and pre-heated in the presence of acids and/or oxidants for detection by atomic spectrometry. The on-line FIA system with solid sample introduction, digestion, treatment and determination of Fe, Zn and Cu in multi-vitamin tablets reported by Sweileh [42] and that for the simple, rapid FIA–HG atomic absorption spectrometric determination of lead in wine, other beverages and fruit developed by Cabrera et al. [43], constitute two salient examples. In the former system, the solid sample powder is inserted into a special chamber and carried by the digestion solution to a thermally heated PVC coil (Figure 3). The analyte metal, in the form of a chlorocomplex, is retained on a coarse-particle anion-exchange resin mini-column. The resin beads are held between two plastic screens, which allow the insoluble residue to pass through to waste. After a brief column wash, the analyte is eluted with dilute HNO3 and determined spectrophotometrically or by atomic absorption. The novel configuration of the multi-channel pinch valve allows slurries to be handled without tube blockage or valve damage. In the latter system, lead hydride was generated in an HNO3–H2O2 medium using NaBH4 as reducing agent. To determine lead in beer, juice and fruit, a microwave oven was coupled on-line to the HG atomic absorption spectrometric system; with fruits, however, the lead hydride was generated from slurries of fresh sample. No matrix effect was observed in the determination of lead and the method afforded the direct determination of the metal in untreated samples by using an aqueous calibration graph. The detection limit was 10 mg L1 in wine and other beverages, and 1.0 ng in fruits. An FIA system similar to that reported by Haswell and Barclay was used to prepare environmental samples for the determination of Pb by using isotope dilution ICP-MS [44]. Samples of leaves, air filters, urine, sludge, dust and paint were introduced as slurries and microwave irradiated in the presence of an acid mixture (HClO4/HF/HNO3). One other heating technique comparable with microwave-assisted heating in performance is electromagnetic induction heating. This is an effective choice for processing samples, saving energy and reducing processing times [45] In fact, this technique was previously used to prepare samples for the determination of Hg in fish [46] (Figure 4) and mainstream smoke [47]. An on-line flow method of higher efficiency and heating rate was recently developed by using electromagnetic heating [48]. The custom-made electromagnetic heating column (f ¼ 1 mm) consisted of a PTFE outer tube and seven

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S V1

V2

T1

HCI

V4 V3

DU

H2O2

1

W

2 W P

T2 T6

3

T T3

CARRIER T5

KSCN

4 C

T4

5

RC

6 T7

W 7 W SP

INT

PR

Figure 3 Schematic diagram for on-line solid sample digestion and flow injection analysis with spectrophotometric detection. P, peristaltic pump; HCl, carrier digestion solution; H2O2, oxidant; CARRIER, eluent carrier, 0.10 M HNO3 solution; V1, V2 and V3 are three-way solenoid valves; S, solid sample insertion device; DU, thermal digestion unit; V4, multi-channel pinch solenoid valve; T, programmable timer; C, anion exchange mini-column; RC, reaction coil; SP, spectrophotometer; INT, integrator; PR, printer; W, waste; line thicknesses indicate relative internal diameter size, and dashed lines are electrical connections. Reprinted from Ref. [42]. Copyright (2000), with permission from Elsevier B.V.

EMIO digester

HCI / A

AFS

K2S2O8 Quartz cell Argon GLS KBH4 Waste Pump

Figure 4 Schematic diagram of the AFS with on-line digestion manifold: A, analyte; EMIO, electromagnetic induction oven; GLS, gas–liquid separator; AFS, atomic fluorescence spectrometry. Reprinted from Ref. [46]. Copyright (2006), with permission from Elsevier B.V.


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compactly packed PTFE coil-coated iron wires. The pre-digested sample solution, prepared by mixing 0.5–0.1 g of tea leaves with an acid decomposition solution (HNO3+HClO4), was pumped through the electromagnetic heating column and directly transferred to a flame atomic absorption spectrophotometer. An analytical throughput of 72 samples per hour was thus obtained. According to its proponents, this heating technique features a high efficiency by virtue of the heating energy being concentrated in a thin layer beneath the surface of the heated material and it may prove a useful alternative to existing digestion heating techniques.

3.1.2 Liquid samples On-line digestion systems for the determination of elements occurring as or forming volatile species (e.g., mercury) and hydride-forming elements (e.g., As, Sb, Se, Sn, Pb) are widely used to facilitate and accelerate digestion, and also to avoid losses during the pretreatment of samples. Microwave-assisted digestion occupies a prominent place in this context. Usually, an aliquot of sample mixed with appropriate reagents is transferred to a microwave oven for digestion, the resulting volatile species being driven to the detection system for measurement. There are several available methods for determining mercury in liquid samples using the above-described method in combination with the CV technique and AAS or AFS. Welz et al. [49] developed and assessed an on-line microwave sample pretreatment system for the determination of mercury in water (river, lake, rain) and urine. The set-up consisted of an atomic absorption spectrometer equipped with a mercury–CV system and amalgamation accessory, a flow-injection system, an autosampler and a microwave digester. Urine and environmental water samples were stabilized with potassium dichromate–nitric acid and mixed with a bromination reagent. The throughput was 30–40 samples per hour without amalgamation and 24 samples per hour with amalgamation. Guo and Baasner [50] conducted this determination in whole blood by using a similar method involving the same detector type. Following dilution of the whole blood and addition of an oxidant, all further treatment and measurements were performed automatically on-line. The throughput was in the region of 45 measurements per hour. HG in FIA systems provides many advantages over conventional approaches including the ability to process smaller samples, higher throughputs, better tolerance to interferences, improved absolute detection limits, lower consumption of reagents and greater ease of automation [51]. However, transforming the analytes into an appropriate oxidation state for HG and destroying organic matter detract from expeditiousness in the process. On-line microwave digestions are ideally suited to FIA–HG systems and have been extensively used to shorten pre-detection steps. As noted earlier, liquid samples are mixed with an appropriate reagent (e.g., a bromination agent or peroxydisulfate to digest urine and waters for the determination of As, Sb, Sn and Pb). The mixture is transferred to the microwave oven and the resulting digest mixed with sodium borohydride to form the corresponding hydride, which is swept to the quartz cell of an AAS detector.

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Arsenic is an especially interesting element on account of its toxicity and has been the subject of various FIA-HG-AAS procedures involving microwaveassisted digestion. Inorganic arsenic levels in blood samples have been determined using the above-described procedure. However, because the digestion/pre-reduction reagent contained l-cysteine in addition to nitric and perchloric acids, the merging-zones mode was used in order to save amino acid [52]. A similar system was used to determine total and toxic arsenic in urine samples with and without oxidation, respectively [53]. Total arsenic was determined by converting arsenicals to arsenate with alkaline peroxydisulfate. This procedure affords speciation. Thus, Burguera et al. [54] developed an on-line FIA–HG/atomic absorption spectrometric method for the preconcentration and selective determination of inorganic arsenic, As(III) and As(V), and its methylated species in aqueous samples. Separation of species was based on a pH selective procedure: arsine from As(III) alone was generated in the presence of citric acid while nitric acid was used to generate the corresponding arsines from total inorganic arsenic, monomethylarsine and dimethylarsine. The previous species were cryogenically trapped in a PTFE coil knotted and sealed inside another tube of a wider diameter placed in a microwave oven through which liquid nitrogen was aspirated by negative pressure. Based on their different dielectric constants, the arsines were selectively released by using a heating cycle of microwave radiation. The HG process for selenium involves reducing Se(VI) or any of its organic forms to Se(IV), which can react with NaBH4 to form SeH2. The sample is first analysed for Se(IV) and then heated with hydrochloride acid to reduce Se(VI). There are several procedures using microwaves to facilitate reduction which also afford speciation and rely on HG-AAS [55–57] or FIA cathodic stripping voltammetry [58] for detection. Flow-injection microwave-assisted systems coupled to separation units facilitate detection for speciation purposes. One method using such a system has enabled the determination of arsenobetaine in canned seafood by coupling HPLC, microwave-assisted oxidation and HG-AAS. The lyophilized sample [59] was mixed with methanol–water and the extract evaporated to dryness, redissolved in HCl and its pH adjusted below 2. The solution was passed through a strong cation exchanger, arsenobetaine sorbed being eluted with phosphate buffer and mixed on-line with alkaline peroxydisulfate before entering the PTFE tube accommodated in the microwave oven. The thermo-oxidized effluent, cooled in an ice-bath, was also introduced on-line into a continuous HG-AAS system. Other systems allow Se to be speciated following chromatographic separation of its different forms. Trimethylselenium, Se(IV) and Se(VI) in tap water have thus been determined by HG-AAS [60] following separation on an anionexchange column and microwave-induced thermooxidation of trimethylselenium in the presence of peroxydisulfate and thermoreduction of Se(VI) to Se(IV) in HCl by heating in a microwave oven. The system proposed by Gonza´lez et al. [61] allows the reliable speciation of selenoaminoacids (viz. selenomethionine and selenoethionine) in urine versus total inorganic selenium in a single injection. Further speciation of the overlapped inorganic Se(IV) and Se(VI) peaks is


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accomplished by using a second injection of the urine sample in order to determine Se(IV) alone by avoiding microwave heating. The experimental set-up consists of a microwave digestion–HG-HPLC system coupled on-line with three atomic detectors (viz. an atomic absorption spectrometer (AAS), an ICP-AES and an ICP-mass spectrometer (ICP-MS)). A number of FIA on-line digestion methods for determining other metals in a variety of matrices have also been reported. Thus, a fully automated computer monitored set-up for the determination of metals in whole blood by on-line microwave-assisted mineralization with FAAS detection was developed by Burguera et al. [62]. A double injection valve was used to introduce, in the merging-zones mode, the sample and reagent into a flowing stream of water. The mixture was passed through a Pyrex coil placed in a domestic microwave oven and the digested plug then driven to the AAS nebulizer in order to measure Fe, Cu and Zn. The residence time of the sample–reagent mixture in the oven was long enough for the sample to be mineralized while avoiding the production of abundant acid fumes and water vapour during digestion. Subsequently, the previous authors reported a similar, albeit more automated system for determining Cu and Zn in whole blood [63]. Electromagnetic induction-assisted heating has been used for on-line oxidation coupled to atomic fluorescence spectrometry in order to prepare samples for the determination of total and inorganic mercury in fish samples [46]. The injected sample was a previously centrifuged acid extract of a fish homogenate. Potassium peroxodisulfate was used as oxidant in order to decompose organomercury compounds and, depending on the temperature employed, inorganic or total mercury was determined. A potassium tetrahydroborate (III) solution was used to reduce Hg(II). UV-assisted digestion has been used as an alternative to microwave-assisted digestion. Thus, Wurl et al. [64] developed an FIA device for on-line digestion affording the determination of total Hg in the River Elbe by using FIA cold vapour atomic absorption spectroscopy. Three different digestion methods (viz. chemical, microwave- and UV-based) were tested, and the best results were obtained using UV-assisted digestion, with each complete analysis taking only 4 min. Point et al. [65] proposed an integrated approach to the accurate determination of total, labile and organically bound dissolved concentrations of trace metals (Cd, Cu, Mn, Ni, Pb, U and Zn) in the field. Two independent automated platforms consisting of an UV on-line unit and a chelation/preconcentration/ matrix elimination module were specifically developed to process samples onsite in order to avoid the need to store them prior to analysis by inductively coupled plasma mass spectrometry (ICP-MS). The influence of UV photolysis on organic matter and its associated metal complexes was investigated by using fluorescence spectroscopy and the ensuing method validated against natural samples spiked with standards of humic substances. The speciation scheme thus developed was applied to two natural freshwater and seawater samples that were processed in the field. Finally, Comber et al. [66] used a simple on-line stopped-flow photolysis system including a UV lamp to determine copper complexes in natural waters. The system was validated in the stopped-flow mode

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by digesting glycine, nitrilotriacetic acid and EDTA complexes of copper. Recoveries were always higher than 90%.

3.2 Organic analysis Monitoring water quality entails determining various parameters including total and organic nitrogen and phosphorus, and chemical oxygen demand (COD), in order to take appropriate actions if necessary. This requires using an expeditious method to mineralize samples. While on-line digestion meets the requirements, some samples such as wastewater usually contain high levels of suspended solids, which call for highly robust methodologies. Detection is usually colorimetric and also performed on-line with digestion.

3.2.1 Organic and total nitrogen Determining organic nitrogen entails digesting the sample in order to convert nitrogen-containing organic compounds into inorganic forms of this element. The digest can be used to determine total nitrogen directly and organic nitrogen by difference. Sample digestion is the most labour-intensive and time-consuming step of the analytical process; hence, it has attracted especial interest from researchers with a view to its automation. Samples can be digested by using the Kjeldahl method or by photochemical oxidation, oxidation with alkaline peroxydisulfate or high-temperature combustion (HTC). The Kjeldahl method, which is the recommended manual choice for this purpose, provides a widely used parameter in the characterization of water, viz., Kjeldahl nitrogen. This parameter can be determined by using an automated segmented-flow system [67] where the sample is digested in a coiled reaction at a controlled temperature and the metal catalyst used in the original method is replaced with a mixture of sulfuric and nitric acids. Detection is performed on-line, using the Berthelot reaction. The ensuing method has been used to determine total nitrogen in various types of water other than wastewater. In fact, its application to wastewater meets with problems such as easy clogging of the digester and low recoveries as a result. This has led to semi-automatic methods being preferred for wastewater. Such methods use a conventional digester in combination with a flow system for treatment and/or reaction [68]. Additionally automating the distillation step complicates things even further, so much so that many researchers have sought effective alternatives to the Kjeldahl method. One is on-line UV photo-oxidation in the presence of an oxidant such as hydrogen peroxide or sodium peroxydisulfate (the APHA method). In this way, organic nitrogen and ammonium ion are converted into nitrite and nitrate ions within a few minutes and determined spectrophotometrically using the Griess reaction (Figure 5). Digestion reactors are usually made of quartz or Teflon. The latter, which are less fragile and easier to handle, have been successfully used to treat samples with widely variable matrices including wastewater in FIA [69,70], SIA (Table 1) [71] and SFA systems [72]. Alkaline oxidation with peroxydisulfate, which is the basis for the Koroleff method [73] is one other highly effective choice for sample digestion. Samples are

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Resin Sample

DB

Photorreactor

Sample

VS1

R1 Thermostatic bath (40°C) VS2 R2 H2O

RC1 V1

RC2

Detector

H2O

Waste

R3

Figure 5 FIA manifold for the determination of nitrates, nitrites and total nitrogen in wastewater. DB, debubbler; VS1 and VS2, switching valves; IV, injection valve; R1, peroxydisulfate reagent; R2, hydrazine reagent; R3, Shinn’s reagent; RC1 and RC2, reaction coils. Reprinted from Ref. [70]. Copyright (1996), with permission from the Royal Society of Chemistry.

Table 1 Comparison of the results obtained with the Kjeldahl method and the SIA-UV method when applied to real wastewater samples [71] Samples

N-Kjeldahl (NK) (mg L1)

NO 2 +NO3 (mg L1 N)

NK+NO 2 +NO3 1 (mg L N)

SIA-UV (mg L1 N)

Difference (mg L1 N)

ALA-2E ALA-2S ALA-3E ALA-3S ALA4S ALA5S E2295 P2295 S2295 E1295 LAG-1 LAG-2 LAG-3

19.6 32.2 39.4 — 15.7 16.5 45.870.5 58.5 50.4 58.8 17.1 12.6 8.7

35.2 10.8 — 34.1 3.9 — — — — — 0.3 0.5 5.3

54.8 44.0 39.4 34.1 19.6 16.6 — — — — 18.3 14.3 14.1

49.872.6 52.773.2 33.071.9 37.072.0 24.670.9 16.571.5 48.872.6 54.373.1 51.071.1 61.972.4 17.4 13.1 14.0

+5 8.7 +6.4 3.0 5.0 +0.1 3.0 +4.2 0.6 3.1 +0.9 +1.2 +0.1

mineralized in an autoclave at 1201C at 2 bar in order to convert nitrogencontaining compounds into nitrate ion. As with the Kjeldahl and photo-oxidation methods, the recoveries for compounds containing N–N or HN ¼ C bonds are usually low [74,75]. Replacing the autoclave with a microwave oven has

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Figure 6 Cross-section of a PTFE microwave reactor: (a) PTFE body, (b) screw cap, (c) connectors, (d) inlet, (e) outlet and (f) glass beads. Reprinted from Ref. [76]. Copyright (1997), with permission from Elsevier B.V.

facilitated the development of an FIA system for determining total nitrogen (Figure 6) [76]. The whole sequence of steps is performed on-line and takes less than 2 min to complete — the throughput is ca. 45 samples per hour. The method was applied to wastewater samples, which were digested while circulating inside a microwave oven. The resulting nitrate was reduced to nitrite with hydrazine sulfate at the oven exit for spectrophotometric detection using the Griess reaction. The joint use of oxidation with alkaline peroxydisulfate and heated capillary reactors furnished with Pt catalysts in flow systems has also enabled the efficient, expeditious determination of total nitrogen in wastewater at a rate of 15 samples per hour [77]. Finally, digestion can be accomplished by HTC of the sample in the presence or absence of a Pt catalyst. This allows all nitrogen forms to be determined with a high sensitivity at a rate of 10–30 samples per hour by using an automatic configuration. Nitrogen compounds are converted into NO, which is detected by the chemiluminescence of its reaction product with ozone. The equipment needed for HTC is more sophisticated than that used in above-described digestion methods. However, the procedure is more effective and can be applied to wastewater containing refractory organic nitrogen compounds [78,79].

3.2.2 Organic and total phosphorus The determination of filterable organic phosphorus (FOP), total filterable phosphorus (TFP), total phosphorus (TP) or total organic phosphorus (TOP) requires the prior digestion of the sample in order to convert organic phosphates into the orthophosphate reactive species. This can be accomplished by using thermal (wet chemical, HTC and fusion and microwave), UV photo-oxidation and combined thermal hydrolysis–photo-oxidation methods.


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The determination of total phosphorus is a very usual practice that can also be performed by FIA. Thus, Korenaga and Okada [80] developed an FIA system for the continuous determination of total P with spectrophotometric detection of molybdophosphate where samples were digested on-line by heating following injection into a stream of peroxydisulfate. Two APHA methods for phosphorus have been proposed both of which rely on Molybdenum Blue chemistry for the spectrophotometric detection of the resulting orthophosphate that differ in the way digestion is performed, namely: by hand [81] or on-line [82]. In the manual method, polyphosphates and organic phosphorus are converted into orthophosphates by digestion with sulfuric acid and peroxydisulfate, respectively. The on-line method additionally requires UV radiation to convert organic phosphorus. Dissolved organic phosphorus can be determined by using the Molybdenum Blue method following mild on-line photooxidative decomposition with a lowpressure mercury lamp [83] or a UV tube [84], using acid or alkaline peroxydisulfate. Low- and high-molecular weight (i.e., dissolved inorganic and organic) phosphorus species have been successfully speciated by using an FIA gel filtration approach [85]. The determination of total dissolved phosphorus using the previous chemistry requires the prior acid hydrolysis of condensed phosphates (viz. pyro-, meta- and polyphosphates) into orthophosphate. Because these species are not susceptible to UV photodecomposition, thermal- [86] and microwave-assisted digestion [87] were used in combination with FIA or hyphenated techniques to obtain recoveries above 85%. The disparate reaction conditions needed to convert simple organic phosphates and condensed phosphates to orthophosphate were achieved using a two-stage procedure involving UV photo-oxidation/thermal digestion with a combined oxidizing/ hydrolysing reagent [88]. One salient feature of the latter approach is the ability to quantify particulate- and colloid-associated phosphates, and hence total phosphorus. With regard to thermal decomposition, the mineralization step can be expedited by using a capillary digester containing a platinum wire acting as catalyst for the in-line oxidation of organic species. A selective determination method for orthophosphate and total inorganic phosphate in detergents [89] exists that involves a prior acid hydrolysis step. Orthophosphate was directly determined in the presence of other phosphates by using the kinetic discrimination capabilities of FIA and total inorganic phosphate was measured following on-line hydrolysis of polyphosphates in 2.5 mol L–1 sulfuric acid at 1451C for 50 s. Sodium dodecylsulfate (SDS) was added in order to mask the interference of non-ionic surfactants. The FIA determination of specific fractions of phosphorus using enzymatic reactions with immobilized alkaline phosphatase [90,91] or 3-phytase [92] in combination with the Molybdenum Blue method has potential use as an indicator of the pool of biologically available and unavailable dissolved organic phosphorus, respectively. Alkaline phosphatase immobilized on a cellulose nitrate membrane has been used for the rapid spectrophotometric determination of monofluorophosphate [93] and the simultaneous spectrophotometric determination of orthophosphate and monofluorophosphate [94] in toothpaste following on-line hydrolysis in a flow-injection system.

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With regard to electrochemical techniques, the development of an FIA potentiometric method [95] which uses a second-species cobalt wire ISE to determine cobalt phosphate following precipitation as orthophosphate from wastewaters and an FIA amperometric method for the determination of total phosphorus in domestic wastewater by using continuous microwave ovenassisted decomposition and subsequent detection of orthophosphate [96]. Other flow systems have also been used for the spectrophotometric determination of total phosphorus. Originally, Lima et al. [97] proposed the colorimetric determination of phosphorous in milk by FIA using thermal/UV-induced digestion to convert all forms of P into orthophosphate on-line. Subsequently, they adapted the previous methodology to SIA [98]. Almeida et al. [99] proposed a multi-syringe method based on the Molybdenum Blue reaction where the sample and digestion solution are dispensed simultaneously delivered to a digestion vessel placed in a domestic microwave oven. The digested sample is then merged with appropriate reagents for the colorimetric determination. The ensuing method was applied to wastewater samples and the results obtained were consistent with those of the reference method. Repeatability was good and the throughput 12 samples per hour. Pons et al. [100] determined dissolved orthophosphate and dissolved organic phosphorus in wastewater samples by using a multi-pumping flow system. On-line UV photo-oxidation was used to mineralize organic phosphorus to orthophosphate prior to detection using the vanadomolybdate method. A solenoid valve allowed the flow to be diverted to a UV lamp in order to determine organic phosphorus and an injection throughput of 11 samples per hour for the procedures involving UV photo-oxidation was achieved (Figure 7). Using the multi-pumping

UV-lamp M4 DB

P C3 ON

Reaction coil

M1 S OFF

C1

D

W

C2

V1

M3 M2 C

R

Figure 7 Experimental MPFS for the determination of orthophosphate and organic phosphorus. S, sample; C, carrier; R, chromogenic reagent; P, peroxydisulfate solution; DB, debubbler; W, waste; D, detector; M1–M4, micro-pumps; V1, commutation valve; C1–C3, cross-junctions. Reprinted from Ref. [100]. Copyright (2006), with permission from Elsevier B.V.


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flow system avoided the need for time-consuming steps in the reference methods. Also, reagent consumption was minimal by effect of each micro-pump being operated individually to propel the fluids and reagents, which are inserted into the system only when needed.

3.2.3 Protein digestion Proteomic research has focused on the identification and characterization of proteins encoded by the genomes as efficiently and rapidly as possible. Usually, this is accomplished by using mass spectrometry (MS); however, because fragmentation and ionization are scarcely efficient, the results are of dubious value with a view to unequivocally establishing sequences. This shortcoming is usually circumvented by degrading the protein concerned. To this end, the protein is subjected to in-gel or in-solution proteolytic digestion prior to peptide mapping and fragmentation with electrospray ionization tandem MS [101] or matrix-assisted laser desorption/ionization MS [102]. Proteolytic digestion can be done with one or several proteases. Trypsin is highly efficient and widely used for this purpose. Also, in-solution digestion, which is more common than in-gel digestion, requires using a low enzyme concentration and long incubation times in order to avoid the self-digestion of trypsin, which would hinder the unambiguous assignment of the protein sequence. Immobilizing trypsin reduces autolysis and allows higher enzyme concentrations to be used; this shortens the digestion time, increases the enzyme stability against chemical denaturants and organic solvents, and facilitates its isolation and removal from the protein digest before the MS technique is applied. The enzyme can be immobilized on a porous silicon matrix, glass, a gel or a porous monolithic material. Calleri et al. [103] constructed a trypsin-based bioreactor for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support that was subjected to a single reaction step. The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins using electrospray ionization tandem mass spectrometry. The efficacy of the reported on-line bioreactor for tryptic mapping was evaluated by using somatostatin and myoglobin as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared with others obtained by traditional off-line digestion. The sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of the myoglobin sequence) was comparable to that provided by the off-line protocol (18 peptides, 76.47% coverage). Porous monolithic materials have been regarded as ideal supports for the immobilization of enzymes and fast conversion of substrates A monolithic enzymatic microreactor prepared in a fused-silica capillary by in situ polymerization of acrylamide, N-acryloxysuccinimide and ethylene dimethacrylate in the presence of a binary porogenic mixture of dodecanol and cyclohexanol was found to exhibit very low back-pressure and afford the fast digestion of proteins [104]. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at a high flow rate. In-solution digestion and on-column reaction were compared using a nano-high performance liquid

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chromatography–mass spectrometry system. A mixture of four standard proteins was digested and analysed using on-line digestion in combination with the nano-HPLC–MS system and the results exposed the promise of such a system for the analysis of protein mixtures. A method for integrating nano-electrospray mass spectrometry with a microreactor for on-line digestion and fast peptide mass mapping from dilute protein samples was proposed by Zhao et al. [105]. Fused-silica capillaries were employed as digestion microreactors and the nano-electrospray emitter constructed by immobilizing trypsin onto the surface of the inner wall of the fused silica capillary tubing. The procedure was demonstrated by using solutions of angiotensin II, cytochrome c, hemoglobin and b-casein. Because the inner walls of the capillaries were modified by covalent chemical bonds, adsorption of peptides and proteins on them was efficiently suppressed. The method allowed the generation of tryptic peptides with sequence coverages of up to 90% within minutes. Also, no trypsin autolysis was detected and the immobilized enzyme was easily cleaned in order to reuse the microreactor for nano-electrospray MS. The miniaturization of analytical systems has aroused vast interest. A miniaturized on-line digestion system was used by Hedstrom et al. [106] for the sequential identification and characterization of the proteins chloroperoxidase, staphylococcal enterotoxin B and protein A. The system used a fused silica capillary packed with a zone of trypsin-modified Eupergit C beads and a second zone of reversed-phase C18. The proteins were first digested in the trypsin reactor portion of the column and the resulting peptides trapped in the C18 part, shaped as an electrospray emitter. Following washing of the capillary, the peptides were eluted by using an increased concentration profile of organic solvent created by a dual syringe pump system in order to facilitate the release of bound peptides for their identification by electrospray ionization MS–MS. Microfluidic systems are especially suitable for the high-throughput detection and characterization of large sets of proteins necessary for proteomics. Recent efforts in the field of micro total analysis systems (mTAS) for proteomic applications have included the development of separation devices for proteins and peptides; interfacing microchips to electrospray ionization-mass spectrometry (ESI-MS) [107]; and coupling with previous separation by capillary electrophoresis [108,109]. Some authors have integrated enzymatic digestion in a microchip for the subsequent MS identification of proteins from peptide maps stored in databases. Thus, Peterson et al. [110] used trypsin immobilized on porous monoliths; Wang and coworkers [111,112] employed packed beads immobilized between weirs in a channel; and Wells et al. [113] succeeded in electroimmobilizing trypsin through a conductive membrane. Magnetic particles are easy to handle by using magnets or magnetic coils. Magnetic beads have been used in microfluidic chips for purposes such as immunoassay, DNA hybridization and reverse transcription-polymerase chain reaction (RT-PCR). One of the most interesting properties of suspended super paramagnetic particles is their ability to self-organize in a magnetic field. In fact, they can form organized structures in a direction normal to the magnetic field, their morphology depending on the container geometry, particle density and


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magnetic field history. Such structures have been used as sieving matrices to separate log DNA in a microchip. If the particle concentration is high, the organized structure is lost and a ‘‘labyrinth-like’’ structure formed instead. Slovakova et al. [114] have used grafted trypsin magnetic beads in a microchip to effect protein digestion. Their device uses strong magnets to create a magnetic field parallel to the flow with a strong gradient pointing through the centre of the chip channel. This allows the formation of a low-hydrodynamic resistance plug of magnetic trypsin beads that serves as a matrix for protein digestion. This device provides an inexpensive means of constructing a multi-open tubular-like column of appropriate pore size for proteins. Also, the bead matrix is very easily washed out and replaced. Similar sequence coverage (about 44%) was achieved with MS analysis of the products after 10 min on-chip and after 4 h with soluble trypsin in bulk.

3.2.4 Other on-line digestion processes Oxidizing organic matter for COD determinations, which take about 2 h in a conventional digester, entails using the potassium dichromate–sulfuric acid mixture. The oxidation step can be expedited by using a spectrophotometric flow-injection assembly including a microwave oven [115]. The manifold comprises two lines (one for a water carrier and the other for potassium dichromate–sulfuric acid mixture as reagent), three coils — of which the central one, wound around a specially strong microwave absorber support, acts as reaction coil —, a membrane degassing unit and a spectrophotometric detector. The system was used with well water, river water with low COD levels and wastewater, the results being consistent with those provided by the manual reference method. Cuesta et al. [116] proposed one other FIA method where the sample, held in a coil, was digested on-line with sulfuric acid in a microwave oven at a high throughput (up to 50 determinations per hour); however, this method is based on the measurement of Cr(VI) not reacting with organic matter in the sample by flame AAS. After digestion, evolved gases are condensed by cooling, which is followed by retention on an anionic cartridge and elution; this additionally facilitates the removal of bubbles before the sample is delivered to the detector. The method has been applied to various types of water. Analogously the reactions of noble metals such as Pd, Rh, Ru, Os, Ir and Pt with the organic chromogenic ligand 4-(5-chloro-2-pyridylazo)-1,3-diaminobenzene to be substantially expedited [117] and facilitates the saponification of vitamin A to retinol [118] in an on-line FIA microwave-assisted system. One other interesting application is the determination of dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC) in freshwater by sequential injection spectrophotometry with on-line UV photo-oxidation (Figure 8) [119]. By on-line sample acidification, DIC is converted into CO2, which subsequently diffuses through a PTFE membrane into a basic Cresol Red acceptor stream, the resulting absorbance decrease being directly proportional to the DIC concentration. DIC+DOC was determined following on-line irradiation of the sample in the presence of an acid and peroxydisulfate, CO2 thus formed being determined as described above and DOC by subtraction. The throughput of the automated

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To HC Sample Cresol red reagent

H2SO4

Waste

Std. / Sample

AcidPeroxydisulfate reagent

Sulfuric reagent

570 nm

P

RC 2 6

HC

5 MV

4 3

7 IN

OUT

8

1

Waste

GD RC1

2

Carrier SY Waste

Waste UV reactor

To UV reactor Sample

Acid-peroxydisulfate

Figure 8 SIA manifold for the sequential determination of DIC and (DIC+DOC), SY, syringe pump; MV, multi-position valve; GD, gas diffusion unit; P, peristaltic pump; UV reactor, PTFE tube coiled around UV lamp (15 W); HC, holding coil PTFE tube; RC1 and RC2, reaction coils consisting of coiled PTFE tube; carrier, ultrapure water. Reprinted from Ref. [119]. Copyright (2005), with permission from Elsevier B.V.

system was 8 samples per hour for both DIC and DOC, and reagent consumption quite low. A range of model carbon compounds and river water samples were analysed for DIC and DOC, the results being quite consistent with those of a high-temperature catalytic oxidation reference method. Microwave radiation has also been used in flow-injection systems for the on-line microwave pretreatment of natural water samples with a view to determining urea as ammonia by colorimetry [120]. The reduction step was facilitated by using microwave radiation, which resulted in substantially shortened analysis times. Urea present in the samples was reduced on-line to ammonium ions, which were then reacted with sodium hydroxide to release gaseous ammonia; passing the gas through a permeable membrane caused a pH change in a Bromothymol Blue indicator stream that was detected colorimetrically. Each sample took 14 min to analyse and the results surpassed those of a conventional batch preparation methodology. Dan et al. [121] developed a merging-zones/stopped-FIA system coupled with on-line digestion for the fast catalytic spectrophotometric determination of


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iodine in urine. Urine samples were digested with a KMnO4–K2CrO4–H2SO4 mixture. The throughput was about 130 samples per hour.

3.3 Pharmaceutical analysis FIA spectrophotometry has been used in pharmaceutical analysis for various purposes compiled in a recent review by Tzanavaras and Themelis [122]. Digestion is usually intended to release the inorganic portion of the sample or break its molecules down into more simple components for subsequent reaction with a colorimetric reagent. Phosphorus-containing compounds can be digested by using various methods that cleave C–P or C–O–P bonds and facilitate the determination of the resulting orthophosphate ions using the Molybdenum Blue reaction. The treatment of choice in each case depends on the strength of the bonds to be broken. Thus, C–P bonds can be cleaved on-line by thermal-induced digestion in the presence of peroxydisulfate ions. This procedure has been used for the precise, fast determination of the antibiotic fosfomycin in urine and pharmaceutical samples previously dissolved in water [123]. This procedure is highly precise, allows up to 60 samples per hour to be processed, and provides high recoveries (96.4–102.5%). Compounds containing C–O–P bonds can be hydrolysed either by using the previous procedure or enzymatically (with alkaline phosphatase). Thus, diethyl stilbestrol diphosphate (fosfestrol) in pharmaceutical formulations has been determined following on-line thermal-induced digestion of the analyte with peroxydisulfate [124], and also with alkaline phosphatase, using the ‘‘chasing zones’’ mode [125]. The procedure involving digestion with peroxydisulfate afforded 60 sample injections per hour and allowed the analyte to be determined in a pharmaceutical formulation; the results differed from those certified by the manufacturer (Asta Medica, Inc.) and those provided by the US Pharmacopoeia’s recommended method by only +0.8% and 0.3%, respectively. The average recoveries of known amounts of the analyte ranged from 97.9% to 100.8%. With enzymatic digestion, the throughput was 40 samples per hour and the method afforded accurate determinations of fosfestrol in a pharmaceutical formulation, with relative errors of only +0.6% and 0.5% relative to the value stated by the manufacturer (Asta Medica, Inc.) and the concentration determined by using the method of the US Pharmacopoeia XXI, respectively. The average recoveries of known amounts of the analyte ranged from 99.2% to 101. 2%. Pretreatments based on on-line hydrolysis reactions are very frequently used prior to spectrophotometric measurements. For example, Capella-Peiro et al. [126] used an on-line hydrolysis pretreatment for the spectrophotometric determination of nicotinic acid. The FIA system was used to hydrolyse the analyte with cyanogen bromide in acetic–acetate buffer at pH 5. Glutaconic aldehyde formed was injected into a stream containing aniline at pH 7 in a micellar medium of the cationic surfactant N-cetylpyridinium chloride that was intended to raise the sensitivity of the reaction in order to obtain the polymethine. The sampling rate was 95 h1 and the ensuing method was used to analyse

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commercial pharmaceuticals containing nicotinic acid with recoveries in the range 96.7–101%. The cephalosporins cefadroxil and cefotaxime have also been determined following on-line hydrolysis in an alkaline medium [127]. Hydrogen sulfide produced was determined photometrically by reaction with either N,Ndiethyl-p-phenylenediamine and Fe(III), or p-phenylenediamine and Fe(III). The method was successfully applied to the analysis of various pharmaceutical formulations, particularly of the injectable and capsule types. Recoveries were quantitative and the results agreed with those obtained with alternative methods. The on-line hydrolysis of paracetamol to p-aminophenol in a reaction coil placed in the cavity of a Microdigest 301 system and its subsequent reaction with 8-quinolinol (oxine) in the presence of potassium periodate has been used for its determination from the absorbance of resulting dye (Indophenol Blue) [128]. The method was used for the analysis of real pharmaceutical formulations (syrup samples, tablets, capsules and suppositories), using alkaline solutions of paracetamol as standards. The throughput was 70 samples per hour. Numan et al. [129] used photodiode array spectrophotometry to study the photochemical activity of six sulfur compounds (viz. sulfacetamide, sulfadiazine, sulfaguanidine, sulfamerazine, sulfamethoxazole and sulfamethizole) under different experimental conditions as regards photolysis time, solvent and pH, and found ethanol and no pH adjustment to be the optimum photolysis conditions. An FIA method has been proposed for the determination of sulfamethoxazole in the presence of trimethoprim. The method, which is based on the on-line photolysis of the analyte using a UV lamp, has the advantages of requiring no reagents, using a simple manifold and providing increased selectivity relative to direct UV methods. It afforded a recovery of 99.7% of sulfamethoxazole from pharmaceutical tablets.

ABBREVIATIONS AND DEFINITIONS AAS AFS BCSS-1 CFA COD CV DIC DOC DORM-2 ESI-MS ET-AAS FOP HG-AAS HTC ICP-AES

Flame atomic absorption spectrometry Atomic fluorescence spectrometry National Research Council of Canada marine sediment certified reference material Continuous flow analysis Chemical oxygen demand Cold vapour Dissolved inorganic carbon Dissolved organic carbon National Research Council Canada certified reference material Electrospray ionization-mass Electrothermal furnace atomic absorption spectrometry Filterable organic phosphorus Hydride generation atomic absorption spectrometry High-temperature combustion Inductively coupled plasma-atomic emission spectrometry


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ICP-MS LUTS-1

Inductively coupled plasma-mass spectrometry National Research Council of Canada lobster hepatopancreas tissue certified reference material MS Mass spectrometry nano-HPLC-MS Nano-high performance liquid chromatography-mass PACS-1 National Research Council Canada certified reference material RT-PCR Reverse transcription-polymerase chain reaction SDS Sodium dodecylsulfate SIC Sequential injection chromatography TFP Total filterable phosphorus TOP Total organic phosphorus TP Total phosphorus

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CHAPT ER

7 On-Line Sample Pretreatment: Extraction and Preconcentration Shoji Motomizu and Tadao Sakai

Contents

1. Introduction 2. Liquid–Liquid Extraction (Solvent Extraction, SE) without Membrane 2.1 Wetting film extraction-FIA system without segmentation and phase separation 2.2 On-line liquid–liquid extraction with a segmentor and a phase separator 2.3 On-line liquid–liquid extraction in a chromatomembrane cell 3. Liquid–Solid Extraction (Solid Phase Extraction, SPE) of Organic and Inorganic Substances 3.1 SPE based on hydrophobic interaction for organic compounds 3.2 SPE with hydrophobic or less hydrophobic sorbents for inorganic species 3.3 SPE based on ion exchange for inorganic species 3.4 SPE based on a chelation mechanism for inorganic compounds 4. Gas–Liquid Extraction Based on Mass Transfer 4.1 Gas denuders and diffusion scrubbers 4.2 Chromatomembrane cell 5. On-Line Pretreatment System, Including Computer-Controlled Automated Systems Abbreviations References

159 160 165 169 170 171 172 180 182 184 188 191 192 196 198 199

1. INTRODUCTION This chapter focuses on on-line sample pretreatment for chemical analysis prior to measurement, which is based on flow injection, sequential injection, and other related techniques. Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00607-7


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160

Shoji Motomizu and Tadao Sakai

In chemical analyses, various kinds of detection method have been used; they are spectrophotometry, fluorophotometry, chemiluminescence (CL) detection, spectroscopy (AAS, ICP-MS, ICP-AES), electrochemical detection, etc. In most cases, some pretreatment procedures prior to the measurement are requisite for improving sensitivity, reproducibility, and accuracy. Extraction and preconcentration are well-used and powerful techniques for chemical analysis, and in this chapter, the following pretreatment techniques are described: liquid–liquid extraction, liquid–solid extraction with octadecylsilyl silica (C18) and its analogues, ionexchange resins, chelating resins, and other adsorbing materials such as polytetrafluoroethylene (PTFE), organic, and inorganic materials. gas–liquid extraction for the mass transfer of analytes between two phases, for the preconcentration of analytes and for the removal of interfering substances or the matrix, and on-line pretreatment system, including computer-controlled automated systems.

2. LIQUID–LIQUID EXTRACTION (SOLVENT EXTRACTION, SE) WITHOUT MEMBRANE Liquid–liquid extraction or solvent extraction (SE) is frequently used for sample pretreatment in order to separate an analyte from interfering substances in the sample matrix, to preconcentrate the analyte for enhancing sensitivity, or to improve the limit of detection. This technology is very useful, and has been widely used in pharmaceutical, environmental, agricultural, and industrial analyses. Batchwise SE, however, is very tedious and time-consuming and needs a large amount of organic solvent for extraction. Furthermore, batchwise procedures are usually carried out in open space leading to health issues associated with volatile organic solvents. Flow injection analysis (FIA) is one of the most useful and versatile techniques for automated analysis, and has been widely applied to routine analyses in various fields. By the combination of SE with the flow injection technique (SE-FIA), SE can be performed on-line in a flowing stream with smaller amounts of extraction solvents and with minimal solvent release to the laboratory atmosphere. Therefore, SE-FIA has become an essential analytical tool for the separation and preconcentration of analytes. Karlberg and Thelander [1] first proposed the performance and construction of a SE-FIA system for the determination of caffeine in acetylsalicylic acid preparations. In the SE-FIA system, sample solutions are introduced into an aqueous carrier stream by a rotary valve (12 or 25 mL), and a chloroform stream chilled in an ice bath is mixed with the aqueous stream. A specially designed segmentor is used as a mixing connector and makes it possible to obtain a regular pattern of alternate aqueous and organic segments. A phase separator is used: it consists of a T-connector with PTFE fibres twisted together to a thread and

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161

inserted in the bend from the inlet down into the outlet directed towards to the flow cell. Phase separation is performed on the basis of the hydrophobic interaction between an organic solvent and PTFE fibres. However, special care must be taken to prevent droplets of water from entering into the flow cell and contaminating it, since its removal by rinsing is very tedious and time consuming. The segmentation and phase separation of the aqueous and organic phases in SE-FIA are very important for efficient recovery of the organic phase, to improve the extraction efficiency and the sensitivity, and to obtain reproducible analytical results. A number of effective tools have been developed to achieve this. Several techniques without phase segmentation and phase separation have also been proposed for simple on-line SE processes in FIA. Kina et al. [2] proposed a novel SE-FIA technique for the determination of potassium at a microlitre scale. In this system, an aqueous sample is injected into a continuously flowing organic stream containing an extracting reagent such as dibenzo-18-crown-6 and anilinonaphthalenesulfonate (ANS). Dibenzo-18crown-6 can react selectively with the potassium ion, and the complex formed can be extracted into 1,2-dichloroethane as an ion associate with ANS as the pairing anion. ANS is non-fluorescent in water, while it becomes strongly fluorescent when extracted into the organic phase. In this system, it is unnecessary to separate the aqueous and the organic phase. This is a first SE/ on-tube detection system without any phase separation. Thommen et al. [3] used a capillary flow cell of 1-mL volume and a computerbased data acquisition method for measuring directly the segmented stream of an aqueous and an organic phase without any phase separation. A novel flowthrough cell, which utilizes an optical fibre for transmitting light through a capillary from and to a photometric detector, was designed. The system has several advantages: (1) simultaneous monitoring of an organic and an aqueous phase is possible, (2) a phase separation step is unnecessary, and (3) fast and simple detection compared to routine SE-FIA is possible. Motomizu et al. proposed capillary flow cells for absorbance and fluorescence measurement in the determination of anionic surfactants [4]. The capillary flow cell for spectrometry is made of Pyrex glass tubing (0.8 mm i.d., 2 mm o.d.), and is fixed on an aluminum body. Visible light passes through a 0.3 mm diameter pinhole and reaches a silicon photodiode. In the flow cell for fluorescence measurements, excitation light passes through a slit (0.5 mm wide 4 mm high) and the fluorescence intensity is measured at right angles. The capillary flow cells used are shown in Figure 1. In the proposed system, a regular-segment flow is required in order to stabilize the background noise. The segment regulator (Figure 2) is designed to obtain a regular-segment flow. The flow rates of the carrier stream and the organic solvent stream are identical at 0.4 mL min1. The flow system can be applied to the determination of anionic surfactants (AS). Rhodamine B (RB) is readily extracted into benzene as its colorless lactone form, which can form an ion associate with AS (RB+ AS), and the colored ion associate is extracted into benzene. The absorbance of the ion associate is measured at 560 nm. When samples containing AS and 0.05 M H2SO4 are injected into the carrier stream (water), a larger background appears because

162


Segment out

(b)

(a)

4

Segment out 4 1

1

2 Exciting line 2 Fluorescence Segment in

3 Segment in 1 cm

3

Figure 1 Capillary flow cells. (a) for absorbance measurements and (b) for fluorescence measurements. 1, Aluminum body; 2, capillary flow cell made of Pyrex glass (0.8 mm i.d. 2 mm o.d.); 3, PTFE tubing (0.5 mm i.d. 1.5 mm o.d.); 4, Tygon tubing. Reprinted from Ref. [4]. Copyright (1992), with permission from Elsevier B.V.

some part of RB is transferred into the aqueous phase. Fortunately, cationic RB extracted into benzene shows fluorescence, and hence the fluorometric determination of AS (2 106 – 1 105 M) is possible. An SE-FIA system without phase separation based on dual-wavelength spectrophotometric detection was proposed by Liu and Dasgupta [5]. The absorbance is read in PTFE tubing, which constitutes a part of the reaction coil. With an illuminated detector (volume: 60 nL), the optical aperture is shorter than the length of the segment, enabling the detector to measure signals for each phase. The detector consists of an LED-based dual-wavelength spectrophotometric system utilizing personal computer-based data acquisition and processing. The detection system can be applied to anionic surfactant determination using ion associate formation with Methylene blue (MB). The LOD is 0.03 ppm of C-12 alkylbenzenesulfonate for a 65 mL-injected sample. Kuban and Ingman [6] designed a dual-channel dropping segmentor for the simultaneous introduction of aqueous solutions containing a sample and an organic analytical reagent directly into a continuous flow of an immiscible organic solvent (Figure 3). The proposed segmentor consists of a PVDF screw with multiple capillary inlet channels (0.3 mm i.d.) for the simultaneous introduction of an aqueous sample and a reagent solution and a segmentor body with a conical confluence chamber containing inlet and outlet capillary channels (0.7 mm i.d.) for the delivery of organic phase and drainage of the segmented flow stream. The measurement system is shown in Figure 3. The system can be used for the determination of Cu(II) with APDC (ammonium pyrrolidinedithiocarbamate) and chloroform.


163

Segment in

1

2

1

Segment out

Figure 2 Detail of segment regulator. 1, PTFE tubing (0.5 mm i.d. 1.5 mm o.d.); 2, PTFE tubing (1.5 mm i.d. 3 mm o.d.). Reprinted from Ref. [4]. Copyright (1992), with permission from Elsevier B.V.

EC

MT D

W

B

C

G O

LC

B

PC

A S

P1

R

P2

Figure 3 Measuring system manifold with a multi-channel dropping segmenter. O, organic phase; R, reagent solution; S, sample solution; LC, LC pump with pulse damper, pressure indicator and restrictor column; P1, P2, peristaltic pumps; A, PVDF screw with dual-channel inlet system; B, PVDF body of the multi-channel dropping segmenter; G, thick-walled glass tube; C, conical compartment with outlet capillary channel; EC, extraction/equilibrating coil; MT, transparent measuring tube (FEP); D, ‘‘on-tube’’ fast-reading detector; PC, personal computer; W, waste. Reprinted from Ref. [6]. Copyright (1991), with permission from Elsevier B.V.

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An automated liquid–liquid extraction process in an unsegmented flow system without a segmentor or separator was proposed by Valcarcel et al. [7]. This method utilized the continuous monitoring of an injected plug of the organic phase during several forward and reverse flow cycles at the detector. The usefulness of this approach was demonstrated by applying it to the determination of anionic surfactants with MB and chloroform. Figures 4 and 5 show the FIA manifold incorporating liquid–liquid extraction. The detector is installed in the loop of the injection valve (Figure 5a), which is filled with the organic solvent. The aqueous phase containing the analyte and the reagent is used as the carrier. When the injection valve is switched to the empty position, the organic phase kept in the loop is introduced into the carrier and two liquid–liquid interfaces are created (Figure 5b). The plug of the organic phase flows into the loop forward until the rear interface reaches the flow cell (Figure 5c). At this point, the flow is changed to the reverse direction, and this flow is maintained until the other interface is close to the detector (Figure 5d). This process is repeated several times to achieve a suitable extraction of the ion associate between two liquid–liquid interfaces. The extraction efficiency is improved by the formation of a thin film of an organic phase on the inner wall of the PTFE tubing due to the hydrophobic affinity of the organic solvent with PTFE. In Figure 4, an aqueous sample merges with a reagent solution, and these are mixed in L1. The loop is filled with chloroform and the photometric detector installed in loop L0. The calibration

Computer control Spectrophotometer (λ=652nm)

L0 Sample L1

MB

L0

I.V.

L2

Methanol S

Water W

CHCl3

Figure 4 Solvent extraction-FIA system without segmentation and separation for anionic surfactant determination. L1 and L2, reactors; L0, subloops into which the detector divides the loop of the injection valve; S, selection valve; W, waste. The back and forward flow was controlled by a PC. Reprinted from Ref. [7]. Copyright (2006), with permission from the American Chemical Society.

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a)

D

D

b) o.p.

o.p.

W(o.p.)

a.p.

a.p.

W(a.p.)

W(a.p.) I.V.

I.V. c)

o.p.

D

D

d) o.p.

o.p.

a.p.

a.p. a.p .

a.p. I.V.

I.V.

Figure 5 Behavior of the liquid–liquid extraction system at the detector located in the loop of the injection valve. (a) filling position of the valve with the organic phase and establishment of the base line; (b) emptying position with circulation of the organic phase through the flow cell; (c) conditions immediately after reversal of the flow in one; and (d) the other direction. o.p., organic phase; a.p., aqueous phase; W, waste; D, detector; I.V., injection valve. Reprinted from Ref. [7]. Copyright (1988), with permission from the American Chemical Society.

graph is linear over 0.1–0.4 mg mL1, with a relative standard deviation (RSD) of 3.0% and a sampling frequency of 50 h1. Burakham et al. [8] have developed a micro-extraction-sequential injection analysis (SIA) system. In this system, SIA-LAV (lab-at-valve) is used for introducing samples, reagents, and an organic solvent, and a conical-type phase separator like a separating funnel is attached to the selection valve of a SIA system. Absorbance of the organic phase is measured in the lower section of the conical-type phase separator which is also used as the detector cell (Figure 6). This system has been applied to the determination of anionic surfactants with MB. A sample, reagents, and an organic solvent are sequentially aspirated into an extraction coil (holding coil) connected to the selection valve, where the extraction process can be carried out by several flow reversal cycles. The aqueous and organic phases can then be separated in the conical-type separating device attached to one port of the valve.

2.1 Wetting film extraction-FIA system without segmentation and phase separation Lucy and Yeung proposed an SE-FIA system without phase separation using differential flow velocities in a segmented flow [9]. The system depends on the

166


Separating vessel Spectrometer

Light source

Computer with a controling program

Extraction coil

10 Selection valve

7

Carrier

Syringe pump

Waste Sample/reagent

Sample/reagent

Figure 6 Schematic flow diagram of the SIA-LAV system for on-line micro-extraction.

wetting film formation of the organic phase on the inner wall of PTFE tubing. The wetting film of the organic solvent acts as a stationary phase, and it can reduce the average linear velocity of the organic phase flow. An analyte extracted into the organic phase requires more time to pass through the extraction coil than the sample zone does. When hexanol is used as the extraction solvent, the differential flow velocities can yield a zone separation between the sample zone and the extracted analyte zone. Methanol is added to homogenize the segmented flow and the absorbance is measured spectrophotometrically without a phase separator. Figure 7 shows the schematic diagram of the SE-FIA manifold based on differential solvent velocities. This system has been applied to the analysis of motion sickness tablets; T1 was replaced with a 0.8 mm i.d. connector that allowed the addition of 2 M NaOH into the distilled water as a carrier stream, and the extraction coil, E, was increased to 300 cm of coiled 0.5 mm i.d. PTFE tubing. The analytical conditions were: 0.123 mL min1 of distilled water, 0.039 mL min1 of 2 M NaOH, 0.036 mL min1 of hexanol, and 1.91 mL min1 of methanol. The method was used to assay diphenhydramine hydrochloride and 8-chlorotheophilline in motion sickness tablets, as well as for a synthetic mixture of o-nitrobenzoic acid and o-nitroaniline. In the latter example, when both analytes were injected into a pH 12 aqueous carrier stream, o-nitrobenzoic acid was fully deprotonated at pH 12, and only o-nitroaniline was extracted quantitatively into the organic phase. First, a sharp peak for the unextracted


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Methanol V1 I He

Aqueous solution

V2

V3 T2

Organic solvent V1

T1

D

P

W

M

E

wetting thin film of organic phase

Aqueous phase

Organic phase

Figure 7 Schematic diagram of the SE-FI manifold based on the differential solvent velocities within an extraction coil. Reprinted from Ref. [8]. Copyright (2006), with permission from The Japan Society for Analytical Chemistry.

o-nitrobenzoic acid appeared, followed by a broad peak for the extracted and dispersed o-nitroaniline. In conventional SE-FIA methods, an aqueous and an organic phase flow continuously during the whole process, and therefore the consumption of organic solvent, carrier, and reagent solutions is relatively large, and as a result, a larger volume of solvent waste will be accumulated. The enrichment factors achieved in SE-FIA are dictated by the ratio of the flow rates of aqueous and organic phases. This ratio is generally not so large compared with batch methods, because large volume ratios of aqueous to organic solvent cannot be achieved in the latter. To overcome such disadvantages, Luo et al. developed a novel thin filmforming system, where an organic solvent film is formed on the surface of PTFE tubing wall by using an SE-FIA or SE-SIA system [10]. When a hydrophobic surface like a PTFE tubing is used in a flow system, an organic solvent can act as a film-forming liquid in a similar manner as to that shown in Figure 7; the film thus formed can surround an aqueous phase as is shown in Figure 8. In this system, the driving force for the film formation is the minimization of the interfacial energy at the solid–liquid interface, which is determined by the relative magnitude of the surface tension of the wall of the inner tubing in contact with the liquid and the interfacial tension of the liquid. Figure 8 shows the schematic diagram of the SE-FIA/SIA manifold. A coating solvent (a thin film-forming solvent), an aqueous sample solution, and an eluting solution, which are separated by air, are sequentially introduced into a PTFE extraction coil. The

168


H2O C

V1

(Benzene)

EC

Air S (BTB)

D Air

DB

E

(50% EtOH /2MNaOH)

V2 W

P

A wetting film B

Aqueous solution

Air

Organic solvent

H2O

Figure 8 (A) Schematic diagram of the SE-FIAA/SIA manifold. P, pump; DB, displacement bottle; D, sandwich cell detector; EC, extraction coil; V1, selection valve; V2, injection valve; C, coating solvent; S, sample solution; E, eluting solution; W, waste. (B) Expanded schematic diagram of flows in the extraction coil. Reprinted from Ref. [10]. Copyright (1994), with permission from the Royal Society of Chemistry.

stationary organic thin film is formed on the wall of the extraction coil, and an analyte in the aqueous phase can be transferred to the organic thin film and is retained on the tubing wall. The extracted analyte is eluted with microlitre volumes of a 50:50 mixture of methanol and 2 M sodium hydroxide; the absorbance of the analyte zone in the effluent is measured using a flow-through sandwich cell, where extracted and unextracted analyte zone can be separated. The flow cycle is started by flowing water for 15 s through the valve V1 (Figure 8), the extraction coil (EC), and the detector (D) to wash the flow line. The valve V1 is then switched to port C to aspirate the organic solvent into the extraction coil to coat the inner wall of the PTFE tubing for 10 s. After that, air is introduced into the extraction coil for 5 s in order to prevent the direct contact of the organic solvent and the aqueous solution. Then a model analyte solution (BTB, bromothymol blue) is aspirated through the port S and introduced into the extraction coil. The analyte, BTB, in the aqueous phase is then extracted into the organic thin film. The eluting solution is loaded into an injection loop of the valve V2 , while the sample solution flows into EC. After the sample solution flows out, V1 is switched to the port E, and V2 is switched to the inject position. The airsandwiched eluting solution is then introduced into the extraction coil to backextract the extracted BTB from the thin film. The system can be operated at sample frequencies of 30 and 10 h1 for 1 and 5 mL sample solutions, respectively, with LODs of 50 and 12.5 ppb of BTB, respectively. The consumption of the organic solvent and the reagent, as well as experimental wastes, are extremely small.


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Nakano et al. also reported the application of sequential injection-wetting film extraction to the photometric determination of vanadium(IV) and vanadium(V) [11]. This system is constructed by using a six-port selection valve (V1) connected to reservoirs (R1–R5), an injection valve V2 attached to a reservoir (R6), an extraction coil (0.8 mm i.d., 3.5 m long), a fibre-optic spectrophotometer with a 546 nm interference filter, and a sandwich flow cell. The chelate of V(V) with Ncinnamoyl-N-(2,3-xylyl)hydroxylamine (CXA) can be extracted into the benzene film formed on the inner wall of a PTFE tubing. After washing of the film with 6 M hydrochloric acid, the V(V)-CXA complex can be eluted with 50 mL of benzene and the absorbance is measured at 546 nm. Vanadium(IV) can be oxidized to V(V), which can be measured at concentrations as low as 106 M at a sampling rate of up to 15 h1. Using a similar system, chromium(VI) is complexed with 1,5-diphenylcarbazide, and its ion associate formed with perchlorate is extracted into an organic thin film consisting of octanol and isobutylmethylketone (IBMK). The extracted analyte can be eluted with 100 mL acetonitrile. Also, Cr(III) can be measured after oxidation to Cr(VI) [12]. Such a system can also be applied to the determination of molybdenum(VI), which reacts with thiocyanate to form anionic Mo(V) and/or Mo(VI) complexes. The Mo(VI) complex thus formed can then be extracted into a toluene film as the ion associate with the tetraheptylammonium ion. The relative standard deviation was 2.5% for 50 ng mL1 of molybdenum(VI) at a sampling rate of 25 samples h1 [13].

2.2 On-line liquid–liquid extraction with a segmentor and a phase separator On-line SE-FIA is one of the most important and versatile techniques for the enrichment of analytes. Several unique methods without any segmentor and/or phase separator have been proposed; some of them are outlined in the previous section. For the improvement of phase separation efficiency, enrichment efficiency, and reproducibility and accuracy of analysis, a phase segmentor and a phase separator are very important devices in on-line SE-FIA. As a phase segmentor, a commercially available T-shaped connector is one of the useful devices [14], and a phase separator with a PTFE membrane filter offers a versatile and efficient means of phase separation. A number of research papers concerning membrane phase separation have been reported so far. Kawase et al. proposed a phase separation system with a PTFE porous membrane [15]. Later, Imasaka et al. [16] and Ogata et al. [17] reported newly designed phase separators using PTFE membranes. Motomizu et al. also proposed a simple and highly efficient separator with a PTFE membrane [14,18], in which the membrane chamber is designed to have as small a volume as possible. The body is made of poly(chlorotrifluoroethylene) (CTFE), and the grooves of both bodies are designed to be narrow (2 mm) and sloped. Sakai et al. subsequently developed a modified phase separator with double membranes (Figure 9) [19,20], in which the efficiency of the phase separation was greatly improved in order to prevent small droplets from passing through the

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membrane. Motomizu and Korechika developed a new cylindrical cavity-type phase separator [21]; they used this to evaluate several kinds of membrane filters; a pore size of 0.8 mm was recommended. On-line SE-FIA with a phase separator has been applied to the determination of various kinds of analytes; for example, phosphate extracted as the ion associate of molybdophosphate with Malachite green [14], potassium extracted as the ion associate of dibenzo-18-crown-6 complex with tropaeolin OO [18], anionic surfactant extracted as the ion associate with cationic azo dye [22] and MB [23], Ca2+ extracted as dicyclohexano-24-crown-8 complex with Propyl orange [24], and berberine extracted as the ion associate with perchlorate ion [19]. SE-FIA with membrane phase separators is described in more detail in Chapter 9.

2.3 On-line liquid–liquid extraction in a chromatomembrane cell The chromatomembrane cell (CMC) is one example of continuously operating extraction devices. The CMC consists of a biporous PTFE membrane block. It was proposed by Moskvin et al. for the transfer of analytes from one phase to another [25], and has proved to be a very useful separation and enrichment device for sample pretreatment prior to measurement. The CMC has been used for on-line collection and concentration by liquid–liquid extraction [25,26] and gas–liquid

Figure 9 Phase separator with double membrane. Seg, segment; aq, aqueous phase; w1, aqueous waste; w2, organic waste; w3, organic phase; MF1, MF2, membrane filter (pore size: 0.8 mm). Redrawn from Ref. [19]. Copyright (1993), with permission from Elsevier B.V.


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extraction. In the CMC, the PTFE membrane block contains both micro and macro-pores. Hydrophobic organic solvents can act as a stationary phase and are present in the micro-pores of the PTFE block, whereas a hydrophilic phase like water acts as a mobile phase and occupies the macro-pores, and can move through the CMC. Thus analytes can be transferred from the mobile phase into the stationary phase and are enriched in it. The principal advantages of CMC are: (1) a high mass transfer efficiency from water to the organic phase because the surface area of the membrane is very large and the two phases can contact directly with each other in the CMC, (2) a very high efficiency of analyte enrichment from the mobile phase to the stationary phase, (3) only a very small volume of an extracting solvent and an organic waste are involved, and (4) the dead volume and cylindrical cell volumes (12 mm i.d. 14 mm height) are very small. The principle of CMC is described in detail in Chapter 9.

3. LIQUID–SOLID EXTRACTION (SOLID PHASE EXTRACTION, SPE) OF ORGANIC AND INORGANIC SUBSTANCES Olsen et al. first reported the use of on-line solid phase extraction (SPE) in FIA [27], and subsequently similar technologies have often been applied to the enrichment of analytes and the removal of the matrix. On-line SPE techniques in FIA, SIA, SIA-LAV, and SIA-LOV (lab-on-valve) are clearly advantageous in performing sample-pretreatment processes such as sample clean-up, analyte preconcentration, and removal of matrices and/or interfering substances. Use of on-line SPE-FIA methods has resulted in the improvement of simplicity, accuracy, reproducibility, and ease of automation. A number of books and reviews have been published in recent years which deal with the hyphenation of on-line SPE-FIA with various detectors [28], on-line column preconcentration-atomic spectrometry for trace metal determinations [29], on-line elemental speciation in aqueous solutions [30], separation and concentration [31], sample-pretreatment schemes for trace-level determinations of metals by inductively coupled plasma-mass spectrometry (ICP-MS) [32], sequential injection-bead injection-LOV extraction and preconcentration of heavy metals for the determination by electrothermal atomic absorption spectrometry (ETAAS) and ICP-MS [33], SIA for on-line sample handling and pretreatment [34], and the use of on-line renewable SPE-liquid chromatography bead injection-LOV [35]. By incorporating SPE into flow systems, both organic and inorganic species can be collected, preconcentrated, and separated prior to the detection. FIA, SIA, and SIA-LOV coupled with on-line sample-pretreatment procedures offer benefits such as (1) high sample throughput, (2) reduced reagent consumption and waste production, (3) reduced sample contamination, (4) lower LOD and/or LOQ (limit of quantification), (5) automation by programmed control, and (6) hyphenation with many kinds of detectors. In general, the adsorption behavior of substances on sorbents depends mostly on the nature of the analytes and sorbents, as well as the solutes and solvents in solutions. Substances collected on a solid phase can be classified

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as follows: (1) hydrophobic or less hydrophobic substances, (2) bulky or less bulky molecules, (3) charged or non-charged metal chelates, and (4) ion exchangeable or less exchangeable substances. From the point of view of adsorption mechanisms, sorbents used for on-line SPE are classified as follows: (1) hydrophobic polymer gel and long alkyl-chain functionalized silica gel, (2) ion-exchange resins for cations and anions, (3) chelating resins for metals, and (4) others such as molecularly imprinted polymer (MIP) gel and solid particles adsorbing substances based on hybridized adsorption mechanisms. For the efficient collection and enrichment of analytes and for the efficient separation and the removal of interfering and matrix substances, a suitable pair of analytes and sorbents must be selected. Also, suitable eluents must be selected for the efficient desorption of analytes from the sorbents, since the adsorption mechanism and the strength of adsorption are very different for each pair of sorbents and analytes. In on-line SPE-FIA, moderate strength of adsorption and desorption of analytes is preferable. Tables 1–3 show examples of SPE with hydrophobic sorbents for organic compounds, inorganic species, and ion-exchange resins for inorganic species, respectively.

3.1 SPE based on hydrophobic interaction for organic compounds A combined FI-SPE-capillary zone electrophoresis (FI-SPE-CZE) system was designed for the determination of trace pseudoephedrine in human plasma. A C18 micro-column was used and the adsorbed analyte was eluted with acetate buffer/acetonitrile (40/60 v/v) to give a detection limit of 12 mg L1 and a sample throughput of 10 h1 [37]. Phenols can be preconcentrated on an Amberlite XAD-4 column at pH 2 and separated from the sample matrix. Analytes collected on the column are eluted with alkaline solution (pH 13). In this procedure, aromatic amines are not retained and do not interfere with the determination of phenols. The reaction products obtained by the 4-aminoantipyrine (4-AAP) reaction are detected by spectrophotometry at 510 nm (Figure 10a) [41]. At lower concentrations of phenols, 4-AAP derivatives are extracted into chloroform and the absorbance of the extract is measured at 460 nm (Figure 10b). Anionic surfactants at low levels in river water and wastewater are determined using automated FIA with a tubular flow-through ion-selective electrode. The FIA system involves an SPE procedure for purification and preconcentration of analytes using the polymer sorbents, such as Sep-Pak C18 (SP), LiChrolut EN (LEN), and LiChrolut RP-18e (LC) from Millipore and Merck. As little as 0.03 mg L1 dodecyl sulfate can be detected after elution with 50 mL of 25/75 (v/v) water/acetonitrile [42]. Nitrite ions can react with a modified Griess reagent to form an azo dye, which can be adsorbed onto C18, and detected with an optosensor to give a LOD of 0.46 ng mL1 and an enhancement factor of 93 [43]. Figures 11 and 12 show the FIA-SPE optosensing setup and the associated flow-through cell for the determination of nitrite. After detection with the optosensor, the adsorbed azo dye is eluted from the C18 with 20/80 (v/v) water/methanol.

Table 1

Solid phase extraction using hydrophobic sorbents for organic compounds by FIA and SIA

Analyte

Sorbent

Eluent

Sample

LOD (LOQ)

Calibration range/ Detector/reagent Enrichment factor

Reference

Salicylate

Silica-gel with quaternary ammonium C18

0.05 M HNO3

Urine serum

—

0.05–20 mg mL1

[36]

40/60 (v/v) acetate buffer (pH4.0)/ acetonitrile 0.05 M HNO3

—

0.03 mg mL1

180-fold with 4 min loading

Tablets

—

2–50 mg mL1

Human plasma

12 mg L1

—

Pseudoephedrine

Silica-gel with R4N

Pseudoephedrine

C18

Naptalam

C18

Phenol

Amberlite XAD-4

Anionic surfactants Nitrite

Sep-Pak C18 Li Ch rolut EN Li Ch rolut RP-180 C18

Nitrite

C18 disk

Cimetidine

C18

Salbutamol

Silica gel with carboxylic acid

[37]

Spectrophotometry with Folin Ciocalteau Capillary electrophoresis

[38]

Fluorimetry with hydrolysis Spectrophotometry with 4-amino antipyrine Potentiometry

[40]

River

2.9 106 M

up to 6 105 M

Tap, river water, soil

0.2 ng mL1

0.5–60 ng mL1

25/75 (v/v) water/ acetonitrile

River, waste water

5 107 M

20/80 (v/v) water/ methanol 20/80 (v/v) water/ methanol

Tap, rain water

0.46 ng mL1

0.03–2.66 ppm 1 1071 105 M 1–100 ng mL1/93

Tap, ground, harbor, aquarium Human plasma

0.1 ng mL1

3-40 ng mL1/140

8 mg mL1

0.05–2.0 mg mL1/ 15%

Capillary electrophoresis

[45]

Human urine

0.1 mg mL1

1-15 mg mL1

Spectrophotometry with FolinCiocalteau reagent

[46]

40/60 (v/v) acetate buffer (pH3.6)/ acetonitrile 0.01M HNO3

Spectrophotometry with Griess reagent Spectrophotometry with Shinn reagent

[39]

[41]

[42]

[43] [44]

173

40/60 (v/v) acetate buffer (pH3.5)/60% acetonitrile Small volume of acetonitrile Aqueous soln. at pH13


Captopril

Spectrophotometry with Fe(NO3)3 in HNO3 Capillary electrophoresis

174

Table 1 (Continued ) Sorbent

Eluent

Sample

LOD (LOQ)

Calibration range/ Detector/reagent Enrichment factor

Dodecylamine

Aminopropyl isolute cartridge


Diesel fuels

2.9 mg L1

2.9–50 mg L1

Thiamine

C18-poly[styrene/ divinylbenzene]

Microbeads

Pharmaceutical tablets/syrup

0.03 mg mL1

0.06–8 mg mL1

Ascorbic acid (AA), Rutin trihydrate (RT)

C18

Methanol/water (28/72)

Tablets

Phenylephrine

Dowex 50WX8

0.1 M NaOH

Pharmaceuticals

1 mg mL1 (AA) 1.5 mg mL1 (RT) 5.8 mg L1 (LOQ)

10–100 mg mL1 (AA) 2–20 mg mL1 (RT) 30–160 mg L1

b-estradiol

Molecular imprinted polymer

50% acetonitrile

1.12 mg L1

Tetracycline

Molecular imprinted polymer C18 disk

20/80 (v/v) 0.01 M HNO3/acetonitrile

Pond, well water raw drinking water Fish

1 mg L1


River water

10/90 (v/v) water/ methanol

Tap water upper reaches

2.5 mM for 3 mL r10 mM (0.07 mg L1)/ (2.66 mg L1) 40 0.25–10 ng mL1 0.1 ng mL1

Anionic surfactants Phenols

OASIS HLB cartridge

Reference

CL peroxyoxalate/ [47] sulforhodamine 101 CL reaction [48] Fluorimetry with hexacyano ferrate(III) oxidation [49] Spectrophotometry at 262 nm

[50]

4-80 mg L1

Spectrophotometry with 4-AAP/hexa cyanoferrate(III) Fluorimetry

4-400 mg L1

CL

[52]

Potentiometry

[53]

Spectrophotometry with 4-AAP

[54]

[51]


Analyte

Table 2 Solid phase extraction using hydrophobic sorbents for inorganic species by FIA and SIA Sorbent

Eluent

Sample

LOD (LOQ)

MeHg, EtHg PhHg, Hg(II)

C18 S

Water/acetonitrile methanol

Urine

Pd

Ag-PDC on-line displacement C18 Pb-DDAP

Methanol

Rock

MeHg, 9.3 mg L1 MeHg 95,000 mg L1/787 EtHg, 5.5 EtHg 6– 5,000 mg L1/858 PhHg, 10.4 PhHg10– 5,000 mg L1/751 Hg(II), 7.6 Hg(II)8– 5,000 mg L1/952 1 3.3 ng L 0.02–5 mg L1/71

Methanol

Seawater

Pb

Cu Pb

Cu

Zn

PTFE turnings Cu-APDC PTFE Pb-PDC

C18 Cu-5,7dichloro quinoline-8-ol C18 Zn-TAN

1BMK 1BMK

Soil

Detector/ Reference reagent CVAAS

[55]

ICP-MS

[64]

LEI

[57]

0.15–15 mg L1/340

FAAS

[59]

0.8 mg L1

1.6–100 mg L1/170 for 60s

FAAS

[60]

0.05 mg L1

0.2–40 mg L1/100

FAAS

[61]

0.15 mg L1

0.5–50 mg L1/120

FAAS

[62]

0.61 ng mL1 for 5 mL sample 0.0033 ng mL1 for 15 mL sample 0.05 mg L1

250–1,000 ng mL1 6.25–1 ng mL1

175

Acidified methanol (pHW2) Acidified methanol (pH~2)

Top, river coastal seawater Coastal seawater marine, sediment mussel tissue Seawater

Calibration range/ enhancement factor


Analyte

176

Analyte

Sorbent

Eluent

Sample

LOD (LOQ)

Calibration range/ enhancement factor

Detector/ Reference reagent

Co, Ni

C18Co, Ni-DHN

Soil human hair

0. 1 mg L1 for 1 min loading

0.5–20 mg L1/ Co:725, Ni:600

FAAS

P

Acidified methanol (pH~2) 0.3 M H2SO4 ethanol

Sep-PakC18 MoP-CTAB ion associate PCTFE Cu, IBMK Pb-DDP Complex PTFE turnings As- 2 M HCl PDC complex 0.1 M HCl Sephadext G-25gel DHNS-B complex 0.01 M HCl Sephadex G-25gel oxidation of Chromotropic acid

Seawater

0.002 mM

0.005–0.194 mM

Luminol CL [65]

River, lake, seawater

0.67 mg L1 for 90s sampling

0.24–20 mg L1/250

FAAS

[66]

Natural water

0.04~5 mg L1/10

HG-AAS

[67]

Iron

0.02 mg L1 for 60s sampling 0.005 mg L1

0–206 ng mL1

Fluorimetry [58]

Natural water

0.02 ng mL1

0–2.5 ng mL1

Photometry [56]

Cu, Pb

As(III) B

V

[63]



Table 3

Solid phase extraction using ion exchange resins for inorganic species by FIA and SIA Sorbent

Eluent

Sample

LOD (LOQ)

Calibration range/ enrichment factor

Detector

Reference

Pb, Cd

Cation exchange resin

1 M malic acid

River water soil

0.1 mg mL1

Photometry, TMPyP

[73]

Bi

Dowex 1 8 anion exchange resin Amberlite XAD-4 2,6-diacetylpridine

0.5 M H2SO4 0.1 M HNO3

Steel

0.005–0.3 mg mL1

Spectrophotometry Iodide complex FI-ICP-MS FI-FAAS

[74]

As(III)(arsenite) As(V)(arsenate)

Muromac Cl-from

2 M HNO3

Pb:0.001 mg mL1, Cd:0.002 mg mL1 With 2.5 m sample loop 0.2 mg mL1 (LOQ) FIA-ICP-MS (mg L1) Cd:0.33, Co:0.094, Cu:0.34 Mn:0.32, Ni:0.30, Pb:0.43 U:0.067, Zn:0.20, FIA-AAS (mg L1) Cd:22, Co:60, Cu:10, Ni:4.8 0.1 mg L1

ICP-AES

[76]

Se(IV)(selenite)

Muromac Cl-from

98:2 (v/v)

River water, bottled drinking water Tap, pond water, 0.009 mg L1 bottled drinking for 1 mL water sampling

Cd, Co, Cu, Mn Ni, Pb, U, Zn

Se(VI)(selenate)

1 M HNO3/ methanol

Soil, sediment

1–5 mg L1

0.5–20 mg L1 for 20 mL sample

0.02–1 mg L1 ICP-MS (selenite, selenate) 0.1–1 mg L1 (total selenium)/20

[75]

[77]


Analyte

177

178


P1 -1 mL min 2.0

SV1 SV2

0.1M NaOH 4-AAP K2S2O8 P1 mL min -1 2.0

XAD-4 column (4 cm 2.5 mm)

1.0 0.15 m

0.40 1.0

510 nm R1

ml min -1 P2

0.1M NaOH 4-AAP (pH 11)

K2S2O8 H2O

SV1

4.8

W

W

XAD-4 column (4 cm 2.5 mm)

1.0 0.15 m

0.40

mL P2

0.8 mm

R1

W

R2

SV3

min-1

0.8 mm 2m

1.0 0.55

W

D

(a)

SV2 Sample

0.8 mm

1m

0.01M HCl

W

4.8

Restrictor

Sample

0.3 mm

0.01M HCl

H2O

Displacement bottle

CHCl3

PS 460 D nm W

(b)

Figure 10 Manifolds for phenol determination. (a) Detection in the aqueous phase and (b) detection in CHCl3. D, spectrophotometer; PS, phase separator; P1, P2, peristaltic pumps; R1, reactor; R2, extraction coil; SV, switching valve. Reprinted from Ref. [41]. Copyright (1996), with permission from the Royal Society of Chemistry.

The determination of salbutamol (a b-adrenergic receptor agonist) with FolinCiocalteau reagent has been proposed. Salbutamol can be extracted onto silica gel modified with carboxylic acid, and the analyte on the column is eluted with 0.01 M HNO3 and measured by spectrophotometry at 750 nm. The system can be controlled by a computer program written in LabVIEWt [46]. The fluorometric determination of thiamine has been reported. The sensitivity of the method depends on the conversion efficiency of the analyte to fluorescent thiochrome by the oxidation with alkaline hexacyanoferrate. The thiochrome adsorbed on octadecyl-alkylated poly[styrene/divinylbenzene, C18-PS/DP] can emit strong fluorescence at lex ¼ 385 and lem ¼ 433 nm. A SIA system coupled on-line to a chip-based flow-through cell is employed to handle a chemical reaction [48]. Phenylepherine hydrochloride was measured on a micro-column packed with Dowex 50W X8 ion-exchange resin after removing co-formulated substances using an eluent of 0.1 M NaOH. The Emerson reaction with 4-AAP and potassium hexacyanoferrate(III) was used for color development [50]. A FIA method for the fluorometric determination of b-estradiol was proposed; it involves the use of a MIP packed in a micro-column.

179


Pump 1 mL min-1 Eluent (80% MeOH)

0.8 Detector

Conditioning solution

1.2

Griess R.

1.2

W

C18 column

SV

2.5 mL Water

W

1.2 IV1

Sample (NO2-)

IV2

RC

100

On-line azo-dye formation

L

W

h LED

2

Pump 2

Figure 11 Flow injection-sorbent extraction optosensing setup for the determination of nitrite. IV, injection valve; SV, switching valve; GR, (0.01% sulfanilamide + 0.005% N-naphthylethylenediamine). The conditioning solution is 0.3M HCl + 0.5% MeOH. Reprinted from Ref. [43]. Copyright (2001), with permission from Elsevier B.V.

Flow in

C18 material 1 mm Optical fiber outlet

Optical fiber inlet

1 mm

Flow out

Figure 12 Side view of the prismatic flow-through cell for determination of nitrite. Reprinted from Ref. [43]. Copyright (2001), with permission from Elsevier B.V.

180


Microwave-assisted extraction can be used to remove the template from MIP. The carrier and the eluent are 97.5:2.5 (v/v) and 50:50 (v/v) water/acetonitrile, respectively [51]. A molecularly imprinted polymer solid phase extraction (MIP/SPE) combined with FIA CL detection was used for the determination of tetracycline [52]. MIP particles are packed in a PTFE tube, which is installed in the injection loop of a switching valve. An eluent of 0.01 M HNO3/acetonitrile (20/80 v/v) is used for the elution of adsorbed tetracycline which enhances the CL reaction between Ce(IV) and RB. On-line preconcentration and determination of phenols at sub-ppb levels were achieved using a OASISt HLB extraction cartridge [54]. Buffered sample (pH 4) is loaded onto the column at a flow rate of 2.0 mL min1. The analyte collected onto the column can be eluted with 10/90 (v/v) water/methanol, the effluent merged with 0.15 % 4-AAP in NH4Cl-NH3 buffer at pH 10 and 0.6% K3[Fe(CN)6 as shown in Figure 13. The calibration graph for phenol is linear over the range of 0.25–10 ng mL1.

3.2 SPE with hydrophobic or less hydrophobic sorbents for inorganic species Octadecylsilyl silica (C18), PTFE particles or turnings, Sephadext, Silica gel, alumina, and activated carbon can be used for metal separation and preconcentration. OH OH OH Concentrated OH

SPE W

P1 (pH 4)

V2

MeOH

ON P3

V1

OFF

H2O H3C H2N

W

CH3 N N O (pH 10)

K3[Fe(CN)6]

V3

RC1 RC2 D

P2 W

Figure 13 Schematic diagram of the flow system for on-line preconcentration of phenols. Reprinted from Ref. [54]. Copyright (2005), with permission from The Japan Society for Analytical Chemistry.


181

Methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and inorganic mercury at ng L1 level were determined by cold vapor atomic absorption spectrophotometry (CVAAS), in which SPE-FIA preconcentration was incorporated in an HPLC system [55]. A C18 reversed phase material can also be used for the collection of mercury complexes formed on-line with pyrrolidinedithiocarbamate (PDC); the mercury complex adsorbed on the column is eluted with an ethanol/acetonitrile/water mixture. Phosphate was determined by using SP cartridges to extract the ion associate of molybdophosphate (MoP) with cetyltrimethylammonium (CTA) ion [65]. Since CL emission is generated via the MoP reaction with alkaline luminol, the CL intensity can be measured for the determination of phosphate with a LOD of 0.2 nM. Vanadium(V) was collected with a column and determined: it can be adsorbed onto a small column (4 mm i.d. 7 cm) packed with Sephadext G-25 gel, desorbed with 0.01 M HCl and detected using the catalytic oxidation reaction of chromotropic acid with bromate at pH 3 [55]. A Sephadex column can be also used for in-line preconcentration and the separation of boron as boric acid. The fluorescent complex of boric acid with 1,8-dihydroxy-naphthalene-3,6-disulphonic acid (DHNS) is utilized for the detection of boron [58]. PTFE particles packed in a mini-column can be used as a sorbent material for SPE [59]. The copper complex with PDC is easily adsorbed onto PTFE particles; it can be eluted with IBMK. Copper in the effluent is detected by flame atomic absorption spectrometry (FAAS) with a sample frequency of 40 h1 for a preconcentration time of 1 min. On-line displacement SPE-FIA can be effectively applied to minimize mass interferences (the isobaric effect) for the determination of palladium by ICP-MS [64]. The method depends on the on-line complexation of Ag+ with PDC and the displacement reaction between Pd2+ and the presorbed Ag-PDC. The retained Pd2+ is eluted by ethanol and the effluent is analyzed by ICP-MS. Inorganic sorbents, such as fibrous alumina and activated carbon, can also be used for on-line preconcentration of metal ions, such as lead and nickel. Preconcentration is based on the collection of the metal ions on a fibrous alumina packed in a mini-column, followed by elution with 250 mL of 1 M HNO3 to give a LOD for lead of 0.7 mg L1 by AAS [68]. The method can be applied to real water samples. A conical mini-column packed with activated carbon can be used for on-line nickel preconcentration using 20% HNO3 as the eluent. The detection of nickel is performed by inductively couple plasma-atomic emission spectrometry (ICPAES): with an LOD of 82 ng L1 [69]. On-line chromium preconcentration can be performed using a conical mini-column containing100 mg of activated carbon; the retained chromium is eluted with 10 % (v/v) nitric acid, and is determined by ICP-AES [70]. A glass capillary tube and PTFE filter tube can be used for on-line preconcentration of zinc and/or arsenic. The method is based on the adsorption of metals on the inner surface of the tubing. Zinc is concentrated for 5 min, and after elution it is measured by spectrophotometry with 4-(2-pyridylazo)resorcinol

182


[71]. Arsenic coprecipitated with beryllium hydroxide at pH 10 on the tubing can be eluted with 1 M HNO3; the effluent is mixed with ammonium molybdate to form molybdoarsenic acid (molybdenum yellow), which is reduced with ascorbic acid. The absorbance of the arsenomolybdenum blue is measured at 840 nm; LOD of 0.7 ppb and LOQ of 2 ppb [72].

3.3 SPE based on ion exchange for inorganic species The simultaneous determination of lead and cadmium in water and soil samples by FIA with a column of cation exchange resin was performed, using a malic acid solution as an eluent [73]. The photometric detection system exploits the complex formation of lead and cadmium with 5,10,15,20-tetrakis(N-methyl-pyridinium4yl)-21H,23H-porphine, tetrakis(p-toluene sulfonate) (TMPyP) at pH 10.1. A linear calibration graph using a 2.5 m sample loop is obtained in the range of 0–0.1 ppm for lead and cadmium. A Dowex 1 8 anion exchange resin can be used for the collection of bismuth in 0.5 M HCl, which can be desorbed with a small volume of 0.5 M H2SO4; bismuth is detected as the iodide complex [74]. A mini-column packed with anion exchange resin can be installed in the flow line with an ICP-MS system, which can be used for the determination and speciation of selenite and selenate at the sub-mg L1 level [77]. An anion exchange resin, Muromac Cl form, can be used for the separation of charged selenate and uncharged selenite at different pHs. The system for the selenium speciation is shown in Figure 14. At pH 1.5, selenate is present as HSeO 4 , whereas selenite is present as an uncharged species, H2SeO3. Only the charged selenate species can be collected on the column; selenite species is collected on a second column at a higher pH. A mixture of 1M HNO3 and 2% methanol can be used as the eluent. The enrichment factor is 20-fold by using 10 mL of sample. The calibration graphs are linear over the range from 0.02 to 1.0 mg L1 of selenite and selenate. Chromium speciation can be carried out by using small-sized thin solid phase column resin reactors packed with cation exchanger for Cr(III) collection and with anion exchanger for CrO2 4 collection [78]. The detection of both species is performed by ICP-AES. Silica gel can also be used as a cation exchanger. A silica gel (100–200 mesh) column packed in a PTFE column (1 mm i.d. 30 cm) can be used for the separation of sodium and potassium ions as their crown ether complexes. Both alkali metal ions in the effluent are detected by spectrophotometry coupled with on-line SE as an ion associate with tetrabromophenolphthalein ethyl ester (TBPE) or an azobenzenesulfonate derivative [79,80]. The calcium ion can also be determined by on-line SPE-FIA coupled with spectrophotometry [24]. A cation exchange resin, AG 50W-X2 (100–200 mesh, H+ type; Bio-Radt), was used for the determination of trace amounts of cobalt by solid phase spectrometry (SPS) [81]. SPS is based on the direct measurement of the absorbance or reflectance of a solid phase when an analyte is concentrated on the packing material [82,83]. As is shown in Figure 15, the flow system is very simple; the solid phase is packed in a ‘‘black’’ flow-through glass cell, which has a light

183


DRC ICP-MS Eluent P3 SL

SPERC-1

1M HNO3/2% MeOH

SPERC-2

Waste V2

V3

MC

V4

P1

NH3 Solution

V1

P2

Sample (pH 1.5)

Conditioning / Washing Solution dil HNO3, pH 1.5

ModuleControlling Software

SPERC: Solid Phase Extraction Resin Column SL: Sample Loop V: Valve P: Pump

Serial & TTL Communication

MC: Mixing Coil

HSeO 4

Laptop

HSeO 3

Figure 14 Preconcentration of and using anion exchange resin (SPERC1,2). Reprinted from Ref. [77]. Copyright (2007), with permission from Elsevier B.V.

Figure 15 Schematic diagram for FI-SPS. A, pump; B, six-way switching valve for sample introduction; C, six-way switching valve for desorbing agent introduction; D, detector packed with ion exchanger in spectrophotometer (wavelength: 575 nm). Carrier solution: (1) 1 M H2SO4; (2) 0.1 M 2(N-morpholino)ethanesulfonic acid (MES), 104 M ethylenediamine-N,Nu-dipropionic acid, dihydrochloride (EDDP), 0.01 % (v/v) Triton X-100 (pH 6.0). Flow rate: (1), (2) 0.6 mL min1. Reprinted from Ref. [81]. Copyright (2006), with permission from The Japan Society for Analytical Chemistry.

pathlength of 10 mm in length and 1.5 mm in diameter (volume about 0.02 cm3). The solid phase beads are retained in the flow-through cell by a polypropylene filter chip at the exit of the cell; the length of the solid phase must be less than 5 mm. The analyte or its derivatized product can be accumulated on the solid

184


Figure 16 Absorption profile of cobalt complex on cation exchange resin measured by FI-SPS. Sample volume: 4.0 mL. A, blank; B, 1.0 mg L1 Co; C, 2.0 mg L1 Co; D, 3.0 mg L1 Co; E, 3.0 mg L1 Co without Triton X-100; F, desorbing age. Reprinted from Ref. [81]. Copyright (2006), with permission from The Japan Society for Analytical Chemistry.

phase, and hence the solid phase must be cleaned with a washing solution before another measurement can be initiated. For cobalt determination, a cationic cobalt(III) chelate with 2-(5-bromo-2pyridylazodiethylaminophenol can be used; the chelate adsorbed on the resin can be eluted with 1 M NaOH after the measurement. An LOD of 40 ng L1 was obtained using 4 mL of sample ((Figure 16)). Chromium(III) can also be determined; after preconcentrating Cr(III) on a cation exchange resin (Amberlitet IR 120(H)) packed in a mini-column (2 mm i.d. 20 mm), which is installed in the loop of a six-way switching valve, Cr(III) is eluted from the column with 2 M HCl, and Cr(III) in the effluent can react with EDTA to form a colored chelate, which is measured at 530 nm by spectrophotometry [84].

3.4 SPE based on a chelation mechanism for inorganic compounds Metal ions may adsorb on chelating resins, as well as ion-exchange resins. The adsorbing mechanism is based on the chelation of analytes with functional groups of the resins. In general, alkali metal ions, as well as alkaline earth metal ions, are less adsorbed on chelating resins than cation exchange resins. Therefore, for the collection of trace metal ions in highly saline samples, chelating resins are recommended. In spectroscopic analysis, such as ICP-MS and GF(ET)AAS, a saline matrix can cause serious problems in the determination, as well as damage to the instruments. With ICP-MS, for example, a saline matrix may damage the mass spectrometer and sampling cone, and can interfere with the measurement by an isobaric effect. Therefore, in ICP-MS, the use of SPE is more important for the removal of matrix substances in sample solutions than it is for analyte enrichment.


185

In most cases of the spectroscopic measurement of metals, high selectivity of metal analyte in the collection/enrichment by SPE is not so important, compared with the need for high efficiency and reproducibility in the adsorption process, because each spectroscopic method already has adequate selectivity. ICP-MS and ICP-AES, for example, can measure about 60 elements at once. In SPE, various kinds of chelating resins can be used. In Table 4, examples of on-line SPE-FIA are summarized [85]. One of the most frequently used chelating resin types has an iminodiacetate or iminodiacetic acid moiety as a chelating functional group. Chelext-100 and Muromact A-1 have such a group, and are commercially available. It is said that the fundamental adsorption characteristics of these resins are very similar, but the former is more prone to swell and shrink than the latter. On-line SPE used in conjunction with spectroscopic measurements usually requires various kinds of pretreatment, such as column conditioning, pH adjustment, sample loading, washing, and elution and washing. Because of such tedious and time-consuming procedures, the use of the more versatile SIA mode is preferable to the flow injection mode. In Figure 17, the adsorption behavior of 60 metal ions on Muromac A-1 is summarized [85]. It can be seen from Figure 17 that while a number of metal ions can be collected on Muromac A-1, the efficiency of collection and recovery is incomplete for some metals. However, the percentage recoveries of less adsorptive metals are usually quite reproducible, provided that the tedious pretreatment procedures are performed reproducibly using an automated system. By using such a system, many kinds of metals can be measured simultaneously. An automated pretreatment system for ICP-AES can be used for the simultaneous determination of metals in water samples [109]. Such an automated pretreatment system is shown in Figure 18. The system is controlled by a computer program, and the detector responses for cadmium show the possibility of the determination of Cd at sub-ppb levels. Table 5 shows the characteristics of the method. The LODs for nine metals are improved by about tenfold using 5 mL samples. Using this method, nine metals in river water samples from Okayama, Japan were determined (Table 6). To improve the sensitivity and the performance of the pretreatment, a fully computer-controlled pretreatment system (Auto-Pret AES) can be used. The system consists of a syringe pump, a selection valve, and a switching valve with a mini-column (2 mm i.d. 40 mm) packed with Muromac A-1 (Figure 19) [85]. The preconcentration cycle steps are shown in Table 7. By using Auto-Pret AES, 13 metals have been determined with enrichment factors of 5–12 for 5 mL of sample, giving LODs in the range of 0.008 ng mL1 (Mn) to 0.18 ng mL1 (Pb) (Table 8). Tables 9 and 10 show the analytical results for standard reference materials performed in order to evaluate the accuracy and precision of the method and for real water samples, respectively. The results show that Auto-Pret AES method can be used effectively for the simultaneous determination of toxic heavy metals in environmental water samples.

186

Table 4 On-line SPE for the determination of trace metals in various samples [85] Matrix

Sorbent

Preconcentration method

Detection

LOD ng mL1

Sample frequency (h1)

Reference

Se

AAS

0.2

30

[86]

Flow mode

AAS

0.14–2.1

13

[87]

Flow mode

GF-AAS

2 104

8

[88]

Cu, Mo

SRM seawater

Flow mode

GF-AAS

0.009–0.06

14

[89]

Bi, Cd, Pb

urine

Flow mode

GF-AAS

0.002–0.013

10

[90]

Cd, Co, Ni

—

Flow mode

GF-AAS

0.0001–0.033

6–15

[91]

Co, Ni, Cu

SRM seawater

Flow mode

GF-AAS

[92]

Seawater

Flow mode

ICP-MS

1.5 104– 0.0012 0.001–0.076

—

Trace metals

7.5

[93]

Mn, Co, Cu, Zn, Cd Cr

SRM seawater

Flow mode

ICP-MS

0.01–0.04

25

[94]

Seawater

Flow mode

ICP-MS

0.02

12

[95]

REEs

Seawater

Flow mode

ICP-MS

[96]

Biological RM, waste water —

SFb

ICP-AES

4 105– 2.5 104 0.05

9

Cd

Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1 Muromac A-1

SF-HGAASa

Cd

Cu alloys, Ni sponge SRM: biological, silicate SRM river water

25

[97]

Flow mode

ICP-AES

0.08–1.5

17

[98]

Trace metals

Cr, Ti, V, Fe, Al


Elements

Trace elements Pb Cu, Hg Pb Cd

Trace metals

Muromac A-1 Chelex-100

Flow mode

ICP-AES

SIA

Chelex-100 AG-1 8 PTFE

1–2

[99]

Spectrophotometry 25

17–24

[100]

SIA SIA SIA

Spectrophotometry 0.63–0.25 Spectrophotometry 25 GF-AAS 0.0013

21 10 16

[101] [102] [103]

PTFE

SIA

ICP-MS

0.003–0.006

18

[104]

Chelating disk Muromacs Muromacs Muromac A-1

Flow mode

ICP-MS

9 104–2.5

10

[105]

Flow mode Flow mode Flow mode

ICP-AES ICP-AES ICP-AES

1 0.1 0.08–0.15

5 4 6

[106] [76] [107]

Flow mode

ICP-MS, ICP-AES

Flow mode

ICP-AES

49 105–0.8 — 0.3–28 0.003–0.3 6

As(III), As(V) Freshwater As(III), As(V) Freshwater Cr(III), Cr(VI) River water, tap water, wastewater Trace Seawater Muromac elements A-1 Trace metals Tap and river water Chelating disk a

Suction flow-HG-AAS. Suction flow.

b

0.001–0.2

[108] [109]


Cd, Pb

Seawater, river water Natural, waste water River water Drinking water SRM: sea lettuce, river sediment, natural water Urine, river water, SRM: natural water, sea lettuce Natural waters

187

188


1

2

3

4

5

6

7

8

9

Be

1 11

12

13

14

15

16

17

18

Cu

H

He Be

Li 2 Na

100

% Recovery

% Recovery

1

10

50 0

Mg

K

Ca

Sc

Rb

Sr

Y

Cs

Ba

50

B

0 1 2 3 4 5 6 7 8 9 pH

3

100

V

Cr

Mn

Co

N

O

F

Ne

Si

P

S

Cl

Ar

As

Se

Br

Kr

I

Xe

At

Rn

Al

1 2 3 4 5 6 7 8 9 pH

Fe

C

Ni

Cu

Zn

Ga

Ge

Pd

Ag

Cd

In

Sn

Pt

Au

Hg

Tl

Pb

Ti

4

Zr

Mo Nb

5 Hf La-Lu

6

La

Tc

Ta Ce

Pr

Re Nd

Rh

Sb Bi

Os

Ir

Sm

Eu

Gd

Tb

Dy

Ho

Er

Tm

Pu

Am

Cm

Bk

Cf

Es

Fm

Md

Po Yb

Lu

Pm

Lanthanoid Th Actinoid

Ru

W

Te

Ac

U Pa

Np

No

Lr

Figure 17 Adsorption behavior of trace elements at various pHs on the Muromac A-1 resin using Auto-Pret AES system (off-line measurement). Sample volume, 1 mL; concentration of alkaline, alkaline earth and Fe is 100 ng mL1 (measured by ICP-AES); the concentration of other metals is 10 ng mL1 (measured by ICP-MS); eluent, 1 mL of 2 M nitric acid. Reprinted from Ref. [85]. Copyright (2007), with permission from The Japan Society for Analytical Chemistry.

The Auto-Pret AES with a resin selective for lead, Analigt Pb-01 (GL Sciences, Tokyo), can be used for trace amounts of lead in river water samples with an LOD of 50 pg mL1 [110]. The Auto-Pret system can be used for various kinds of analytes by changing a solid phase packed in the mini-column. Chitosan resins containing chelating functional groups are packed in a mini-column, and used for on-line SPE-ICP-AES for the simultaneous determination of metals in water samples with Auto-Pret AES [111–113].

4. GAS–LIQUID EXTRACTION BASED ON MASS TRANSFER The mass transfer of analytes from a gaseous phase to a liquid phase has traditionally been performed by bubbling the gas phase into a liquid phase. This classical approach is very simple and the collection efficiency can often reach almost 100%, although it needs a long time to achieve a high enrichment factor, and is difficult to miniaturize and incorporate a bubbling system in a flow analysis system. To overcome the disadvantages of the gas bubbling methods, several gas–liquid extraction methods have been developed; these are a gas denuder (DN), a diffusion scrubber (GDS), and a CMC.


189

RP 1

0.5 M CH3 COONH4 Å@

Waste

Waste (a) RP 1 Chelating disk Waste

V1

Sample

RP 2 V2

Chelating resin (φ 50Å`100 μmÅj

ICP-AES

Eluent Å@ÅiHNOÇRÅj

3 mm

5 ppb 3 ppb 1 ppb (c)

(b)

Chelating disk (φ6 mm)

Cd Åi=228.802 nmÅj, 1~5 ppb

Figure 18 Automated pretreatment system (a) with a mini-column RP, peristaltic pump; V, six-way switching valve; (b) a mini-column packed with two chelating disks (3 M Empore extraction disk) and chelating resin (Muromac A-1); (c) an example of flow signals for Cd. Reprinted from Ref. [110]. Copyright (2006), with permission from The Japan Society for Analytical Chemistry.

Table 5

a

Enrichment factors and limits of detection (LOD) [109]

Element

Enrichment factor

Collection efficiency, %

LOD ng mL1

ICP-AES LODa ng mL1

Bi Cd (1) Cd (2) Co (1) Co (2) Cr Cu Mn Ni (1) Ni (2) Pb V

2.67 11.68 10.03 9.15 9.79 5.95 11.73 11.50 8.07 9.00 5.33 9.05

101.9 134.9 114.3 125.3 120.2 77.3 120.3 141.9 115.5 105.9 130.4 100.1

0.31 0.015 0.022 0.062 0.050 0.043 0.025 0.003 0.051 0.090 0.25 0.048

3.0 1.5 2.1 — 4.5 2.2 1.8 0.3 — 10.0 25.0 3.5

LOD of normal measurement by ICP-AES obtained from Seiko Instruments.

190

Table 6


Analytical results for water samples [109] Found, ng mL1

Element

Bi Cd (1) Cd (2) Co (1) Co (2) Cr Cu Mn Ni (1) Ni (2) Pb V

Tap water A

Tap water B

River water C

River water D

1.15 0.05 0.28 0.38 0.31 1.00 1.02 0.30 0.71 0.83 0.57 0.81

1.06 0.07 0.30 0.34 0.30 0.94 1.95 0.09 0.57 0.60 0.63 1.15

1.66 0.11 0.38 0.52 0.37 0.86 0.72 0.12 0.65 0.51 0.27 1.15

1.52 0.11 0.34 0.50 0.39 0.86 0.60 0.10 0.48 0.58 0.62 1.18

Sampling details: A, Faculty of Science, Okayama University; B, Venture Business Laboratory, Okayama University; C, Asahi River, Okayama city; D, Zasu River, Okayama University.

HC ICP-AES P2

P1

6 5

1

4 SV3

2 SW

PP

SP Waste

Water

2M HNO3 Buffer Standard Sample Waste

Water

Figure 19 Schematic diagram of the automated pretreatment system for ICP-AES (Auto-Pret AES system). SP, two-port syringe pump; SV, six-port selection valve; HC, holding coil (2.5 mL); SW, six-port switching valve; PP, peristaltic pump equipped to ICP-AES system; (—), loading stage; (y), eluting stage. Reprinted from Ref. [85]. Copyright (2007), with permission from The Japan Society for Analytical Chemistry.


Table 7

Preconcentration cycle steps of Auto-Pret AES system [85]

Step Operation description

1

2

3

4

5

191

Cleaning (a) 2 M HNO3 (b) Ultrapure water (UPW) (c) Cleaning Conditioning (a) 0.1 M buffer (b) Conditioning Preconcentrating (a) Sample/standard (b) Loading Matrix removing (a) UPW (b) Washing Eluting (a) 2 M HNO3 (b) Eluting

SPa

SVa SWa Flow rate, Volume, mL s1 mL

Aspb (P2) Aspb (P1)

6 0c

Disb (P2)

2

Aspb (P2) Disb (P2)

5 2

Ld

Action

70 300

1 0.5

Aspirate Aspirate

50

1.5

Dispense

Ld

70 50

0.5 0.5

Aspirate Dispense

Aspb (P2) 3/4 2 Disb (P2)

Ld

70 50

2.5 (2 ) Aspirate 2.5 (2 ) Dispense

Aspb (P1) 0a,c 2 Disb (P2)

Ld

300 50

0.5 0.5

Aspirate Dispense

Aspb (P2) Disb (P2)

Ed

70 8

0.5 0.5

Aspirate Dispense

6 2

a

SP, syringe pump with ports P1 and P2; SV, selection valve with six ports; SW, 6-way switching valve equipped with a mini-column. Asp, aspiration from the port P1 or P2; Dis, dispense from the port P2. c SV inactive. d L, loading stage; E, eluting stage. b

4.1 Gas denuders and diffusion scrubbers In a conventional gas DN, absorbing materials, such as viscous or less viscous liquids and solids, are coated on the inner wall of a tube. When a gas sample flows through this tubing, mass transfer of analytes occurs from a gas phase to a liquid phase or vice versa by gas diffusion. After the collection of analytes, the inner wall of the tube is washed with a suitable solution, and the analytes collected are eluted with or dissolved in a suitable solution. Such a DN can be used for the discrete sampling and the collection/concentration of analytes. A similar principle is used in paper meter gas detectors, in which a ribbon paper impregnated with absorbing reagents is used. However, collection methods using DN and the ribbon paper are less useful for liquid flow-based analysis systems. In GDS, a liquid absorbing solution is kept on one side of a gas-permeable membrane, while a gas sample flows over the other side of the membrane. A gas sample can be continuously flowed over the diffusion membrane, while an analyte in the gas sample can be transferred into the absorbing liquid. Therefore GDS can be favorably applied to continuous flow measurement methods, such as FIA, SIA, IC, and HPLC. Dasgupta reported GDS in 1984, for the first time, and

192


Table 8 Analytical characteristics obtained by ICP-AES coupled with Auto-Pret AES system [85] Elements

Ba Be Cd Co Cr Cu Fe Mn Ni Pb Sc V Zn

Wavelength, nma

Linear range, ng mL1

Linearity, R2b

Enrichment % RSDd factorc

493.408 313.042 226.502 228.615 205.560 324.754 259.940 257.610 231.604 220.353 361.383 292.401 213.856

0.1–10 0.001–10 0.01–10 0.1–10 0.1–10 0.1–10 0.1–10 0.01–10 0.05–10 0.1–10 0.01–10 0.1–10 0.1–10

0.9973 0.9946 0.9991 0.9987 0.9998 0.9909 0.9968 0.9997 0.9971 0.9980 0.9916 0.9986 0.9955

5 10 16 9 14 15 13 10 12 16 19 17 12

4.6 5.1 6.7 9.6 4.6 2.1 2.7 4.4 6.7 5.5 4.5 2.9 8.7

LOD, ng mL1 AutoPretc,e

Direct ICP-AESf

0.02 0.001 0.018 0.10 0.09 0.08 0.05 0.008 0.16 0.18 0.01 0.09 0.02

0.4 0.4 0.6 1.5 1.3 1.5 1.3 0.4 2.4 4.4 0.3 1.4 1.3

a

Emission wavelengths, are based on US-EPA Method 200.7 32. R , coefficient of determination. c Mean n ¼ 7. d Obtained by using 5 mL of the mixed standard containing 0.5 ppb of each metal ion (n ¼ 7). e Limit of detection, corresponding to 3 S/N ¼ 3. f Instrumental detection limit, corresponding to 3 sn1 for 0.01 M HNO3 (n ¼ 10). b 2

later he and his colleagues developed a number of GDS-based analytical systems [114–120]. GDS is one of the most useful devices for on-line gas-liquid extraction, which can be miniaturized and used in portable systems, and installed in various kinds of flow-based analytical systems. A number of applications to various kinds of gaseous analytes in real samples have been reported by Dasgupta, Toda, and their colleagues, and these are summarized and reviewed elsewhere [121,122], and in detail in Chapter 22.

4.2 Chromatomembrane cell A type of gas collector that uses a different approach from those of DN and GDS is the CMC, which was first reported by Moskvin in 1994 [123]. It was later improved by Moskvin, Simon, and their colleagues, who developed a number of applications in liquid–liquid [25,26] and gas–liquid [124–126] extraction of analytes. The CMC is made of a biporous PTFE block, which has two kinds of pores, micro-pores (0.1–0.5 mm) and macro-pores (250–500 mm), and the specific interfacial area is about 65 cm2 per cm3 [127]. The macro-pores are filled by polar liquids, whereas the micro-pores are occupied only by non-polar phases, such as

Table 9 Analytical results of river water reference materials obtained by ICP-AES coupled with Auto-Pret AES [85] SLRS-4a

Elements

a

JSAC 0302c

Certified values ng mL1

Auto-Pret ng mL1


Auto-Pret ng mL1


Auto-Pret ng mL1

12.270.6 0.00770.002 0.01270.002 0.03370.006 0.3370.02 1.8170.08 10375 3.3770.18 0.6770.08 0.08670.007 — 0.3270.03 0.9370.10

ORd 0.00970.001 0.01470.005e 0.04070.012e 0.3370.02 1.9270.13 ORd 3.3370.16 0.6570.02 0.09270.002e (0.02670.003)f 0.3470.04 0.9270.08

0.6070.02 — 0.002370.0007 — 0.1570.01 0.5770.07 4.770.3 0.12570.007 — (0.005)f — — 0.1970.03

0.5570.03 (0.00670.001)f 0.00570.002e (0.04070.010)e,f 0.1870.02 0.6170.05 4.670.5 0.1370.03 (0.08270.013)e,f NDd (0.03670.008)f (0.4770.04)f 0.1770.03

0.6070.01 0.9970.04 1.0170.01 — 10.170.2 10.370.2 5671 5.070.1 9.970.2 10.170.2 — — 10.270.3

0.5870.01 1.0270.01 0.9270.06 (0.03270.013)e,f 9.870.2 10.770.3 ORd 4.970.1 9.470.3 9.870.6 (0.01570.004)f (0.4970.04)f 9.870.3

River water reference material for trace metals issued by National Research Council Canada. River water reference material for trace metals issued by The Japan Society for Analytical Chemistry (unspiked). c River water reference material for trace metals issued by The Japan Society for Analytical Chemistry (spiked), diluted 2 times with 0.01 M HNO3. d OR, over range; ND, cannot be detected. e Sample volume: 15 mL. LODs of Cd, Co, Ni and Pb were 0.005, 0.03, 0.05, and 0.06 ng mL1, respectively, when 15 mL was used. f Figures in parentheses were information values. b



JSAC 0301-1b

193

194

Elements

Artificial RWa % recovery

Asahi river ng mL1

Zasu river ng mL1

Nishi river ng mL1

Tap water 1b ng mL1

Tap water 2c ng mL1


111.5 98.7 92.9 96.8 105.9 94.5 103.4 105.0 106.6 96.9 97.9 92.8 101.9

2.670.2 0.00270.001 0.04270.003 0.04670.010e 0.2470.02 1.770.1 ORd 5.270.6 0.2970.02 0.2570.06 0.0570.00 1.270.2 2.370.1

1.770.2 0.00370.001 0.02370.008 0.2070.03 0.1770.00 1.870.3 ORd 3.770.1 0.3670.01 0.2370.04 0.0470.01 1.070.1 0.870.1

9.670.4 0.00970.001 0.09070.006 0.1570.04 0.3670.04 1.270.0 ORd ORd 0.9470.10 0.4870.10 0.1370.01 1.870.3 6.871.0

5.070.8 0.00370.001 0.00570.002e 0.03570.018e 0.1270.01 2.570.2 ORd 0.2570.01 0.7370.07 0.0870.02e 0.0570.01 0.9470.05 0.870.1

6.370.4 0.00370.001 0.00670.002e 0.04970.013e 0.1370.03 7.370.0 3.770.1 0.2070.02 0.7570.11 0.1070.04e 0.0870.00 1.270.1 0.870.0

Sample volume: 5 mL. a Artificial river water samples spiked with mix standard solution, concentration of each metal ions was 0.5 ng mL1. b Tap water, Faculty of Science, Okayama University. c Tap water, VBL Okayama University. d OR, over range e Sample volume: 15 mL


Table 10 Analytical results for trace metals in tap and river-water samples obtained by ICP-AES coupled with Auto-Pret AES [85]

195


an organic solvent or a gas: this can occur since the capillary pressure of polar liquids prevents their penetration into the micro-pores. A schematic illustration of CMC and its collection mechanism is shown in Figure 20. In the general use of a CMC for gas–liquid (aqueous phase) extraction, an absorbing aqueous solution first fills the macro-pores as a stationary phase, and then the gas sample containing gaseous analytes is flowed continuously through the CMC, where the gas phase passes through the micro-pores and goes out of CMC (Figure 21). The mass transfer of the analytes in the gaseous sample occurs while the sample is flowing through the biporous PTFE block. Rapid, complete transfer of analytes can be easily achieved because the interfacial area between the two phases is very large; therefore a high enrichment can be easily achieved because a small volume of an absorbing solution is retained in the macro-pores as the stationary phase, while a large volume of gas sample flows through CMC. In the CMC method, the volume ratio of a gas sample to a stationary phase is very large, and usually several hundred-fold enrichment can be achieved. Furthermore, the collection efficiency of analytes from air is almost 100%, which means that gaseous standards are not necessary for the calibration of a system since aqueous standard solutions can be used as standards. On-line preconcentration of analytes in atmospheric samples can be realized by coupling CMC with FIA or other flow-based methods. Gas–liquid extraction can be performed with an automated CMC system which consists of a six-way switching valve, a liquid pump for filling an absorbing solution, a gas pump for aspirating a gaseous sample and delivering it to CMC, a degassing device, and a CMC device packed with biporous PTFE block (12 mm o.d. 14 mm) and a

Air in

Abs. soln. in

CM

Abs. soln. out

Illustration in CMC

Peak profile

Air out Volume

C C C C C

Conc.

C

V1

V2

V3

Air

V

V

V

Figure 20 Illustration of the principle of the distribution of an analyte in a CMC.

Time

196


CMC Air Air

P3

Figure 21 Schematic flow injection system coupled with a CMC system for the determination of sulfur dioxide in air. RS, reagent solution (4 105 M pararosaniline+4.5 102 M formaldehyde at pH 1.4); CS and AS, absorbing solution (2 g l1 triethanolamine (TEA) solution); P1, double-plunger pump (each flow rate 0.2 mL min1); P2, peristaltic pump (0.5 mL min1); P3, syringe type pump (5 mL min1); V1 and V2, six-way valves; SL, standard sample loop; S, sample; M, mixing joint; DG, degassing unit; RC; reaction coil (0.5 mm i.d. 200 cm). Reprinted from Ref. [132]. Copyright (2004), with permission from The Japan Society for Analytical Chemistry.

micro-porous PTFE filter (Figure 21). The CMC device is connected to the switching valve. The CMC-FIA system can be applied to the determination of nitrogen dioxide in air (LOD: 0.9 ppbv) [128–130], sulfur dioxide in air (0.5 ppbv) [131], and formaldehyde in air (0.03 ppbv) [132]. Further details on the CMC are provided in Chapters 9 and 22.

5. ON-LINE PRETREATMENT SYSTEM, INCLUDING COMPUTERCONTROLLED AUTOMATED SYSTEMS Various kinds of on-line sample-pretreatment devices for preconcentration have been applied to FIA and SIA. In general, the preconcentration process needs tedious and time-consuming procedures. If the preconcentration process is carried out on-line by conventional FIA, the system will become very complicated, several pumping systems and switching valves are necessary to assemble the system, and large volumes of a carrier and reagents are necessary. As a result, large amounts of waste are generated. Figure 22 shows an example of a system based on FIA for the speciation of Cr(III) and (VI) in river water and seawater samples [107,108]. Such a

197


P2

ICP-AES H2O

P1

C1

E V4

C2

H2O

W V2 S

RC

V3

RS

V1 R

Figure 22 Flow diagram of on-line dual-column FIA coupled with ICP-AES system. P1 and P2, peristaltic pump; C1 and C2, column, PTFE tubing 5 cm 2 mm i.d. packed with iminodiacetate chelating resin (Muromac A-1); S, sample, pH 3.3, flow rate of 1.0 mL min1; E, eluent, 2M HNO3, flow rate of 1.0 mL min1; R, reducing agent, 100 mM hydroxyammonium chloride; RS, reduction-switching unit, flow rate of 0.2 mL min1; RC, reaction coil, PTFE tubing 5 m 0.5 mm i.d. Reprinted from Ref. [107]. Copyright (2005), with permission from Elsevier B.V.

Cr(VI)

Cr(III)

0.2M NH4OAc pH 3.5 Standard/ sample SL

Holding coil (5 m x 1.6 mm i.d)

1

8

2 3

7 6

Water (W1)

ICP AES

Air

Cr (VI)

SV1

Cr (III)

waste

2M HNO3

5 4

water SV2

Syringe pump Volume: 10 ml

waste

PP

waste

Figure 23 Automated pretreatment system for the speciation of Cr(III) and Cr(VI). Columns size: 40 mm 2 mm i.d. [134].

preconcentration system based on FIA can be replaced by a system based on SIA. One of the most powerful features of SIA is the ability to perform computer control of flows. This enables arbitrary timing of aspiration or delivery of several kinds of solution at arbitrary flow rates under a previously determined program. By utilizing this function, much more versatile preconcentration systems, as well

198


as pretreatment systems, can be designed and assembled by coupling SIA with several switching valves. In Figure 23, a fully automated computer-controlled pretreatment system, Auto-Pret system, for double mini-column is shown [133]. The system can be simplified by removing one switching valve, when a 10-port switching valve is available. The Auto-Pret AES system can be applied to various kinds of sample preconcentration, and is coupled with ICP-AES [85,110–113]. The Auto-Pret system can be combined with an auto-sampling system, a spectroscopic detection system (ICP-AES, -MS, and AAS) and a data processing system to constitute a computer-assisted flow chemical analysis system (CAFCA system), by which total chemical analysis from sampling to data processing can be performed automatically under a computer control [134].

ABBREVIATIONS 4-AAP ANS CL CMC CTAB CTFE CVAAS CXA DDAP DHN DHNS DN EDDP FAAS GDS HG-AAS IBMK ICP-AES ICP-MS LAV LEI LOV MB MES MIP MoP PCTFE PDC PTFE RB SE

4-aminoantipyrine anilinonaphthalenesulfonate chemiluminescence chromatomembrane cell cetyltrimethylammonium bromide poly(chlorotrifluoroethylene) cold vapor atomic absorption spectrophotometry N-cinnamoyl-N-(2,3-xylyl)hydroxylamine ammonium diethyl dithiophosphate 2,3-Dihydroxynaphthalene 1,8-Dihydroxy-3,6-naphthalenedisulphonic acid denuder ethylenediamine-N,Nu-dipropionic acid, dihydrochloride Flame atomic absorption spectrometry Gas diffusion scrubber Hydride generation-atomic absorption spectrometry isobutylmethylketone inductively couple plasma-atomic emission spectrometry inductively coupled plasma-mass spectrometry Lab-at-valve laser-enhanced ionization detector Lab-on-valve Methylene blue 2(N-morpholino)ethanesulfonic acid molecularly imprinted polymer Molybdophosphate Polychlorotrifluoroethylene pyrrolidinedithiocarbamate polytetrafluoroethylene Rhodamine B solvent extraction


SPS TAN TBPE TMPyP

199

solid phase spectrometry 1-(2-Thiazolylazo)-2-naphthol tetrabromophenolphthalein ethyl ester 5,10,15,20-tetrakis(N-methyl-pyridynium-4yl)-21H,23H-porphine, tetrakis(p-toluenesulfonate)

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CHAPT ER

8 Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation Maria Dolores Luque de Castro

Contents

1. 2. 3. 4. 5. 6.

Introduction The General Membrane-Based Separation Module The Continuous Manifold Detectors Chemical Reactions Involved Dialysis 6.1 Generalities 6.2 Membranes 6.3 The module, the FIA manifold and other units 6.4 Applications of on-line dialysis 7. Microdialysis 7.1 Types of microdialysis probes 7.2 Detectors and microdialysed samples 7.3 Salient improvements in microdialysis 7.4 Calibration in microdialysis 7.5 Coupling MD to high-resolution separation techniques through dynamic approaches 8. Gas Diffusion 8.1 Applications of continuous gas diffusion 9. Analytical Pervaporation 9.1 The pervaporator 9.2 The auxiliary manifold 9.3 Detectors coupled to FIA–pervaporation 9.4 Coupling a pervaporator to a gas chromatograph: an alternative to headspace sampling 9.5 Coupling a pervaporator to capillary electrophoresis equipment Abbreviations References


204 204 207 211 211 212 212 213 214 214 218 219 221 221 222 222 223 223 226 226 227 229 229 230 231 232


203

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Maria Dolores Luque de Castro

1. INTRODUCTION Sample preparation (SP), a crucial step and the bottleneck of most analytical processes, is gaining due recognition as a specialized area in analytical chemistry [1]. An increasingly important consideration when developing SP methods is the ability to automate the entire analytical process. In this respect, flow injection analysis (FIA) is a key tool for SP as it facilitates the implementation of the steps required to obtain the ‘‘analytical sample’’ [2]; that is, the solution to be led to the detector or injected into a high-resolution equipment. Although the latest generations of flow-based approaches possess attractive features for use in this field, their short lives have allowed for only modest development to date. The variety of techniques used to prepare liquid samples (or solids that are previously dissolved in part or in full) are primarily employed for cleanup and/ or preconcentration. Those involving the use of a membrane are the subject matter of this chapter and the next. All membrane-based separation techniques have the advantage that they can be incorporated in dynamic (mainly FIA) manifolds for continuous operation. Table 1 lists such techniques — which differ mainly in the physical state of the membrane (i.e., solid or liquid) — and states the material transferred through the membrane and the driving force of mass transfer through it in each case. Based on the different materials that are transferred and also the different driving forces, existing dialysis modes (viz. conventional or passive, Donnan, electrodialysis, microdialysis) are listed separately in the table. Liquid membranes, which are widely used at present to implement a variety of liquid–liquid extraction techniques or modes, are the subject matter of the next chapter, which follows the discussion of conventional continuous liquid–liquid extraction. Continuous filtration, which can be implemented by using a membrane or filter, but also filterlessly — by exploiting special geometric characteristics of the dynamic system or with the assistance of ultrasound — is discussed also in the next chapter. In most membrane-based separation processes the membrane separates two miscible or immiscible fluid phases that can be static or mobile. Most frequently, the two fluids are liquids, one constituting the liquid sample (often referred to as the ‘‘feed’’ or ‘‘donor solution’’) and the other the ‘‘receiver’’, ‘‘strip’’ or ‘‘acceptor solution’’. The membrane prevents mixing and, very often, direct contact between the two solutions, the latter function being particularly important when the donor and acceptor solutions are miscible. One major feature of solid, porous membranes — those used in the techniques discussed in this chapter — is the so-called molecular weight cut-off (MWCO), which is governed by pore size. The boundaries between membrane techniques, which have been established from particle size, as proposed by Porter [3], provide an indication of the relationship between MWCO and pore size.

2. THE GENERAL MEMBRANE-BASED SEPARATION MODULE Figure 1 shows the two main types of membrane separation modules: sandwich and tubular or hollow-fibre. The former consists of two blocks made of Perspex,

Membrane-Based Separation Techniques: Dialysis, Gas Diffusion and Pervaporation

Table 1

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Features of continuous membrane-based separation techniques

Technique

Material transferred

Driving force

Material retained

Ions and lowmolecular-weight compounds

Concentration difference

Charged compounds Idem

Ionic strength gradient Electrical field

Microdialysis

Ions and lowmolecular-weight compounds

Concentration difference

Gas diffusion

Gases and vapours

Pressure difference

Analytical pervaporation

Gases and vapours

Pressure difference

Dissolved and suspended material with high molecular mass No-charged compounds No-charged compounds Dissolved and suspended material with high molecular mass Membrane impermeable gases and vapours No gas or vapour compounds

Soluble compounds in the immiscible phase Idem Compounds with reversible change of charge Soluble compounds in the immiscible phase Idem

Partition coefficient

Dialysis Conventional, passive dialysis

Donnan dialysis Electrodialysis

Continuous liquid–liquid extraction Conventional

Without typical units Supported liquid membrane extraction (SLME) Microporous membrane liquid–liquid extraction (MMLLE) Polymeric membrane extraction (PME) or membrane extraction with sorbent interface (MESI) Continuous filtration With filter

Idem Change of partition coefficient Partition coefficient Idem

Solvent and dissolved species

Gravity

With knotted-tube

Idem

Centrifugal

Ultrasound-assisted

Idem

Standing ultrasonic waves

Insoluble compounds in the immiscible phase Idem Compounds with permanent charge Insoluble compounds in the immiscible phase Idem

Suspended material variable particle size cut-offs Suspended, precipitate material Idem

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(1)

Acceptor stream

(A)

To detector Membrane

Waste Donor stream (sample)

(2) (a)

(b)

(c)

Figure 1 Different types of membrane-based units. (A) Sandwich-type (1), with different chamber designs (2): (a) parallelepipedal, (b) winding and (c) spiral. (B) Tubular or hollow-fibre type.

Teflon, aluminium or some other material having identical internally engraved conduits (usually semicircular, triangular or rectangular grooves 0.1–0.5 mm deep and 0.5–2 mm wide) that make up the inner chamber, the geometry of which varies from model to model. The membrane is placed between the two blocks, which must be joined tightly in order to avoid leakage. Each engraved microconduit has two holes on its ends that connect it with the manifold tubing. The tubular module consists of two concentric tubes, the inner one being a porous tube of an appropriate polymer through which the donor stream (the sample) is circulated internally while the acceptor stream is circulated externally or vice versa. The main variables influencing the performance of a membrane-based separation module are as follows: (i) membrane surface, which is a function of the particular membrane material; (ii) membrane path length, which should be as long as possible; (iii) membrane porosity, which is crucial in some techniques; (iv) membrane thickness, which should be a compromise between mass-transfer efficiency and membrane life, and (v) membrane geometry, which should result in a large contact area.


(B)

207

Waste

Acceptor (or donor) stream

Membrane

To detector

Donor (or acceptor) stream

Figure 1 (Continued).

The best relative position of the donor and acceptor chambers depends on the particular technique. Thus, dialysis is favoured by placing the acceptor chamber below the donor chamber, and the opposite holds for gas diffusion and pervaporation; in liquid–liquid extraction, however, the best position depends on the relative density of the two immiscible phases.

3. THE CONTINUOUS MANIFOLD Coupling a continuous FIA manifold to a membrane-based separation module has opened up a host of prospects for conditioning the donor and/or acceptor solution to optimize mass transfer or implement post-mass transfer reactions in order to prepare the analytical samples for the next step of the overall process. With these aims in mind, injection valves can be used to insert samples (Figure 2A) and switching valves (Figure 2B and C) to select the appropriate

208


(A) PP S

IV DS MSM

R1

W1

AS D

R2

(B)

W2

PP

R1

S/DS SV

MSM W1

C IV AS

R2

D

W2

Figure 2 Continuous flow configurations coupled to a membrane-based separation module (MSM) and, optionally, auxiliary reagents (R). (A) Continuous operational mode with injection of the sample into the donor stream. (B) Intermittent operation for preconcentration (the MSM is located in the loop of an injection valve IV). (C) Stopped-flow configuration with intermittent operation of one of the programmable pumps PP for preconcentration. AS, acceptor stream; C, carrier; D, detector; DS, donor stream; S, sample; SV, switching valve; W, waste.

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(C)

PP1

R1

S/DS SV W1 MSM

C

PP2 AS R2

D

W2

Figure 2 (Continued).

stream for each operational stage. The membrane-based separation module is usually placed in the transport–reaction zone (Figure 2A and C) or within the loop of an injection valve (Figure 2B). The sample can be injected into the channel through which the carrier acting as donor solution is circulated (Figure 2A) or, alternatively, can be continuously inserted by aspiration (Figure 2B and C), in which case the inserted volume is controlled through the flow rate and aspiration time. A higher efficiency of mass transfer entails stopping the acceptor stream, while the donor solution is continuously passing through the separation module for an accurately preset time, or stopping both streams if the sample is scant. This approach can be implemented in two ways, the simpler one being the use of a six-way valve as depicted in Figure 2B. The valve loop can be isolated in order to hold a static portion of acceptor liquid while the acceptor stream is driven directly to the detector during the enrichment mass-transfer time. The other choice is based on intermittent pumping, which entails synchronizing sample injection or aspiration with the stop-and-go sequence of pump PP2 to establish the flow of acceptor stream, while pump PP1 is allowed to work uninterruptedly (Figure 2C). In this way, a static microvolume of acceptor stream collects the analyte from a large sample volume over a programmed stop period. For small sample volumes, the donor, or both streams, can be stopped for proper mass transfer. In SIA manifolds, the donor and acceptor solutions are connected to two different ports of the multiposition selection valve and the acceptor line is connected to the detector [4]. The sample is aspirated into the holding coil and delivered to the donor port, where mass transfer to the acceptor solution occurs. Then, the acceptor solution is propelled to the detector. In order to expand the scope of these approaches, one can use more sophisticated manifolds containing

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additional components. For example, the acceptor stream can be driven by a second pump in a hybrid FIA–SIA manifold [4]. More drastic sample pretreatments such as hydrolysis or reagent addition require the use of additional pumps [5], which obviously complicates the experimental setup. No membrane-based separation modules have so far been coupled to lab-on-valve (LOV) devices. The variables most markedly influencing performance in continuous manifolds are as follows: (a) the flow rate, which dictates the membrane-solution contact time; (b) the composition of the donor — buffered, to avoid different behaviour of samples and standards — and acceptor phases, the latter of which should be selected as a function of the nature of the former [6]; (c) the relative direction of the flows (concurrent or countercurrent); (d) the temperature, which has a strong influence on some membrane-based techniques (particularly on those involving vapour formation). Other specific variables affect some techniques only and are discussed in the pertinent sections. Monitoring mass-transfer kinetics is crucial with a view to optimizing membrane-based systems. The main goals here are to identify the influential factors and establish an appropriate transfer time in order to ensure a high throughput without appreciably detracting from sensitivity. The kinetics of mass transfer can be monitored in two ways, namely: (a) by integrating separation and detection in a single module [7], which can be done by placing either a probe-type sensor (whether electroanalytical or optical) inside the acceptor chamber, with its active surface facing the membrane, or interfacing the acceptor chamber with an external detector via fibre optics, with optical detection; (b) by integrating, following separation, a retention step with detection, which can be accomplished by using a flow-cell packed with a suitable material (an ion-exchanger or sorbent) inside a non-destructive detector to quantify the continuous retention of the separated analyte or its reaction product. When mass transfer through the membrane fails to provide adequate concentrations of the target analytes, the process can be forced by increasing the flow rate of the acceptor stream in order to continuously deliver a clean acceptor for increased mass transfer and inserting a preconcentration module (e.g., a sorption column) downstream of the acceptor chamber [8]. When the concentrations of the target analytes in the sample exceed the upper limit of the linear range of the calibration curve, then a dilution or pseudo-dilution step must be used in order to fit the unknown concentration to this portion of the calibration curve and increase the precision of the measurements as a result. A number of alternatives to the usual prior sample dilution are possible and can be based on: (a) using a smaller loop for the injection valve; (b) changing the chemical conditions to a less favourable situation if a derivatization reaction is required, in order to reduce the yield of the monitored species; (c) using a thicker membrane; (d) increasing the flow rate of the donor and/or acceptor; (e) using a lower temperature in the donor chamber (with gas diffusion or pervaporation); and (f) enlarging the air gap between the sample and membrane by inserting an appropriate number of spacers (in pervaporation) [9].


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4. DETECTORS Most types of detectors have been used in combination with membrane-based separation modules, albeit to a different extent depending on both the type of analyte and separation technique involved. Thus, photometers are by far the most frequently used detectors to monitor the intrinsic absorbance of either the separated compounds or the products of their derivatization (for which FIA manifolds can be of great help) [10,11]. However, fluorescence or chemiluminescence-based molecular detection techniques are scarcely used in this context [12]. Atomic spectroscopic absorption and emission detectors are mainly used after liquid–liquid extraction. However, the use of electroanalytical techniques is usually associated with dialysis (particularly potentiometry and, to a lesser extent, conductometry and amperometry) [9,10]. Mass spectrometers are mainly used after microdialysis — the complexity of the compounds calls for such a sophisticated type of detector [13–15] — and also, occasionally, following dialysis or gas diffusion [16,17].

5. CHEMICAL REACTIONS INVOLVED Some membrane-based separation processes require a (bio)chemical reaction to be effective; such a reaction can take place outside the continuous system, in the donor or acceptor stream (or donor or acceptor chamber) or in both. Using a derivatizing reaction prior to separation in dialysis is uncommon, but subjecting the dialysate to some reaction is a frequent step. Derivatizing reactions in the acceptor chamber have a two-fold purpose. First, in taking place at the interface, they facilitate transfer of the analyte by preserving a favourable concentration gradient, thus increasing the rate of the separation process. Second, the derivatizing reaction can also help adapt the transferred analyte to the particular detector used, thereby frequently increasing the selectivity and sensitivity of the continuous method. Gas diffusion and pervaporation frequently require derivatizing reactions prior to separation, usually to generate volatile products from the analyte. Acid– base reactions are by far the most frequently used in this context: an acid carrier is incorporated into the carrier stream to cause the release of gases such as CO2, SO2, HCN, H2S, Cl2 or NO2 from their respective analytes; alternatively, a basic carrier stream induces the formation of ammonia gas from samples containing the ammonium ion, a very common product of enzymatic reactions. Concerning derivatizing reactions in the acceptor chamber, in addition to the two-fold purpose of dialysis, absorption of the transferred gas analyte by the acceptor solution is substantially increased if the analyte has acid–base properties. Thus, gases such as CO2, SO2, HCN and NO2 can be readily dissolved in a basic acceptor, and so can NH3 in an acid solution; then, a variety of analytical indicators can be used to monitor the separation process. However, one can also monitor the pH change caused by the analytes potentiometrically or use more complex derivatizing reactions depending on the analyte–detector combination.

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Another alternative is direct transport of the analyte by a carrier gas to the detector (e.g., with hydride generation or cold mercury vapour generation). In liquid–liquid extraction, reactions prior to or in the donor chamber intended to favour mass transfer, allow the analyte to be converted into a nonionic or ionic product depending on whether it is extracted to an organic or aqueous phase, respectively. When the analyte is in the acceptor, the goals of derivatizing reactions are similar to those in dialysis. Filtration can require a prior reaction either to form the solid species or facilitate its filtering; a subsequent dissolution reaction is usually necessary in direct methods if the analyte is contained in the solid phase.

6. DIALYSIS 6.1 Generalities In conventional, passive dialysis, separation between solutes is based on a concentration gradient between two liquid miscible phases, of the species liable to cross the membrane. Miscibility between the donor and acceptor phases clearly distinguishes dialysis from liquid–liquid extraction, and also from other membrane-based separation techniques such as osmosis or ultrafiltration. In osmosis, it is the solvent rather than the solute which crosses the membrane — it is unclear, though, whether this phenomenon occurs concomitantly with dialysis. In ultrafiltration, occasionally referred to as ‘‘reverse osmosis’’, a solution is forced under pressure across a membrane with concomitant separation of its components. The driving force in this case comes from the pressure difference and it is the solvent, rather than the solute, which crosses the membrane against the concentration gradient. The theoretical principles of dialysis are beyond the scope of this chapter and are described in detail elsewhere [10]. Dialysis can be classified according to the dynamic state in which one or both phases are involved. The process may or may not be allowed to develop until mass-transfer equilibrium is reached: if the two phases are quiescent, the process must develop to completion; however, if one or both phases are in motion — or even if either is stopped over a given interval to increase the efficiency of the process — equilibrium is never reached and strict timing must be employed. In the latter instance, the process can be carried out with agitation of a constant volume of the two phases or by using streams of the two phases which may flow concurrently. This latter alternative is the more commonly used in analytical chemistry (mainly with FIA systems) as it affords separation with precision of 1–2% RSD (i.e., much better than that achieved with air-segmented or non-segmented continuous flow systems) [11]. Dialysis has been implemented in FIA manifolds in its four modes, namely: passive or conventional dialysis, active dialysis in both the Donnan and electrodialysis modes and microdialysis — which is also passive — the first and last being the most widely used. Depending on the way the sample reaches


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the donor compartment (viz. continuously or after splitting into several aliquots), operation can be continuous or pulsed — alternatively, the flow can be halted at the donor compartment. The driving force in dialysis is the analyte concentration gradient across the membrane (i.e., passive dialysis); if an external electric field is applied, electrodialysis occurs. When the analytes are charged species, they can be separated from the sample matrix and preconcentrated in an acceptor solution by Donnan dialysis. In this case, an appropriate ion-exchange membrane (e.g., Nafion) is used to separate the sample from the acceptor solution, of smaller volume and higher ionic strength. Ions of a given charge as a function of the membrane type are transported from the acceptor solution into the sample solution as a result of the existing ionic strength gradient, while co-ions from the sample solution, including analyte ions, diffuse in the opposite direction in order to maintain electroneutrality. Unlike passive dialysis, electrodialysis and Donnan dialysis afford preconcentration of the analyte as well.

6.2 Membranes Dialysis membranes are hydrophilic and mainly of the microporous, homogeneous and ion-exchange types. Microporous membranes are structurally similar to a conventional filter and operate basically on the same principle. Pore sizes are typically 1–10 nm and hence much smaller than those of conventional filters. Typically made of cellophane, cellulose acetate, polycarbonate, polysulfone, polyvinylidene fluoride, copolymers of acrylonitrile and vinyl chloride, polyacetal, polyacrylate, polyelectrolyte complexes, cross-linked polyvinyl alcohols and acrylic copolymers such as Nafion, porous membranes, which are the most widely used, are filled with both the donor and acceptor solutions. Homogeneous membranes are made of homogeneous films with interfaces distributed uniformly throughout. Mass transfer across them occurs via molecular diffusion, dialysis efficiency depending on the solubility and diffusivity of species across the membrane interface. However, homogeneous membranes have scarcely been used in FIA–dialysis approaches. Ion-exchange membranes have a submicroporous structure with no conventional macroscopic pores, but consist of film-forming polymers with positively or negatively charged ions attached to pore walls. An important, yet often ignored parameter in dialysis is the MWCO, which usually ranges from 14,000 to 3,000 (i.e., 0.003–0.01 mm). Ensuring optimal separation between macromolecular matrix components and the target analytes entails choosing a pore size providing the best possible compromise between a high membrane flux of analytes and adequate removal of interfering compounds. The membrane thickness and porosity (i.e., the number of pores per unit membrane area) are often not reported in the scientific literature even though they can have a significant influence on the dialysis efficiency, since obtaining high analyte fluxes obviously requires using thin, highly porous membranes [18].

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6.3 The module, the FIA manifold and other units Usually, dialysers are of the type shown in Figure 1, which were formerly marketed by Technicon and Tecator, and later by Gilson; however, FIA practitioners used to design their own modules for proper fitting to their needs, which led to the development of a number of units with only slight differences between them. Conventional FIA manifolds for coupling to dialysers are as depicted in Figure 2. Some special designs are commented on in Section 6.4. In addition to the indispensable units of an FIA–dialysis system, some approaches require other units to facilitate application of the particular analytical methodology. Such units are also occasionally of separative nature (e.g., a dialysis–liquid chromatograph assembly in which the dialyser is used as a precolumn system [19], an ion-exchanger column intended to increase the sensitivity [20] or a gas diffusion module for the simultaneous or sequential determination of two analytes in the same sample [21] or the insertion of gaseous reagents in addition to liquids into the main flowing stream). The detectors most frequently used in this context are optical in general and photometric in particular. Detection is usually preceded by a derivatizing reaction between the dialysated analyte and a reagent dissolved in the acceptor stream. This increases the efficiency of the separation process by effect of the dialysis equilibrium being gradually shifted as the free analyte is removed from the acceptor stream. Optical techniques such as fluorimetry and atomic absorption spectrometry have also occasionally been used in this context — the scant use of atomic techniques can be a result of organic colloidal or suspended materials in the sample usually not interfering with most determinations. In regards to electrochemical detectors, on-line dialysis is very useful with a view to increasing the selectivity, stability and lifetime of ion-selective and voltammetric electrodes. No FIA–dialysis–GC-coupled systems appear to have been reported; also, FIA–dialysis has been only occasionally used with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) [20,21].

6.4 Applications of on-line dialysis The earliest applications of FIA–dialysis (passive dialysis) were developed by Ruzicka and Hansen for the determination of inorganic phosphate and chloride in blood serum [22]. Despite their early implementation, applications in this field have been rather scant compared to other separation techniques used in combination with FIA. This may have resulted from dialysis being slow relative to most FIA operations, and also from dialysis efficiencies usually being quite low. Under typical experimental conditions, solute transfer in FIA–dialysis is at best less than 15% — occasionally less than 1%; this can be ascribed to the short time available for solute transfer in FIA. Only in the scantier used Donnan dialysis and electrodialysis can preconcentration or enrichment factors be


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considered; some authors, however, use these terms as synonym for efficiency in passive dialysis. The concept of permselectivity, which refers to the preferential permeation of one molecule through a membrane with respect to diffusing molecules in a mixture, is frequently misunderstood. Permselectivity is defined as the ratio of the final concentration of molecules to their initial concentration. Therefore, the adjective ‘‘semi-permselective’’ is absurd. In addition to the methods for highly concentrated analytes in biological fluids such as blood, urine or milk [10,11], where dialysis avoids the need for dilution and complex deproteination steps, some more recent methods warrant a brief description here. L-(–)-malic and L-(+)-lactic acids in wine have been simultaneously determined as depicted in Figure 3, by coupling dialysis–enzymic derivatization with photometric or fluorimetric detection in order to monitor the reduced form of the coenzyme nicotinamide adenine dinucleotide (NADH). Discrimination between the two analytes was accomplished by splitting the dialysate into two aliquots, a portion of the solution being trapped in the loop of an auxiliary injection valve and each aliquot being led to one of the bioreactors by means of a switching valve. The ensuing method is fast and simple; also it provides results consistent with those of official methods and can be easily used to monitor malolactic fermentation in wines. A study of dialysis selectivity and efficiency as a function of membrane features such as pore size and material was previously carried out by using an FIA–dialysis system on-line connected to a liquid chromatograph [18]. The FIA–dialysis combination was used as early as 1984 for non-determinative purposes such as examining drug–protein (sulphonamide–bovine serum albumin, BSA) binding interactions [23] in a manifold, which included a sandwich-type dialyser. After more than 20 years, similar studies involving carbamazepine–BSA have been carried out by dialysis sampling using a hollowfibre unit (Figure 4A) placed in the loop of the injection valve (which operates as shown in Figure 4B) and coupled on-line with a solid-phase extractor prior to fluorescence detection [24]. The evolution of the FIA–dialysis coupling over the PP

IV2

(1)

Buffer

D IV3 IV1

H2O Sample

R1

SV (2)

R2

W2

M W1 DU

Figure 3 Flow-injection manifolds for the simultaneous determination of malic and lactic acids in wine. D, detector; DU, dialysis unit; IV, injection valve; M, membrane; PP, peristaltic pump; R, enzymatic reactor; SV, selection valve; W, waste. Reprinted from Ref. [18]. Copyright (2001), with permission of Elsevier B.V.

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S (A) PTFE tube

Epoxy resin

C

Microdialysis tubes

(B) W

W

3

3 2

4

3’

C

1

2

4’

2’

5’

1’

4

2’ 3’

1’ 5

D

C

1

8’

DU

4’

5

D

DU

8

7’

6 7

S Sampling

5’

7’

6’

8’

8

6’

6 7

S Injection

Figure 4 (A) Hollow-fibre dialyser and (B) its location in the loop of the sampling valve and operation. C, carrier; D, fluorescence detector; DU, dialysis unit; S, mixed sample solution; W, waste. Reprinted from Ref. [24]. Copyright (2005), with permission of Elsevier B.V.

past 20 years can be envisaged by comparing the two methodologies. In biochemical analysis, it is the preferred choice for determining iron bioavailability by simulating gastrointestinal digestion in a continuous flow–dialysis assembly coupled on-line to an electrothermal atomic absorption spectrometer (ETAAS) and pH-meter as in Figure 5 [25]. A comparison of the tubular dialysers in Figures 4 and 5 reveals that the two operate in the opposite manner as regards to the donor and acceptor streams. Donnan dialysis has scarcely been used in combination with FIA even though when used in 1989 it provided a 100-fold enrichment factor for cations [26]. This was later on improved to values over 200 for an 8-min dialysis time [27]. A more recent application of this approach to the determination of lead in spiked


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Figure 5 Scheme of a continuous manifold with the hollow-fibre dialyser operating in an opposite manner to that in Figure 4A. The pH measurement cell and ETAAS autosampler are also shown. Reprinted from Ref. [25]. Copyright (2005), with permission of Springer-Verlag.

sweeteners provided recoveries greater than 90% [28]. The main reason for such limited application, which contrasts with the high efficiency achieved, is the steep ionic strength gradient required to facilitate the process, which accounts for the fact that Donnan dialysis has only been used prior to optical atomic detection and never with biological fluids [29]. Electrodialysis has also scarcely been coupled to FIA manifolds despite the apparent simplicity of the approach and the efficiency of the process, e.g., more than 37% for chloride [30] and 94% for copper(II) [31]. Possibly, the presence of high concentrations of other species with charge of the same type causes interferences in most cases. Less frequent, but also worth mentioning here, is the use of dialysis for reagent insertion into streams without dilution as proposed by Hwang and Dasgupta [32]. Finally, one completely ignored application of dialysis in continuous approaches is in membrane-protected flow-through electrodes to avoid deterioration and poor performance by eliminating direct contact between macromolecules and the active surface of the sensor [33]. Sequential injection analysis (SIA) has also been coupled to dialysis for purposes similar to those in FIA (i.e., analyte dilution and removal of highmolecular-weight sample components), albeit with substantially reduced sample and reagent consumption. Thus, chloride in milk has been dialysed, then conductometrically determined with an excellent precision (RSDo1%), but using sample and reagent volumes per analysis similar to those required by FIA

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computer detectors S DV

660 nm

1 RE

HC W pump

C

H- SE

Cl- SE

W

D

W

Figure 6 SIA–dialysis approach for the simultaneous determination of pH, chloride and nickel. C, coil; Cl–SE, chloride selective electrode; DU, dialysis unit; DV, stream directional valve; HC, holding coil; H+SE, pH selective electrode; Ref., AgCl/Ag reference electrode. According to Ref. [4]. Copyright (2004), with permission of Elsevier B.V.

(viz. 142 and and 750 mL, respectively) [34]. Simultaneous monitoring of pH, nickel and chloride has also been accomplished with the SIA–dialysis manifold in Figure 6, where, following monitoring of the sample pH, the sample is mixed with a derivatizing reagent to monitor nickel photometrically and finally dialysed, the dialysate being led to a chloride selective electrode for potentiometric monitoring [4]. Like FIA–dialysis [10,11], the SIA–dialysis couple constitutes an excellent tool for monitoring evolving systems. Thus, Lapa et al. developed a method for the simultaneous biosensing of glucose and ethanol in beer fermentation by which portions of the dialysate were driven to bioreactors containing immobilized glucose oxidase and alcohol oxidase, and the resulting H2O2 was quantified amperometrically at 50 samples per hour [35]. In an SIA determination of reducing sugars in wine by the neocuproine/ Cu(II) method, the dialyser served the two-fold purpose of diluting and separating the sugars from high-molecular-weight coloured compounds [36]. No LOV–dialysis combination has so far been reported.

7. MICRODIALYSIS Microdialysis (MD) warrants separate discussion from dialysis on account of its special characteristics and the devices used for implementation. In MD, the dialysate constitutes the sample itself, so the place where the sample has been taken should be the sampled system, which can be a living system or even a large sample portion where the MD probe is inserted for sampling. The fibre is perfused slowly with a sampling solution (the perfusate), the ionic composition and pH of which should be close to those of the system under study.


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The dialysis membrane, which can be microporous (e.g., polytetrafluoroethylene, PTFE, or polypropylene of 0.1–1 mm pore size), but also homogeneous non-porous (polydimethylsiloxane, latex), is permeable to small molecules, but not to macromolecules such as proteins [37]. Compounds which can diffuse through the membrane are swept to either a collection vial or an on-line connected manifold for appropriate treatment or detection. The key features of MD, which are the basis of its wide field of applications, include: (1) the ability to collect samples with minimum disturbance of the sampling site; (2) the improved selectivity by effect of high-molecular-weight species and particulate matter being removed from the sample medium; (3) no need for a purification step prior to analysis — as no cellular matter is diffused, and hence absence of enzymatic breakdown of dialysated substances; (4) the ability to continuously monitor reactions in near-real time; and (5) the ability to calibrate probes and estimate absolute concentrations in extracellular fluids in some cases. This last is a major concern as both in vitro and in vivo calibration methods are frequently unable to mimic the dialysis of species from the sampling site. Although MD is widely believed to be a relatively recent sampling technique, the earliest dialysis probe was developed back in 1972 [38]. Originally, MD was designed for sampling brain tissue; subsequently, however, it has been used to sample other organs and fluids including blood, adipose tissue, muscle tissue, liver, glands or even plant tissues such as those of bananas. Also, MD has been employed in physiological, pharmacological, toxicological and behavioural studies to remove endogenous substances such as neurotransmitters and their metabolites or exogenous substances such as drugs and toxicants with both in vivo [39] and in vitro sampling [40]. MD-based sampling is a powerful tool for studying in vivo pharmacokinetics and drug metabolism, and also for monitoring bioprocesses as it allows the concentrations of target analytes in blood or other tissues to be determined with minor changes in their composition. The main advantages of MD sampling are as follows: (1) sample collection is expeditious — long-time sampling (over several days) from freely moving animals is possible; (2) multiple MD sampling in the same animal allows the distribution of a drug in different pharmacokinetic compartments to be established [38]. A new and promising trend in this field is the use of MD as an in-situ sample-processing technique for environmental research [41]. A very different application of MD sampling is as passive dosimeter for on-site, real-time monitoring of chemical contaminants in pore soil solutions [42]. Below are briefly described the typical MD experimental setup, ancillary equipment; the types of probes, continuous manifolds, and detectors used; major achievements; calibration and the main combinations of MD with other techniques (namely: MD–HPLC and MD–CE).

7.1 Types of microdialysis probes There are four types of MD probes, namely: the stainless steel concentric cannula design, flexible side-by-side cannula design, linear design and flow-through

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(A)

(B)

FT

(C) Extension of FS

DM

DM FS

(D)

PET DM

Figure 7 Microdialysis probes: (A) concentric, (B) flexible, (C) linear and (D) flow-through cannulas. DM, dialysis membrane; FS, fibre skeleton; FT, flexible tubing; PET, polyethylene tubing.

design [43]. The first one is a concentric cannula, also known as ‘‘pin-style, rigid cannula’’ (Figure 7A) consisting of an inner and outer length of stainless steel tubing. The inner cannula extends beyond the outer cannula and is covered with the dialysis membrane. This probe is mechanically stable and permits precise placement in the brain, but is not amenable to implantation in peripheral tissues [44]. The second design, which is used for intravenous implantation and is called the ‘‘pin-style flexible cannula’’ (Figure 7B) is based on a modification of the concentric cannula involving the use of side-by-side pieces of fused silica. In this design, one piece of fused silica extends beyond the other and is covered with the dialysis membrane [44,45]. The third design is the most useful probe for sampling peripheral tissues (Figure 7C). In it, a hollow-fibre dialysis membrane is connected to a small-bore tubing to form an inlet on one end and an outlet on the other. This probe can be implanted simply by threading through the target tissue. The fourth probe (Figure 7D) is


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constructed by inserting an MD fibre into a length of polyethylene tubing [46]. MD probes and membranes are commercially available under different trade names (e.g., Bioanalytical Systems Inc., CMA/MD, Enka Glantzoff, ESA). The continuous manifolds used to interface probes to detectors or highresolution equipment are invariably very simple.

7.2 Detectors and microdialysed samples MD probes have frequently been coupled to electrochemical detectors [47], highly sensitive radioimmunoassay detectors [48] and, especially, to mass spectrometers (MS) and tandem mass spectrometers (MS/MS) [49,50]. Biosensors have enabled direct monitoring of glucose [51–53] or glucose and lactate by using an integrated array consisting of glucose and lactate enzyme biosensors [53]. Glucose and lactate have also been determined with an amperometric enzyme biosensor consisting of an enzyme bilayer oxidase/osmium poly(vinylpiridine) redox polymer, a horseradish peroxidase ring and a split-disk plastic film carbon electrode [54].

7.3 Salient improvements in microdialysis The performance of this membrane-based microtechnique has been improved by the use of cyclodextrins (CDs), an addition to perfusate which has salting-out effect for biological samples and allows low-molecular-mass components to be separated from biopolymers. CDs are also involved in the selective transport of the target analytes in chelated form [55]. Traditional MD shortcomings such as the need for time-consuming calculations in order to compensate for partial recovery of some analytes and depletion near the sampling site have been circumvented by developing a new sampling mode known as ‘‘ultraslow MD’’ [53], in which the flow rate is reduced from 2 mL/min to 100–300 nL/min. One effective alternative to conventional MD for complex systems is dual MD, which requires a more complex dynamic manifold than single MD. A complex biological sample is brought into contact with the first stage of MD in order to remove high-molecular-weight contaminants. The dialysate, which contains only medium-molecular-weight target compounds and low-molecular-weight contaminants, is then connected on-line to the second stage of MD to remove low-molecular-weight contaminants. A clean fraction of the sample, containing medium-molecular-weight target species, is thus obtained that can be directly analysed with an appropriate technique [50]. One other dual MD approach involves two MD membranes sandwiched between three polymer layers. In this case the sample passes sequentially through the first and second MD stage [49]. Continuous subcutaneous glucose monitoring is also possible by coupling an MD probe to a miniaturized thermal flow-through biosensor. To this end,

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the perfusor outlet is directly connected to the sample loop of the FIA miniaturized thermal biosensor [52].

7.4 Calibration in microdialysis Calibration is one of the most important issues in MD. At the perfusion rates typically used in MD, no equilibrium across the dialysis membrane is reached. The concentration of analyte collected in the dialysate is a fraction of the concentration in the sampled system. The efficiency of the microseparation step (viz. relationship between the concentration in the dialysate and that in the sampled system) is dependent on the type of membrane used and its length, the geometry of the probe, the perfusion flow rate, the sample matrix and the physical properties of the analyte [43]. Various approaches based on retrodialysis [56], extrapolation to zero flow rates [57], point of no net flux [58] and slow perfusion rates [45] have been developed to overcome these drawbacks.

7.5 Coupling MD to high-resolution separation techniques through dynamic approaches Reversed-phase or ion-exchange HPLC is the preferred choice for analysing MD samples [59]. Usually, short (r5 cm) microbore (1 mm i.d.) columns have been advocated for the analysis of microdialysates on the grounds of their combined high sensitivity and throughput. Conventional HPLC columns are also convenient to use here as they require no special devices such as low flow-rate pumps, low dead-volume injectors or reduced-volume detection cells. Microdialysates are protein-free and hence amenable to direct injection into a chromatograph or CE system. This enables direct coupling of MD to either techniques via a very simple dynamic manifold [60]. On-line MD–HPLC or MD–CE systems are useful for pharmacological studies. Near-real time information on the concentration of several analytes can be obtained simultaneously, and the separation and detection systems can be optimized for the target analyte. However, MD is a continuous sampling method and HPLC and CE require discrete samples; therefore, the dialysate must be collected over a fixed time interval in order to deliver the required sample volume, and each sample represents an average concentration obtained over the preset interval. As a result, temporal resolution is dependent on the sample requirements of the chromatographic system. Further improving temporal resolution and dialysis efficiency entails analysing submicrolitre volumes on-line. For this reason, CE is suitable for the analysis of MD samples [43]. Three types of on-line interfaces have so far been used to couple MD to HPLC or CE, namely: flow gap [61], flow gated [62] and attachable electrode interfaces [63]. Most of the ensuing applications use FIA configurations to develop the steps required in between MD and HPLC or CE separation; however, no applications involving SIA or LOV have been reported to date, even though these approaches are best suited to the small volumes provided by MD.


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8. GAS DIFFUSION The gas diffusion technique relies on the permeability of microporous membranes to gases. The process is typically kinetically controlled, each gas having its own diffusion constant, which is related to the so-called coefficient of permeability through the solubility constant in Henry’s law [64]. The mechanism of transport across a gas diffusion membrane comprises three consecutive steps, namely: (1) sorption of volatile components at the membrane surface; (2) diffusion of sorbed components through the polymer matrix and (3) evaporation from the polymer into the vapour phase on the permeate side of the membrane. The efficiency of the gas diffusion process is governed mainly by the intrinsic properties of the polymers used for membrane preparation. Glassy polymers (e.g., cellulose) have been shown to be less permeable than rubbery polymers (e.g., polydimethylsiloxane). Also, because the contribution of solubility to permeability prevails in non-glassy polymers, permeability increases with increased molecular mass of permeants. Selectivity depends on the molecular dimensions of the permeating species. Thus, hydrophobic gas diffusion membranes are usually made of PTFE and similar to those used in processes such as ultrafiltration and pervaporation. The gas diffusion module can be of the two types shown in Figure 1; i.e., sandwich or tubular. Similarly to dialysis — they only differ in the type of membrane used — modules have traditionally been supplied by Technicon, Tecator and, more recently, Gilson, and incorporated into both FIA and SIA manifolds. However, some users prefer to design or even make their own modules. Such is the case with the module recently reported by Iida et al., of the tubular type and very small dimensions, in which the ultrathin hollow-fibre — gas-permeable tubing of poly-4-methyl-1-pentene — is 190 mm in i.d. and 250 mm in o.d. [65]. The typical FIA manifolds coupled to gas diffusers are similar to those in Figure 2, and those typically used in SIA systems very much like that in Figure 6. No LOV–gas diffusion approaches have so far been reported. When a gas phase of analyte is formed in LOV, it is removed from the sample matrix by means of a gas–liquid separator [66].

8.1 Applications of continuous gas diffusion The earliest applications of FIA–gas diffusion were in the clinical field and involved the determination of carbon dioxide in plasma by using a straightforward manifold where the biological fluid was injected into a sulfuric acid stream and the 2 diffused analyte accepted by an HCO buffer containing an acid–base 3 /CO3 indicator [67]; that of ammonia in whole blood and plasma with direct potentiometric sensing [68] or photometric detection with the aid of an acid– base indicator [69] and the determination of ammonia in undiluted urine [70].

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Enzymatic systems with integrated separation and detection have been employed to determine analytes such as creatinine in undiluted blood serum [71]. The most frequent field of application of FIA–gas diffusion is environmental analysis, where it has enabled the determination of chlorine dioxide in water with chemiluminescence detection [72]; inorganic arsenic species in surface seawater by hydride-generation–ICP [73]; sulfide in water with an S2-selective electrode [74] and ammonia in water by photometry with an acid–base indicator [75,76], UV-molecular absorption [77] and chemiluminescence [78], to name a few. Recent applications of the FIA–gas diffusion combination include the determinations of the previous analytes in addition to novel methods for determining chlorine dioxide based on the fluorescence quenching of chromotropic acid [79]; creatinine using creatinine deiminase immobilized on chitosan [80] and urea in alcoholic beverages (rice wine) using the above-described microgas diffuser to separate CO2 by urease catalysis and photometric detection using an indicator [65]. The multisyringe FIA mode [81] has been used in combination with gas diffusion to separate sulfide from urban wastewaters containing suspended solids without the need for batch sample treatment. The stagnant acceptor solution was a mixture of N,N-dimethyl-p-phenylene diamine and Fe(III) which, in the presence of the diffused analyte, forms Methylene Blue, the dye being transferred to a miniaturized flow-through light-emitting diode-based fibre optic plug-in spectrophotometer for quantitation [82]. SIA systems have also been coupled to gas diffusers to isolate volatile analytes. One of the earliest methods of this type was that developed for the photometric determination of ammonium in environmental samples using an indicator [83]. More recent is the method for free chlorine involving the formation of a coloured compound with o-dianisine, which allows, with minor changes in the operating conditions, the obtainment of two dynamic ranges, namely: 0.6–4.8 mg ClO/L, which is suitable for determining the analyte in water; and 0.047–0.188 g ClO/L, which is suitable for bleaches. The throughput was 15 and 30 samples per hour, respectively, [84]. The SIA–gas diffusion system of Figure 8A has been proposed for the determination of free and total sulfur dioxide in wines based on the formation of the well-known coloured product of the analyte with formaldehyde and p-rosaniline. An acid solution was added to the sample prior to its passage through the donor chamber of the gas diffuser in order to facilitate SO2 formation. For the free SO2 determination, the sample was directly aspirated into the holding coil; for the total SO2, the sample was processed following in-line hydrolysis of bound SO2 with an alkali solution [85]. A similar method based on FIA–gas diffusion based on the same reaction and in-line hydrolysis of the bound analyte in the manifold of Figure 8B was previously developed [86], that facilitated comparing FIA and SIA coupled to gas diffusion. Both methods were found to provide similar linear ranges for the determination of the two analyte forms, namely: 5–300 and 2–35 mg/mL with FIA and 25–250 and 2–40 mg/mL with SIA for total and free SO2, respectively; the sampling rate was 50 and 20 samples per hour (total and free analyte) for the former method and 16 samples

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(A)

PP1 R3

W2

GDU

HC

R1

D

W3 W4

SV

1

2 W1

PP2

7

3

PP3

MC

8

4

6

X

R2

5 S Y

DC

S

Z

N

W5

(B) R3 RC2 R2

GDU

RC3

W2

D

W3

W1 MC

BR S

IV

C

RC1 R1

PP

Figure 8 Determination of free and bound SO2 in wines. (A) SIA–gas diffusion manifold. D, photometric detector; DC, dilution coil; GDU, gas diffusion unit; HC, holding coil; PP, peristaltic pump; R1, 0.8 M hydrochloric acid; R2, 4 M hydrochloric acid; R3, colour-forming reagent (p-rosaniline+formaldehyde); S, sample; SV, switching valve; W, waste; X, Y, Z, merging points. (B) FIA–gas diffusion manifold. BR, basic reagent; C, carrier; IV, injection valve; MC, mixing chamber; N, basic solution; R1, acid solution; R2, p-aminoazobenzene (chemically similar to p-rosaniline, but having single amino group); R3, formaldehyde; RC, reaction coil (other abbreviations as in Figure 8A). According to Refs. [85,86]. Copyrights (2001) and (1991), with permission of Elsevier B.V. and the American Chemical Society, respectively.

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per hour (both analyte forms) for the SIA method. The reproducibility, expressed as RSD, was 0.4% and 1.2% with FIA and SIA, respectively. Finally, the higher sophistication and automation in SIA affords lower reagent consumption.

9. ANALYTICAL PERVAPORATION Analytical pervaporation can be defined as a combination of continuous evaporation and gas diffusion through a gas permeable membrane. Both processes take place in a single step and in the same unit. The volatile analyte (or its volatile reaction product) present in a heated donor phase evaporates through a porous membrane and is collected in an acceptor stream for appropriate detection. An air gap is used between the sample in the donor chamber and the membrane to avoid clogging of the membrane pores when processing dirty samples and also to permit the presence in the donor chamber of species such as high-molecular-weight components, acids, bases, organic solvents, etc., which could damage the membrane on contact with it [87–92]. This is the most salient advantage of analytical pervaporation over its industrial counterpart and other membrane-based non-chromatographic techniques such as dialysis and gas diffusion.

9.1 The pervaporator An analytical pervaporation module differs from the general devices in Figure 1A in that the donor chamber is larger in order to contain both the sample and headspace in contact with the membrane. Basically, it consists of the following parts (Figure 9A): (a) an upper acceptor chamber fitted with inlet and outlet orifices through which the acceptor stream (liquid or gas) is circulated and in which the pervaporated analyte (or its volatile reaction product) is collected; (b) a thin membrane support; (c) spacers of varying thickness, if necessary, to increase the volume of the corresponding chambers; and (d) a donor chamber (lower part of the unit) for circulation of the feed stream [87–92]. The spiral rather than round shape of the acceptor chamber allows its volume to be reduced from 1 mL to 150 mL (Figure 9B). The conventional pervaporation unit is usually made of methacrylate [92], which is a transparent material facilitating continuous checking of the performance of the device. The different parts of the pervaporator are aligned by inserting metal rods in the drilled orifices — see (f), (g) and (i) in Figure 9A— and closer contact is achieved by screwing them together with four screws between two metallic supports — (e) in Figure 9A. Typical connectors, (h), are used to integrate a pervaporator in an FIA manifold. Integrating pervaporation with detection requires altering the acceptor chamber in order to place a probe sensor.


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(A) (e)

(a) (i)

(g)

(f)

(h)) (b)

(c) (i) (d)

(e)

(B)

Figure 9 (A) Components of a pervaporator (for details, see text). (B) Schematic of the spiral acceptor chamber.

The efficiency with which several components in a liquid mixture can be separated is a function not only of differences in vapour pressure, but in their permeation rate through the membrane.

9.2 The auxiliary manifold Analytical pervaporation can be carried out in a continuous way if the sample reaches the pervaporator via a continuous manifold or in a discrete mode if the sample is introduced by injection (with the aid of a valve or a syringe).

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The former choice option allows simplification and miniaturization in the preliminary operations and results in markedly improved analytical quality and productivity. After separation, the pervaporate is always either subjected to other steps preceding detection or driving to the detector via a continuous FIA manifold in both cases [93]. The simplest way of making pervaporation to work as a continuous separation technique — and the only way used so far — is by inserting a pervaporator into an FIA system. With liquid samples, coupling via a dynamic approach is essential to drive the sample either by injection or aspiration to the donor chamber. In addition, (bio)chemical and/or physical steps (namely, reactions converting the analyte into the most suitable form for evaporation, physical dispersion, etc.) can also be performed in the manifold, before the sample reaches the donor chamber; a detector can be placed behind the pervaporator in order to monitor non-volatile species [94,95]. With solid samples, the donor chamber operates as a multifunctional device in which the sample is weighed and, following adjustment to other parts of the pervaporator, is subjected to leaching, masking and (bio)chemical derivatization [96–104]. The acceptor chamber also requires in-line coupling to a dynamic manifold for driving the pervaporated species to the detector, a preconcentration unit or a high-resolution system. Even when the detector is integrated in this chamber [105], coupling with a continuous manifold is desirable in order to flush the chamber between samples. Derivatizing reactions, preconcentration steps, etc., also require the continuous manifold–upper chamber arrangement [106–108]. Discrimination between species can thus be readily achieved [89,94,109–113]. Placing the upper chamber in the loop of a conventional low-pressure injection valve as in Figure 2B allows the acceptor fluid to remain still during pervaporation if the valve is kept in its loading position. After a preset time long enough to ensure adequate enrichment of the acceptor fluid with pervaporated analyte, the valve is switched to its inject position and the stream drives the loop content to the detector. When the detector is integrated with the pervaporator through a probe [105] placed at the top of the acceptor chamber, facing the membrane, the valve allows monitoring mass transfer through the membrane (i.e., the kinetics of the pervaporation process). In addition to typical variables of continuous membrane-based separation techniques, pervaporation is influenced by the following factors: (a) Sample agitation. Magnetic stirring facilitates the removal of gases from the donor stream, and so does ultrasound, even more strongly — however, it is not recommended because it can cause leakage and losses of the gas phase as a result. (b) The presence of chemically inert beads of appropriate size in the donor chamber boosts the transfer of volatile species into the air gap with both liquid and solid samples because the residence time, in the former instance is increased by effect of the winding path the liquid has to go through and the beads separate the particles of solids, thus providing an increased surface area to remove the gas from the sample to the air gap. (c) Volume of the air gap. The smaller the gap is, the smaller the amount of analyte required to go through the membrane will be.


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With liquid samples, the best way to decrease the volume of the air gap is by lengthening the waste line in order to increase overpressure in the donor chamber, which raises the solution level.

9.3 Detectors coupled to FIA–pervaporation The best way of monitoring pervaporated species is by inserting a probe-type sensor in the acceptor chamber with its active side facing the membrane. The most salient advantages of integrating pervaporation and detection are as follows: (a)

(b)

(c)

The response time is shortened with respect to the typical use of the sensor behind the separation module as there is no need to transport the analytes to the detector. The kinetics of mass transfer across the membrane can be monitored to obtain a better understanding of the pervaporation process and facilitate optimization. The system can be dramatically miniaturized as leaching, derivatization, separation and detection can be done in a single module for solid samples.

Pervaporators are amenable to coupling to any type of detector via an appropriate interface such as a transport tube, a microcolumn packed with adsorptive or ion-exchange material, etc. The acceptor stream can be either liquid or gaseous depending on the characteristics of the particular detector. The detectors most frequently used here are of the spectroscopic (atomic or molecular), electroanalytical (potentiometric, voltammetric), electron capture and flame ionization types. The low selectivity of some of them is offset by the high selectivity of the pervaporation step, which makes the overall analytical process selective enough for analyses in complex matrices. However, the potential of pervaporation for sample insertion into water-unfriendly detectors such as mass spectrometers or devices such as those based on microwave-induced plasma remains unexplored.

9.4 Coupling a pervaporator to a gas chromatograph: an alternative to headspace sampling Coupling a pervaporator to a gas chromatograph is one of the most promising uses of pervaporation and it warrants a more detailed discussion based on the advantages of pervaporation over both static and dynamic headspace sampling. In the static approach, the sample is placed in a closed chamber and heated until the volatile compounds in the headspace reach equilibrium with the sample. Then, a portion of the vapour phase is injected into the chromatograph for analysis. The dynamic headspace or purge-and-trap mode requires continuous removal of the gas phase from the chamber. Because separating the volatile analytes from the sample is a slow process, an intermediate trap is needed in order to concentrate the analytes before introduction into the chromatograph.

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Pervaporation provides a number of advantages over headspace techniques that can be summarized as follows: (a)

(b) (c)

(d) (e)

The thin air gap above the sample requires very small amounts of the analytes to establish equilibrium with the sample and mass transfer across the membrane. Continuous removal of volatilized analytes displaces the equilibrium and increases the separation efficiency. Continuous removal of the pervaporated analytes to a preconcentration column, if used, allows fresh portions of acceptor gas to come into contact with the diffused species, thus displacing the mass transfer equilibrium. The separation step can be totally or partially automated (with liquid and solid samples, respectively) with minimal purchase and maintenance costs. Unlike the purge-and-trap mode, no water vapour condenser is required; nor is a hydrophobic sorbent as no water crosses through the hydrophobic membrane at the usual working temperatures.

9.5 Coupling a pervaporator to capillary electrophoresis equipment Recently, pervaporation was coupled on-line to CE for the individual separation of volatile analytes or products in the pervaporate. This combination has been used with liquid samples (wines) to determine volatile acidity and sulfur dioxide [114]; solid foods (fish, meat and sausage) to determine biogenic amines [115]; and slurry samples (yoghurt, juice and yoghurt–juice mixtures) [116] to determine four aldehydes plus acetone. The FIA manifolds used varied depending on the physical state of the sample. An FIA–CE interface designed by the authors was employed. Figure 10 shows the experimental setup required for each method and a detail of the FIA–CE interface, which consists of a methacrylate vial with an internal volume lower than that of commercial vials (0.45 mL vs. 3.5 mL) and two orifices to which the ends of PTFE tubes were glued. The upper orifice is the inlet and the orifice at the bottom of the vial – the outlet; the latter is used to both unload the liquid in the vial after injection into the capillary and to flush the vial between successive loads of pervaporate. As can be seen in Figure 10, the acceptor chamber is placed in the loop of an injection valve in order to facilitate halting the acceptor stream in order to enrich the stagnant solution as required — usually a compromise between sensitivity and sample throughput must be made in this respect. A switching valve preceding the injection valve allows a water stream to flush the upper dynamic manifold, including the interface, from which the stream circulating through it is aspirated for more reproducible performance. The FIA manifold used as an auxiliary of the donor chamber does not differ for liquid or slurry samples; however, it is not required for solids as these are weighted in the donor chamber and the reagents, if required, are injected through appropriate septa. Pervaporation invariably results in preconcentration and cleanup. The former is a function of the volatility and vapour pressure of the target analyte or reaction


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SR

PP

IV WS A OCV

SV

PU M

S R WB

MST

CE

W

Figure 10 Scheme of the pervaporator–CE coupling for solid samples (solid lines only), slurries (with additional dotted lines), liquid samples (solid, dotted and dashed lines). A, acceptor; CE, capillary electrophoresis equipment; IV, injection valve; M, pervaporation membrane; MST, magnetic stirrer; OCV, open–closed valve; PP, peristaltic pump; PU, pervaporation unit; R, reagent; S, sample; SR, syringe; SV, switching valve; W, waste; WB, water bath.

product, and the latter is a consequence of the removal of analytes from the sample matrix. No pervaporator appears to have been coupled to an SIA or LOV system to date.

ABBREVIATIONS BSA CD CE ETAAS FIA GC HPLC ICP LOV MD MS MWCO NADH SIA SP

Bovine serum albumin Cyclodextrin Capillary electrophoresis Electrothermal atomic absorption spectrophotometer Flow injection analysis Gas chromatography High-performance liquid chromatography Inductively coupled plasma Laboratory on valve, lab-on-valve Microdialysis Mass spectrometry Molecular weight cut-off Reduced form of nicotinamide adenine dinucleotide Sequential injection analysis Sample preparation

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CHAPT ER

9 Membrane-Based Separation Techniques: Liquid–Liquid Extraction and Filtration ´ lvarez-Sańchez M.D. Luque de Castro and B. A

Contents

1. Introduction 2. Membrane-Based Continuous Liquid–Liquid Extraction 2.1 Conventional continuous liquid–liquid extraction 2.2 Continuous liquid–liquid extraction without the conventional units 2.3 Ultrasound-assisted continuous liquid–liquid extraction without the conventional units 2.4 Liquid membrane-based extraction techniques 3. Continuous Filtration 3.1 Continuous filtration with filters 3.2 Continuous filtration using knotted reactors 3.3 Continuous filtration with ultrasound assistance 3.4 Applications of continuous filtration 3.5 The choice of precipitate collectors Abbreviations References

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1. INTRODUCTION The two continuous membrane-based separation techniques dealt with in this Chapter (liquid–liquid extraction and filtration) rely on rather different principles (Table 1, Chapter 8). Thus, while filtration requires the presence of two different states (solid and liquid) in the system (either originally or after some conversion) and their physical separation, liquid–liquid extraction is mainly based on the solubility of the target species in a phase immiscible with the sample; on mass Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00609-0


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transfer of the analytes between two immiscible phases which can be brought into contact either directly or through a membrane. Whereas the two liquid phases in dialysis processes must be separated by a membrane, the use of such a membrane is always optional in liquid–liquid extraction. This chapter is concerned with the different ways in which continuous liquid– liquid extraction (CLLE) can be implemented (viz. conventionally, without the typical units, with supported liquid membranes (SLM), microporous membranes or non-porous membranes). Conventional continuous filtration is also discussed here, as are filterless filtration modes such as those based on the use of knotted reactors or some special effects of ultrasound (US) [1].

2. MEMBRANE-BASED CONTINUOUS LIQUID–LIQUID EXTRACTION CLLE across membranes has been implemented in both flow injection analysis (FIA) and air-segmented flow systems. However, FIA manifolds have proved more suitable for this purpose in technical and practical terms. There have been many recent attempts at coupling CLLE with sequential injection analysis (SIA) manifolds, contact between phases being facilitated in most cases by the presence of a segmented merging stream or the use of ‘‘wetting film extraction’’. These modalities are also explained further in this chapter. CLLE has experienced a great resurgence of interest after the development of liquid membranes. Although they emerged in 1986, they reached a widespread use and a more ambitious prospect in the past decade. Traditional membranebased units have been used in combination with FIA and SIA manifolds to implement CLLE in various ways as described in this section. The use of hydrophobic biporous polytetrafluoroethylene (PTFE) membranes (chromatomembranes [2]), which has allowed the development of special liquid– liquid and gas–liquid extraction modes, is discussed in depth in Chapter 7 by Motomizu and Sakai. Because the type of dynamic manifold required to implement CLLE depends strongly on the particular mode, it is discussed separately in each section. Because metals have so far been the most commonly extracted species, the detectors most frequently used with CLLE are atomic and molecular spectrometers [3,4]. Less common applications are briefly described in the sections dealing with the extraction techniques used with the detectors concerned.

2.1 Conventional continuous liquid–liquid extraction Conventional continuous liquid–liquid extraction (CCLLE) has successfully overcome the traditional drawbacks of batch liquid–liquid extraction (LLE) methods (viz. high reagent consumption, the need to handle hazardous and expensive organic solvents, tediousness and risk of formation of stable emulsions). A CCLLE process involves the same sequence of steps as a batch process, namely, dispensing appropriate volumes of the immiscible phases, bringing the phases into contact in order to facilitate mass transfer and separating

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the phases prior to determining the analytes. Initially, this sequence was only feasible in the presence of specific components [3,4] some of which were subsequently found to be dispensable. This allowed operational setups to be simplified and increasingly useful analytical information to be obtained. An FIA conventional continuous liquid–liquid extraction (FIA–CCLLE) setup should include the three basic elements shown in Figure 1: (i) a phase segmentor allowing organic and aqueous streams to converge in order to obtain regular alternate segments of organic and aqueous phase; (ii) an extraction coil (EC) effecting the transfer of analytes; and (iii) a phase separator continuously separating the two immiscible phases prior to determining the analytes. A general description of each unit is provided below. The phase segmentor. The two streams carrying the immiscible phases come into contact at a merging point or a mini-chamber. As a result, a single stream consisting of regular segments of the two phases emerges from the phase segmentor and is delivered to the EC. The most extensively used segmentors are based on triple merging points in a variety of configurations (T, Y and W, see Figure 1A) where phases meet frontally or laterally at different angles. Because these units are typically made of Teflon,

AP

(A)

OP

SP

AP OP

AP

OP

(C) SP

SP

AP

SP

(B)

membrane OP

Figure 1 Basic components of a continuous liquid–liquid extractor. (A) Segmentor producing regular, sequential plugs of organic phase (OP) and aqueous phase (AP) constituting the segmented phase (SP). (B) Extraction coil (glass or Teflon), where mass transfer takes place. (C) Membrane-based separator giving two outlet streams, one of which, completely free from the other, is led to the detector.

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Daiflon, polypropylene, polyetheretherketone (PEEK) or chlorotrifluoroethylene (CTFE, Kel-F), organic solvents easily wet their walls and preservation of segments is facilitated by interfacial forces. The length of the segments is crucial for mass transfer to be effective, and is a function of the inner volume of the mixing chamber and the ratio between the flow-rates of the organic and aqueous phases. Shorter segments lead to more efficient mass transfer and hence to decreased extraction times. With low organic-to-aqueous flow-rate ratios organic segments are so small that they tend to adhere to the tubing walls. Some CCLLE modes use no phase segmentor as only one segment of acceptor phase is inserted in between two segments of the donor phase by means of an injection valve [5] or, alternatively the acceptor is contained in a conventional photometric cell where the donor phase is bubbled [6]. The EC. This unit consists of a tube (usually as a helical coil) where mass transfer occurs for a given time dictated by its length and the flow-rate (Figure 1B). The material of the tube has a strong influence on the profile of the segmented flow by the effect of the disparate wettability of the tube walls. Thus, organic phases form a thin film on the walls of Teflon coils, whereas the aqueous phase is repelled by them and circulates as bubbles into the organic flow. On the other hand, aqueous phases wet glass coils and organic phases form bubbles in them. The sample phase should be that forming bubbles in order to avoid or minimize crosscontamination. Because liquid–liquid extraction usually occurs from an aqueous phase to an organic one, Teflon coils are the preferred choice. The phase separator. This component of the flowing system receives the segmented flow from the EC and provides separate phases downstream. This element, when included, is a key part of the extractor and must operate efficiently and rapidly in order to ensure a high throughput and reproducible results. Phase separators can be classified according to the phase separation mechanism involved, namely, membrane-based and gravity-based phase separators. The former are discussed here and the latter are discussed in Chapter 7. Figure 1C shows a typical membrane-based phase sandwich segmentor, quite similar to the common general membrane module in Figure 1A in Chapter 8, having a single inlet (for the segmented phase) and two outlets, one for a clean extract (acceptor phase enriched with the analyte) on the other side of the membrane, and the other for the donor phase, usually containing some short segments of the acceptor phase. Because the sandwich-type membrane-based separator is easy to fabricate, it exists in a variety of designs, which can be either of the grooved type or cylindrical cavity type [3]. The former is simpler and was the first to be marketed by Tecators; however, the latter provides increased efficiency as its mass transfer surface is circular rather than parallelepipedal, and hence larger. Problems arising from the mechanical stability of the circular membrane surface can be avoided by using an appropriate inert support. The position of the segmentor depends on the relative density of the immiscible phases and should facilitate passage of the acceptor phase through the membrane. The hydrophilic or lipophilic nature of a microporous semi-permeable membrane to be used for CCLLE depends on the nature of the donor and

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acceptor phases. Thus, the membrane should be able to selectively permeate the receiving phase (two clean phases, aqueous and organic, can be obtained by using both hydrophilic and lipophilic membranes in contact with the segmented phase [7,8]). Lipophilic membranes are generally more appropriate as the organic phase is usually of interest, so the membrane should be able to exclude water. PTFE membranes have so far been the preferred choice on account of their ready availability and resistance to organic solvents. The dynamic manifold. The CCLLE can be placed at two different positions in an FIA manifold (viz. in front of or behind the injection valve, as shown in Figure 2 for aqueous samples). In the former case (Figure 2A) the sample or blank, depending on the position of the switching valve, is continuously introduced by aspiration and mixed with the reagents before reaching the solvent segmentor. The outgoing extract is used to fill the loop of the injection valve, through which a carrier, continuously reaching the detector, is circulated. In the inject position, the extract in the loop is driven to the detector. In the manifold in W1

(A) (2)

(1)

AQUEOUS

SV

(3)

CONTINUOUS EXTRACTOR

CARRIER BLANK ORGANIC SOLVENT CARRIER

D

W2

W1

(B)

AQUEOUS

IV

CARRIER CONTINUOUS EXTRACTOR

D

CARRIER W2 DF

Figure 2 Two representative types of FIA–CLLE arrangements in which separation takes place prior to (A) or after injection (B). In the former case, the sample is aspirated and the main points of the dynamic system in which a derivatizing reaction can take place are: (1) before extraction; (2) in the extractor, either in the solvent segmentor of in the extraction coil; (3) behind the extractor. D, detector; DF, displacement flask; IV, injection valve; SV, switching valve; W, waste.

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Figure 2B, the sample is injected into an aqueous carrier and then merges with the organic extractant, propelled from a displacement flask, at the segmentor and the outgoing phase is continuously monitored. In SIA systems coupled to liquid–liquid extractors, the separator is usually not of the membrane, but rather of gravity-based, type [9,10]; this allows enrichment factors close to 20 to be obtained. SLMs, which are discussed later on [11], appear to hold more promise in this context. (Bio)chemical reactions. Chemical and biochemical reactions can be implemented in CCLLE manifolds for one or two purposes, namely, to obtain a reaction product capable of facilitating mass transfer and/or continuous detection with a view of increasing the sensitivity and/or selectivity. Figure 2A shows the different possible locations for implementation of a derivatizing reaction in the overall manifold. Applications. CCLLE has been used prior to the spectrometric determination of metals, either by molecular (photometry, fluorometry, chemiluminometry) or atomic techniques (emission-flame or plasma as atomization-excitation source-or absorption-flame or electrothermal atomization) for more than 25 years. Such applications have been reviewed elsewhere [3,4], as so have indirect methods using atomic detection techniques for the determination of anions such as perchlorate, nitrate and nitrite [3]. Organic compounds such as surfactants [12], amines [13], vitamins [14] and drugs [15] have also been extracted in this way prior to their determination. Typically, concentration factors provided by the LLE step range from 20 to 60, depending mainly on the sample volume and phase volume ratio (which can be from 4 to 32). Precision, as RSD, typically ranges from 1 to 6% [4]. Although LLE work in dynamic systems has been refocused on more recent modes such as membrane-supported and microporous membrane LLE, a few additional methods have recently been reported such as those involving the twostep extraction of heavy metals, which are concentrated 50–100 times by extraction as dithiocarbamates into Freon 113 and then stripped into a dilute aqueous mercury(II) solution to obtain a throughput of 30 h1 and RSD values between 1.5% and 2.7% [16]. Once again iron has been the primary target analyte. This metal has been determined at concentrations down to 9 10–7 mol L1 in tap water following ion-pairing between tetraphenylarsenium ion and tetrathiocyanatoferrate(III), and extraction into chloroform, the volume of which can be dramatically reduced by using the merging-zones mode [17]. Although CCLLE methods perform quite well, ensuring adequate repeatability and accuracy, it requires skilled operators; also, the phase separator is regarded as the Achilles’ heel of a CCLLE system owing to its vulnerability to fouling and carry-over. Coupling CCLLE with separation techniques. Most non-chromatographic continuous separation techniques, but particularly sorption and hydride generation [3], have been coupled to CCLLE. Concerning high-resolution equipment, CCLLE has been preferentially coupled with liquid chromatographs, which is technically easier than coupling with gas chromatographs (GC) or capillary electrophoresis systems. CCLLE


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systems can be coupled to liquid chromatographs in a pre- or post-column arrangement for the same or different purposes. In the former case, the outgoing extract stream is used to fill the loop of the chromatographic injection valve, which is switched in synchrony with passage of the most enriched portion of the extract through the loop. The post-column arrangement of the extractor is more common, and is used for two main purposes, namely, (a) to concentrate the analyte in the eluate in order to reduce band broadening and (b) to enable otherwise impossible analytical detection when excess reagent interferes by giving the same signal as the reaction product (e.g., with ion-pairs) or the solvent must be changed (e.g., with mass detectors). Obviously, CCLLE and GC can only be coupled in a precolumn arrangement. The first such coupling was carried out in 1985 for the determination of traces of aliphatic and aromatic hydrocarbons in wastewater [18]. A number of variants have subsequently been reported and the interface improved [19]. Microfluidic chips for liquid–liquid extraction have been designed and coupled to capillary electrophoresis equipment [20].

2.2 Continuous liquid–liquid extraction without the conventional units Attempts at circumventing the shortcomings of CCLLE have been made by avoiding the use of some or all of the three basic units of the extractor, but mainly the phase separator (i.e., by avoiding phase separation). Simplified modes have been implemented in various ways, namely, (a)

(b)

(c)

By using none of the three usual devices. The sample being bubbled through a cell containing the extractant instead. When the solute is to be transferred to an organic phase, such a phase must be the heavier one; the sample is bubbled at the bottom of the cell and mass transfer occurs while the drops travel through the organic layer [6,21]. This mode is not very effective and the use of an organic phase heavier than the aqueous phase is a highly restrictive condition. By using both a segmentor and an EC. In this way, longer, more extensive contact between the sample and extractant is achieved, and mass transfer is accomplished by using a coil as long as required. The enrichment of the extractant with the solute can be monitored by increasing the length of the segments in an expansion chamber [22], thus enabling transient alternate passage of only organic phase and only aqueous phase through the flow-cell. By using an EC and iteratively changing the flow direction. In this way, only the extractant phase (a single, long enough segment inserted into the sample by switching the injection valve to the inject position) is monitored by changing the flow direction when the interface between the two immiscible liquids is close to the detection point, but has not yet reached it [5,23]. The detector can be accommodated in the loop of an injection valve or in the transport tube (Figures 3 and 4). The extraction process is thus similar to the conventional shaking in separation funnels except for the major differences that it can be fully automated and allows continuous monitoring of solute transfer.


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PC IL AP W IV C1

OP

D

UP SV C2

PL

W PP WB

EC

Figure 3 Dynamic manifold for USALLE without phase separation, mass transfer from an aqueous to an organic phase or vice versa, and monitoring of one of the phases. AP, aqueous phase; C, coil; D, detector; IL, injection loop; IV, injection valve; OP, organic phase; PC, personal computer; PL, propagating liquid; PP, peristaltic pump; UP, ultrasonic probe; W, waste; WB, water bath. Reprinted from Ref. [28]. Copyright (2003), with permission of Elsevier B.V.

The manifolds in Figures 3 and 4, which differ in the chemical system involved and whether one or both phases are monitored, are based on the last approach. Thus, the manifold in Figure 3 can be used when only one of the phases, either organic or aqueous, is to be monitored. The procedure is as follows: initially, the injection loop (IL) of valve IV is filled with sample and the extractant phase is circulated through the manifold to establish the detector baseline. Switching of IV to the inject position is synchronized with the iterative change of the flow direction in such a way that the injected zone (the sample) never reaches the flow-cell in the detector — thus avoiding parasitic signals resulting from changes in refractive index or viscosity — but approaches it as much as possible; as a result, the extract is monitored at the most analyteenriched zone to obtain a multi-peak recording. Circulation of the liquids through the flow system is accomplished by using the peristaltic pump in propelling–aspiration cycles to ensure reproducible operation. After extraction, the system is unloaded and rinsed through switching valve SV to avoid passage of organic solvent through the pump tubes. The manifold in Figure 4A is an available choice for the sequential monitoring of the two phases in a chemical system involving extraction from an aqueous phase to an organic one. The operational procedure is as follows: the loop of the


(A)

243

PP W

SV

UP EC2

PL

AS

IV

WB

EC1 W SL1

SL2 OP

D PC (B)

PP W

C1

SV C2

MV OS W

EAP

W AV

UP PL

D

WB

EC2

EC1

PC

Figure 4 Dynamic manifolds for USALLE without phase separation, mass transfer from an aqueous to an organic phase (A) and from an organic to an aqueous phase (B), and monitoring of both interfaces. AS, aqueous sample; AV, auxiliary valve; C, coil; D, detector; EAP, extractant aqueous phase; EC, extraction coil; IL, injection loop; MV, main valve; OP, organic phase; OS, organic sample; PC, personal computer; PL, propagating liquid; PP, peristaltic pump; SL, sub-loop; SV, switching valve; UP, ultrasonic probe; W, waste; WB, water bath. According to Ref. [29]. Copyright (2003), with permission of Elsevier B.V.

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injection valve accommodating the flow-cell (IV) is filled with a clean organic extractant phase in order to establish the detector baseline and the aqueous sample is then propelled by the peristaltic pump to fill the manifold. Then, extraction is started by switching IV to the inject position and the process monitored in the same flow direction until each phase is as close as possible to the detection point — without reaching it to avoid parasitic signals — in order to monitor virtually the entire organic plug. The manifold in Figure 4B allows the sequential monitoring of the two phases of a chemical system involving extraction from an organic phase to an aqueous one. The key element of this manifold is an internally coupled valve system that serves two main purposes, namely, (1) to enable passage of virtually all aqueous extractant contained in the loop of the auxiliary valve to the detection point while avoiding that of the interfaces and (2) to dispense with the need to circulate the organic sample phase through the peristaltic pump and thus avoid the use of special pump tubes. It is worth noting that SIA–CLLE uses a similar procedure to bring organic and aqueous phases into contact, namely: go-and-back changes of the flow direction are programmed in order to favour contact between phases and hence mass transfer between them. However, continuous monitoring of the enriched phase has been unfeasible to date and information about the kinetics of mass transfer, which is of great help with a view to selecting the time to finish the LLE step [10,11], is lost. A gravity-based phase-separator has been used as an alternative. Also, SIA–lab-at-valve (LAV) coupled systems used for liquid–liquid microextraction (called ‘‘mesoextraction’’ by some authors) have been employed with a conical gravity-based phase separator (in fact, a plastic micropipette) [24,25].

2.3 Ultrasound-assisted continuous liquid–liquid extraction without the conventional units Although the use of auxiliary energies such as microwaves was tested in batch LLE more than 10 years ago [26], no further contributions have been reported despite the excellent results claimed at the time. US was also successfully used to assist CCLLE by the authors’ group in the 1980s [27], but was not used again until recently, when the manifolds in Figures 3 and 4 were employed to identify the types of chemical systems amenable to improvement by using this type of energy. The manifold in Figure 3 was used for the ultrasound-assisted liquid–liquid extraction (USALLE) of paracetamol from suppositories [28], using a ultrasonic probe. Hydrolysis of the analyte prior to reaction with o-cresol in an alkaline extractant medium was also favoured by US — the entire sample plug was irradiated in the EC. Hydrolysis and formation of the reaction product displaced the extraction equilibrium, thus favouring extraction into the aqueous phase. The influence of the variables related to the dynamic manifold (namely, flow-rate and sample volume), chemical variables (namely, NaOH and o-cresol concentrations) and temperature was studied using the univariate method on account of their mutual independence; on the other hand, those related to US (namely, probe


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position, radiation amplitude and pulse duration) were the subject of a multivariate study in which the latter two exhibited an insignificant, but positive, effect. Positioning the probe closest to the EC was found to maximize the extraction efficiency. The favourable effect of US on extraction and analyte hydrolysis provided the overall enhancement illustrated in Figure 5A, which shows the results obtained in the presence and absence of US. The time required for the development of the method was significantly shorter than that for the US Pharmacopeia (USP) method. In addition, the latter produces emulsions that need about 30 min for phase separation after extraction.

(A) 120

Efficiency (%)

100 80 60 40 20 0 2

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5 Cycle number

6

7

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Absorbance

0.6 0.5 0.4 0.3 0.2 0.1 0 0

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400 600 Time (s)

800

1000

Figure 5 Influence of US on CLLE. (A) Efficiency of the extraction step with (E) and without (’) ultrasonication [28]. (B) Multipeak recording obtained by monitoring the ultrasonicated and non-ultrasonicated phases for the extraction of polyphenols from olive oil into a basic aqueous phase. Reprinted from Refs. [28] and [29]. Copyright (2003), with permission of Elsevier B.V.

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Sequential monitoring of the two phases in each cycle as afforded by the manifolds in Figure 4 has facilitated the simultaneous monitoring of extraction with and without US assistance by using a sample plug long enough to ensure irradiation of only one phase; however, simultaneous monitoring of extraction under assistance by different types of energy (e.g., US and microwaves) would also be possible. The manifold required — and used for USALLE — differs depending on the nature of the donor and acceptor phases; in both cases, however, the extractant plug must be long enough to allow one of the phases reaching reactor EC1 to be ultrasonicated while the other is outside the transmitting liquid and far enough from it to avoid its influence. Each phase is circulated through a tubing length ( ¼ EC+sub-loop (SL)) large enough to avoid the loss of liquid in the cycles and to facilitate monitoring of both phases. Absorbance changes in the organic phase, which result from extraction of the analytes, are monitored at an appropriate wavelength in order to obtain multipeak recordings consisting of alternate peaks for the US-assisted and non-USassisted phase (see Figure 5B). This allows the processes occurring at the two phases to be compared in a single experiment. The manifold in Figure 4A has been used to develop two methods for the extraction of two analytes from an aqueous phase, with or without a chemical reaction (viz. extraction of Fe(II) into a dichloromethane/o-phenanthroline phase with formation of the well-known red complex, and extraction of I–3 into dichloromethane) [29]. US-related variables (namely, probe position, radiation amplitude and cycle duration) were optimized by using a multivariate approach and the temperature was kept constant throughout. The results for iodine revealed that the presence of US resulted in poorer extraction of this analyte. On the other hand, those for the water Fe(II)/o-phenantholine dichloromethane system revealed that the presence of US improved the extraction after several cycles; however, the improvement was rather modest, so it did not justify the use of US. The manifold in Figure 4B, which was designed for the sequential monitoring of the two phases of a chemical system involving extraction from an organic phase to an aqueous one, operates as follows: the loop of the auxiliary valve is filled with the aqueous phase modified as required (e.g., pH adjustment, derivatizing reagents) and the detector baseline is established. Then, the loop of the main valve is filled with organic sample (preferably with a syringe, hypodermic needle and adapter to avoid the use of special pump tubes) and an aqueous phase acting as carrier in the go-and-back movement of the donor and acceptor plugs is circulated through C. Next, both valves are switched to the inject position and, simultaneously, the programme controlling the pump is started. EC1 is sonicated while it contains one of the phases. After each run, the manifold is flushed. To this end, the loop of the main valve is filled with an appropriate organic solvent, which is circulated in both directions through the manifold before going to waste through selection valve SV — which avoids passage through the pump tubes. Finally, the manifold is rinsed with water. This manifold and procedure have been used for the extraction and determination of polyphenols from extra virgin olive oil [29]. The method is


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based on the standard approach for this type of analytes and samples, which involves extraction of organic compounds into a Folin–Ciocalteu reagent solution, with monitoring of the product at 725 nm. Figure 5B shows the multi-peak recording obtained, which demonstrates that US is more effective than no sonication. The treatment to be applied to the peaks in order to obtain an appropriate analytical signal can differ depending on the sensitivity of the monitored product. Thus, if the absorbance is high enough, measurements of the first peak close to each interface can be used. With low absorbances, the combined absorbance of a number of peaks can be used as the analytical signal. The peristaltic pump in all these manifolds is operated in a propulsion– aspiration mode in order to ensure more reproducible flow-rates. From the previous results it follows that (1) US does not always favour mass transfer between two immiscible phases; (2) transfer from an organic phase to an aqueous one is more markedly favoured by US than is transfer from an aqueous phase to an organic one; (3) the effect of US can be more significant when a chemical reaction occurs simultaneously with extraction — the influence of US on mass transfer and chemical reaction should be discriminated in order to select the latter; and (4) available information is too scant to allow the formulation of general rules on the performance of chemical systems under USALLE to be established, but a door has been opened to further research with a view to improving some types of CLLE.

2.4 Liquid membrane-based extraction techniques New membrane-based separation techniques have been developed in the last two decades on the basis of liquid–liquid extraction principles and the use of a liquid membrane. These techniques replace conventional units in continuous liquid–liquid extraction with a single membrane-based device quite similar to those used in dialysis or gas diffusion, but where an immiscible liquid acts as a physical barrier for two liquid phases which can be miscible or immiscible. Liquid membranes — also known as ‘‘non-porous membranes’’ — are formed by impregnating a porous hydrophobic solid, which acts as a support for an organic solvent held in its pores by capillary forces. Because the sample is usually aqueous, no applications of aqueous liquid membranes immobilized in a hydrophilic support have so far been reported. There are three main modes of liquid membrane extraction [30], namely, supported liquid membrane extraction (SLME), microporous membrane liquid– liquid extraction (MMLLE) and membrane extraction with a sorbent interface (MESI). Table 1 (Chapter 8) summarizes their features. Liquid membrane-based approaches are easily implemented in continuous flow manifolds and have gained popularity in recent years by virtue of their high selectivity, potentially high enrichment factors, sample clean-up capabilities and automatability. Most uses of membrane-based continuous liquid–liquid separation have been developed in FIA manifolds, even though the technique is well suited to other dynamic approaches such as SIA or lab-on-valve (LOV).

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2.4.1 Supported liquid membrane extraction Principles. The most important liquid membrane technique, SLM, has found many applications in FIA manifolds ever since its inception in 1986 [31]. An SLME module operates with three phases, two of which are aqueous (the donor and the acceptor solutions) and an organic solvent (the liquid membrane proper). Therefore, SLME can be considered a combination of two LLE steps and an intermediate dialysis step. The analytes are extracted from a donor phase into the liquid membrane, which is followed by back-extraction into the acceptor phase [32]. A reaction prior to extraction is mandatory in order to obtain a neutral form, which can be transported through the liquid membrane. Finally, the analyte must be converted into an ionic product in order to facilitate transfer to the acceptor phase and avoid back-extraction [33]. The most suitable molecules for extraction by SLM are highly polar and ionizable compounds such as acidic and basic species. It is possible to chemically tune the membrane by addition of carrier molecules selectively forming chelates or ion-pairs with the target analytes [34]. Typical SLME modules. The SLM extractor can be of the sandwich or hollowfibre type. The characteristics, construction and geometry of these modules have previously been described in Chapter 8 and illustrated in Figure 1 in Chapter 8. Although the scheme shows the fibre in a linear form, in practice the membrane module can be spiralled to make it compact [35]. The solid support of a liquid membrane must be impregnated with organic phase prior to insertion in the module. The internal volume of the acceptor and donor phases in the extraction module depends on the dimensions of the dynamic system, differing from a custom-made design to another. Typically, volumes are in the range 1–2 mL with hollow fibres and from 10 mL to 1 mL with sandwich-type devices. The main differences between these modules when used for dialysis or gas diffusion and for SLM is that, in the latter case, the liquid membrane serves as the solid membrane. It acts as a solid inert support soaked with the organic solvent prior to clamping between the engraved blocks or into the tubular module in sandwich- and hollow fibre-type devices, respectively. The continuous manifold. FIA and SIA manifolds are well suited to both types of modules. Although the sandwich-type is the more widely used, some completely continuous manifolds with time-based sample insertion have also been reported. The flow-through membrane unit can be constructed by connecting the dynamic manifold at both ends of each groove, perfectly sealed to prevent leakage. Before use, the excess organic solvent must be removed from the solid support by passing a washing solution, which can be either the acceptor or the donor solution, through the lower and upper chambers of the separation module. This operation should be repeated after each membrane replacement or between samples, in order to prevent carry-over problems. Figure 6 illustrates a typical FIA–SLM approach where the sample, usually aqueous, is introduced into a carrier stream by means of an injection valve and then merged with a solution at an appropriate pH to convert the analytes into neutral species before they are led to the donor chamber of the extraction unit, where mass transfer occurs, and finally propelled to waste. Alternatively, the

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SAMPLE PP1 CARRIER MC

W

DETECTOR

ACID / BASIC SLM

SOLUTION PP2 BASIC / ACID SOLUTION

SOLID−PHASE TO NEXT STEP

EXTRACTION UNIT AUTOSAMPLER

HPLC, GC or CE

Figure 6 FIA–supported liquid membrane extractor arrangement and possible subsequent steps. PP, peristaltic pump; W, waste.

sample may be aspirated — time-based sampling. The acceptor can be continuously circulated or kept stagnant — either by halting the propulsion system or by accommodating the acceptor chamber in the loop of an injection valve as described in Chapter 8 — this provides increased enrichment factors at the expense of lower throughput. The pH of the extract may be subsequently adjusted by incorporating an acidic or basic stream behind the extraction module. If required, the manifold may also include a point of merging for an appropriate reagent to enhance mass transfer. After extraction, the extract can be collected manually and delivered to an appropriate detector, a sorption column for concentration, an autosampler or a high-resolution equipment, as required [36]. The variables influencing the extraction process are those related to the dynamic manifold (i.e., the flow-rate, composition of the donor and acceptor phases, relative flow-direction of the phases and temperature) and those pertaining to the separation technique (i.e., composition and pH of both aqueous phases, which is crucial for the extraction process and should thus be carefully selected). The presence of a reactive carrier and the nature of the organic solvent are two key factors. The choice of organic solvent will be dictated by its amenability to immobilization in the membrane pores, immiscibility with water and stability. A low viscosity, toxicity and tendency to form emulsions are among the desirable properties of the liquid membrane phase [37]. The nature of the sample, analytes and potentially interfering species should be taken into account in selecting the organic membrane. Typically, the liquid membranes used in this context consist of long-chain hydrocarbons such as n-undecane or kerosene, more polar compounds such as dihexyl ether, dioctyl phosphate or mixtures of the two types of compounds [30]. Memory effects, carry-over and the risk of organic solvent leakage by effect of partial dissolution in the aqueous phases — mainly with relatively polar organic solvents — are the main shortcomings of SLME as they severely restrict the durability of the membrane and hence its usability in continuous systems. The lifetime of liquid membranes with non-polar organic solvents such as n-undecane is typically several weeks. However, the use of additives or more polar organic

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solvents compromises membrane stability [38]. For this reason, it is essential to study the stability of the liquid membrane used in each new application. Below are described briefly some key issues to be considered in optimizing a liquid membrane-based extraction step. A more comprehensive description can be found elsewhere [34,38]. Enrichment. In an SLM, the effectiveness of the extraction process is expressed in terms of extraction efficiency or enrichment factor. In practice, the extraction efficiency should be maximized in terms of the phase volumes (i.e., the extraction time and flow-rate of the donor and acceptor phases) and other physical variables, such as the length and width of channels, nature of the liquid membrane, organic support and phase composition. Extraction time. The most efficient extractions are achieved at low donor flowrates, albeit at the expense of increased extraction times. The available sample volume also has a key influence on the extraction time. If the sample volume is limited (e.g., plasma), a complete extraction of the analyte in the sample is desirable and the extraction time must be longer; if the sample volume is not a limiting factor (e.g., with urine or water), then it is more efficient to maximize the concentration enrichment by changing the donor flow-rate in order to decrease the extraction time. Therefore, the extraction time is highly dependent on the sample volume. Selectivity. Selectivity is a key feature in the extraction of trace analytes from complex matrixes (e.g., biological samples). In this sense, SLME provides good selectivity in comparison with other sample pretreatment techniques such as solid-phase extraction [39], which compensates in part for its low sensitivity. Selectivity can be greatly improved by using additives for the membrane phase or acceptor phase, and also by using the optimum pH in both aqueous phases. Calibration curves. Enrichment has been found to increase linearly with time (or volume) over a wide range in many cases. Limits of detection below the microgram-per-litre are thus easily achieved. Coupling an SLM extractor to analytical equipment. On-line coupling SLME with capillary electrophoretic or chromatographic equipment through an appropriate interface is indeed possible [40]; however, directly coupling to HPLC can in principle be so easy as filling an injection loop with the acceptor phase after the completion of the enrichment step in the SLM module (the extract). The volume of the acceptor chamber should be adjusted as required for introduction of its content into the analytical equipment since, if the volume of the extract is large relative to the injection volume, most of it will be lost. There are several reported uses of SLM for sample clean-up and/or enrichment prior to HPLC–MS or HPLC–UV [41–43]. As shown in Figure 6, a typical SLME configuration includes a liquid membrane module, pumps to deliver the carrier (donor) and acceptor (extractant) streams, an injection valve to insert the sample into the carrier stream and the liquid chromatograph. The SLM module must be small enough to ensure that the final volume of the extract is not too large so the entire volume can be transferred to the injection loop [44,45]. Alternatively, a precolumn can be inserted in front of the loop of the high-pressure injection valve [46]. An acidified


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solution is passed through the precolumn while enrichment in the SLM module takes place. After extraction, the content of the acceptor channel is propelled further on by switching the appropriate valve and the ionized analytes are neutralized and driven to the precolumn. A polymer phase (PLRP-S) or an octadecylsilica phase can be used as a precolumn sorbent. Alternatively, the acceptor phase can be enriched in an ion-exchange phase. On-line coupling of SLME with capillary GC requires the use of an interface to remove water and to transfer the analytes to an organic solvent, which can be accomplished by retaining them on a solid-phase precolumn; then, a nitrogen stream is passed through the precolumn to remove the water and, finally, the analytes are desorbed with hexane, transferred to a large-volume injection loop and injected into a capillary column system comprising a retention gap, a retaining precolumn and an analysis column [47]. However, other membranebased extraction techniques such as microporous liquid–liquid membrane extraction (MMLLE) are more appropriate for direct coupling to GC since hexane is typically the acceptor phase. An SLM module has also been on-line coupled to capillary zone electrophoresis (CZE) by using the double stacking procedure; the analytes were concentrated in the injector end of the capillary for CE separation [48]. Coupling an SLM module to an electrothermal atomic absorption spectrometer (ETAAS) for the analysis of trace metals has been accomplished [49–51] simply by pumping the extract to a vial placed on an ETAAS autosampler. The acceptor solution can also be introduced into a flame or an inductively coupled plasma (ICP) nebulizer [52]. An experimental setup for fully automated speciation analysis of chromium uses two connected in-series membrane devices as shown in Figure 7 [53]. The sample solution is delivered by pump PP1 to the SLM module, where Cr(III) is extracted into the acceptor solution, and further through

PP1

Fraction collector

W3

Sample

Fraction collector

SV1 M2

PP3

M1 SV4

0.1M HNO3

PP2

SV2

SV5

SV3

Eluents 0.75 M HNO3

W1

W2

Figure 7 Experimental setup with connected in-series SLM units for the selective extraction of anionic and cationic chromium species. PP, peristaltic pump; SLM, supported liquid membrane module; SV, switching valve.

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the second module, which contains an appropriate cationic carrier to extract 2 2 anionic Cr(VI) species (HCrO 4 , CrO4 or CrO7 ). After enrichment, pump PP2 is used to propel the selectively enriched analytes in the stagnant acceptor solutions from the two-membrane devices into two different vials in the fraction collector. Pump PP3 is used to empty the tubes that lead the extracts to the vials. Applications of continuous supported liquid membrane extraction (CSLME). SLME endows methods with sample clean-up capabilities, which usually result in high selectivity. With this aim, CSLME has been extensively applied to complex or dirty matrices such as biological samples (mainly blood plasma [53], urine [54] and manure [55]). Some sample treatment is required in most cases that may include filtration and/or centrifugation steps. The method for the determination of haloacetic acids constitutes an excellent example of good performance in an SLME–HPLC combined system; in fact, it provides an enrichment factor of 500 and an extraction efficiency of 54% [54]. The overall system consists of an autosampler tray, a robotic arm, an injection port, a six-port injection valve and two syringe pumps in addition to the liquid chromatograph. The system operates as follows: initially, the samples are loaded in vials and placed on the autosampler. The first syringe propels a volume of solution containing buffer and reagents that is added to the sample in order to neutralize the analytes. After mixing, an aliquot is transferred to the injection port and into the donor channel of the SLM module, where the analytes pass through the membrane and are collected in the acceptor channel — which contains an appropriately buffered solution that remains stagnant during extraction. Finally, the content of the acceptor channel are transferred to the injection loop of the high-pressure injection valve by means of the second syringe pump and injected into the chromatographic column. No precolumn is necessary since the acceptor volume (80 mL) is lower than the injection loop volume (100 mL). Equilibrium sampling through membrane (ESTM) is the name given by some authors to SLME when the extraction step proceeds until equilibrium is established. This mode, which allows the determination of free species in fluids by their partition coefficient — and of bound species as the differences between their total amounts in the sample and those of their free forms — has been applied to the determination of free drugs and drug–protein binding in blood plasma using time-based sampling in a simple continuous manifold with postextraction HPLC separation and UV detection [56,57]. Speciation studies of copper in trace amounts have been carried out by using a similar continuous flow approach but with off-line ETAAS detection instead [58]. An SIA–SLM assembly has been used in combination with immunoassay on magnetic beads to determine progesterone in saliva samples [59]. This approach, known as ‘‘magnetic particle-based immuno-supported liquid membrane assay’’ (m-ISLMA), is based on a competitive immunoassay of the antigen (Ag), progesterone, following transfer through the liquid membrane but prior to backextraction into a stagnant acceptor containing antibodies on magnetic beads held at the acceptor chamber by a magnet. The formation of antigen-antibody (Ag–Ab) complexes facilitates selective enrichment with the antigen prior to chemiluminescence detection.


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2.4.2 Microporous membrane liquid–liquid extraction Principles. MMLLE is based on the same chemical principle as conventional liquid–liquid extraction with one of the liquid phases located in the pores of an inert support. MMLLE can thus be considered a variant of LLE using a membrane-type sandwich module where the two immiscible phases are physically separated by a solid support, the two phases flow through the module and leave it separately. The greatest difference from SLME is that MMLLE involves only two phases, one of which partially impregnates an inert solid support. MMLLE is more suitable for highly hydrophobic compounds, which can be easily extracted from water into an organic solvent (e.g., cyclohexane, n-hexane) for direct injection into a GC column. A detailed discussion of the principles of MMLLE can be found elsewhere [30,34,38]. Interfacing MMLLE to other devices. As noted earlier, one of the main benefits of membrane-based extraction techniques is that they can operate in an automated way and the extract can be directly transferred to chromatographic or electrophoretic equipment. MMLLE has been preferentially coupled to GC. Online coupled MMLLE–GC–FID (flame ionization detector) systems have been described for the determination of pesticides in red wines [60] and organic pollutants in water samples [61]. In this approach, the whole extract is transferred to the loop of the GC injection valve. An FIA–MMLLE approach has been applied to the extraction of organotin compounds in biological samples prior to their determination by GC–MS (mass spectrometry) [62]. The analytes in the injected sample are derivatized and then pumped to the membrane device. The acceptor channel and the membrane support hold isooctane as the extractant, which is manually collected after enrichment and introduced in the autosampler of the GC. The MMLLE–SLME combination has also been used to facilitate the extraction of ionizable and non-polar compounds in a single run. Such is the case of the determination of thiophanate-methyl and derivatives, where the former is extracted by MMLLE and its polar metabolites by SLME [63]. The system used comprises four syringe pumps, a six-port valve, a ten-port two-way valve and a liquid chromatograph. The procedure is as follows: after the donor and the acceptor channels of the SLME module are flushed, the acceptor phase stream is stopped while the sample is pumped through the donor channel of the SLME unit and the MMLLE module is simultaneously being flushed with an organic phase. The extract in the SLME module is transferred to the loop of the HPLC injection valve for chromatographic analysis, during which a new sample aliquot is subjected to MMLLE under conditions similar to those of SLME. In this way, while a portion of the sample is being extracted by the SLM, the MMLLE extract is analysed. The modules used in each extraction mode possessed rather disparate mass transfer surface areas, which precluded comparison of their efficiencies.

2.4.3 Alternative non-porous membrane extraction modes As mentioned before, SLME is by far the most widespread non-porous membrane separation mode, followed by MMLLE, as reflected in the number of reported applications relative to other techniques such as polymeric

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membrane extraction (PME) or membrane extraction with sorbent interface (MESI). Extraction with a polymeric non-porous (continuous) membrane of a material such as a silicone rubber involves three steps. First, the aqueous sample is brought into contact with the membrane and a portion of the analytes is extracted into the membrane; then, they diffuse across it to the membrane-extractant interface and are finally dissolved in the extractant. Mass transfer is governed mainly by the partition coefficients between the sample and the membrane, the diffusion rates of the analytes in the membrane and the partition coefficients between the extractant and membrane [64]. The dynamic manifolds to be connected to the extraction module are essentially the same as in SLME. The membrane consists of silicone rubber and is quite thin in order to facilitate diffusion. This extraction mode has been used for the separation of neutral and non-ionized compounds such as pyridine, benzene and their derivatives [65]. The main differences from SLME are that mass transfer is slower through silicone rubber membranes, which can be offset by using a smaller thickness, and also that the membranes are more stable and can be used for several years. Finally, MESI [30] is based on mass transfer from a liquid or gaseous sample to a gaseous acceptor phase via a membrane (usually silicone rubber membrane). The analytes are then trapped into a cooled sorbent, and, finally, thermally desorbed for transfer to a GC [66]. Any type of dynamic manifold, whether continuous, FIA, SIA or LOV, can be used to transport the fluids to and from the extraction device.

3. CONTINUOUS FILTRATION Filtration can be implemented in a continuous manner with the aid of a filtering or filterless device; in the latter case, the filter is replaced with either some type of energy facilitating solid–liquid separation (US) or a sustained centrifugal force in the stream carrying the precipitate (with knotted reactors). Although filtration can be considered an outdated separation technique, it has been revitalized for use in continuous flow systems. The most common purposes of using continuous filtration are as follows: (a) to remove undesirable particles in a liquid sample resulting from a prior sampling or from an evolving medium, the filtration step thus constituting a sampling step as well; (b) to remove particles from the leachate of a solid sample– leachant system having escaped from a coarse filter in a continuous leacher or formed while the leachate was outside the leaching working conditions; (c) following precipitation, to (i) remove interferents; (ii) isolate the target analyte as a solid compound in order to facilitate its indirect determination; (iii) isolate the analyte as a solid compound to be subsequently dissolved for detection, thus avoiding matrix interferences and facilitating preconcentration. Filters are used for purposes (a) to (c), US – for (a) and (b), and knotted reactors – mainly for (iii). The design and dimensions of the solid collection device have a critical influence on the overall performance of the manifold, particularly for purpose (c).


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The ideal on-line collector in this case should meet the following requirements: (a) it should be able to collect a few milligrams of precipitate without developing prohibitive flow impedance — which could reduce the sample and reagent flow to 4–5 mL min1; (b) its geometric design should be such that the collected precipitate is easily accessible by the flushing solution and dissolution reagent, if required; (c) its design and dimensions should facilitate radial dispersion and restrict axial dispersion of the dissolved analyte in the case of precipitate dissolution — this criterion is especially important in trace analyses, which require the highest sensitivity; (d) it should be made from inert materials in order to be able to hold various reagents and solvents, and minimize contamination from the collector itself; (e) it should possess robust structure remaining stable under long-term continuous use; and (f) it should be able to collect different precipitate forms. The ideal placement of the collection device in a continuous manifold obviously depends on the particular purpose and increases in proximity to the detector from (a) to (c).

3.1 Continuous filtration with filters In continuous filtration with a filter, the filtering unit can be made of various materials including stainless steel, disposable membranes and packed beds. Stainless steel filters, which can be cylindrical or planar, have so far been the most frequently used. Cylindrical stainless steel filters (Figure 8A), which were originally designed as cleaning devices for HPLC, possess a large filtration area (ca. 3 cm2) and also a relatively large dead volume (usually higher than 500 mL). On the other hand, the planar filters (Figure 8B) possess a variable inner volume (100–800 mL), and a much smaller area (as small as 7 mm2); this requires pore sizes larger than 1 mm in order to avoid diminishing the flow-rate when the precipitate is collected. Disposable membrane filters are usually of nylon or cellulose. Their short lifetime and the need to adopt a compromise between sensitivity and trouble-free manipulation frequently results in poor precision, so they tend to be avoided [67]. Packed-bed filters made from 3–4 mm i.d. PTFE or Tygon tubing and packed with polystyrene granules, cotton, or filter paper pulp, have been only sparingly used as they have limited capacity to collect precipitates and the added disadvantage of the difficulty in controlling and reproducing the tightness of the packing [68].

3.2 Continuous filtration using knotted reactors Knotted reactors (KR, Figure 8C) were first introduced in FIA to reduce dispersion and facilitate thorough mixing by promoting radial dispersion and limiting axial dispersion via their insertion as transport units and sample loops [69]. They have enabled near-quantitative on-line precipitate collection [70] on the reactor walls presumably by the effect of the centrifugal force exerted by secondary flow in the three-dimensionally disoriented configuration of the reactor. This retention


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A

FLOW

B

FLOW

C

Figure 8 Filter and filterless devices for filtration. Stainless-steel filters: cylindrical (A) and planar or disk-shaped (B). Knotted reactor (C) and filterless ultrasound-assisted filtration chamber (D). A and B reprinted from Ref. [4]. Copyright (1993) with permission of VCH. C and D reprinted from Ref. [1]. Copyright (2006), with permission of Elsevier B.V.

mechanism, which relies on the fact that straight or even coiled reactors made of the very same piece of tubing are much less effective in collecting precipitates, is favoured by the fast mixing of sample and precipitating reagents, and limited dispersion, typical of these devices, and affords high enrichment factors in the preconcentration of traces. These devices feature a relatively large capacity for precipitate collection by virtue of their large tube wall area — which is particularly important in coprecipitation applications, where the amount of


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Concentrated particles out D

Clarified medium out

Clarified medium out

Stream of particles leaving the sound field

Stream expansion

Transducer forming standing wave

Ultrasound concentrates particles into a single band Laminar flow stabilization

Sample in

Figure 8 (Continued)

precipitate form tends to be quite large. The main advantages of a KR are: low back pressures, even at high flow-rates; negligible losses in sensitivity by effect of dispersion; little risk of contamination thanks to the inert material they are made of; easy, inexpensive construction; and long, almost permanently useful life time.

3.3 Continuous filtration with ultrasound assistance The use of US energy to assist solid–liquid separation processes has been explored in different ways, mostly with the aim of obtaining drying effects; however, the compressive and expansive effects of ultrasonic waves result in a pumping effect that acts as a separation tool via collisions in individual particles and their sticking to a solid surface. This is accomplished by creating points where the particles are held in the fluid solely by ultrasonic forces, and by agglomerating with other particles forming temporary flocs [1]. Thus, when ultrasonic standing waves of megahertz frequency and laminar flow are combined in a chamber such as that in Figure 8D, effective solid–liquid separation is accomplished (so much that the chamber has been named a ‘‘filter chamber’’ [71]). The standing wave pressure on suspended particles drives them towards the centre of the ultrasonic pathlength, and the clarified suspending phase from the region closest to the filter wall is drawn away through the downstream outlet. Effective US-assisted filtration relies on appropriate choice of US variables such as frequency and intensity, power and propagation direction. At the very-low size-scale, US action allows non-invasive and non-contact cell manipulation and separation integrated in microfluidic systems [72], which,

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in fact, constitute an extreme case of filterless filtration assisted by ultrasonic standing waves.

3.4 Applications of continuous filtration The uses of filtration in a dynamic analytical process depend on the particular purpose and, specifically on the way solid–liquid separation is accomplished. Sample filtration. This step, which is indispensable to ensure proper operation in continuous manifolds when the sample contains suspended matter (e.g., in bioprocess control), can be easily implemented by placing a filter either on the aspiration end of the probe in the sampled system — in which case a stainless steel cylindrical filter is the best choice — or in between the aspiration probe and injection valve. Such is the case of the direct screening of lyophilized biological fluids for bile acids by using a liquid chromatograph equipped with an evaporative light scattering detector [73] where a glass column packed with cotton wool was used to retain undissolved particles. Another example is the determination of Folpet and Metalaxyl in pesticide formulations by Fourier transform infrared spectrometry [74] using a nylon filter of 0.22 mm pore size. These approaches have allowed the development of a variety of determinations depending on the concentration of the particles in the system, deposition of which can cause clogging of the filter and result in overpressure in the overall manifold. One way of circumventing this shortcoming is by placing the filter in the loop of an injection valve; this allows the unit to be flushed by passing a rinsing solution in opposite direction to the sample between injections. On-line FIA–filtration can also be used for the two-fold purpose of cleaning the sample for solid particles and separating an insoluble fraction from a soluble one. This fact has facilitated the determination of total chromium and that in the soluble fraction according to Hernańdez-Co´rdoba and co-workers [75]. US-assisted filtration has scarcely been used in FIA systems, despite the interesting demonstration of its usefulness by Wang et al. [76] in different types of samples such as mammalian cell cultures, whole blood or surface water samples. Each filtration unit was subject to an US resonance field in order to cause particle aggregation without the need for any physical filter surface. One of the main reasons why analytical chemists have been reluctant to use this effective type of energy to assist a number of analytical steps including filtration [1] is their poor knowledge of its features or the absence of such knowledge. A multisyringe–FIA approach involving on-line filtration and potentiometric detection has been proposed to determine exchangeable potassium in soils following leaching, at a sampling frequency of 4 h–1 [77]. Filtration in sample preparation. Particles in the extracts from static [78], dynamic [79] or static–dynamic subcritical-liquid extractors [80] are usually formed after cooling by effect of supersaturation, a phenomenon which is also present in continuous microwave-assisted [81] and US-assisted extractors [82]. Figure 9 illustrates a system for the determination of nitrated polycyclic aromatic hydrocarbons in soils by using continuous subcritical water extraction in

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PV OVEN SV

EC PH

HPP

C IV2

ER PP 1

IV1

W

WR

W

N2

MC

H2O

F IV3

AIR V

W

EL

A

PP 2

GC-MS-MS

Figure 9 Approach for the determination of nitrated polycyclic aromatic hydrocarbons in soils based on continuous subcritical water extraction, filtration, preconcentration, chromatographic separation and in-tandem mass-spectrometry detection. A, acetonitrile; C, cooler; EC, extraction chamber; EL, elution loop; ER, extract reservoir; F, filter; HPP, highpressure pump; IV1, IV2, IV3, injection valves; MC, minicolumn; PH, pre-heater; PP1 and PP2, peristaltic pumps; PV, pressure valve; SV, switching valve; V, vial; W, waste; WR, water reservoir. Reprinted from Ref. [79]. Copyright (2003), with permission of the Royal Society of Chemistry.

combination with filtration, preconcentration, chromatographic separation and UV detection [78]. A 0.25 mm filter, placed in the loop of an injection valve received the extract from the reservoir. The filtrate was sent to a minicolumn packed with sorbent material — also accommodated in the loop of an injection valve in order to facilitate complete removal of the liquid phase and elution in the opposite direction to retention — while the filter was cleaned by circulating water in the opposite direction to filtration. On-line precipitation–filtration. Precipitation in a dynamic system for the three above-described purposes was implemented more than 20 years ago by using sample or reagent injections or alternating continuous aspiration of the sample and dissolving solution — which is especially effective with highly diluted samples (Figure 10). Continuous precipitation–filtration without precipitate dissolution is used either for interference removal or for indirect determination of analytes by its precipitation with a reagent producing a signal at the detector that is diminished by precipitation. Thus, a negative signal is obtained that is proportional to the concentration of the target species [3]. Inorganic anions such as halogenides [83,84] or sulfate [85] have been determined in this way by using Ag(I) or Pb(II), respectively, as precipitants, and so have organic compounds such as sulphonamides [86] and local anaesthetics [87] using Cu(II) and Co(II), respectively. The main problem with these methods arose from the need to frequently clean the filter; this problem has been solved by placing the filter in the loop of an injection valve as shown in Figure 9.


260

(A)

PP

H2O

REAGENT (SAMPLE)

SV

PRECIPITATION

SAMPLE (REAGENT)

FILTER AD

COIL

DISSOLVING REAGENT

(B) PP SOLVENT REAGENT

SV

PRECIPITATION

FILTER AD

SAMPLE W

COIL

(C) PP2 SOLVENT PP1 REAGENT PRECIPITATION

FILTER AD

SAMPLE COIL

Figure 10 Dynamic manifolds for implementation of continuous precipitation–filtration with precipitate (dashed lines) or without dissolution. (A) With injection of sample or reagent, with continuous aspiration of sample and precipitating reagent and change to the dissolution medium by means of a switching valve (B), or with intermittent pumping (C). AD, atomic detector; IV, injection valve; PP, peristaltic pump; SV, switching valve; W, waste. Reprinted from Ref. [3]. Copyright (1991), with permission of the Royal Society of Chemistry.

Continuous precipitation–filtration–dissolution with a filter was first used for the preconcentration of metal cations prior to their atomic detection; a number of methods were thus developed for different metals [3,4] (Figure 10). Later on, the approach was used to develop immunochemical methods involving the formation of high-molecular weight immunocomplexes such as that for the determination of anti-canine immunoglobulin G (IgG) by formation of a sandwich-type heterogeneous non-competitive reaction as a result of the affinity


261

interaction between streptavidin and biotincanine anti-mouse IgG, and the immunoreaction between canine IgG and mAb anti-canine IgG [88]. The most common way of implementing filterless continuous precipitation– filtration–dissolution is by using knotted reactors, which were first reported by Fang et al. in 1991 [70], and subsequently employed by other FIA users [89] but not extended to SIA or LOV. These reactors have been used mainly to retain metal cations following the formation of a complex of appropriate stability with a selective enough ligand [89] for their determination with an atomic technique. Recent applications have focused on the retention of ion-pairs [90] for the determination of Pd or on immobilization of the ligand on the inner walls of the KR, which has enabled the simultaneous separation–preconcentration of traces of Ag, Cd, Co, Ni, Pb, U and Y for their subsequent detection by ICP–time of flight (TOF)–MS [91].

3.5 The choice of precipitate collectors The following few guidelines are intended to help in selecting the best precipitate collector for various purposes: (a)

(b)

(c) (d)

When sensitivity or concentration efficiency is not important, and the method and reagents or sample solutions are not extremely corrosive, large-volume cylindrical stainless-steel filters may be the best choice. For trace analyte preconcentration by precipitation — particularly by coprecipitation — the filterless KR should be used whenever possible; otherwise, a packed-bed column containing coarse granules in combination with a small-diameter membrane filter appears to be the best choice, even though sample loading rates may be limited by the resulting backpressure. Packed-bed filters and small-diameter membrane filters may be used when moderate sensitivity or preconcentration effects are required. The high potential of US-assisted filterless filtration should be investigated at large in order to establish the best working conditions for use in different dynamic approaches on account of its proven high flexibility [1,76].

ABBREVIATIONS CCLLE CLLE CTFE CZE EC ETAAS ESTM FIA FID

Conventional continuous liquid–liquid extraction Continuous liquid–liquid extraction Chlorotrifluoroethylene Capillary zone electrophoresis Extraction coil Electrothermal atomic absorption spectrometer Equilibrium sampling through membrane Flow injection analysis Flame ionization detector

262

HPLC ICP IgG KR LLE LOV MESI m-ISLMA MMLLE PEEK PME PTFE SIA SL SLM SLME US USALLE USP


High-performance liquid chromatography Inductively coupled plasma Immunoglobulin G Knotted reactor Liquid–liquid extraction Lab-on-valve Membrane extraction with sorbent interface Magnetic particle-based immuno-supported liquid membrane assay Microporous membrane liquid–liquid extraction Polyetheretherketone Polymeric membrane extraction Polytetrafluoroethylene Sequential injection analysis Sub loop Supported liquid membrane Supported liquid membrane extraction Ultrasound Ultrasound-assisted liquid–liquid extraction US Pharmacopeia

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CHAPT ER

10 Chromatographic Separations Petr Solich

Contents

1. Introduction 265 2. Separation Columns Used in Flow Analysis 267 2.1 Monolithic columns 271 2.2 Solid-phase extraction and restricted access material columns 273 3. Pharmaceutical Applications of Sequential Injection Chromatography 277 3.1 Liquid samples 277 3.2 Topical semisolid formulations 277 3.3 Tablets and capsules 278 3.4 Automation of simultaneous release tests 278 4. Comparison of Sequential Injection Chromatography and High Performance Liquid Chromatography 278 5. Other Chromatographic Approaches 280 6. Future Trends 281 Abbreviations 284 References 284

1. INTRODUCTION Flow methods have become an attractive tool in analytical laboratories worldwide. Among them sequential injection analysis (SIA) offers several important advantages: the instrumental setup is very flexible, the components undergo little wear and the hydrodynamic variables can be controlled with high efficiency. On the other hand, SIA has an important drawback — it lacks the ease to carry out separation procedures and analysis of the multicomponent samples. Overcoming this limitation will make SIA a powerful tool in both scientific research and routine analysis. A sample is often composed of numerous compounds, and most conventional analytical methods such as spectroscopy are suited to detect one compound at a Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00610-7


265

266

Petr Solich

time. As in many cases when the concentration of one compound only is of interest, this limitation does not pose a substantial problem. However, if the concentrations of more sample components are to be determined, one has to use separation techniques allowing physical separation of the different compounds of interest from each other, followed by their detection. Low-pressure flow methods offer the possibility to substantially increase analytical workload in laboratories as a result of automation, miniaturization, great versatility and low-sample and mobile-phase consumption. These benefits were so strong that during the last two decades flow methods have become a favourable analytical tool in many laboratories, mostly orientated to scientific research. However, these methods are usually suitable for the analysis of one individual compound in a sample and do not offer the possibility of carrying out multicomponent analysis in a single step. In recent years several attempts have been made to rectify this problem. Various strategies have been tested [1] involving the use of multichannel manifolds, selective detectors, chemometric treatment of multivariate data, solid-phase extraction (SPE) and state-of-the-art chromatographic approaches as depicted in Figure 1. The approach based on the use of multichannel manifolds is often used in the simultaneous quantification of two components. The analytical procedure often involves several injections of the sample into various conduits, where each component is determined individually. Flow splitting and delay coils are also used. The split streams follow lines of different lengths, finally all channels join at a common point prior to the detection. As a result, various successive peaks are obtained from a single sample injection. Selective detectors can be used for the determination of multicomponent samples in two ways. Either the detectors are sensitive to all chemical species of Flow techniques

Solid phase extractions

SIC and approaches

Multichannel manifolds

Selective detectors. Hyphenations

Chemometrics

Multicomponent analysis

Figure 1 Strategies used in flow systems for multicomponent analysis. Reprinted from Ref. [1]. Copyright (2007), with permission from Elsevier B.V.

Chromatographic Separations

267

interest or each detector provides a signal specific to a particular chemical species only. A typical example of the utilization of selective detectors for multicomponent analysis is the determination of the complex formed between naproxen and b-cyclodextrin in pharmaceutical preparations using fluorescence detector without any previous separation, extraction or other sample manipulation [2]. Another example is the monitoring of glucose and penicillin during cultivations using specific reactions and chemiluminescence detection [3]. Organic acids and sugars in soft drinks were determined by Fourier transform infrared spectroscopy (FTIR) [4]. It has been well known for a long time that processing multivariate data with chemometric tools can be used for the determination of several components simultaneously in a single analysis. Multivariate signals can be divided into two major groups — first-order data and second-order data. First-order data are usually generated with multichannel detectors or fast-scan detectors. To obtain second-order data, multichannel detectors must be used, and another order of data must be generated simultaneously. A typical example of the use of chemometrics in simultaneous multicomponent analysis was described by Pasamontes and Callao [5]. They utilized multivariate curve resolution with alternating least squares for the simultaneous determination of analytes in the presence of interferents without the need of sample pretreatment. SIA utilizing SPE or column separation is the most promising tool for multicomponent analysis in low-pressure flow methods and will be discussed in more detail separately. A detailed overview of applications of multicomponent analysis is given in Table 1.

2. SEPARATION COLUMNS USED IN FLOW ANALYSIS From its introduction in the beginning of the twentieth century, chromatography has gained a strong popularity in the area of analytical chemistry. Numerous types of chromatographic systems and chromatographic columns have been developed and utilized in different areas, ranging from environmental to bioanalytical sciences. Nowadays high performance liquid chromatography (HPLC) and related chromatographic techniques are the analytical separation techniques mostly utilized worldwide in both scientific research and routine analysis in a variety of fields (e.g., quality control of drugs, determination of pollutants and food analysis). The ‘‘heart’’ of a chromatographic system is its separation column, which enables the separation of compounds based on their affinity to the stationery phase. A typical chromatographic column consists of a tube made of stainless steel, glass or polymers and filled with usually 3–5 mm porous silica microparticles. The separation performance of such a column is mainly determined by the particle size, their size distribution and the structure of packing within the column. Separation science needs to provide means for faster, more selective and more comprehensive separation and detection. Research in the field of

268

Table 1

Petr Solich

Multicomponent analysis in flow systems based on different strategies [1]

Analytes

Sample type

System

Strategy

Detection

Ascorbic acid and rutin trihydrate Furosemide and triamterene Sacarine and aspartame Vitamins B2 and B6 Hg and methyl Hg

Pharmaceuticals

SIA

Spectrophotometry

Pharmaceuticals, urine and serum Sweets and drinks

MCFIA

Pharmaceuticals

MCFIA

Solid-phase extraction Solid-phase extraction Solid-phase extraction Solid-phase extraction Solid-phase extraction

Sb(III), Sb(V)

FIA

Certified reference FIA materials (natural water, rice flour and pork) Bottled drinking Segmented water FIA Pharmaceuticals SIA

Methylparaben, propylparaben and sodium diclofenac Methylparaben, propylparaben and triamcinolone acetonide Ambroxol hydrochloride and doxycycline Ambroxol, methylparaben and benzoic acid Naphazoline nitrate and methylparaben Salicylic acid and triamcinolone acetonide Amoxicillin, ampicillin and cephalexin Nitrites and nitrates Cu, Zn

Water, soil and FIA biological samples Pharmaceuticals FIA

Mo, W

Steel alloys

Citrate and piruvate Cr(III), Cr(VI)

Pharmaceuticals, Stopped-flow urine and serum Surface waters and BI-LOV standard reference materials

Solid-phase extraction SIC and approaches

Fluorescense optosensor Spectrophotometry Fluorescence optosensor Fluorometry

HG-AAS Spectrophotometry

Pharmaceuticals

SIA

SIC and approaches

Spectrophotometry

Pharmaceuticals

SIA

SIC and approaches

Spectrophotometry

Pharmaceuticals

SIA

SIC and approaches

Spectrophotometry

Pharmaceuticals

SIA

SIC and approaches

Spectrophotometry

Pharmaceuticals

SIA

SIC and approaches

Spectrophotometry

Pharmaceuticals

MSFIA

SIC and approaches

Spectrophotometry

Multichannel manifold Multichannel manifold Multichannel manifold Multichannel manifold Multichannel manifold

Spectrophotometry

FIA

Spectrophotometry Spectrophotometry Chemiluminescence ET-AAS


269

Table 1 (Continued ) Analytes

Sample type

System

Strategy

Detection

Malic and lactic acid Ammonia and phosphate Fe(II), Fe(III)+nitrite, nitrate Cu, Fe, Zn

Wine

FIA

Photometry

Surface waters

SIA

Serum

FIA

Cu, Ni

Plant digests

FIA

pH, Cl, Ni

Electrolytic bath

SIA

Multichannel manifold Multichannel manifold Multichannel manifold Multichannel manifold Multichannel manifold Selective detectors

Cd(II), Pb(II)/Ni(II), Co(II)

Fertilizer and ore

FIA/SIA

Selective detectors

Cysteine, glutathione

Plasma and blood

FIA

Phenolic compounds

Industrial wastewater and surface water Ocean seawater

FIA

Selective detectors Selective detectors

Standard reference materials Certified reference materials and natural water Certified reference materials and natural water River, tap and wastewater Water

FIA

Co, Ni, Cu, Zn, Cd, Pb Ge, As, Se V, Cr, Mn, Co, Ni, Cu, Zn, Cd, Pb Ag, Pd, Au, Ga, In, Nb Cr(III), Cr(VI) Cr(III), Cr(VI) Seven anions

River and sea water MCFIA

FIA

FIA

Spectrophotometry Spectrophotometry Spectrophotometry Potentiometry — two ion-selective electrodes Anodic and adsorptive stripping voltammetry Biosensor Biosensor

ICP-MS ICP-MS ICP-MS

FIA

Selective detectors

ICP-OES

FIA

Selective detectors Selective detectors Selective detectors Selective detectors

ICP-AES

FIA

Water, juice and milk Synthetic water samples

FIA

Honokiol and magnolol Four anthraquinones

Pharmaceuticals

FIA

Pharmaceuticals

FIA

Sn, Ge, Mo

Food

FIA

Co(II), Cu(II)

Water

FIA

Eight amines

Selective detectors Selective detectors Selective detectors

Fluorometry

SIA

Selective detectors Selective detectors Chemometrics (PLS) Chemometrics (PLS)

ICP-OES Capillary electrophoresis Capillary electrophoresismass spectrometry Capillary electrophoresis Capillary electrophoresis Spectrophotometry Chemiluminescence

270

Petr Solich

Table 1 (Continued ) Analytes

Sample type

System

Strategy

Detection

Fe, V

Alloys

MPFIA

Spectrophotometry

Amoxicillin and clavulanic acid Rifampicin, isoniazid Cu(II) Ni(II)

Pharmaceuticals

Stopped-flow

Pharmaceuticals

FIA

Electrolytic bath

Two inhibitors

Pharmaceuticals

FIA-stoppedflow FIA

Cr(III), Cr(VI)

Tanning wastewater SIA

Three acid dyes

Tanning wastewater SIA

Xanthine and hypoxanthine Four dyes

Urine

Stopped-flow

Fruit-drink powder

FIA

Chemometrics (PLS) Chemometrics (PLS) Chemometrics (ANN) Chemometrics (ANN) Chemometrics (MCR-ALS) Chemometrics (MCR-ALS) Chemometrics (MCR-ALS) Chemometrics (N-PLS) Chemometrics (BLLS)

Fluorescence Chemiluminescence Spectrophotometry Spectrophotometry Spectrophotometry Spectrophotometry Spectrophotometry Spectrophotometry

Note: SIA, sequential injection; MCFIA, multisyringe flow injection analysis; MPFIA, multipump flow injection analysis; FIA, flow injection analysis; BI-LOV, bead injection-lab-on-valve; HG-AAS, hydride generation-atomic absorption spectrometry; ET-AAS, electrothermal-atomic absorption spectrometry; ICP-MS, inductively coupled plasma-mass spectrometry; ICP-OES, inductively coupled plasma-optical emission spectrometry; PLS, partial least squares; MCR-ALS, multivariate curve resolution-alternating least squares; N-PLS, N-way-partial least squares; BLLS, bilinear least squares.

liquid chromatographic columns has accelerated tremendously during recent years. Innumerable types of chromatographic columns (e.g., sub-2-micron columns, columns based on zirconium oxide and polymeric stationary phases) have been developed to solve particular separation problems. The recently introduced instrumentation and columns with sub-2-micron microparticles for ultra performance liquid chromatography (UPLC) is a dream which has come true for many chromatographers. This kind of equipment allows a 5–10-fold reduction in time of analysis, with the addition of increased sensitivity [6]. The high pressure required with columns packed with small particles, such as those used in HPLC and UPLC, makes these columns non-applicable to low-pressure flow methods. Therefore, research has also been focused in the opposite direction, i.e., to find materials for columns which could be more permeable for the mobile phase, thus requiring lower pressures while keeping the separation efficacy sufficiently high. Monolithic columns, firstly introduced for HPLC applications by Tanaka and co-workers in 1996 [7], represent another way to achieve the high quantum of analysis in specific time, largely exceeding the limits of conventional particle-based separation columns.


271

2.1 Monolithic columns Monolithic columns are prepared from either organic polymers, such as polymethacrylates, polystyrenes or from inorganic polymers, such as silica (‘‘silica rods’’) [8]. The polymerization process can run either in situ in a column tube, or in a column mould in which the monoliths can later be replaced. In contrast to columns made of organic polymers, the silica-based monolithic column in typical analytical size cannot be prepared in-situ because of the significant shrinkage accompanying the solidification during hydrolytically initiated polycondensation of tetraalkoxysilane in the presence of poly(ethylene glycol) porogen [9]. Due to the shrinkage, the straight rods are no longer than 15 cm. The bimodal porous structure of monoliths, consisting of small pores within the monolith skeleton surrounded by larger through-pores or transport channels, in combination with high mechanical stability of the matrix, provides high permeability and low back pressure from the through flow of the liquid phase [10]. Another advantage is that these columns have the ability to maintain sufficiently high column efficiencies at mobile-phase flow rates much higher than those used in conventional HPLC. As the monolithic columns do not contain interparticular voids, all the mobile phase must flow through the stationary phase. As a result of this convective flow, the mass transfer is accelerated. In contrast to diffusion, which is a typical driving force for mass transfer within the pores of particulate stationary phases during a chromatographic process, convective flow through the polymer of the monolithic column enables a substantial increase in the speed of separation of large molecules such as proteins [8]. Several brands of monolithic columns suitable for low-pressure flow methods are available commercially nowadays — Mercks Chromolitht first introduced to the market in 2000 and Phenomenexs Onyxt some years later — in usable lengths of 25 or 50 mm with silica-based ODS–C18 sorbent [11,12]. These monolithic columns consist of a single piece of high-purity polymeric silica gel rod with porosity exceeding 80% and a bimodal pore structure incorporating macropores and mesopores. Macropores (average size 2 mm) dramatically reduce the column back pressure and allow the use of higher flow rates. The mesopores (average size 13 nm) form the fine porous structure and create the large uniform active surface area enabling high performance chromatographic separation. Monolithic rods exhibit very high mechanical stability and long operative lifetimes, in most cases far exceeding the lifetime of particulate columns. Monolithic columns also exhibit similar chromatographic properties with respect to retention and selectivity as particulate columns of the same specific surface area and pore diameter. Since monolithic columns have become commercially available, they have been used in many different fields [8]. Another type of column based on the monolithic principle is so-called Convective Interaction Media (CIMs) [13], a new polymeric macroporous material produced by radical co-polymerization and recently used for the separation of biomacromolecules. However, monolithic columns have also several disadvantages. Their general drawback is their relatively lower efficiency compared to silica-based particulate

272

Petr Solich

columns. Most polymers can swell or shrink in organic solvents, which could dramatically affect the chromatographic performance of the corresponding monolithic columns, and can also lead to lack of mechanical stability. Furthermore, the structure of porous polymers often contains micropores that negatively affect column efficiency and peak symmetry [8]. Monolithic columns permit high flow rates of mobile phase at low back pressures without losing efficiency and can be used as a new separation tool in flow analytical methods. This feature allowed the integration of these columns into SIA manifolds in 2003 [14], which are low-pressure flow systems with pump back pressure up to about 2.5 MPa. The technique was called sequential injection chromatography (SIC) and it has been already successfully applied in the analysis of relatively simple multicomponent samples — mainly in the field of pharmaceutical analysis. SIC was originally built on classical SIA manifolds produced by Alitea– FIAlabs 3000 or FIAlabs 3500 analysers, each equipped with a syringe pump. The chromatographic component was a short (e.g., usually 25 or 50 mm) commercially available monolithic column (with or without monolithic precolumn of 5 or 10 mm length) placed between the multiposition valve and the Z detector flow cell. Detection was provided by a fibre optics UV–VIS diode array detector (DAD). The scheme of the SIC manifold is depicted in Figure 2. Early applications of SIC were focused on the analysis of pharmaceuticals (e.g., solutions, drops, syrups). These are relatively simple multicomponent mixtures (2–5 compounds of interest) without substantial interferences and they can be determined directly only by diluting the samples by the mobile phase. Other samples (topic creams, tablets, capsules, etc.) have to be pre-treated

DAD UV ZFC

1 MV 6

SV out

MC SI

2

S2

W

5 3

4

S3

SP FLAlab® MP

PC

Figure 2 Scheme of an SIC setup for multicomponent analysis. DAD, diode-array detector; MC, monolithic column; MP, mobile phase; MV, 6-port multiposition valve; SP, syringe pump; SV, solenoid valve; S1, S2 and S3: sample 1, 2, 3; PC, computer; UV, UV lamp; W, waste; ZFC, Z-flow cell. Reprinted from Ref. [32]. Copyright (2007), with permission from Elsevier B.V.


273

(e.g., extraction into an organic solvent). The length of the separation column was chosen depending on the chromatographic features of all substances in the sample. The mobile phase was usually methanol- or acetonitrile-based and only a volume sufficient to elute all substances from the column was used. The volume of the syringe pump used in these SIA systems was usually 5.0 or 10.0 mL, with the smaller syringe being more preferable due to the possibility to achieve higher working pressures. The flow rate of the mobile phase was optimized according to the length of the column and peak shape and it was usually less than 1.5 mL min1. Detection was based on a UV–VIS DAD coupled with a Z flow cell with 10 mm active optical path length. The use of two or three wavelengths allowed increased detection sensitivity. The sample volume used was usually 10–20 mL depending on the column length. The whole system was controlled by commercial FIAlabs software with predefined sequence. SIC has developed into a powerful technique allowing fast chromatographic determination of relatively simple multicomponent mixtures with possibility of easy automatic sample handling and pretreatment. The latter feature decreases the possibility of human errors and enables determination of aggressive or biohazardous samples. SIC combined with a Franz cell and a peristaltic pump has been used successfully in liberation tests of semisolid pharmaceuticals through artificial skin or in in vitro tests where monitoring the concentration of a substances was required for prolonged periods of time [15]. Table 2 summarizes relevant applications of SIC in pharmaceutical analysis.

2.2 Solid-phase extraction and restricted access material columns Nowadays, SPE represents the favourite sample pretreatment method which is often used in flow methods [16]. Columns packed with various types of materials that can selectively retain, adsorb or absorb components of the mixture are often used. Like in chromatography, separation is achieved due to the different affinity of the analytes to the solid phase. A generally adopted strategy is based on the fact that one of the analytes passes freely through the solid support in the SPE column and is detected in the flow-through cell. The other analyte is retained on the solid phase, and is eluted later by changing the composition of the carrier. Direct incorporation of an SPE microcolumn as a separation element in an SIA manifold was firstly described by Solich and co-workers [17]. This system was used for the simultaneous determination of ascorbic acid and rutin. The SPE microcolumn was used for retention of rutin, while ascorbic acid was eluted with the solvent front phase and complete separation was achieved within 3 min (Figure 3). This application illustrates one of the main advantages of this SPE configuration, i.e., the simplicity of the manifold. On-line coupling of classical SPE with SIA offers possibilities for automation of the separation process and higher sampling rates. Furthermore, a preconcentration step can also be carried out using SPE. The most important drawback of the SPE approach is that usually only two components can be determined in a single step. Another application of the SIA–SPE technique was developed for the determination of salbutamol in urine [18]. The sample was dispensed to the

274

Matrix

Analytes

Column (mm)

Flow rate (mL min1)

Mobile phase

Detection UV (nm)

Pretreatment of Time sample (min)

Pharmaceutical syrups and drops

Ambroxol, methylparaben, benzoic acid

50+10

0.48

245

Extraction into the mobile phase

o11

Pharmaceutical drops

Naphazoline nitras, methylparaben

25+5

0.9

220; 256

Dilution of drops

o4


Triamcinolone acetonid, salicylic acid

50+5

0.9

240

Dilution of drops

o7


Betamethasone, chloramphenicol

25+5

0.48

Acetonitrile: tetrahydrofuran: water (10:10:90, v/v/v) pH 3.75 adjusted with triethylamine and acetic acid Methanol:water (40:65, v/v), pH 5.2 adjusted with triethylamine 0.8 ml mL1and acetic acid Acetonitrile:water (35:65, v/v), pH 3.2 adjusted with acetic acid Acetonitrile:water (30:80, v/v)

241; 271

o8

Topical cream

Triamcinolone acetonid, methylparaben, propylparaben

25+10

0.6

Extraction into methanol with 1% H3PO4 Extraction into methanol

Acetonitrile: methanol: water (35:5:65, v/v/v), pH 2.5 adjusted with 0.05% nonylamine and H3PO4

243

o6

Petr Solich

Table 2 Application of SIC fort analysis of pharmaceuticals [19]

240

Extraction into methanol

o7

275

Extraction into methanol

o8

213

25

0.6

Acetonitrile:water (10:90, v/v), pH 4.05 adjusted with 98% H3PO4

210

25

0.6

Acetonitrile:water (40:80, v/v), pH 7.1 adjusted with 0.01% triethylamine and H3PO4

212

Exctraction into o9 methanol with 1% of H3PO4 Extraction into o7 methanol and mobile phase Automated o7 system for release testing of semisolid dosage forms

Salicylic acid, methylsalicylate

50

Topical cream

Diclofenac natrium, methylparaben, propylparaben

25

Pharmaceutical capsules

Ambroxol hydrochloride, doxycycline

25

Pharmaceutical tablets

Paracetamol, coffein, acetylosalicylic acid

Topical cream

Lidocaine, prilocaine

0.6


Acetonitrile:water (35:60, v/v), pH 2.5 adjusted with 98% H3PO4 0.48; 0.9; 1.2 Acetonitrile:water (40:70, v/v), pH 2.5 adjusted with 0.05% triethylamine and H3PO4 0.48 Acetonitrile:water (20:90, v/v), pH 2.5 adjusted with 98% H3PO4

Topical cream

275

276

Petr Solich

0.7 A AA

Absorbance (262 nm)

0.6 0.5 0.4

RT

0.3 0.2 0.1 0.0 0

30

60

90

120

150

180

210

240

270

300

330

360

180

210 240

270

300 330

360

0.6 B Absorbance (262 nm)

0.5

RT

0.4 0.3 0.2 0.1 0.0 0

30

60

90

120

150

Figure 3 (A) SIA–SPE trace for duplicate injection of ascorbic acid and rutin. (B) SIA–SPE trace of duplicate injection rutin in the absence of ascorbic acid. Reprinted from Ref. [17]. Copyright (2003), with permission from Elsevier B.V.

SPE microcolumn; salbutamol from the sample was adsorbed onto the sorbent, which was successively washed with the carrier stream and thus the interfering substances were removed to the waste. In recent years, special SPE supports possessing restricted access properties have been developed to allow the direct injection of untreated biological samples into analytical systems. These restricted access material (RAM) sorbents combine size exclusion of proteins (without destructive accumulation) and other macromolecular matrix components with the simultaneous enrichment of lowmolecular mass analytes, which can be retained and selectively desorbed. Coupling of RAM with SIA produced a new hybrid flow technique [20] for the direct determination of drugs in biological samples at high sampling rates and low costs. This technique can fill in the gap between traditional HPLC and manual sorbent extraction methods. The incorporation of a solid phase C18 RAM


277

column into an SIA system allowed the successful direct determination of furosemide in human serum [21]. In a similar approach a selective anion exchange solid-phase microcolumn was utilized for the simultaneous determination of benzophenone-4 and phenylbenzimidazole sulphonic acid in sunscreen sprays [22]. A recent report on the application of an SIA–RAM system for the determination of propranolol in human plasma illustrates the potential of this approach [23]. In this study a special RAM column containing 30 mm polymeric material (N-vinylacetamide copolymer sorbent Shodexs MSpak) was integrated into the SIA manifold. Propranolol was selectively retained in the column, while the plasma matrix components were eluted to waste with two weak organic solutions and detected fluorometrically. The whole procedure comprising sample pretreatment, analyte detection and column reconditioning was carried out within 15 min.

3. PHARMACEUTICAL APPLICATIONS OF SEQUENTIAL INJECTION CHROMATOGRAPHY As mentioned earlier SIC has been successfully applied to the analysis of various types of pharmaceutical products which will be discussed in the subsequent sections.

3.1 Liquid samples For the determination of compounds in liquid pharmaceutical mixtures it is usually not necessary to do sample pretreatment because of the absence of interferents. The ionic compounds present usually do not interfere with the chromatographic measurement. Several SIC methods for the analysis of such type of samples have been reported. Ambroxol, methylparaben and benzoic acid were determined simultaneously in various pharmaceutical syrups and drops with salicylic acid as the internal standard [24]. Naphazoline nitras and the preservative methylparaben were determined in eye drops using ethylparaben as the internal standard [25]. This method involved for the first time the simultaneous use of two UV wavelengths to increase the selectivity of analysis. Topical solution containing salicylic acid and triamcinolone acetonide was analyzed with propylparaben as the internal standard [26]. In the case of betamethasone and chloramphenicol determination in eye drops with propylparaben as the internal standard, a simple sample pretreatment procedure involving extraction into methanol with 1% of H3PO4 was used [27].

3.2 Topical semisolid formulations Due to interferences in the matrix it is necessary to carry out simple and fast pretreatment of topical semisolid formulations. Extraction with organic solvent had to be done before the determination of triamcinolon acetonid and two conservants (methylparaben and propylparaben) in a topical cream [28].

278

Petr Solich

Ketoprofen was used as the internal standard. Extraction also preceded the determination of salicylic acid and its ester methylsalicylate in topical pharmaceutical preparations [29] and sodium diclofenac and the conservants methylparaben and propylparaben in a topical emulgel [14]. The internal standards in these two methods were propylparaben and butylparaben, respectively.

3.3 Tablets and capsules SIC was also used for the chromatographic determination of ambroxol hydrochloride and doxycycline in pharmaceutical capsules with ethylparaben as the internal standard [30] and for the simultaneous determination of paracetamol, caffeine and acetylsalicylic acid in common antipyretic and antiflogistic tablets with benzoic acid as the internal standard [31]. In both methods a simple and fast pretreatment step involving extraction into an organic solvent was required.

3.4 Automation of simultaneous release tests Franz diffusion cell is often used as a standard vessel for controlling the liberation of active compounds from topical preparations [32]. It consists of two parts — a donor and an acceptor compartments — that are separated by a membrane. The donor compartment holds the drug preparation and the acceptor compartment the receiving medium. For release experiments, artificial membranes are usually used to physically separate the donor and acceptor compartments. The membrane should allow the active ingredient to diffuse readily to the receiving medium as it is being ‘‘released’’ from the dosage form. The diffusion across the membrane should not be the rate-limiting step in the overall mass transfer process. Connection of an SIC manifold to a Franz diffusion cell has enabled to create a fully automated system for the in vitro release testing of composed semisolid dosage forms [15]. This system was used for the determination of two active substances in a topical pharmaceutical formulation composed of lidocaine and prilocaine. Trimecaine was used as the internal standard. Samples were taken in 10 min intervals during a 4 h release test.

4. COMPARISON OF SEQUENTIAL INJECTION CHROMATOGRAPHY AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SIC can be viewed as a new generation of SIA, which offers an attractive alternative to HPLC for fast analysis of relatively simple multicomponent samples, containing preferably 2–5 analytes. The use of commercial monolithic silica columns and detection at several (2–4) wavelengths often matching the absorbance maxima of the analytes of interest have transformed SIC into a powerful analytical tool for simultaneous multicomponent analysis. Its applications have expanded beyond the area of pharmaceutical analysis [19]. It can be expected that a more accurate assessment of the actual potential of SIC and related techniques will require more time. However, even on the basis of the


279

limited number of applications of SIC so far, it can be concluded that this technique offers efficiency of separation which in most cases is comparable to that of HPLC. This point is well illustrated with Figure 4 where a typical SIC chromatogram of a pharmaceutical mixture (Triamcinolon-IVAX topical solution [28]) is compared with the corresponding HPLC chromatogram. Other advantages offered by SIC, which enhance its attractiveness to multicomponent analysis, are inexpensive instrumentation compared to HPLC, short time of analysis, possibility for on-line use of reagents in all steps of the determination, low production of waste and lower consumption of solvents compared to HPLC. These advantages lower the analysis costs compared to HPLC. In addition, SIC provides easy liquid manipulation not attainable by classical HPLC and possibility for miaturization, thus allowing the construction of portable instruments for ‘‘on-field’’ analysis. On the other hand, at present SIC suffers from disadvantages associated with the use of short monolithic column and limitations regarding the maximal amount of mobile phase available per analysis, which is determined by the

Figure 4 (a) SIC chromatogram of the separation of paracetamol, caffeine, acetylsalicylic acid and benzoic acid (internal standard). Mobile phase: acetonitrile-(0.01 M) phosphate buffer (10 : 90, v/v). (b) HPLC chromatogram of the separation under the same conditions. Reprinted from Ref. [31]. Copyright (2004), with permission from Wiley-Interscience.

280

Table 3

Petr Solich

Comparison of SIC and HPLC [19]

Characteristic

HPLC

SIC

Possibility of separation Flow of mobile phase Direction of flow of the mobile phase Flow rate of the mobile phase Consumption of mobile phase Use of reagents (reactions) Pretreatment of sample Data evaluation Cost of instrumentation Portability of analyser

Yes Continuous Unidirectional

Yes—simple mixturesa Discontinuous Bidirectional+stop flow

Constant High Limitedc Yes (restricted) Sophisticated High No

Variable Low (discontinuous flow)b Yes Yes Simple Lowd Yese

a

From two to five substances. The mobile phase is flowing during the separation only. c Post-column derivatization. d 2–3 times cheaper. e Small-sized box. b

volume of the syringe pump barrel (5.0 or 10.0 mL). These drawbacks together with back-pressure problems at high flow rates, reduce the robustness of SIC compared to classical HPLC. Data processing in SIC, currently based on peak height only, is less sophisticated than that in HPLC and can be considered as another drawback which is expected to be rectified in the future. Table 3 summarizes the main similarities and differences between HPLC and SIC.

5. OTHER CHROMATOGRAPHIC APPROACHES A growing interest can be seen in the last two years for the implementation of monolithic columns to various types of applications and in different low-pressure flow methods. Recently Garcıá-Jimeńez et al. [33] and Adcock et al. [34] employed monolithic columns in FIA for the first time and these hybrid systems were used for the analysis of several antioxidants and preservatives in food and cosmetics and six opiates and four biogenic amines in urine, respectively. Zacharis et al. [35] incorporated a monolithic strong anion-exchanger disk (CIMs) into an SIA manifold for on-line drug–protein interaction studies. Two detection modes were used — the free fraction was monitored by its intrinsic fluorescent properties while UV monitoring was used for the retained Bovine serum albumin (BSA). For the first time, sequential injection affinity chromatography was used for drug–protein interaction studies [36]. The analytical system used consisted of an SIA manifold directly connected to a CIM monolithic epoxy disk modified by ligand-immobilization of a protein. The non-steroidal, anti-inflammatory drug


281

naproxen (NAP) and BSA were selected as the model drug and protein, respectively. The SIA system with fluorescence and spectrophotometric detection was used for sampling, introduction and propulsion of the drug towards the monolithic column. Competitive binding on BSA was also studied using naproxen and alendronate. Cerda´ and co-workers [37,38] demonstrated the great potential of connecting a short monolithic column to a multisyringe flow injection analysis (MSFIA) manifold (Figure 5) with the simultaneous determination of three antibiotics — amoxicillin, ampicillin and cephalexin. They named this hybrid technique multisyringe liquid chromatography (MSC). The experimental system consisted of a multisyringe module, three low-pressure solenoid valves, a monolithic Chromoliths Flash RP-18e column and a diode array spectrophotometer (Figure 5). AutoAnalysist software was used for instrumental control and automated data collection. The results obtained with the MSC system, outlined above, were compared with those obtained with an HPLC system under similar conditions (Figure 6). The main advantages of this hybrid system (e.g., low-cost, flexibility and simplicity of design and operation) are similar to those offered by SIC manifolds.

6. FUTURE TRENDS The growing interest of scientific laboratories in the implementation of chromatographic columns in low-pressure flow systems suggests that we can MP

On

Off V1

S

V2 sc Off

M

RP-18 DAD

V3

E1

E2 W

S1

S2

MS

Figure 5 MSC system for isocratic separation: MS, multisyringe burette; M, manometer; V1–V3, solenoid valves; E1, two-way connector; E2, three-way solenoid valve; MP, mobile phase; S, sample; W, waste; RP-18, monolithic column; DAD, diode array detector and SC, sample coil. Reprinted from Ref. [37]. Copyright (2007), with permission from Elsevier B.V.

Petr Solich

Abs A

0.4000

2

299.1

0.5000

74.5

282

1 0.3000 360.7

0.2000 0.1000

3

0.0000

time (sec)

-0.1000 -0.2000

0.06 0.04

1 2

193.4

0.08

B

154.8

0.1

Abs 35.0

0.12

3

0.02 0 -0.02

time (sec)

-0.04 -0.06

Figure 6 MSC separation of amoxicillin (1), cefalexin (2) and ampicillin (3) using a Chromolith RP-18e column as stationary phase (mobile phase: methanol–sodium acetate buffer pH 6.2, 0.1 mol L1 (10:90); flow rate: 2 ml min1; detection at 250 nm). (A) MSC chromatogram, (B) HPLC chromatogram. Reprinted from Ref. [38]. Copyright (2007), with permission from Springer.

expect a lot of new applications in this area in the near future. It can be expected that future research in this field will be focused on shortening the analysis time. Another emphasis will be on the development of appropriate hardware configurations and suitable software, being equally sophisticated to that currently used in HPLC techniques. Along with the effort to achieve the desired high performance, attention will also be paid to the reduction of the instrumentation costs which lead to cheaper analysis. The benefits of using flow analytical methods such as automation of analysis, miniaturization of instrumentation and low-sample and mobile-phase consumption can be extended further by the implementation of a separation step in the analytical procedure. An excellent example of such implementation is the use of short monolithic chromatographic columns in SIA systems. This has opened a new area in flow analysis (i.e., SIC) involving on-line chromatographic separation


283

of multicompound samples in low-pressure flow systems with the added benefits of flow programming and possibility of sample manipulation. Recent developments in this area have resulted in the commercially available SIC instrument SIChromt — Accelerated Liquid Chromatography System (FIAlabs instruments, USA). This instrument overcomes some of the shortcomings of the first generation SIC systems. It is equipped with a powerful syringe pump allowing higher flow rates and therefore it can use longer monolithic columns. This instrument also incorporates a chemically resistant lab-on-valve unit for variable sample handling and pretreatment [39]. The software allows easy data processing. SIChrom represents the first commercial liquid chromatography instrument based on SIA concepts thus allowing the performance of liquid manipulations not attainable by traditional liquid chromatographs. It can be expected that future developments in flow methods for multicomponent analysis will involve extensive use of chemometric treatment of the analytical data, especially in cases when overlapping peaks are encountered. These mathematical treatments could be able to substantially improve the separation capacity of SIC and related techniques. Recently reported utilization of the second-order multivariate regression models based on multivariate curve resolution-alternating least squares (MCR–ALS) allowed the corresponding SIC approach to outperform current chromatographic methods in terms of resolution efficiency [40]. The proposed SIC–MCR–ALS method involving sequential injection separation on short monolithic columns along with isocratic elution is expected to provide ultrafast reversed-phase separation of complex multicomponent mixtures regardless of the analytes’ retention times. This feature of SIC–MCR–ALS was convincingly demonstrated with the simultaneous determination of five phenolic species commonly used in disinfectant products and featuring similar UV spectra and close retention times [40]. A short reversedphase silica-based monolithic column was used and all five model compounds were determined in 1 min using mobile-phase flow rates of Z2 mL min1. Notwithstanding the fact that the five phenolic derivatives coeluted in a single chromatographic band, thus rendering resolution values ranging from 0.05 to 1.11, the concentration profiles and the pure spectra of each individual phenolic species could be concurrently obtained. Quantitative validation of the SICchemometric method demonstrated both the reliability of this approach and the possibility of enhancing the resolution by chemometric means. There was no need of thorough optimization of the separation conditions. Further research could also be focused on the automation of pretreatment of biological samples (on-line extraction) and on the use of other types of monolithic columns (different monolithic sorbents, lengths and diameters). Coupling of SIA with other selective detection techniques (e.g., mass spectrometry detection, fluorescence detection) is another promising area of development which will result in increasing the sensitivity and selectivity of the analytical determinations. The construction of portable field SIC instruments is another possible area of future development. It can be concluded that the introduction of a chromatographic separation step in low-pressure flow methods can extend substantially the attractiveness of

284

Petr Solich

FIA, SIA and similar flow techniques not only to scientific research but also to routine analysis. The potential of SIC and related techniques is high, but its future development depends very much on finding practical applications of these techniques for routine analysis.

ABBREVIATIONS BI-LOV BLLS BSA CIMs DAD ET-AAS FIA FTIR HG-AAS HPLC ICP-MS ICP-OES MCR-ALS MPFIA MSC MSFIA N-PLS PLS RAM SIA SIC SPE UPLC

Bead injection-lab-on-valve Bilinear least squares Bovine serum albumin Convective interaction media Diode array detector Electrothermal-atomic absorption spectrometry Flow injection analysis Fourier transform infrared spectroscopy Hydride generation-atomic absorption spectrometry High performance liquid chromatography Inductively coupled plasma-mass spectrometry Inductively coupled plasma-optical emission spectrometry Multivariate curve resolution-alternating least squares Multipump flow injection analysis Multisyringe liquid chromatography Multisyringe flow injection analysis N-way-partial least squares Partial least squares Restricted access material Sequential injection analysis Sequential injection chromatography Solid-phase extraction Ultra performance liquid chromatography

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CHAPT ER

11 Flow Injection Analysis–Capillary Electrophoresis Pavel Kubańˇ and Peter C. Hauser

Contents

1. Introduction 2. Fundamentals of Capillary Electrophoresis 3. On-Line Coupling of FIA and CE 3.1 Electrokinetic injection 3.2 Hydrodynamic injection 3.3 Dual opposite end injection 3.4 Sample pretreatment 3.5 Derivatization for detection 3.6 FI-Microchip CE 3.7 Fast separations in short capillaries 4. Electrokinetically Pumped Flow Analysis 4.1 Electrophoretically mediated microanalysis in capillaries 4.2 Miniaturized systems 5. Conclusions Abbreviations Acknowledgments References

287 288 291 291 295 296 297 298 299 300 302 302 303 305 305 306 306

1. INTRODUCTION The combination of flow analysis and capillary electrophoresis (CE) is a fruitful one and a multitude of systems have been designed to achieve quite a variety of goals. Chiefly, two main approaches can be distinguished. The first group concerns the coupling of conventional flow injection analysis (FIA) or sequential injection analysis (SIA) manifolds to CE systems. In this combination the entire CE part of the instrumentation may be considered as a detector for flow analysis. The main advantage is the inherent multi-analyte capability of the separation Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00611-9


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method. In flow injection analysis selectivity is often achieved by adding specific reagents such as colorimetric complexing agents or enzymes, or the use of specific detection systems such as chemical sensors or an atomic absorption spectrometer. It is difficult though to find specific reagents for all potential analytes, chemical sensors are only available for certain species and powerful spectrometers are expensive. These approaches usually also imply that only a single analyte can be determined at one time. Multi-analyte FIA-systems are mostly fairly complex as they usually rely on sample splitting into several lines, with special sample treatment on each branch, or the incorporation of several specific detectors in series. An alternative possibility to achieve at the same time selectivity and multi-analyte capability is the coupling of FIA to a separation method such as chromatography or electrophoresis. Detectors which otherwise do not show sufficient selectivity are made accessible, and concurrent determinations of more then one species is then the norm. The approach is also attractive from the point of view of CE. Sample injection in electrophoresis is a rather delicate operation as the volumes are in the nanolitre range. An FIA manifold may be employed to automate injection into an electrophoresis capillary to achieve high precision and accuracy. Furthermore, on-line preconcentration or matrix clean-up steps may be implemented in this way. The second approach in which flow analysis and CE can be integrated is the employment of the electrokinetic phenomena of electrophoresis and electroosmosis as transport mechanisms instead of using conventional pumps. The technique is often termed electrophoretically mediated microanalysis (EMMA) and is attractive for two reasons. Capillaries employed in CE have to have narrow diameters. Out of necessity the volumes of sample and reagent solutions used are therefore small, which makes the methods attractive when the available sample volume is limited as in biochemical applications, or when expensive reagents are required. Electrokinetic pumping is simpler than conventional pumping as no moving parts are required to propel solutions. The high voltages needed can be obtained from inexpensive modules, which are readily available commercially. The method may be implemented on conventional CE instruments but special EMMA-systems have also been designed, mainly with the aim of achieving a higher degree of miniaturization.

2. FUNDAMENTALS OF CAPILLARY ELECTROPHORESIS A schematic drawing of a CE system is shown in Figure 1. Separations are achieved by applying voltages of 10–30 kV to a capillary of about 50–100 cm in length. Different modes of electrophoretic separations are used, but the most common approach is zone electrophoresis. Analyte ions move in the electric field produced by application of the separation voltage, and differences in their electrophoretic mobility, in dependence of their size and charge, lead to a separation. In order to obtain an even electric field along the length of the capillary, a background electrolyte (BGE) solution (of typically about 10–50 mM

Flow Injection Analysis–Capillary Electrophoresis

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Capillary D HV +/-

Samples

Buffer

Buffer

Figure 1 Capillary electrophoresis. Voltages of up to 30 kV (HV) are applied across capillaries. Samples are introduced by placing the injection end of the capillary into the sample container. Detection (D) is carried out on-column or in the case of amperometric detection at the capillary end.

Mobility of anions

Mobility of cations Electroosmotic flow

+

Net mobility of cations Net mobility of anions

Figure 2 Illustration of the effect of the normal electroosmotic flow (EOF) on the migration of cations and anions.

concentration) is used. This solution commonly also incorporates buffer species to control the pH-value. The capillaries are usually made of fused silica. On the surface of this material silanol groups are found, which dissociate in aqueous solution in dependence of its pH-value leaving fixed negative surface charges. This in turn leads to the development of an electroosmotic flow (EOF), a small bulk flow of solution towards the cathode brought about by loose binding of the water molecules to the mobile counter ions. This secondary electrokinetic phenomenon present in electrophoresis is usually not desired but has to be taken into consideration when carrying out separations. The situation is illustrated in Figure 2. The electrophoretic movement of cations towards the cathode is accelerated by the EOF. All but the fastest anions are also pushed towards the cathode, but show a much-reduced effective mobility. For optimization of the separation, the EOF is often deliberately modified with an auxiliary reagent in order to permanently or dynamically coat the surface of the capillary. Such a coating may minimize charges on the surface for suppression of the EOF, or a reversal, i.e., a flow towards the anode, can be achieved by coating the surface with a cationic

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surfactant. This then allows the determination of all anions by applying a positive potential at the detection end of the capillary. The capillaries used in electrophoresis have to have small internal diameters between about 25 and 100 mm. Larger tubing would lead to excessive flow of current (due to the buffer ions as charge carriers). As the electrical power consumed (the product of separation voltage and current) is transformed into heat, efficient cooling would then not be possible. As a result, the volumes of BGE solutions and of samples injected are small. While the use of small amounts of reagents and samples has obvious benefits, this imposes challenges on detection and injection. As in HPLC, the most commonly used detection method in CE is the absorbance measurement with ultraviolet light. The determination of organic, biochemical and other analytes having strong UV-absorbing moieties can be readily accomplished. However, absorbance detection in CE is not as sensitive as in HPLC as the small available detection volumes do not allow standard optical pathlengths. Indirect absorbance detection, utilizing charged dyes, is used for determination of non- or poorly UV-absorbing species, such as inorganic anions and cations. Higher sensitivity can be achieved for direct UV detection compared to the indirect mode. Fluorescence detection is often used as an alternative. Due to its high sensitivity this method does not suffer significantly from the limited detection volume. It has found wide use for biochemical applications because peptides, proteins and DNA fragments can be derivatized to render them fluorescent. The coupling of a separation capillary to a mass spectrometer via electrospray sample injection and ionization is also a powerful combination for research in the life sciences. In contrast to the older ion-separation method of ionchromatography, where conductometry and amperometry are the norm, electrochemical detection techniques have not been as popular for CE as the spectroscopic methods. Conductivity detection is however attractive as it is a universal method for ion detection (regardless of their optical properties) and amperometry has good detection limits for electroactive species. The reasons for the limited adoption are twofold. It is first of all difficult to construct cells with electrodes of a size that match the internal dimensions of the separation capillaries, and the high voltage present for electrophoretic separation imposes constraints on the cell geometry. However, these limitations can be alleviated for conductivity measurements by carrying them out in a contactless manner, and capacitively coupled contactless conductivity detectors have recently become available commercially. The second challenge due to the small dimensions in CE is the injection. This cannot be carried out with conventional injection valves as the sample volumes in the low nanolitre range are too small, and due to complications arising from the separation voltage. In standard CE instruments the end of the capillary is therefore placed directly into a sample vial for injection. A small amount of the sample is then forced into the capillary by either momentarily applying air pressure to the vial, or by creating a vacuum at the other end. A third possibility to achieve hydrodynamic injection is to temporarily lift the sample vial together with the capillary end to a fixed height in order to achieve a siphoning effect.


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Alternatively, electrokinetic injection is used. Hereby, a voltage is applied directly to the sample solution to achieve insertion of analyte into the capillary by electrophoretic and electroosmotic transport. Care has to be taken in this approach to eliminate or compensate a bias that arises due to differences in the electrical conductivity of different solutions in dependence of their total ionic compositions. In either method the capillary end has to be returned to a vessel containing the buffer solution before commencing the electrophoretic separation. It is difficult to achieve high precision for these injection methods, and to eliminate carry-overs, and the necessary movements of the capillary end or of the vials sets a limit to the maximum speed at which this first step of the procedure can be carried out.

3. ON-LINE COUPLING OF FIA AND CE It is possible to couple an FIA manifold to a commercial CE instrument by using a robotic system equipped with syringes to collect fractions from the effluent of the FIA manifold and place these into the vials of the autosampler of the electrophoresis instrument. This approach has been used successfully to bring the benefits of automated sample pretreatment operations to CE [1,2]. Less complex, and from the point of view of electrophoresis more desirable is the online coupling of the two manifolds. A plug of the sample is then delivered directly to the sampling end of the separation capillary by means of a flowing stream of a carrier solution. No handling of vials for sample and buffer solutions is involved and no physical disruption of the separation system occurs. Only a small part of the sample plug is then introduced into the capillary by hydrodynamic or electrokinetic injection modes. Note that as far as the insertion of sample into the capillary is concerned it makes no difference if the front end consists of a conventional FIA or a SIA manifold.

3.1 Electrokinetic injection A schematic drawing of a hyphenated FIA–CE system is shown in Figure 3. The carrier corresponds to the BGE solution used in the electrophoretic separation with which the capillary is filled. A sample plug of typically tens of mL is transported through a short piece of tubing to the interface with the CE part of the set-up. In order to avoid possible sample diffusion during the transport, the internal diameter of the tubing and of the flow-through channel in the FIA–CE interface is kept minimal. An interface developed by Kubańˇ et al. [3] is shown in Figure 4. It consists of a polymeric block with approximate dimensions of 3.5 2.5 2.5 cm which includes a horizontal flow-through channel, to which the outlet tubing of the FIA system is connected. Two additional channels perpendicular to the flowthrough channel serve as joints for connecting the injection end of the separation capillary and for holding the ground electrode of the CE system. Excess solution flows to waste.

292


D HV +/Buffer FI-CE-interface Sample

Buffer

To waste

Figure 3 FIA–CE combination. A conventional FIA or SIA manifold is coupled on-line to a CE system and serves as an injection system.

Figure 4 FIA–CE interface according to Ref. [3] made from polymethyl methacrylate. Connections are made with standard fittings.

A different interface, as depicted in Figure 5, has been developed by Fang and coworkers [4]. The device does not require machining facilities and was made of a plastic vial. This was punctured at the bottom and on the side. The conical end of a plastic micropipette tip was sealed into the lower hole, and the connecting tubing from the FIA manifold was connected to it. The inlet end of the separation capillary was placed into the lower part of the pipette tip and was positioned approximately 2 mm above the bottom of the tip; the ground electrode of the high voltage power supply was placed into the pipette tip as well. A constant level of liquid in the FIA–CE interface was ensured by the second hole drilled in the sidewall of the vial, which was connected to a FIA pump and any excessive solution above the level of the hole was automatically pumped out of the interface. In either of the FIA–CE interfaces, the sample plug is transported past the inlet of the capillary towards waste. During this passage a small portion of the original sample volume enters the separation capillary in electrokinetic injection mode. To ensure this, the separation voltage of the CE system is continuously applied across the capillary. The electrode in the FIA–CE interface is usually kept at ground potential while the second electrode at the detector end is set to the high voltage. This is opposite to the usual arrangement in CE and avoids complications that could arise from exposing the FIA part of the manifold to


Figure 5

293

FIA–CE interface according to Ref. [4].

high voltages. It is also important to design the FIA–CE interface in such a way that no significant backpressure is produced at its outlet in order to avoid pressure driven flow of liquid into the separation capillary. Similarly a siphoning effect through the capillary is avoided by adjusting the level of the electrolyte solution in the vessel at the detector end to the same height as the outflow from the FIA–CE interface. The continuous carrier flow washes the major part of the sample, which is not injected, to waste and replaces the solution in the flow-through channel of the FIA–CE interface with fresh BGE solution. A constant high voltage is applied to the CE system during the entire injection and separation procedures. Electrokinetic sample injection in conventional CE is usually performed at much lower voltages than employed during separation owing to the fact that longer and more reproducible injection periods can then be applied. Using different voltage settings for injection and separation steps is not necessary in hyphenated FIA–CE systems as the precision is determined by the operation of the FIA manifold. The use of a constant voltage setting for the sequence is beneficial for the electrophoretic separation as the stability of the current-voltage relationship leads to a well equilibrated system especially with regard to the Joule heating produced inside the separation capillary. The FIA–CE systems developed enabled rapid sample injections into the FIA system. High analytical throughput was achieved and up to 150 analyses per hour could be performed for the determination of chloride, nitrate and sulfate in standard solutions [3]. Excellent reproducibility was obtained for the determination of the three anions and RSD values less than 2.3% and 6.3% were reported for 20 consecutive injections of a standard solution for peak height and peak area measurements, respectively. Multiple FIA injections and CE separations of up to nine anions were demonstrated and the high potential of the hyphenated system was further extended to repeated determination of common inorganic anions in tap and environmental water samples with a sample throughput of

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10 mgL-1 1 3 2

60 mV

5 mgL-1 Drainage Tap 1 mgL-1

3

4

5

6

7

Surface1 Surface2 Rain

8 9 10 11 12 13 14 15 Time (min)

Figure 6 Determination of chloride (1), nitrate (2) and sulfate (3) using an FIA–CE manifold with electrokinetic injection. The injection of a standard mixture was followed by samples of tap, drainage, rain and surface run-off water samples. Each was injected in triplicate. Reprinted from Ref. [61]. Copyright (2003), with permission from Wiley-VCH.

approximately 60 samples/h. The automated analysis of standards and samples is illustrated in Figure 6. In the electrokinetic mode of injection, the exact amount injected into the capillary is dependent on the length and dispersion of the sample plug, the flow rate of the BGE solution and on the separation voltage applied to the capillary. Adjustments of the amount injected can be made mainly by altering the size of the sample plug through changing the size of the loop on the injection valve of the FIA manifold. Electrokinetic injection in FIA–CE systems has the same main limitation as in conventional CE systems, namely an effect of the conductivity of the sample on the amount of analytes moved into the capillary end. This bias may be suppressed by dissolving the sample in the BGE solution to normalize the conductivity, or corrected for by using internal standardization. A comprehensive study on the calibration principles for electrokinetic injection in FIA–CE systems has been published by Kubańˇ et al. [5] showing that linear calibration curves over 2 orders of magnitude can be achieved if one of the two above mentioned methods is used. Note that the additional step of sample dissolution in BGE solution or addition of internal standard to the sample may be automated in the FIA manifold. The injection bias associated with the electrokinetic mode may also be used to advantage by exploiting the stacking effect occurring for samples of low conductivity for preconcentration. Kaljurand and coworkers have described a pneumatically driven sampling device for on-line stacking [6,7]. Different injection parameters were considered, including the residence and flush time of the sample and electrolyte solution, electrophoretic current and CE system


295

backpressure and substantial reduction in detection limits could be achieved for optimized conditions.

3.2 Hydrodynamic injection As in conventional CE, the injection bias of the electrokinetic mode may be overcome by hydrodynamic injection. Independently of the sample matrix and analyte properties, identical amounts of all analytes are injected into the separation capillary. However, this mode of injection is somewhat more difficult to implement then electrokinetic injection also in on-line coupled systems. In FIA–CE, hydrodynamic injection is achieved with the help of a blocking valve at the waste outlet of the interface for temporary pressurization. The resulting manifold is illustrated in Figure 7. It is important to precisely control the timing of the pressurization following the launch of the sample plug into the carrier stream. Furthermore the separation voltage is turned off and only turned on again after flushing of the interface with BGE solution has occurred. The amount injected is determined by the magnitude and duration of the pressure pulse created. Precise control and synchronization of the timing of events from introducing the sample into the carrier stream until reapplication of the separation voltage is mandatory. The principles and applications of hydrodynamic injection in FIA–CE hyphenated systems were for the first time described in detail in 1999 by Kubańˇ et al. [8]; however, the method has not reached the same popularity as electrokinetic injection. This can be very likely ascribed to two reasons. Firstly, the additional instrumental and electronic set-up required represents a barrier for implementation of hydrodynamic injection. Secondly, the total number of injections is reduced due to the fact that the high voltage of the CE system must be switched off during every injection step. This usually causes artifacts on the detector signal. Consequently, longer time intervals must be selected between successive injections. Nevertheless, linear calibration curves are achieved for wide concentration ranges

D HV +/Buffer FI-CE-Interface

Buffer

Sample

To waste Pressurization valve

Figure 7 FIA–CE manifold with pressurization valve at the outlet of the interface (as shown in Figure 4) for hydrodynamic injection.

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without additional sample treatment, which would be necessary for electrokinetic injection.

3.3 Dual opposite end injection In CE it is normally only possible to determine either cations or anions in a single determination. If the analysis involves species from both groups it is therefore usually necessary to carry out two separate runs for which the polarity of the applied separation voltage, and often also the buffer solution has to be changed. A technique for the simultaneous determination of cations and anions is to inject the sample at both ends of the capillary. The ions of different charge then move in opposite direction, and the detector is positioned approximately at the midpoint of the capillary. Dual opposite end injection has also been automated using two separate FIA manifolds at each end of the capillary as illustrated in Figure 8. The challenge in this approach lies in the fact that it is not possible to avoid the exposure of both FIA manifolds to the high voltage of the CE system. It has to be considered that the buffer solutions involved are electrically conducting and any parts upstream or downstream from the high voltage electrode which are in contact with the solution will not only be exposed to the high tension but also might provide an unwanted electrical path to ground. This hurdle could by overcome by employing gravity flow from a suspended, electrically isolated container to avoid the use of a conventional pump [9]. Such a system was successfully demonstrated in automated on-site analysis of inorganic soil nutrients in the effluent of a pasture during a rainfall [10]. The concentration profiles obtained with this system are shown in Figure 9 for just two of the ions, namely ammonium and nitrate, which show distinctly different behaviour in their movement through the soil.

D

HV +/-

FI-CE-Interface buffer

FI-CE-Interface To waste

Figure 8 FIA–CE manifold for dual opposite end injection of samples to enable concurrent determinations of cations and anions.

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4

10

8

6 NH4+ photometric

2

NH4+ CE

4

NO3- photometric

NH4+ [mg N L-1]

NO3-[mg N L-1]

3

-

NO3 CE

1

2

0 12:00

16:00

20:00

24:00

4:00

0 8:00

Figure 9 Automated monitoring of ammonia and nitrate in a run-off from a pasture during a rainfall. The ions originate from the manure used to fertilize the field. Reprinted from Ref. [10]. Copyright (2004), with permission from the Royal Society of Chemistry.

3.4 Sample pretreatment A very important application of the FIA–CE coupling is the implementation of an on-line sample pretreatment step in the flow-injection manifold prior to the capillary electrophoretic determination. Two main reasons for this can be distinguished, namely matrix elimination in order to achieve selectivity, and preconcentration for the purpose of lowering the limit of detection. Often both of these functions are concurrent. On-line coupling of dialysis to remove small analyte ions from samples containing macromolecules and suspended solids [11], and of gas diffusion for the separation of the weak acids sulfite, carbonate and acetate from complex matrices [12], are sample pretreatment methods that have been applied in an automated manner. More complex systems have been demonstrated. Ruiz-Jimeńez and de Castro, for example, constructed a system for the determination of volatile small amines from solid food samples such as meat by sampling with a flow-through pervaporation unit [13]. Microdialysis probes are important tools for in vivo sampling in pharmacological studies. Due to the small sample volumes, CE is well suited, and in order to carry out the studies on-line, FIA-CE is an obvious approach. Ruiz-Jimeńez has provided a review of this technique [14]. A few further examples of on-line flow injection sample processing prior to CE separation are mentioned as follows. Arce et al. have described the analysis of soil samples by using an extensively automated FIA system, which took care of the leaching of the analytes from the solids as a front end to a CE instrument [1]. Valca´rcel and coworkers demonstrated the preconcentration of inorganic ions on a short ion-exchange column for the determination of trace contaminants in a water purification plant [2]. A similar system, shown in Figure 10, was also employed for the extraction and

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Capillary c-18 90% methanol 0.1NNaOH /10% ethanol H2O Sample 1 Sample 2 10% ethonol H2O

DAD SV1 IV

Mechanic arm Waste

SV2

CE Electronic interface

Figure 10 FIA–CE system featuring preconcentration on a C18-column and a conventional commercial CE system. The interface between the FI-manifold and the CE part was based on a robotic arm. Reprinted from Ref. [15]. Copyright (1999), with permission from Wiley-VCH.

preconcentration of chlorophenols in urine samples [15] and of mycotoxins from animal feed [16] on a C18-column, and of myo-inositols on a anion exchange column [17]. Buscher and coworkers have described on-line preconcentration of adenosine and inositol phosphates by electrodialysis [18,19]. The driving force for the development of such hyphenated systems for automated sample pretreatment and separation was the possibility of using one common on-line arrangement for sample treatment, transport of the pretreated sample to the injection device with subsequent injection into the separation capillary and finally for the CE separation. No manual handling of samples was necessary when the samples were treated in the FIA part and problems with sample contamination and sample loss that can be often encountered in off-line pretreatment techniques were avoided. The main advantage of on-line coupled sample treatment techniques in FIA–CE can be seen in their applications in determination of samples with complicated matrices, as for example in body fluids and solid sample extracts. The aspects of sample pretreatment in FIA–CE have been comprehensively reviewed by Kubańˇ et al. in 1998 [20] and by Fang et al. in 2000 [21].

3.5 Derivatization for detection Species which are not absorbing UV-light are difficult to detect in CE and for this reason analytes are often chemically derivatized to make them accessible by standard detectors. A flow-injection manifold may be employed for pre-column derivatization and is desirable since the whole process is then automated and better overall precision of the procedure may be achieved than by a batch-wise approach. On-line derivatization for UV-detection has been used for the determination of pharmaceutical compounds [22], amino acids [23–26] and for the enantiomeric separation of D- and L-carnitine [27].


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Fluorescence detection shows lower detection limits than UV-detection and therefore the use of fluorophores as labelling reagents is desirable. In the life sciences, amino acids and peptides are often determined after reaction with fluorescein isothiocyanate (FITC). However, this reagent requires 12–24 h reaction time and is therefore not suitable for on-line labelling. On-line derivatization for detection by laser-induced fluorescence (LIF) of amino acids and peptides using dichlorotriazinylaminofluorescein (DTAF) as a derivatization agent was demonstrated in a sequential injection–capillary electrophoresis (SI–CE) system by Zacharis et al. [28]. Using DTAF enabled a relatively fast reaction and the whole on-line derivatization procedure was completed in approximately 30 min. Wu et al. [29] described the on-line derivatization of insulin for fluorescence detection using a lab-on-valve SI-CE system with a total derivatization time of less than 10 s.

3.6 FI-Microchip CE Miniaturization allows the analysis of small sample volumes, saves consumables and often leads to high sample throughput. It has become a major trend in the development of instrumentation for analytical chemistry, and has often found expression in the form of integrated manifolds created by micromachining techniques such as photolithography in planar glass or plastic substrates. When separation is desired, electrophoresis is generally used, rather than chromatography, for its inherent simplicity. While a lot of effort has been expended on developing such CE microchips, little attention has been paid to the sample introduction step. The use of a flow-injection manifold for this purpose is a useful approach that deserves more attention, in particular when considering completely automated instruments for routine analysis or on-line systems for process analysis. The common configuration of an electrophoresis microchip is that of two embedded channels with intersect each other and form an elongated cross as shown in Figure 11.The sample is placed into one of the wells at the ends of the Separation channel

Buffer HV +/Buffer Buffer HV +/Sample

Injection channel

Figure 11 Schematic drawing a microfabricated electrophoresis chip. The intersection between the two embedded channels defines the injection volume. Injection and separation are effected by applying voltages to the electrodes placed in the wells at the ends of the channels.

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injection channel while the rest of the manifold is filled with the background buffer used for the electrophoresis process. Once the sample is contained on the chip, the proper injection into the separation channel is carried out by electrokinetic pumping of the sample across the sample channel by application of a voltage. In this process the intersection formed between the two channels is filled and its geometry defines the injection volume. Subsequently, the injection voltage is turned off and the separation voltage is applied between the ends of the longer separation channel. Fang et al. [30] designed a system for automatic placement of the sample into such a microchip manifold by connecting a short piece of polytetrafluoroethylene (PTFE) tubing to the microchip using epoxy glue, which was used to transfer the sample from a peristaltic pump. The sample overflow, resulting from pumping the sample through the sample reservoir was drained to waste using a set of filter papers and a semi tubular trough. Samples were then introduced into the separation channel electrokinetically by applying a high voltage between the flow-through sample reservoir and a waste reservoir. During this procedure, the sample filled the entire sampling channel and the small portion of the sample, which was located at the intersection of sampling and separation channels, was subsequently separated by switching on the high voltage of the separation channel. Different injection sequences were examined for multiple injections of one sample and for minimum carryover between injections of several different samples. LIF detection was applied to the determination of a set of labeled amino acids. He et al. [31] described a system for direct sample transfer and injection onto an electrophoresis chip using a capillary probe, which was placed directly into sample vials. Sample injection into the sampling channel was followed by automated replacement of the sampling vial with a buffer vial and subsequent electrophoretic separation of the sample, which was located in the intersection of the sampling and separation channels. Sample and buffer vials were fixed on a surface of a purpose-made autosampler platform, which was moved linearly using a computer-programmed sequence. No carryover was observed for injections of consecutive samples from the platform, which was demonstrated on the determination of LIF labeled amino acids. In order to overcome the sampling bias of electrokinetic injection Zhang et al. developed a suction device based on a syringe pump to hydrodynamically draw sample into the injection cross of a separation chip and demonstrated their system using rhodamine and fluorescein as standards for fluorescence detection [32].

3.7 Fast separations in short capillaries One of the prominent features of microfabricated electrophoresis devices is the rapidity of the separations achieved. Analyses are often complete in time spans significantly shorter then a minute. This cannot be matched on conventional CE instruments. First of all, they are designed to be used with capillaries of typically 50 cm in length, and short capillaries, which are of the dimension of the separation channels of chip devices of at most a few centimetres in length cannot readily be fitted. Secondly, computer-controlled switching of voltages for the


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electrokinetic injection on chips is fast and no time delays occur between injection and separation due to movements of capillary or containers as necessary when conventional injection is used with standard capillaries. Therefore any band broadening due to diffusion or adverse effects during the injection sequence are limited. On the other hand, the diameters of standard capillaries as used in CE (25–75 mm) are not significantly larger than the width and depth of the separation channels of the chip devices so that similar results can be expected with short conventional capillaries if a suitable fast injection procedure can be used. This hypothesis could be validated by using an experimental set-up using two syringe pumps for propelling buffer and sample solutions through a gated interface to the capillary, which presented either solution in rapid sequence to the inlet of a capillary of short length for electrokinetic injection [33]. An implementation better suited for routine analysis, which is based on an SIA manifold and an interface as discussed above (Figure 4) was subsequently demonstrated for the determination of inorganic ions using contactless conductivity detection [34]. The fast separation of 5 inorganic cations in 10 s possible in a capillary of 8 cm length, which is comparable to the separation length of microchips, is illustrated in Figure 12. The latter system also allowed hydrodynamic sample injection so that injection biases could be eliminated. Several manifolds using short lengths of capillary mounted on microscope carrier slides employing UV absorbance [26,35,36], laser-induced fluorescence [37,38], amperometry [39], chemiluminescence [40,41], contactless conductivity [42] and atomic fluorescence spectrometry [38] for detection have also been reported. The results in terms of separation time, sensitivity and precision for these systems based on short conventional capillaries were comparable or better then what is achieved with devices based on micro machined short channels. Furthermore, the systems took also care of the total sampling sequence, i.e., the complete transfer from the sample container into the separation capillary. The cost of a short length of fused silica capillary is only a fraction of the cost of glass or plastic microchips.

Ca2+ Na+ Mg2+ NH4+

0

K+

5

10

15

Time (s)

Figure 12 Fast separation of inorganic cations in a short conventional capillary following rapid injection using an SIA manifold.

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4. ELECTROKINETICALLY PUMPED FLOW ANALYSIS The notion of using electrokinetic transport phenomena for propulsion instead of conventional pumps arose concurrently with the trend to miniaturization, the desire to create lab-on-chip systems, in the early 1990s. Conventional pumps are difficult to miniaturize to obtain the desired low flow rates. Miniature membrane pumps bases on piezoelectric membranes still require a high voltage source and are not very reliable, while small piston driven pumps are expensive. CE, which utilizes capillaries with small internal diameters, can already be considered a miniaturized method, and the first investigations in using electrokinetic pumping were carried out on standard CE-instrumentation. This was soon followed by reports on specially designed micro machined systems.

4.1 Electrophoretically mediated microanalysis in capillaries EMMA denotes a CE technique in which generally a sample is mixed with a reagent and the product of a reaction is quantified. The common mode of operation is illustrated in Figure 13. Sample and reagent plugs are injected into a capillary in sequence such that the slower component is injected first. The faster species then overtakes the slower one, so that the two merge and the reaction can occur. Except for the differential migration process, the method is therefore somewhat analogous to SIA. The principles were first demonstrated by Liu and Dasgupta [43]. The authors used fused-silica capillaries with an internal diameter of 75 mm for

Figure 13 One possible implementation of Electrophoretically Mediated Microanalysis (EMMA). (1) First a sample plug is injected into the capillary. (2) This is followed by a plug of reagent. (3) As the reagent has a higher electrophoretic mobility than the analyte, the reagent moves into the analyte zone, and reaction takes place. (4) The reaction product is determined as a separate peak.


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Figure 14 Adenosine kinase inhibition assay as an example of EMMA. A, Control assay without inhibitor. B, Assay in presence of inhibitor. The reduction of the amount of AMP produced is due to the presence of the inhibitor, which is not detected in the electropherogram. Reproduced from Ref. [47]. Copyright (2006), with permission from Wiley-VCH.

spectrophotometric determination of iron via complex formation with phenanthroline. Regnier and coworkers demonstrated the determination of calcium ions in this mode via complexation with o-cresolphthalein and optical detection on a conventional CE instrument [44]. As shown by Andreev et al. [45] for example for the determination of Fe3+ with sulfosalicylic acid, the method may also be implemented by using injection of oppositely charged analytes and reagents from the two ends of the capillary. For more complex methods, the subsequent electrophoretic separation will be necessary to achieve selectivity. The main advantage of this method, besides instrumental simplicity, is the small volumes involved. For the latter reason Bao and Regnier pioneered its application for enzymatic assays [46], and this has become the most popular application of EMMA. As illustrated in Figure 14, Iqbal et al. [47] have used this technique for example for the screening of adenosine kinase inhibitors. The reduction of the amount of adenosine monophosphate (AMP) produced allows the direct quantification of the effect of an inhibitor. Comprehensive reviews on EMMA have been written by Glatz and coworkers [48,49] and van Schepdael and coworkers [50].

4.2 Miniaturized systems It had been shown in the early days of the development of micro machined microfluidic systems employing electrophoretic separation that electroosmotic

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pumping as well as controlled merging of different streams from different branches of a manifold is possible by precise application of voltages at different points [51]. Haswell and coworkers thus designed several mFIA manifolds in glass where classical photometric FIA procedures for phosphate [52,53], nitrite [54] and nitrate [55] were implemented. Rainelli et al. [56] similarly demonstrated the determination of Fe2+ with the phenanthroline method, but concluded that it was generally difficult to find conditions, mainly with regard to ionic-strength and pH-value of the solutions, which match the concurrent requirements for electroosmotic pumping and spectrophotometric derivatization reaction. Perhaps for this reason, the method has seen limited popularity. This restriction can however be overcome by separating the functions of pumping and detection. Liu and Dasgupta constructed an electroosmotic fluid propulsion system from several parallel capillaries which was used as an external pulse free pump for miniature FIA [57] and SIA [58] systems. These systems were implemented with conventional capillaries but Paull and coworkers have recently also demonstrated such a system constructed on a polymethyl methacrylate chip which contained several parallel short monolithic silica columns for creation of the electroosmotic pumping action [59]. Note, that most of the lab-on-chip systems based on electrophoretic separation reported include either an electrokinetic transport step (often for filling the sample into a cross or double-T shaped intersection of two channels which define the injection volume), or an on-line derivatization reaction, and may therefore be seen as miniaturized versions of the hyphenated FIA-CE systems discussed in the previous chapter. A single example is given here to illustrate the possibilities. Lunte and coworkers have described a microchip based analysis system for the on-line determination of amino acids and peptides by LIF as illustrated in Figure 15 [60]. The systems features both, automated sampling and analyte derivatization. The sample is picked up via a microdialysis probe and then transferred onto the chip by pumping with a syringe pump. Only on chip the sample is mixed with a derivatizing reagent in order to render the amino acids and dipeptide of interest detectable by fluorescence. The rapid repeated analysis of a standard mixture with this system is shown in Figure 16.

Perfusate syringe Microdialysis probe Modified cap Sample vial Microchip device

Figure 15 Microchip setup using a microdialysis probe for sampling and on-chip derivatization for fluorescence detection. Reproduced from Ref. [60]. Copyright (2006), with permission from Elsevier B.V.


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Gly-pro 5AFU

Arg Asp

0

20

40

60

80

100 120 Time (sec)

140

160

180

200

Figure 16 Rapid repeated analysis of a standard mixture using the system shown in Figure 15. Reproduced from Ref. [60]. Copyright (2006), with permission from Elsevier B.V.

5. CONCLUSIONS It has been seen that the integration of flow analysis and electrophoresis can take quite diverse forms. The development is on-going and time will show that some approaches are more useful in practice then others. For the routine analysis of conventional samples such as those encountered in industrial environments it is expected that the SIA-CE approach will prove a versatile and robust method. When small sample volumes, such as those encountered in the life sciences, are to be analysed, EMMA carried out in conventional capillaries and on conventional CE instruments is a convenient approach, which is readily possible and deserves wider consideration. Microfabricated manifolds can be tailored to special processes, which cannot be carried out by a linear stacking of sample and reagent plugs in a capillary. For such systems, hydrodynamic pumping with external pumps is required for flushing, and to overcome the limitations of electrokinetic movements alone, and interfacing to the outside for sample introduction is best also carried out with a flow analysis manifold.

ABBREVIATIONS AMP BGE CE DTAF EMMA EOF FITC LIF PTFE

Adenosine monophosphate Background electrolyte Capillary electrophoresis Dichlorotriazinylaminofluorescein Electrophoretically mediated microanalysis Electroosmotic flow Fluorescein isothiocyanate Laser-induced fluorescence Polytetrafluoroethylene

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ACKNOWLEDGMENTS Financial support from the Grant Agency of the Academy of Sciences of the Czech Republic (Grant No. IAA 400310609) and Swiss National Science Foundation (Grant Nos. 2000-67830 and 200020105176/1) is gratefully acknowledged.

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PART III Detection


CHAPT ER

12 Photometry Wolfgang Frenzel and Ian D. McKelvie

Contents

1. Introduction 2. Fundamentals of Spectrophotometric Measurements 3. Instrumental Aspects of Flow-Through Spectrophotometry 3.1 Components of spectrophotometric instruments 3.2 Design of flow-through cells for absorbance measurements 3.3 LED-based photometric detectors 3.4 Optical fibre-based spectrophotometric detection 4. Background Absorbance Correction 5. Refractive Index (Schlieren) Effects 6. Conclusions and Outlook Abbreviations References

311 314 317 317 319 328 331 332 334 338 340 340

1. INTRODUCTION The term photometry is not consistently used throughout the scientific literature. In a general sense it is used to describe the ‘‘measurement of quantities associated with light’’ [1] sometimes restricted only to wavelengths which produce visual stimuli. Photometry may refer to that branch of observational astronomy for the measurement of the apparent magnitude of stars [2]. It may also be used to describe the assessment of the properties of light sources, such as radiation characteristics (especially the luminous intensity), colour temperature and colour rendering index in colour imaging [3]. For analytical chemists photometry is mostly associated with the measurements of absorbance for the purpose of quantification of analytes using specific instruments called photometers. Historically photometry was restricted to measurements in the visible wavelength range (390–800 nm), but near ultraviolet and near infrared regions (190–1,000 nm) of the electromagnetic spectrum are Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00612-0


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now often included. Despite the wavelength range provided by the photometer, absorption measurements with photometers are typically made at a single specific wavelength. Spectrophotometry (sometimes unfortunately used synonymously with photometry) is a specific form of photometry where light is measured as a function of wavelength in a particular range. Accordingly the information gathered by a spectrophotometer consists of an intensity–wavelength plot of electromagnetic radiation, termed the spectrum. Spectrophotometry is one of the many tools that spectroscopists use for studying the interaction of radiation with atoms, ions and molecules. For analytical chemists it is both a means of obtaining qualitative information and assists in the identification of compounds, and can be used for quantitative measurement, thus extending the capabilities of single-wavelength photometry. Colorimetry is yet an additional term that refers to the measurement of transmission of light. It is a non-instrumental approach where a broadband light source (‘‘white light’’) such as sunlight or a tungsten lamp illuminates the sample and the human eye serves as the detector. Semi-quantitative information is obtained by colour comparison with standards of known composition and content. In the general analytical chemical literature and also in some flow-injection analysis (FIA) papers, photometry, spectrophotometry and colorimetry are not always correctly and consistently distinguished, and have been used interchangeably or even in the wrong context. In this chapter the terms photometry and spectrophotometry are used according to the definitions given above, and the term colorimetry is not used at all. In quantitative analysis, spectrophotometric determination typically refers to the measurement of absorbance. However, considering the common instrumental configuration of photometers and spectrophotometers it is not absorbance of the analytes that is actually detected, but rather the attenuation of light compared to that in their absence. All other effects resulting in light attenuation must therefore be carefully considered and corrected for. Despite this fact, absorption spectrometry is an established term and is thus also used in the present chapter. However, as atomic absorption spectrometry and infrared spectrometry are separately treated in this book (see Chapters 14 and 15, respectively), only molecular absorbance measurements in the ultraviolet and visible range are considered here. The roots of UV–Vis absorption measurements can be traced back to the eighteenth century when fundamental studies were carried out, e.g., by Lambert, Beer and Bouguer on the light attenuation caused by coloured solutions. Although at that time no theoretical understanding of the absorption process at a molecular level was existent (at least not what we believe now to be the reasons behind light absorption owed to electromagnetic wave and quantum theory) the relationships between concentration of the coloured compound and the travelling distance within the sample were established. The early findings have proved valid until today, and in the form of the well-known Lambert–Beer law they form the basis for quantitative determination using absorbance measurements.

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With the first true photometers (involving light detection by photosensitive electronic components) becoming commercially available during the midtwentieth century, the basis was laid for the continuous advancement of photometry that persisted over the entire course of the last century and has continued thereafter. The improvement of instrumentation and an ever increasing number of applications with respect to the target species and the sample matrices has led to a hardly countable number of scientific publications of spectrophotometry and an unimaginable number of practical measurements made daily worldwide in routine laboratories. The implementation of spectrophotometric detectors in automated methods of analysis (both batch systems as well as flow-based methodologies) is a logical consequence of the broad applicability of UV–Vis absorbance measurements for routine purposes. Many commercial chemical analysers used in clinical, pharmaceutical, agricultural and environmental laboratories are equipped with photometers as one, if not the only detector. When air-segmented continuous flow analysis (CFA) was introduced by Skeggs in 1957 he launched his ingenious technique for automating photometric determinations [4]. Although other detection principles have since been adapted to CFA photometry, photometry has remained the most used detection principle, with the consequence that a huge number of validated protocols have become official methods of analysis. Considering that FIA was initially developed as an alternative flow approach for automation of wet-chemical procedures (cf. Chapter 1) it is not surprising that in the first papers and patents related to FIA, the underlying concept was demonstrated using mainly photometric detection [5]. With the introduction of second and third generation FIA techniques, i.e. sequential injection analysis (SIA), lab-on-valve (Chapter 2) and mutations such as multi-syringe FIA (MSFIA) and multi-commutated FIA (MCFIA), many earlier FIA applications have been adapted, often with only minor modifications. In these new generation FIA techniques, spectrophotometry dominates as the detection method employed, and in commercial FIA/SIA apparatus, the majority of instruments are equipped with a photometer or spectrophotometer, and in most instances this is the only detector option available. This is further clear evidence of the important role that spectrophotometric detection has in routine applications. In view of the huge amount of existing material on spectrophotometric detection in FIA and related techniques, it is obviously impossible to treat the subject comprehensively in this chapter. However, this is not seen by the author as a serious handicap because a large number of the published papers are very closely related, and many only present applications of well-established spectrophotometric procedures that have been well-documented and employed in batch systems and CFA for a long time. Often the publication of papers has been justified by presenting minor modifications of analytical protocols (e.g., different flow rates, reagent concentrations and/or length of mixing coils) and the application to samples of different origin, yet often constituting the same matrix. A closer inspection of many of the published papers suggests that the adaptation to FIA in some instances has not resulted in significant improvements of the

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analytical performance characteristics such as sensitivity, selectivity, dynamic range, precision and robustness, and in some cases the performance may actually be worse. Readers interested in the determination of particular analytes and in various matrices by spectrophotometric FIA are referred to Chapters 18–23 of this book and available databases (Hansen’s FIA-bibliography [6], Chalk’s Flow Analysis Database [7] and the Journal of Flow Injection Analysis Bibliography published by the Japan Association of Flow Injection Analysis). Nevertheless, some applications of UV–Vis absorbance measurements in FIA are dealt within this chapter, but these focus on more general aspects such as fundamental studies of sample dispersion, systems where chemical derivatisation reactions are required to form a detectable product, kinetic measurements, trace analysis, simultaneous determination capabilities and the implementation of separation techniques. Other oft-emphasised attributes of spectrophotometric FIA procedures are the microanalytical features of reduced sample and reagent consumption, and the high sample throughput. Also, the instrumental cost is claimed to be low for FIA and related techniques, and the feasibility of miniaturisation opens the way to field-deployable systems that can be applied for continuous monitoring. These attributes (presented and discussed repeatedly in different chapters of this book) are some of the fascinating facets of the FIA techniques, but have inherently little to do with spectrophotometry. However, this chapter contains short sections on microanalysis, continuous monitoring and portable FIA apparatus addressing the use of UV–Vis absorbance measurements and the associated features. Photometric methods have been readily adapted to FIA because flow-through absorbance measurements had been used in CFA prior to the advent of FIA. However, it became clear that the conditions required for optimum FIA detection were somewhat different to those for CFA. Also the relatively bulky photometers that can be equipped with flow-through cuvettes are not very compatible with the typically small-sized FIA manifolds. In the course of further development of FIA (and subsequently, various related techniques), the search for alternative detector designs has created many innovations and modifications with respect to the flow-through cell, but also to the overall configuration of the spectrophotometric instrumentation. Since several of these developments have led to improved analytical performance and in many instances have supported the further miniaturisation of FIA and related techniques, a considerable part of this chapter is devoted to the principal considerations of UV–Vis absorbance measurements in miniature and capillary flow systems as well as to the practical solutions developed.

2. FUNDAMENTALS OF SPECTROPHOTOMETRIC MEASUREMENTS It is not the intention in this section to present a pedagogical discourse on the fundamentals of the interaction of electromagnetic radiation in the UV–Vis range with matter, or to deal with the many optical configurations that are used in spectrophotometers. However it is important to address the conditions necessary

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for optimum analytical performance, and to focus on the requirements to achieve a defined relationship between the concentration of the analyte and the detector response, and sensitivity aspects. The Lambert–Beers law comes to mind instantaneously but it is not always recognised by practitioners of spectrophotometry that this is an idealised law, valid only when many experimental conditions are satisfied. In its common form, Lambert–Beers law predicts a linear relationship between absorbance (A) and concentration (c) as well as between absorbance and the pathlength (d) the light travels through the sample. The slope of the calibration curve depends on the wavelength-dependent molar absorptivity el of the absorbing species. A ¼ log T ¼ log

P ¼ l c d P0

(1)

Some of the optical conditions for the validity of Lambert–Beers law are the use of collimated and monochromatic light and a cuvette geometry having orthogonal optical windows relative to the incident beam. Monochromatic light (as such not truly available) in molecular spectrophotometry refers rationally to the bandwidth of the incident light beam in relation to the bandwidth of the absorbance spectrum of the molecule under investigation. The larger the bandwidth of the incident light the more significant is the deviations from Lambert–Beers law. If cuvettes with non-parallel optical windows (e.g., vials or test tubes) are used, the mean pathlength cannot easily be calculated, but an effective pathlength can be obtained experimentally. This configuration has received considerable attention in capillary – flow-through systems, such as capillary electrophoresis, micro-HPLC and also FIA and related techniques. When incoherent light is used (typically the case in spectrophotometry) and in particular, when the light beam diverges, multiple internal reflections may occur within a tubular cuvette causing multi-path behaviour. The consequence is that the apparent sensitivity changes with the degree of light transmission, and hence non-linear absorbance–concentration plots are observed. Another undesirable effect in spectrophotometric measurements that causes deviation from expected linear calibration curves is stray light. By proper optical configuration of the spectrometer and the use of cuboid cuvette geometry, the amount of stray light can be kept very small. However, considering the use of transparent capillary flow cells for example, part of the light can potentially travel longitudinally through the wall of the capillary and reach the detector without attenuation by analyte molecules. There are also several conditions pertaining to the sample solution and the analyte that need to be met so that Lambert–Beers law remains valid. Three main requirements are: (i) the absorbing species must be homogeneously distributed within the cuvette, (ii) the concentration of the analytes should not exceed a limiting value to ensure that mutual interactions of the absorbing species remain insignificant and (iii) there should be no effects other than molecular absorbance that cause light attenuation, such as scattering, interaction with the solvent, refractive index (RI) changes or the presence of impurities that cause a spectral background at the measuring wavelength. In practice, absorbance measurements

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are typically made at analyte concentrations below 0.01 M and calibration is performed with standard solutions that are matched to the sample solution as much as possible with respect to its matrix composition. Spectral background corrections can be effectively made by measuring the absorbance difference of a sample solution at two selected wavelengths, or even better if the entire spectrum is recorded (e.g., by a diode-array spectrophotometer) and the background at the measuring wavelength extrapolated. In dual-wavelength spectrophotometry, two beams of radiation (one typically set to the wavelength of maximum absorbance and the other at a wavelength where absorption of the analyte does not occur) are allowed to pass through the cuvette simultaneously or in fast sequence. From a spectral perspective, the optimum sensitivity of photometric determinations is achieved when measurements are made at the wavelength of maximum absorbance using monochromatic light. However, measurements at other wavelengths are possible and sometimes even preferred because of selectivity considerations. Ideally, monochromatic light should be used, but in practice, light sources such as light emitting diodes (LEDs) with a broader spectral bandwidth may provide quite adequate analytical responses for most applications (see Section 3.3). With respect to the cell geometry, the sensitivity (slope of the calibration curve) can be improved by increasing the pathlength. However, this does not necessarily translate into improved detection limits because in most practical situations the gain in the signal obtained with long-pathlength cells is eventually offset by increasing noise caused by light starvation of the photodetector. Even with bright light sources and highly focussed beams, pathlengths above 10 cm rarely present an advantage regarding the achievable detection limits. The application of laser sources in longer pathlength cells has been reported to provide some sensitivity enhancement [8], but they have not been widely adopted, perhaps because lasers are not readily available at all wavelengths of interest and only a few are affordable. A breakthrough in long-pathlength absorbance cells was the introduction of liquid-core waveguides (LCW). The principle of an LCW is analogous to that of an optical fibre, i.e. a typical LCW consists of a tube (the cladding) with an RI lower than that of the liquid (the fibre or core) inside. If these requirements are met, light entering at one end of the LCW propagates through the fluid core by total internal reflection and exits at the other end. The light loss even for long LCWs (up to 50 m) is reasonably low so that generally a considerable improvement of signal-to-noise ratio is achieved compared to common cells [9]. However, until the 1990s, the application of LCWs was limited to few high RI organic solvents, such as carbon disulfide that could be used as the core liquid with the glass, quartz or polymeric capillaries (all with ZWZwater) that were available at the time [10]. This situation changed when DuPont introduced amorphous fluoropolymers (AF) that had an RI lower than water (e.g., Teflons AF2400, Z ¼ 1.29) [11], and since then this material has found increasing application for construction of long-pathlength flow cells (see Section 3.2). In a later section reference will be made also to this polymer material in the context of gas analysis (see also Chapter 22).

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3. INSTRUMENTAL ASPECTS OF FLOW-THROUGH SPECTROPHOTOMETRY Spectrophotometry, as one of the oldest instrumental analytical methods, has not lost its attraction and is certainly the detection technique most widely employed in practical applications (e.g., following separation and selective reaction). This applies to both batch measurements and the variety of flow-through techniques, e.g., CFA, FIA, SIA and related techniques, as well as in liquid chromatography and capillary electrophoresis. Classical and most contemporary spectrophotometric instruments generally comprise four essential parts that are typically contained in a common housing, i.e. the light source, the sample compartment, a wavelength selector (filters or monochromator) and a light-sensitive detector. In addition lenses, mirrors and slits are arranged along the optical axis. The sample solution is placed in a cuvette that for batch measurements is removed for filling, emptying, re-filling and cleaning. For flow-through measurements a variety of special cuvettes are available furnished with connection tubing that permit admission of the liquid sample solution from outside the spectrophotometric instrument. Spectrophotometric instrumentation can be classified in three categories, i.e. fixed wavelength detectors using filters to isolate a single band of radiation, variable wavelength detectors using continuum light source and a monochromator to select the appropriate wavelengths, and detectors that can rapidly perform a rapid, complete scan over a wide range of wavelengths. The latter can be done with fast scanning instruments, or more commonly these days with a diode-array detector (DAD). To focus on the instrumental aspects of importance for absorbance measurements in flow-through systems such as FIA and related techniques, a brief general description of the configurations and components of spectrophotometers, and the flow-through cells where detection takes place appears indispensable to the author. Recent innovations in optoelectronics have played a key role in the implementation of absorbance measurements in FIA systems, and these are briefly discussed in the following sections.

3.1 Components of spectrophotometric instruments Spectrophotometric instrumentation has undergone several changes since its inception, which largely have been connected with innovations and improvements in optical and electronic components. With respect to light sources, brighter lamps with high radiation power covering the entire useful UV and visible spectral range are now available. They can be modulated electronically, are robust and can be operated with low power consumption. In addition, light sources such as LEDs and lasers which both provide monochromatic light have found application as interesting alternative light sources for photometric measurements. In a later section, LED-based photometric systems are discussed separately because they have received considerable attention as detector components in FIA and related techniques. The classical optical systems for

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selecting the measuring wavelength using absorption filters, interference filters or prisms still exist, even in some modern photometric instruments, but have been largely replaced by the grating monochromator. The latter offers convenient wavelength selection, high spectral resolution and is now available at reasonably low cost. The changes that have had the most significant influence on the design of modern spectrophotometers are certainly connected with the light detectors. Photodiodes and phototransistors, which for a long time have been applied in photometric instruments, have progressively become more sensitive, and as a result of their miniaturisation, they have become an integral part of the signalprocessing circuitry, leading to lower noise levels and higher stability. The classical photomultiplier tube, which is still the most sensitive photo-detection device, has been further developed, and miniature, low-voltage powered versions are now available at a reasonable price. It is beyond the scope of this chapter to discuss the various instrumental and electronic aspects of light-tovoltage conversion involving transduction of photons into electrical signals that are now typically digitalised for further processing. The interested reader is referred to excellent sources published on this subject [12]. A quantum jump in spectrophotometry was the development of diode arrays in the mid-1970s that enabled close spatial arrangement of photodiode segments, permitting quasi-simultaneous detection and processing of the light-induced current of the individual segments (picture elements termed pixels). In combination with a grating monochromator, diode-array spectrophotometers (DADs) have been designed that are capable to record a full absorbance spectrum without any moving parts. The time for recording a full spectrum is connected with the integration time selected to obtain sufficient sensitivity, but to a large extent is also limited by the capability of the software required for operation of a DAD. The current generation of commercially available DADs permits recordings of the whole spectrum in the lower millisecond range with high repetition rates. Improved configurations of the photosensitive surface are charge-coupled devices (CCDs) and charge injection devices (CIDs) that are further miniaturised and provide high quantum efficiency with low noise and even faster response time. The development of optical fibres, which have made possible the breathtaking advances in data transmission, has led to profound changes in several optical detection techniques. Optical fibres are the key component of the optical sensors called opt(r)odes, which are also utilised for detection in FIA systems [13,14] (see also Chapter 17), and have only more recently found application in commercially available spectrophotometers. Some instruments are specifically made for measurements with optical fibres, while others can be equipped with optical fibre accessories as an option. Purpose-made instrumentation for absorbance measurements (e.g., for detection in flow injection and related techniques) also increasingly use fibre optics for light transmission. The beauty of optical fibre-based instrumentation is that it enables transmission of light from one location to another with almost unlimited flexibility and without significant loss of radiant power. A single light source can

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for instance be connected to different measuring sites and the transmitted light re-directed to a photodetector or to the entrance slit of a DAD or CCD. Using multiplexing devices, pseudo-two channel spectrophotometric instruments can be configured without mechanical choppers permitting dual- or multi-wavelength detection for reference measurements and background correction. The freedom to guide light in this manner, together with the miniature size of optical fibres enables realisation of as yet unthinkable configurations. Instead of the common practise of transporting sample solutions to the detector, in situ monitoring of some chemical process or ‘‘event’’ becomes feasible and practical. This approach is widely used for in-situ measurements in process control but is also a very common means of coupling detectors with microanalytical systems such as CE, m-TAS and lab-on-a-chip systems [15].

3.2 Design of flow-through cells for absorbance measurements Spectrophotometric flow-through measurements have a tradition which long precedes the advent of FIA. In air-segmented CFA for instance, spectrophotometric detection was and still is the most common detection technique. However, the design of the overall optical configuration as well as the layout of the flow-through cell underwent several changes following the inception of CFA in the 1950s. This was also the case for absorbance detectors used in liquid chromatography for the direct detection of separated analytes containing UV chromophores, or in systems involving post-column derivatisation, which conceptually resembled the FIA situation. In the latter case, undue band broadening (which is equivalent to the term dispersion, D, used in FIA) must be prevented by appropriate layout of the reactor and the flow-through detector cell. The key issue with respect to the detector cell for absorbance measurements is to exclude dead volumes in the connection tubing and to minimise the volume of the flow cell while keeping the illuminated pathlength sufficiently long to achieve adequate sensitivity. Other important aspects are the susceptibility of absorbance measurements to RI changes (see Section 5), and practical considerations such as the likelihood of entrapment of air bubbles, and the ease of cleansing and dismounting the cell. The common design of the flow-through cuvettes used in HPLC, CFA, FIA, SIA and related techniques, have U- or Z-shaped flow channel geometry, with a typical pathlength in the range 1–10 mm and cell volumes of 5–100 mL (Figure 1) [16]. The flow cells are either made entirely from glass or quartz glass (the latter for measurements in the UV range), or have optically transparent windows when the body is made from stainless steel or inert polymer materials. Such flow cells are commercially available and designed to fit into the cuvette holders of common stand-alone spectrophotometers, including DADs. Coupling of the flow-cells with optical fibre-based photometric instruments is possible using cuvette holders with appropriate connections for the light-guiding fibres. These are commercially available as accessories for such systems, but can also be easily prepared in a mechanical workshop.

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Figure 1 Typical (A) U- and (B) Z-flow cells for use in laboratory spectrophotometers. Reprinted from Ref. [16]. Copyright (2005), with permission from Elsevier B.V.

Many homemade FIA and SIA systems, as well as most of the commercially available FIA instruments use this type of detector configuration for absorbance measurements. The advantages clearly are the possibility of exchanging cuvettes for specific needs, the simple removal of the cuvette for cleaning and inspection, and the ready availability of a detector with a relatively low price (in particular when the photometric instrument is already existent). However, with respect to sample dispersion, dead volume, the alignment of the flow cell along the optical axis and the desirable maximum light transmission, these detectors often cannot be regarded as optimal. The contribution of the dead volume of a detector cell to sample dispersion has been a subject of considerable concern in FIA [17], but considering the typical longitudinal propagation of a sample plug leaving the flow manifold of common FIA systems (and also that typically used in SIA, MCFIA, MSFIA) the problems encountered appear to be overemphasised. This is particularly true when photometric detection is employed following chemical derivatisation in coiled reactor tubes. Here peak widths at baseline can easily reach volumes of several millilitres so that for any flow cell having an illuminated detector volume of less than 10–20 mL, the integrating effect on the transient concentration change is insignificant. Also the influence of the detector cell volume and its configuration on peak maximum concentration, and hence sensitivity, is typically low [18]. However, it is worth considering that the dead volume of a flow cell is more than that represented by the illuminated volume alone. The tortuous entrance and exit channels of some commercially available flow cells (which are often of much larger diameter than the illuminated path of the cell or the manifold tubing) can have a volume several times that of the illuminated volume [19]. Sample dispersion caused by unfavourable cell design deserves special consideration when detectors are connected in series or when a single sample plug repeatedly

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traverses a detector cell as has been proposed for kinetic measurements [20] or in multi-commutation systems with detector relocation [21]. In laboratory spectrophotometers the image of the focused light beam on the cuvette window has a diameter of a millimetre or more which is considerably larger than the desirable diameter of the flow channel in a flow-through cuvette. Hence, a significant loss of light transmission through the flow cell occurs with adverse effects on the signal-to-noise ratio. In addition, after removal of the flowthrough cuvette from the cell compartment it is often difficult to return it to the identical position so that baseline absorbance values may change requiring electronic readjustment of the zero-point absorbance. A common problem with U- and Z-shaped flow cells is that they readily trap air bubbles and microparticles that are difficult to remove without interrupting the analytical run. When trapped in the illuminated volume, or even at the inlet of the cell, bubbles or particles cause a putative absorbance increase and a higher noise level and disturb the normally reproducible dispersion pattern of the sample zone. Adsorption of light-absorbing compounds to the optical windows is an occasional but undesirable phenomenon that creates baseline drift and calls for intermediate cleaning. Generally this is done in situ by pumping one or more solutions in an appropriate sequence through the entire flow manifold. The sheath-flow cell proposed by Dasgupta is an ingenious design that avoids contact between the sample and the cell windows so that long-term baseline stability is achieved irrespective of sample composition (Figure 2) [22]. Finally, it must be considered that many commercial photometric instruments are bulky so that

Figure 2 Sheath flow cuvette designed to avoid window coating for use with samples containing fine particulate or colloidal matter (a) longitudinal view and (b) cross-sectional view. D, detector; L, LED light source; G, glass tube; O, O-ring; P, PTFE tube. Redrawn from Ref. [22]. Copyright (1991), with permission from the American Chemical Society.

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relatively long lengths of tubing are required to connect the FIA manifold with the flow-through cell in the detector. This leads to increased sample dispersion and concomitantly to undesirably reduced sensitivity and longer washout times. Purpose-made spectrophotometric flow-through detectors are also commercially available (mainly for the liquid chromatography market) where the flow cell is mounted in a fixed position along the optical axis. This limits the flexibility to exchange the flow cell and makes maintenance more difficult, but it has the advantage of optimum optical alignment with favourably high light transmission and signal-to-noise ratios. Some instruments are equipped with specially designed cells that account for schlieren effects (e.g., imaging the light source on the exit window with focusing optics or using tapered cell constructions) and are arranged in a way that air-bubble entrapment does not occur. Considering the requirements of absorbance detection in FIA, SIA and related techniques it is surprising that such detectors are only rarely employed since they can be regarded very suitable in this context. The reasons for this are the generally high price of unnecessarily sophisticated HPLC detectors, and that many HPLC detectors operate only in the UV range, whereas many applications of photometric detection in FIA and SIA are typically in the visible range. A strong impetus for the development of photometric flow cells with decreasing cell volumes was the introduction of capillary electrophoresis and more recently micro-HPLC, where peak volumes of the separated compounds are in the lower microlitre range and below. To maintain the fidelity of the separation achieved, transport of the sample solution to an external detector is not acceptable, and minute illuminated cell volumes are required. Optical detection in m-TAS and lab-on-chip systems is another challenging area that has created needs for novel instrumental solutions of optical absorbance measurements. As a result of the particular demands for absorbance detection in capillary flow systems advances in miniaturisation and detector integration have been made and new concepts developed. The same imperatives apply to the development of detector cells for capillarybased flow systems involving integrated microconduits [23,24] or lab-on-valve techniques [25]. Accordingly, a variety of dedicated flow cells for absorbance measurements have been conceived, some of which are made for very specific applications (e.g., extremely short path-length cells for the measurement of highly absorbing solutions without pre-dilution), while others address practical problems associated with the previous designs such as air bubble entrapment and susceptibility to RI changes. A number of published papers present sandwich-type planar flow-through cells. Ruzicka et al. [26] were probably the first to describe such a cell for FIA, where the light conducted by a bifurcated optical fibre is brought into the cell though the common leg, reflected at the (white) bottom of the cell and carried out back to the detector through the other leg of the optical fibre. Thus the effective light path is twice the thickness of the liquid layer, which was variable in the range 1–3 mm. The great virtue of this cell is its simplicity, which in turn results in robustness, ease of construction, cleaning and repair. The versatility of the cell is another feature that allows for instance the implementation of separation membranes or immobilised reagents thus creating

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Figure 3 Two types of fibre optic photometric sandwich cells. (a) bifurcated optical fibre perpendicular to the liquid flow, (b) single stranded optical fibres for illumination and detection in an angular configuration. FB, front block; S, spacer; R, metallised reflector; BB, bottom block. Redrawn from Ref. [27]. Copyright (1993), with permission from Elsevier B.V.

a kind of reversible flow-through sensor. Dasgupta et al. built on this concept and constructed a sandwich cell with improved performance for absorbance detection. The cell is furnished with two single strand optical fibres with angled entrance and a metallised reflector plate (Figure 3) [27]. The same group later fabricated a simple multi-purpose planar flow-through cell furnished with a number of discrete LEDs (including tricolour LEDs) providing multi-wavelength detection for simultaneous determinations and background correction. A flow cell resembling this basic design is also commercially available. A curious, yet ingenious example of a flow cell for absorbance measurements is the windowless liquid drop approach described by Liu et al. [28]. This has found interesting application in single drop liquid–liquid extraction (LLE) and for the determination of gaseous compounds where a hanging liquid drop serves as a sampling and detection device [29] (see also Chapter 22).

3.2.1 Capillary-based flow-through cells With respect to the cell geometry, tubular flow cells made from glass and quartz capillaries as well as transparent polymer tubing have found widespread application in FIA. The design of tubular cells commonly used in capillary electrophoresis [30,31] and also widely used for absorbance measurements in FIA and related techniques [32–34] involves transverse illumination of the capillary, in which the incident light crosses the liquid flow orthogonally, and is detected. Effective cell volumes of about 1 mL are readily achievable, e.g., by transverse illumination of a 1 mm i.d. capillary with a side aperture of 1 mm [35] (Figure 4), yet much lower illuminated cell volumes have been realised, especially in capillary electrophoresis systems. Transverse capillary cells can be easily implemented into the conduits of the capillary flow system with only low dead

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Figure 4 Diagram of a transverse photometric flow cell that utilises a referenced LED as the light source and a photodiode detector.

volumes. The use of a straight tube flow cell also minimizes sample dispersion, which can be of particular importance when other detectors are serially arranged downstream, and overcomes some of the practical problems mentioned above. Air bubbles, for instance, are generally not entrapped and are readily carried through the tube by the liquid stream. It has also been shown that the orthogonal observation of the transient concentration change of the sample zone is less susceptible to interfering RI changes [19,36] (see Section 5 for a detailed discussion). However, the tubular design of flow cells for absorbance measurements also introduces problems and limitations that relate to the arrangement of the flow cell relative to the photometric instrument and the proper illumination of the liquid volume to be probed. In contrast to the cuboid U- and Z-shaped flowthrough cells, transversely illuminated capillary cells are not generally applicable in common photometers made for batch measurements, but rather they require dedicated optical configurations (an exception to this are the prototype cells described by Elsholz [37], Figure 7b). Capillary-based detectors must be designed with small optical apertures in order to avoid stray light losses, which can lead to a decrease in signal-to-noise ratios. Miniaturised all solid-state detectors, e.g., using LEDs as the light source, and light-guiding optical fibre-based systems are two keys to obtaining micro flow-through cells with high-performance characteristics. For a comprehensive treatment of theoretical considerations and the various instrumental aspects of absorbance detection in thin capillaries, the interested reader is referred to literature in the area of capillary electrophoresis, micro-HPLC and lab-on-chip analysis systems. Despite the attractive features of transverse observation in tubular capillary cells, their short optical pathlength adversely affects sensitivity in absorbance measurements. If on-tube detection in a thin capillary (typically 0.1–1 mm i.d.) with transverse illumination is made, the optical pathlength is significantly less compared with the common U- and Z-geometry. Also, in order to reduce stray light, and the refraction that occurs as light passes radially through the capillary [30], very small apertures are required which significantly decrease the radiation power reaching the detector. Thus on-tube detection comes at the expense of the

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sensitivity, with typically a 10–50-fold decrease being observed that if no special measures are taken, results in impairment of the detection limit. To offset these disadvantages, several modifications of the optical and electronic systems have been proposed. The key issues are obviously the design and construction of capillary flow cells with enhanced pathlength, but with acceptably low dead volume, and optimised detector electronics to minimise noise. It must also be considered that despite focussing optics, light propagating through a dense media such as water diverges rapidly and a considerable part of the light is lost to the cell walls. Several innovations in this respect originate from Dasgupta and co-workers who have ingeniously combined the use of LEDs, optical fibres, solid-state detectors and sophisticated electronic components for data acquisition to build a variety of high-performance optical absorbance detectors for application in capillary systems including FIA, SIA and derivative techniques [27,33,38]. One solution is to use flow-cell configurations with rectangular, optically transparent capillaries [39]. It is easier to launch light orthogonally through such cells and they provide some gain in sensitivity (due to a longer light path at similar dead volume compared to a tube), however there are practical problems involved in connecting the capillary to the manifold. Axial illumination of the capillaries is another route that has been investigated for sensitivity enhancement of absorbance measurements. The challenge is to find a geometry that permits launching of the light beam and the liquid into the capillary without any change in the direction of liquid flow thus avoiding the problems commonly associated with U- and Z-shaped cells. In addition, increasing and undesirable light losses occur with increasing capillary length, which leads to higher noise. Using lasers as a light source, long capillary cells (10–15 cm length) have been developed to accommodate the requirements for both high sensitivity and small sample volumes [8], but their application has been restricted due to the limited availability and high price of laser sources covering the required wavelength range. Multi-reflection cells are a particularly interesting alternative since they combine reasonably low light losses with theoretically extended pathlength enabling use of common tungsten lamps or LEDs as light sources. Two typical configurations with the light entering through a sidewall aperture are schematically shown in Figure 5a and b. In both instances the fused silica or glass capillary walls are rendered reflective by internal or external coating with a reflective material, such as metallic silver. Light entering the capillary is internally reflected before exiting the capillary through another aperture located at some distance. Both linear and helical [40] configurations have been devised for capillary lengths of 1–10 cm. Experimental parameters that influence the effective pathlength in multi-reflection cells are the distance between light entry and exit windows, the angle of incidence of the light beam and the inner diameter and wall thickness of the capillary. Results achieved by different authors are seemingly contradictory, in that some report that the effective pathlengths were considerably larger than the geometric ones, indicating that multi-reflection behaviour was occurring [19], whereas others found that the

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Figure 5 Simplified representation of the principle of multi-reflection flow cells, showing the importance of the capillary i.d. and wall thickness on the effective optical pathlength. The optical behaviour is complicated in round capillaries by rotary reflection of light, use of noncollimated incident beams and light transmission through the capillary wall. (a) Capillary i.d.Wwall thickness: effective optical pathlength W length of the capillary. (b) Capillary i.d. owall thickness: effective optical pathlength o length of the capillary. The darker segments show the effective pathlength of a single reflection, and the arrow below each diagram represents the sum of all such illuminated segments that comprise the effective optical pathlength. (c) Schematic diagram of the light behaviour in a liquid-core waveguide detection cell which illustrates the similarity between the effective optical pathlength and the length of the capillary.

values were sometimes less than the physical distance [36]. One explanation for this apparent discrepancy relates to the optical launch angle, inner diameter and the wall thickness of the capillaries used. For example, Ellis et al. [19] used a capillary with an internal diameter and wall thickness of 0.8 and 0.25 mm, respectively. When an acute angle of incidence is employed (e.g., 301 to the normal of the capillary wall), the number of reflections of light from one side of the capillary to the other between entry and exit windows is theoretically large, ca. 17. The distance traversed through the liquid core is therefore greater than the physical distance between the apertures, and a commensurate enhancement of signal is observed (Figure 5a). However, in narrow bore, thick-walled capillaries, the light beams will angularly traverse a greater distance of wall material than they do of the liquid core containing the analyte, and thus the optical pathlength will logically be shorter than the physical distance between apertures (Figure 5b). Mishra and Dasgupta have suggested that in cells of this type there is potential for passage of stray light through or along the glass wall of the capillary, which will affect the estimation of the effective optical pathlength [36].

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A completely different concept for long-pathlength absorbance measurements in FIA and related techniques is based on utilisation of liquid waveguide phenomena (Figure 5c). The experimental arrangements, features and some prominent examples are outlined in the following section.

3.2.2 Long-pathlength, liquid-core waveguide cells The principle of the liquid-core waveguide (Figure 5), which is based on the total internal reflection phenomenon, has been briefly outlined above. For total internal reflection to occur the ultimate requirements are that the RI of the core liquid is higher than that of the capillary material, and that the light enters the capillary with a sufficiently low numerical aperture which in turn is connected with the refractive indices of the core and cladding materials [41]. If these conditions are met, total successive reflections occur within a capillary with low light loss, enabling the construction of very long pathlengths cells with small inner liquid volume (a 1 m long capillary with 0.25 mm i.d. has a cell volume of only 50 mL). For glass or fused quartz capillaries with ZE1.46, the first condition is only fulfilled for some non-aqueous solutions such as benzene (Z ¼ 1.501) or carbon disulfide (Z ¼ 1.627). Consequently, early liquid waveguide detection systems were restricted to chromogenic reactions that could be performed in limited number of organic solvents [10]. However, it is also possible to utilise total reflection that occurs at the outer boundary of the capillary tube in contact with air. In that case it must be considered that part of the incident light may be conducted through the capillary material effectively shortening the interaction distance (optical pathlength) within the core solution in the same manner as described for multi-reflection cells above. Furthermore, any contamination of the outer capillary surface can cause light loss through absorption and scattering. The introduction of the low RI amorphous Teflons AF material by DuPont in 1989 [42] led to increasing application of LCW cells because it enabled the use of aqueous solutions as the liquid core. AF-clad, thin-walled silica tubing has also become available in which total internal reflection occurs either at the water Teflons AF interface in inner-coated capillaries [11], or at the silica-Teflons AF interface for externally clad capillaries. The advantage of the Teflons AF-coated capillaries is that the sample solution is in contact with hydrophilic glass rather than the polymer, thus reducing problems associated with surface adsorption of hydrophobic compounds and entrapment of gas bubbles. Finally, excessive backpressure (a problem especially relevant when long capillaries are used) is avoided. The insertion of capillary LCW cells into the conduits of flow systems requires consideration of the liquid and light connections. Generally T-connectors are used on both ends of the flow cell to accommodate the liquid carrying tubing and the optical windows. Optical fibres are typically used to conduct the light from the source to the cell and from the cell to the photodetector. Alternatively, smallsized LEDs and photodetectors can be mounted within purpose-made connectors in immediate contact with the optical windows. For absorbance measurements the light beam is aligned axially with the capillary cell, care being taken to have sufficiently small apertures and also an as short as possible gap between the

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optical window and the capillary cell. Recently LCW cells became commercially available with common LC fittings and SMA adapters for connecting them to the flow system and spectrophotometer, respectively (e.g., Avantes BV, The Netherlands or World Precision Instruments, Inc., USA). A number of papers utilising LCW cells in FIA and to a lesser extent in SIA have been published in the last decade convincingly demonstrating the advantages over common flow cell configurations. Applications so far are mainly in trace analysis of environmentally relevant species [41,43–48]. Sensitivity (in terms of slope of the calibration curve) for LCW cells is reported to increase proportionally with pathlength (in accordance with Lambert–Beers law) and a considerable gain in detection limit for a given photometric method has often been achieved. It is noteworthy however, that detection limits are inherently a function of signal-to-noise ratios, and due to higher blank levels in practical applications, the gain can be much less than expected from sensitivity consideration alone. In fact, with longer pathlength LCW cells, the background absorbance increases and disturbing effects (scattering by microparticles and gas bubbles as well as schlieren formation) may partially or totally offset any sensitivity gains. Excellent overviews of applications of LCW cells in general and in the field of environmental applications are the papers by Dallas and Dasgupta [49] and Gimbert and Worsfold [43], respectively; both discuss the use of LCW cells in FIA, emphasising their role in spectrophotometric trace analysis.

3.3 LED-based photometric detectors LEDs enjoy considerable popularity for absorbance detection since they constitute a simple alternative to the use of tungsten lamps, filters and optical lenses, and offer several additional attractive features. They are miniaturised narrowly focussed electroluminescent light sources that provide radiation covering the entire visible range from blue to red. LEDs with emission in the near UV are also available (Figure 6) [50]. Switchable multi-colour LEDs (with emitters being closely spaced so that the location of the radiation source can be regarded the same), and white LEDs that provide a quasi-continuum emission offer interesting opportunities in photometric detection. LEDs that are now available are ‘‘super bright’’, have extremely long lifetime and long-term stability, are inexpensive, require low power, and for which the necessary electronics are very simple. In combination with a photodiode or phototransistor for light detection, solid-state devices can be fabricated that are highly robust and battery operated. Initially the charm of LEDbased detectors was that the devices could be built with relatively low skill from readily available low-cost components. Accordingly such detectors were applied in FIA systems for educational purposes or whenever high performance was not required. Subsequent efforts have been made to design instruments for easy exchange of the LED source, or with switchable multiple LED sources to provide the required wavelength for a particular application [51]. It has also been demonstrated that by using more sophisticated electronics with referenced detectors and advanced signal treatment procedures, extremely low noise levels

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Figure 6 Typical emission spectra of a selection of commercially available, single wavelength band LEDs over the UV and visible spectral range [50].

can be achieved that are comparable with expensive state-of-the-art commercial detectors. A discussion of the fabrication of such photodetectors is out of the scope of this contribution but excellent sources are a fundamental review by Dasgupta [33] and a recent review by O’Toole and Diamond [52]. Despite their rapid proliferation and ubiquity, LEDs are not available to cover all possible wavelengths, and their use for absorbance measurement exactly at the absorbance maximum of a coloured derivative is possible only in rare instances (cf. Figure 6). Also the designation ‘‘monochromatic light source’’ is optimistic since the typical spectral bandwidths range from 20 to 100 nm, which at best is that of a common filter photometer. These two characteristics of LEDs can have an adverse influence on absorbance measurements in that the maximum possible sensitivity is generally not attained and the linear dynamic range (over which Lambert–Beers law is obeyed) is sometimes reduced compared with common spectrophotometric measurements using a white light source and a grating monochromator. Experimental data published by Trojanowicz et al. [53] and Hauser [51] have shown decreasing sensitivity when deviations between the maximum wavelength of absorbance and the emission wavelength of the LED increase. Since many practical applications do not require high sensitivity, this is not seen as a serious limitation. Published designs of LED-based absorbance detectors for flow-through measurements are manifold. An excellent review with many options for flow-cell configurations and comparative performance data is provided by Dasgupta et al. [38]. LED light sources and photodetectors have been used in combination with common flow cells using purpose-made holders that accommodate the cuboid cuvette and, in close proximity to the cell walls, the LED and the photodetector (see Figure 7) [37]. Purpose-made flow cells with a variable pathlength incorporating the LED and the photosensitive elements into a single unit have also been built, often with

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(a)

(b)

Figure 7 (a) Multi-LED light source for flow analysis with a U-flow cell. (b) Double pass transverse-capillary cells with the bottom endcaps removed. Photographs courtesy of O. Elsholz, Ref. [37].

easily exchangeable LEDs to permit wavelength changes according to the desired application. In an alternative configuration transverse on-tube detection is accomplished by using PTFE capillary as a pseudo-cuvette. This design provides very low dead volumes and practical problems of air bubble entrapment (common to U- and Z-shaped flow cells) are totally eliminated. A new ‘‘flow cell’’ can simply be created by insertion of a fresh piece of tubing. Similar configurations have been used with quartz or ordinary glass capillaries that have higher transparency and due to their hydrophilic nature are wettable by aqueous solutions, which reduce the possible adsorption of organic compounds with concomitant increase of the baseline absorbance with time. Clearly the short optical pathlength of the transverse detection configuration is unfavourable with respect to sensitivity but in some practical applications this has been advantageous because the dynamic range was extended to higher concentration levels. The idea of a flow cell within an LED originated from Dasgupta [33]. An ingeniously designed micro flow cell with an illuminated volume in the lower nanolitre range was fabricated by drilling a tiny hole through the body of an LED just above the emitter and positioning a photodiode detector in very close proximity (Figure 8). Owing to the miniature format and the low price of the optoelectronic components required, LED detector cells incorporating several differently emitting elements were constructed that permitted multi-component determination of differently absorbing species or the simultaneous recording of different wavelengths for background correction or schlieren compensation. Multi-channel flow systems have also been configured using different LED detectors that are controlled and interrogated by a single computer [54]. Another approach is the use of frequency-controlled integrated multi-diode light sources that provide different colours in fast sequence. They have been applied in the simultaneous determination of two and three component mixtures in combination with chemometric data evaluation [55]. Fibre optics coupled to LED photodiode systems have also been used [51] in order to avoid location of the electronic parts

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Figure 8 A flow cell constructed in the body of an LED. C, emitting chip; H, entrance/exit apertures; P, opaque PEEK sleeve; F, fibre optic leading to detector photodiode. The detector can be referenced by placing another photodiode under the LED. Reprinted from Ref. [33]. Copyright (1993), with permission from Elsevier B.V.

of the detector in close proximity to the liquid carrying flow cell, an arrangement that has its own problems if there are any potential leaks. The favourable characteristics of LED-based detectors have led to widespread applications particularly if measurements are made at fixed wavelength so that a single LED can serve as a light source. This is the case in dedicated flow analysis systems where a given compound is to be determined in large numbers of samples, or in continuous monitoring situations for extended periods. The robustness and low power consumption of LEDs are features that allow the unattended use in field monitors [34] and remotely deployed submersible [56,57] analysers. An impressive example of the latter is the application of a battery powered all-solid state LED-based detector for the determination of total oxidised nitrogen [58].

3.4 Optical fibre-based spectrophotometric detection Optical fibres are an invaluable spectroscopic tool since they enable light guiding over long distances with low light loss and almost unlimited geometric flexibility. Hence, light sources and optical detectors can be decoupled from the reaction zone. This can for instance be of importance in remote sensing applications and process control situations where the ‘‘wet’’ part of the flow system is installed in harsh environments that would deteriorate the system electronics. Through the use of optical fibres for light transmission, miniaturisation of the flow cell and its positioning within the flow conduits of the manifold is possible, without any restrictions and irrespective of the size of the light source and the detection unit. Common spectrophotometers can be furnished with optical fibres, but dedicated

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optical fibre-based instruments are now also commercially available. Both can be used in combination with remotely located flow cells. This is for instance realised in the commercially available FIAlabs system furnished with a CCD detector and a Z-shaped flow cell [59]. The use of optical fibres to transmit light to and from a FIA detection cell was first proposed by Ruzicka and Hansen for absorbance measurements in a microconduit assembly [60]. The advantages inherent in this configuration are that the liquid and the electronic parts of the detection system are separated and there is no necessity to transfer the absorbing liquid from the flow manifold to the detector. Also, the electronic components of the detection system can be exchanged or additional components added without disconnection of tubing and/or re-arrangements of detector cells. The use of optical fibres for ad lib light guidance allows the creation of novel configurations that are difficult to realise with conventional optics. The miniaturised format of the fibre tip permits probing within the manifold at almost any conceivable point, thus omitting the implementation of a true flow cell. Instead detection can be accomplished with optical fibre tips fixed directly at the transmission tubing and even at multiple sites within a single manifold without affecting the flow pattern. An impressive example is the multi-channel flow cell developed by Beck and Weigand [61] which altogether incorporated sixty pairs of fixed waveguides into a meandering microconduit connected to six LEDs that serve as switchable light sources. This system provided information on the dispersion pattern and reaction kinetics in a single step, and when used in combination with multivariate calibration procedures, was suitable for simultaneous multi-analyte determinations. On-column detection in sorbent extraction optosensing or implementation of optical fibres into membrane-based separation units [62] are two other impressive applications of probing the events in situ within the flow manifold (Figure 9) [16]. Finally, the lab-on-valve concept (see Chapter 1) relies to a great extent on the implementation of optical fibres for absorbance measurements.

4. BACKGROUND ABSORBANCE CORRECTION In spectrophotometry the zero and 100% transmittance boundaries are arbitrarily set by the operator. The zero transmittance point is generally obtained by switching off the light source, whereas 100% transmittance is usually defined as the radiation power reaching the photodetector when the cuvette is filled with pure solvent. In many spectrophotometric assays chromogenic reagents are used that exhibit some absorbance at the measuring wavelength. In such cases it is sensible to define the reagent blank as zero absorbance. Considering real sample analysis, the absorbance values obtained are not necessarily that of the analyte or the derivative alone, but it is also possible that sample constituents (coloured compounds or colloidal matter) contribute to the background at the measuring wavelength. This value must be subtracted from the total absorbance obtained, otherwise the analyte concentration will be overestimated. In typical FIA

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Figure 9 (a) Sorbent-packed flow cell designed for sorbent extraction optosensing and (b) optical fibre membrane sensor suitable for gas diffusion, dialysis, membrane extraction, etc., with the capability of either reflectance with a bifurcated optical fibre, or absorbance detection with axially aligned optical fibres. LS, light source; D, detector. Reprinted from Ref. [16]. Copyright (2005), with permission of Elsevier B.V.

procedures, the carrier and reagent solutions are continuously pumped so that the detector baseline corresponds to the reagent blank. The absorbance reading of the sample signal is determined from the peak height or peak area. Corrections for sample blank, if required, can be made by running the samples twice, once in the absence and once in the presence of the chromogenic reagent. However, this is not a convenient procedure and is complicated if the chromogenic reagent also absorbs at the measuring wavelength. Another means of sample background correction is to inject a very large sample volume of sample or to pump sample continuously, and to inject the reagent only into the sample zone (reverse or reagent injection; see Chapter 3). In this mode, the detector is zeroed on sample alone, thus compensating for background absorbance. Quantification is based on the peak response after chromogenic reagent is injected [63]. A potential disadvantage of this approach is that chromogenic reagents may contain impurities that give rise to high blank signals. Worsfold et al. utilised a dual flow cell arrangement to compensate for intrinsic sample colour and turbidity in a reversed FIA system as (Figure 10). In this arrangement the sample solution flowed through the first detector cell before reagents were injected and the reaction product monitored by the second cell. Both detectors were operated under identical conditions and the analytical signal was taken as the net absorbance [34]. Multi-wavelength detection can also be used for correction of background absorbance. This is accomplished by simultaneous measurement at the wavelength of maximum absorbance and at a second wavelength where only background absorbance is measured. However, a requirement for correction is that the background absorptivities are the same at both wavelengths. This is typically the case for turbidity and RI corrections (see Section 5) but may not be fulfilled if the sample contains coloured compounds other than the analyte or the derivative. Diode-array detection enables better correction since the spectral background can be obtained by extrapolation of the baseline.

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Figure 10 Dual-flow detection cell suitable for on-line background correction. Reprinted from Ref. [34]. Copyright (1987), with permission from Elsevier B.V.

A completely different approach to background correction is the use of stopped-flow methods, in which the pump is temporarily stopped when the sample zone has just reached the detector cell, and either the net absorbance change or the absorbance change per unit time is used as the analytical signal. The signal response immediately before halting the flow represents the background absorbance plus the coloured product that forms from the point of injection until the sample reaches the detector cell. Any absorbance increase thereafter is due to the formation of chromophore alone. In FIA, the continuous baseline obtained represents the background signal. However in SIA the punctuated nature of sample and reagent introduction means that no continuous baseline is obtained, and correction for reagent blanks and sample background absorbance for reagent blanks, standards and samples must be obtained from detector responses in the presence and absence chromogenic reagents. This requires at least two, often three measuring cycles for a single determination, which considerably increases the total time for analysis. Alternatively a stoppedflow method can be used, which is easily applied in SIA by flow programming, or by the use of multi-wavelength detection. Serious errors can arise in photometric FIA and SIA methods due to the schlieren or refractive index effect. This has been the subject of considerable scientific concern, with more than 25 papers published which focus on this problem, and warrants brief discussion in the following section.

5. REFRACTIVE INDEX (SCHLIEREN) EFFECTS The refractive index (RI) is an inherent property of transparent materials (solids, liquids and gases). Refraction of light occurs when a collimated light beam

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crosses the phase boundary of two transparent materials at an angle different to the normal. The angle of deviation depends on the difference between the refractive indices of the two phases. This effect is utilised in refractometry to characterise chemical species or to determine the composition of binary mixtures of miscible solutions. Another typical application of refractometry is in liquid chromatography where advantage is made of the non-specific nature of the RI detector, i.e. any change of the mobile phase composition caused by analyte elution is recorded. Static RI effects can be caused by a variable reflective light loss at the interface between the optical window and liquid inside the cuvette, but these losses are generally negligible. However in photometric detection for FIA and SIA, RI or schlieren effects can have serious effects on the accuracy, precision and limits of detection. This is because any change in the RI between the moving carrier and injected sample or reagent causes a change in light transmission through the cuvette. Dias et al. have identified two types of RI or schlieren effect can occur in flow analysis systems [64]. The first effect, which is quite reproducible, occurs when the RI of an injected liquid differs from that of the recipient liquid. Under the prevailing laminar flow conditions, the interfacial boundary between the injected bolus will form parabolic liquid lenses at both the head and the tail of the injected sample zone. Light passing along the optical axis of a flow cell will be refracted by these lenses. Depending on the relative magnitudes of the sample and carrier refractive indices, this has the effect of dispersing or focusing light rays from the source either towards or away from the detector, giving rise to artefact or schlieren peaks (Figures 11a, b and Figure 6 of Chapter 17). This schlieren effect occurs even in the absence of a chromophore, and can be a source of major quantification errors if ignored [65]. The second effect is associated with localised variations in the refractive indices of elements of the injected sample and carrier/reagents that arise because of differences in temperature, viscosity, concentration, which is further compounded by pump pulsation and inefficient mixing of confluent streams. This effect causes striated mixing within the flow cell, and generates random baseline noise (Figure 11c). Both effects are seen clearly in the projected schlieren image shown in Figure 11d [66]. Attempts to minimise the adverse effects that schlieren formation can exert in photometric FIA systems are directed either towards avoidance or reduction of schlieren formation, or to designing detector optics that are tolerant of RI changes. Often both strategies are jointly applied. In FIA manifolds with confluence carrier and reagent streams schlieren formation occurs in the mixing coils and this is often the main reason for baseline fluctuations in absorbance detection, which adversely affects detection limits. Measures to reduce this kind of baseline noise are to match the carrier and reagents streams as much as possible with respect to their refractive indices by adjusting, e.g., viscosity or salt content, improving mixing of the streams by using knotted tubing, packed bead reactors or mixing chambers and to optimise the optical configuration. These strategies are reasonably effective when the sample matrix is constant (as is the case for instance in the analysis of seawater, digestion solutions, extracts or

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Figure 11 Diagrammatic representation of refractive index effects in flow analysis. Figures (a) and (b) show the expected schlieren lensing effect when the RI of the sample zone, Z2, is higher than that of the carrier stream, Z1, while figure (c) illustrates how liquid heterogeneity can cause additional detector noise. Figure (d) is a projected image obtained by shining a 660 nm laser through a conventional flow-through cuvette (1.5 mm i.d., pathlength 10 mm), and injecting water with salinity of 35 g L1 into a carrier of dionised water. The bright spot in the centre of the image corresponds to the aperture of the flow cell. Both the effects of schlieren lensing and liquid striation can be seen. The irregular shape of the image is apparently a function of non-uniform liquid flow within the flow cell [66].

industrial process streams) but they fails when samples with highly varying matrices, such as estuarine waters are analysed. Under such circumstances, matrix matching, or more conventional approaches such as standard addition or preparation of standards in the appropriate matrix are not feasible, especially if large numbers of samples are involved. An avoidance approach for FIA proposed by Yamane and Saito [67] involves the use of large injection volume and selecting integration windows so that the signal response is obtained from the mid part of the peak, thus excluding the schlieren peaks that are confined to the front and rear portions of the peak. This method is used successfully in at least one commercial FIA system for brackish water analysis, but careful setting of the integration windows is critical, and there is the disadvantage that sample throughput is diminished by the use of large injection volumes.

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Auflitsch et al. [68] proposed a ‘‘salinity-compensation’’ FIA manifold to overcome schlieren effects in the analysis of estuarine waters. This involved a combination of matrix matching and reagent injection. Chromogenic reagent was injected into a saline carrier with the same RI, and this mixed stream was merged with a continuously pumped sample stream. Hence no discrete variations in RI occurred throughout the manifold, and no schlieren lensing effects were observed. Schlieren effects are also significantly reduced when the flow is stopped. Although the cessation of flow does not lead to complete mixing of solutions, flow and RI heterogeneities are reduced. Hence the stopped-flow technique used in kinetic measurements and in SIA is less susceptible to RI artefacts. However, it can only be employed for slower derivatisation reactions where colour formation continues after the sample mixed with reagent(s) has reached the flow cell. The schlieren effect in absorbance detection is particularly pronounced when the light beam is collinear with the flow direction (as is the case in commonly used U- and Z-shaped flow cells). Some reduction of the schlieren artefacts can be achieved by focussing the light source on the exit window and using tapered cell configurations. A more efficient alternative is to direct the light transversely across the stream of the flowing liquid. This concept was first proposed by Betteridge et al. [32] who placed an LED source and a photodiode on opposite sides of the transparent manifold tubing of the flow system. However, the short pathlength of transverse illumination is a limiting factor if high sensitivity is required, and this shortcoming can be overcome by the use of reflective or multipath flow cells (described in Section 3.2) which offer moderate pathlength with considerable immunity to schlieren effects [19,69]. A totally different approach to compensate for RI effects involves the use of dual-wavelength detection. This relies on the fact that similar schlieren signals occur over a wide range of wavelengths. Using a detection wavelength at the maximum absorbance of the chromophore and a second, separated wavelength where only the schlieren signal occurs, highly effective schlieren compensation has been attained [70,71]. In practice dual-wavelength detection has been accomplished using frequency selective multi-colour LED light sources [72] or diode-array detectors [71]. The latter have the advantage that the correction wavelengths can be freely selected according to optimum schlieren compensation capabilities. The use of liquid-core waveguide cells enables the use of considerably increased optical pathlength because the light is propagated by total internal refraction. However, the angle of reflection of light from the walls of the capillary is quite large and the beams are almost parallel with the flow. The optical pathlength is therefore only marginally greater than the physical length of the capillary liquid-core waveguide. Consequently, LCW cells tend to exhibit acute schlieren effects, but little enhancement of sensitivity other than that which is attributable to the extended physical length can be achieved. In this respect the multi-reflection cells described by Ellis et al. [19] and Mishra and Dasgupta [36] are a good compromise between enhanced sensitivity and tolerance of the

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Figure 12 Comparison of the sensitivity to refractive index effects for a multi-reflection cell and a transverse, single radial-pass cell. For comparative purposes the transverse cell signal has been multiplied by 20. In each case the first injection is 10 mM alkaline bromothymol blue, and the second injection is the same analyte in a matrix of 100% NaCl. Redrawn from Ref. [36]. Copyright (2007), with permission from Elsevier B.V.

schlieren effect, because the light is introduced across the flow, and the angle of incidence of the light beams introduced in this manner is much more acute with respect to the normal to the flow direction. In this case, a number of reflections will occur along the flow cell length, and the optical pathlength will be enhanced to some degree, while avoiding the worst extremes of schlieren peaks (Figure 12). It is worth noting that the schlieren effect has also been intentionally used in FIA for analyte determination. Using a LED photometer with axial illumination of the flow path Betteridge et al. have demonstrated the high sensitivity and reproducibility of RI signals. A more recent application of schlieren effect measurements was the determination of the alcoholic content of beverages [73].

6. CONCLUSIONS AND OUTLOOK Spectrophotometric detection in its various manifestations is probably the most widely used method of detection in flow-based analysis. The development of cheap, robust detectors based on LEDs and photodiodes, and the availability and increasing utilisation of fibre optics and compact, modular, reasonably priced CCD detection systems further reinforces the role that photometry has as the workhorse detection method in FIA and other emergent flow-based techniques. Numerous FIA manifolds implementing separation, preconcentration and spectrophotometric detection have been devised and applied to the determination of a large variety of compounds in various sample matrices. Commonly the

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separation unit and the spectrophotometric detector are connected in series with unidirectional flow of the carrier solution. However, FIA systems have also been proposed where several different detectors and different separation units are placed within a single manifold and the liquid flow is either circulated, reversed, split and recombined or temporarily stopped, and in some instances several of these options were realised in a single procedure [74]. The introduction of SIA has simplified manipulations of liquids within the flow manifold and the presentation to the detector, owing to the programmed pumping and valve switching options (see Chapter 2). Handling of liquids and even dispersed solid particles became possible with so far unimaginable flexibility and this has led also to some innovative concepts for the implementation of separation and preconcentration techniques and eventually to novel means of detector integration, including spectrophotometry. The incorporation of LLE processes into FIA has attracted considerable attention and many instrumental configurations have been devised with a strong focus on phase segmentation and even more on phase separation [75]. However some advances in photometric flow cell design obviate the need for phase separation [76] resulting in a simpler manifold and more robust system operation. However LLE is fast being surpassed by the use of solid-phase microextraction (SPME), which is increasingly being utilised in flow-based analytical techniques in conjunction with photometric detection (see Chapter 7). However, a common problem associated with spectrophotometric detection post SPME is the drastic change in composition between the sample solution and the eluent used to remove the analyte from the minicolumn, which inevitably causes severe schlieren effects. Again, the usual means of addressing the schlieren problem is dual wavelength detection, but this comes with the cost of an increased noise level that in part offsets the gains due to higher analyte concentration in the eluate. The recent introduction of sequential injection chromatography (SIC) cleverly combines the principles of liquid chromatographic separation with the flexible solution handling of SIA (see Chapter 10). An alternative to spectrophotometric detection of analyte in the eluate, is the incorporation of solid sorbent material in a flow cell or, or even more practically, to optically monitor a zone of the microcolumn where retention takes place. There is now a huge amount of evidence of the benefit and potential of this approach for improvement of sensitivity and selectivity of spectrophotometric protocols [77–79]. While spectrophotometric detection has been one of the most commonly used detection approaches in flow analysis, the rapid development of new photonic materials offers the potential for exciting new photometric detection methods and techniques. Recent developments in LED technology have led to the release of near UV LEDs, and it is only a matter of time until mid-UV versions become available. This would add to the versatility of FIA and SIA systems by enabling construction of cheap, compact UV photometers for direct detection of analytes containing UV chromophores, as well as enabling miniaturisation and integration of processes such as sample photo-oxidation and post separation photoderivatisation. Advances in photonics will likely see more specialised techniques

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such as surface plasmon resonance become more readily available and adopted for routine measurement in wider areas of application. Photometry has been widely used as a robust and versatile detection technique for FIA since the mid-1970s. To a large extent the rapid and phenomenal developments that have occurred in both areas have been synergistic, and there is every reason to believe that this fruitful partnership will continue.

ABBREVIATIONS A AF c CCD CID d DAD LCW LED LLE RI (Z) SPME: el

Absorbance Amorphous fluoropolymer Concentration Charge couple device Charge injection devices Optical pathlength Diode-array detector Liquid-core waveguide Light emitting diode Liquid–liquid extraction Refractive index Solid-phase microextraction Wavelength-dependent molar absorptivity

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I.D. McKelvie, D.M.W. Peat, G.P. Matthews and P.J. Worsfold, Anal. Chim. Acta, 351 (1997) 265–271. I.D. McKelvie and P. Monbet, Monash University, Australia, 2007. Unpublished data. T. Yamane and M. Saito, Talanta, 39 (1992) 215–219. S. Auflitsch, D.M.W. Peat, I.D. McKelvie and P.J. Worsfold, Analyst, 122 (1997) 1477–1480. S. Jambunathan, P.K. Dasgupta, D.K. Wolcott, G.D. Marshall and D.C. Olson, Talanta, 50 (1999) 481–490. H. Liu and P.K. Dasgupta, Anal. Chim. Acta, 289 (1994) 347–353. E.A.G. Zagatto, M.A.Z. Arruda, A.O. Jacintho and I.L. Mattos, Anal. Chim. Acta, 234 (1990) 153–160. I.-Y. Eom and P.K. Dasgupta, Talanta, 69 (2006) 906–913. S.R.B. dos Santos, M.C.U. de Arau´jo and R.A. Barbosa, Analyst, 127 (2002) 324–327. M. Valca´rcel and M.D. Luque de Castro, Flow Injection Analysis-Principles and Applications, Ellis Horwood Limited, Chichester, 1987. V. Kubań, L.G. Danielsson and F. Ingman, Talanta, 37 (1990) 1169–1173. H. Liu and P.K. Dasgupta, Anal. Chim. Acta, 288 (1994) 237–245. R.J. Cassella, L.S.G. Teixeira, S. Garrigues, A.C. Spinola Costa, R.E. Santelli and M. De la Guardia, Analyst, 125 (2000) 1835–1838. A.R. Medina, M.L.F. de Cordova and A.M. Diaz, Anal. Chim. Acta, 394 (1999) 149–158. A. Ruiz-Medina, M.L. Fernandez-de Cordova and A. Molina-Diaz, Talanta, 50 (1999) 277–282.

CHAPT ER

13 Luminescence Paul S. Francis and Conor F. Hogan

Contents

1. Introduction 2. Photoluminescence 2.1 Principles of photoluminescence 2.2 Photoluminescence detection in FIA 2.3 Complexation/derivatisation reagents and analytical applications 3. Chemiluminescence 3.1 Principles of chemiluminescence 3.2 Chemiluminescence detection in FIA 3.3 Chemiluminescence reagents and applications 4. Electrochemiluminescence 4.1 Introduction 4.2 Electrochemical generation of chemiluminescence reagents 4.3 In situ electrochemiluminescence detection 5. Future Directions Abbreviations References

343 344 344 346 347 349 349 353 354 358 358 363 363 367 369 370

1. INTRODUCTION Luminescence is the emission of light from an excited chemical species returning to its ground electronic state [1]. The three modes of luminescence detection that are discussed in this chapter can be broadly classified by the process that creates (or leads to) the excited electronic state from which the emission occurs: absorption of light (photoluminescence), chemical reaction (chemiluminescence) or electrochemical reaction (electrochemiluminescence, ECL). Photoluminescence can be categorised as either fluorescence, a spin-allowed luminescent transition from a singlet excited state to a singlet ground state, or phosphorescence, a luminescent Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00613-2


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Paul S. Francis and Conor F. Hogan

transition involving spin conversion (i.e. intersystem crossing) [2]. Chemiluminescence that occurs naturally in living organisms is known as bioluminescence [3]. Compared to the number of molecules that can be detected by UV-visible absorption spectrometry, relatively few exhibit analytically useful photoluminescence and even fewer will produce light with any particular chemiluminescence reagent. Luminescence detection therefore generally provides greater selectivity, which in some cases reduces the need for physical separation of the analyte from other sample components. The second key feature of luminescence detection is that the emission of light often affords much better limits of detection than absorption. We will focus our discussion on luminescence from ions and molecules in solution, and therefore procedures involving atomic fluorescence or gas-phase chemiluminescence detectors will not be covered. Fluorescence, chemiluminescence and ECL are attractive modes of detection for flow injection immunoassay, and whilst examples of these systems will be included in the tables, a detailed discussion of the principles and various modes of immunoassay are outside the scope of this chapter. By the early 1990s, the number of papers published each year on flow injection analysis (FIA) with fluorescence detection and those published on FIA with chemiluminescence detection had risen to similar levels (30–55 per annum).1 Since that time, the number of papers concerning fluorescence detection has remained reasonably steady, but the number involving chemiluminescence detection has increased dramatically (over 200 papers published in the year 2006). A significant increase in papers on this topic published in Chinese language journals (92 in the year 2006) has contributed to this trend. To date, far fewer publications on FIA with ECL detection have emerged, but we have devoted a section of this chapter to ECL, due to its considerable potential and its growing importance in clinical diagnostics using commercially available automated flow-analysis instrumentation.

2. PHOTOLUMINESCENCE 2.1 Principles of photoluminescence Many molecules can absorb a photon of light and exist for short periods of time in a higher electronic energy state, before returning to the ground state via several possible deactivation pathways (Figure 1). If the molecular and environmental conditions favour a pathway that involves the emission of light, the overall process is referred to as photoluminescence, which is divided into two 1

Based on searches using the CAPLUS database with SciFinder software. Papers on fluorescence detection obtained by searching for fluorescence and fluorometry individually as concepts (to incorporate related terms such as fluorescent, fluorometric, spectrofluorometry, and corresponding terms based on alternative spelling: fluorimetry) and refined to those containing the terms flow injection or sequential injection. Statistics on papers on FIA with chemiluminescence detection obtained as above, using the terms chemiluminescence and chemiluminometric. In all cases, final statistics were obtained after further refining based on an examination of abstracts. It should be noted that papers concerning atomic fluorescence spectroscopy and gas-phase chemiluminescence were excluded due to the focus of this chapter.

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Singlet excited state (S1) VR

Triplet excited state (T1)

ISC

VR

Phosphorescence

Fluorescence

Absorption

EC/ IC

EC/ IC

VR

Singlet ground state (S0)

Figure 1 Some of the transitions that may occur in a luminescent molecule (VR, vibrational relaxation; ISC, intersystem crossing; EC, external conversion; IC, internal conversion).

categories — fluorescence and phosphorescence — depending on the nature of the excited state [1,2,4]. Fluorescence involves a direct radiative transition from the first singlet excited state (S1) to the ground electronic state (S0). This process occurs rapidly; the lifetime of the excited state is between 1010 and 107 s. Examination of Figure 1 reveals that the energy of the emission is less than that of absorption, due to the thermalisation of excess vibrational energy. In practical terms, this means that the emitted photons will have longer wavelengths than those used to excite the molecule. Excitation of higher energy singlet states often leads to the same emission from the lowest vibrational level of the S1 state. Analytically usable fluorescence is most often observed from compounds with highly conjugated or aromatic functionality, which exhibit low-energy p to p transitions. Phosphorescence involves a radiative transition from a triplet-excited state (T1) to the singlet ground state (S0), after intersystem crossing from S1. This process depends upon vibrational coupling between S1 and T1, and is more favourable when the energy difference between these two states is small and the lifetime of S1 is relatively long. The radiative transition from T1 to S0 is spin-forbidden, which has several important consequences. Firstly, the emission rates are relatively slow; phosphorescence lifetimes are typically milliseconds to seconds. Secondly, phosphorescence is infrequently observed in the liquid phase at room temperature, due to competition from non-radiative deactivation pathways. As shown in Figure 1, T1 states are less energetic than S1 and therefore phosphorescence will occur at longer wavelengths than fluorescence for

346


any particular compound. However, the distinction between fluorescence and phosphorescence is not always clear: the excited state tris(2,2u-bipyridyl)ruthenium(II) is formally a triplet, but spin–orbit coupling with the heavy metal ion increases the probability of the radiative transition, resulting in a luminescence lifetime of 0.4 ms [2]. Both fluorescence and phosphorescence are enhanced by structural rigidity, which reduces the energy dissipated by vibration and internal rotation. For example, phenolphthalein is not fluorescent, but fluorescein, which differs only by an oxygen bridge, is intensely fluorescent. The complex of aluminium with 8-hydroxyquinoline-5-sulfonic acid is much more fluorescent than the free ligand [4]. Environmental parameters such as temperature, viscosity, solvent type, pH and dissolved oxygen content can also affect luminescence. The influence of temperature and viscosity can be attributed to the degree of collisional deactivation of the excited state. In some cases, strict control of pH is essential, because the free and ionized forms of aromatic compounds with acidic or basic substituents may have different excitation and emission wavelengths, and the stability constants of fluorescent metal chelates may vary considerably.

2.2 Photoluminescence detection in FIA Fluorescence measurements require instrumentation for irradiation, detection and wavelength selection. Ideally, the light source and detector should be orientated at right angles with respect to the sample, to minimize scattering from the solution and cell walls. One viable option for fluorescence detection in FIA is to use a flow-through fluorescence cuvette in a commercially available spectrofluorometer, containing a continuum light source such as a xenon arc lamp, photomultipliers for detection and monochromators for the selection of excitation and emission wavelengths. Another option is the use of modules designed for post-column HPLC detection. Instrumentation specifically marketed for FIA applications is also commercially available. Examples include a flow cell with W-shape design (Figure 2) that can be coupled to a suitable light source and detector via fiber optic cables, and a self-contained fluorometer that consists of a light source (LED or tungsten lamp, or external light source via fiber optic cable) [5] and photomultiplier tube (PMT) positioned against the entrance and exit

Fiber optic cables connected to light source and detector

Figure 2 A flow cell designed for fluorescence detection.

Solution inlet and outlet

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347

windows of a flow-through fluorescence cuvette [6]. A variety of optical filters can be inserted between the components for wavelength discrimination. Lasers have also been used as excitation sources for fluorescence detection. Although the instrumentation is more expensive and complex, the high intensity of laser light can provide remarkably sensitive detection of fluorescent species. Furthermore, this approach can impart additional selectivity due to the small bandwidth and the ability to excite other less prominent absorption bands of the target analyte, where potential interferents may not absorb.

2.3 Complexation/derivatisation reagents and analytical applications Whilst some molecules can be sensitively detected by exploiting their native fluorescence character, there are many avenues to apply this mode of detection to molecules that exhibit little or no native fluorescence. In some cases, simple reactions such as oxidation or hydrolysis can produce species with far greater fluorescence efficiency. Furthermore, a range of derivatising reagents have been used to attach or form suitable fluorophores. One of the more commonly used reagents is ortho-phthaldialdehyde (OPA), which reacts with primary amines (including amino acids) and thiols such as 3-mercaptopropionic acid to produce fluorescent isoindole derivatives. OPA with sulfite or thioglycolate has been used to detect ammonia with considerable selectivity over amino acids [7,8]. Interference from other fluorescent compounds and the turbidity of some samples can be removed by adding a gas-diffusion step. The determination of ammonia has been used in conjunction with fluorescence-based FIA/sequential injection analysis (SIA) procedures for nitrite and nitrate [9] and phosphate [10] for environmental water analysis. OPA has also been applied to the determination of urea, after enzyme-catalysed hydrolysis of the analyte to ammonia [11]. Alternative derivatisation reagents for primary amines include fluorescamine and dichlorotriazinylaminofluorescein. Ethidium bromide has been used as an intercalating agent for the sensitive determination of double-stranded DNA in relatively small volumes of solution using a lab-on-valve instrument with laserinduced fluorescence detection [12] and to examine the DNA binding characteristics of drugs using FIA in stopped-flow mode with the merging zones technique [13]. Many metal ions have been detected by forming fluorescent complexes with reagents that contain rigid aromatic systems and electron donating functionality [14–19]. The selectivity of complexing agents (such as those shown in Figure 3) is in some cases remarkable and can be strongly dependent on the chemical environment. For example, the complex formed between copper(II) and 5-(4-chlorophenylazo)-8-aminoquinoline is highly fluorescent in slightly acidic media, but the fluorescence intensity of this system is low in strongly acidic or alkaline solution. However, a fluorescent complex between cobalt(II) and the same reagent forms only in alkaline solution in the presence of hydrogen peroxide. Therefore, by applying suitable conditions, both copper(II) and cobalt(II) can be determined in the presence of the other cation [17]. Surfactants

348


COOH

OH N

HOOC

CH3

N N

SO3H

O N

H3CO

I

COOH

II HO

COOH

OH Cl

HO

O N

N

N

OH OH

H2N

O

IV

III

HOOC HOOC

HO

O

OH

N

N

COOH COOH

O V O

Figure 3 Some of the reagents used to create fluorescent complexes with certain metal ions: (I) 8-hydroxyquinoline-5-sulfonic acid; (II) quin-2; (III) morin; (IV) 5-(4-chlorophenylazo)-8aminoquinoline; and (V) calcein.

such as Tween-20 and cetyltrimethylammonium bromide (CTAB) are commonly used to enhance the fluorescence from metal–ligand complexes. Complexation or reaction with highly fluorescent species that results in a decrease in fluorescence intensity can also be used for quantitative analysis. The formation of heteropoly acids from orthophosphate and molybdate is the basis for many approaches to determine traces quantities of phosphorus in the environment [20]. One of the most sensitive of these methods involves the formation of ion associates between molybdophosphate and rhodamine B, which quenches the characteristic fluorescence of rhodamine B, enabling the determination of phosphate down to 1 nM [21]. Fluorescence detection can be used to determine the substrates or products of enzyme-catalysed reactions, by coupling the process to a reaction that produces or consumes a biomolecule with native fluorescence character, such as nicotinamide adenine dinucleotide (NADH), for which the optimum excitation and emission wavelengths are 340 and 455 nm. For example, pyruvate has been quantified using pyruvate decarboxylase (PDC) and aldehyde dehydrogenase (AlDH) immobilised in an on-line reactor [22], based on the reactions shown in Equation (1).

PDC pyruvate ⎯⎯⎯ →

acetaldehyde + CO2 AlDH acetaldehyde + NAD+ + H2O ⎯⎯⎯→ acetate + NADH + H+

(1)

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349

This system has been applied to the determination of acetate, citrate and by coupling with other co-immobilised enzyme systems, such as citrate lyase (CL) and oxaloacetate decarboxylase (ODC) for citrate (Equation (2)). L-lactate

CL ⎯⎯→ oxalacetate + acetate citrate ←⎯⎯ ⎯ODC ⎯ ⎯ → pyruvate + CO2 oxaloacetate ←⎯ ⎯ ⎯

(2)

As previously discussed, relatively few species are phosphorescent in solution at room temperature. Consequently, this mode of detection has the potential to provide high selectivity for certain analytes. Background fluorescence from the sample matrix can be avoided by adding a short delay before the relatively longlived phosphorescence is measured. Approaches to increase the intensity of room temperature phosphorescence from species in solution include the use of organized media, the temporary immobilisation of the analyte on solid-phase supports and the addition of ‘heavy atoms’ [23,24]. As a further illustration of the wide range of applications involving FIA (or related techniques) with photoluminescence detection, numerous examples that have recently appeared in the open literature are summarised in Table 1.

3. CHEMILUMINESCENCE 3.1 Principles of chemiluminescence Chemiluminescence is the emission of light (normally in the visible and/or nearinfrared regions) from an electronically excited intermediate or product of a chemical reaction [42–45]. The general process is shown in Equation (3), where species A and B react to form C, some fraction of which is formed in an electronically excited state (C) that can subsequently relax to the ground state by emitting a photon. A þ B ! C þ other products C ! C þ light

(3)

In some cases, the excited intermediate (C) transfers energy to a suitable fluorophore, which may then emit its characteristic fluorescence (Equation (4)). This phenomenon is referred to as indirect or sensitised chemiluminescence. C þ ½fluorophore ! C þ ½fluorophore ½fluorophore ! ½fluorophore þ light

(4)

Once the final excited state is reached, the emission process is identical to that of other modes of luminescence, and for practical applications can often be considered instantaneous. However, unlike photoluminescence, the production of the excited state in chemiluminescence depends on the physical processes of solution mixing and the kinetics of the chemical reaction. Therefore, the transient emission is initiated as soon as the reactants are merged and can last for seconds, minutes or even hours!

350

Table 1


Selected analytical applications of fluorescence and phosphorescence detection

Analyte /application Native fluorescence Labetalol in urine and pharmaceuticals 2-Phenylbenzimidazole-5sulfonic acid in urine Fluorescent complexes Aluminium(II) in natural waters Calcium(II) in alpine ice cores Cobalt(II) and copper(II) in foods

Magnesium(II) in commercial drinking waters Cationic surfactants

Instrumentation and reagents

Limit of detection

Reference

3 mg L1 SIA. Analyte retained on a flow-through cell filled with C18 silica gel solid support SIA. Separation from inter12 mg L1 ferences with a strong anion exchange micro-column

[25]

SIA. Morin in the presence of a non-ionic surfactant (Tween-20) FIA. Quin-2

3 ppb

[14]

15 ppt

[15]

FIA. 5-(4-chlorophenylazo)-8- 10 mg L1 aminoquinoline. Selectivity dependent on the pH of the medium SIA. 8-hydroxyquinoline-512 mg L1 sulfonic acid; EGTA as a masking agent for Ca(II) and CTAC as an enhancer FIA. Enhancement of 0.02 mg L1 (CTAB) fluorescence from Eu(III)thenoyltrifluoroacetone complex

Fluorescence quenching Chlorine dioxide in bottled FIA with gas-diffusion device. waters Chromotropic acid Mercury(II) in natural FIA with cation-exchange resin waters micro-column. Murexide Phosphate in H2O2 solutions FIA. Ion associate between molybdophosphate and rhodamine B Dissolved oxygen in FIA with liquid–liquid natural waters extraction. Iodine liberated in Winkler’s method reacts with 2-thionaphthol Reaction/derivatisation Ammonium in seawater FIA with gas-diffusion device. Derivatisation with OPA/ sulfite Urea in alcoholic FIA. Urea converted to beverages ammonia with immobilised enzyme (acid urease). Derivatisation with OPA/ thioglycolate

[26]

[17]

[16]

[27]

0.03 mg L1

[28]

1 mg L1

[29]

1 109 M

[21]

5 107 M

[30]

7 109 M

[8]

0.06 mg L1

[11]

Luminescence

Table 1 (Continued ) Analyte /application


Limit of detection

Reference

Protein in serum

SIA. Derivatisation with fluorescamine Pretreatment with solid-phase extraction, then FIA with PbO2 reactor to oxidise analyte FIA with on-line solid-phase extraction and photochemical reduction (in SDS micelles) FIA. Scavenging effect on hydroxyl radical (from H2O2 and Co(II)), which reacts with ninhydrin LOV with laser-induced fluorescence detection. Ethidium bromide intercalation. Only 0.6 mL sample required FIA with heated reaction coil. 5,5-Dimethyl cyclohexane-1,3-dione and ammonium acetate SIA used to automate reaction with a fluorescein derivative for a CE procedure with laserinduced fluorescence detection

0.1 mg L1

[31]

5.5 mg L1

[32]

0.05 mg L1

[33]

8 108 M ( OH)

[34]

9 mg L1

[12]

0.9 ppb

[35]

30 mg L1 (for an enkephalin)

[36]

5 106 Ma

[22]

2 107 M

[37]

3 ng L1

[38]

Thioridazine in human plasma

Phylloquinone in fruits/ vegetables

Antioxidant capacity of foods

Double-stranded DNA

Formaldehyde in indoor air

Amino acids and peptides

Enzyme-catalysed reactions Acetate, citrate and L-lactate FIA. Analytes converted to pyruvate, which undergoes further enzymatic reactions using pyruvate decarboxylase and aldehyde dehydrogenase. NADH monitored D-Gluconate in foods FIA. Gluconate kinase and 6-phosphogluconate dehydrogenase. NADH monitored Hydrogen peroxide in FIA. Horseradish peroxidase natural waters and 3-(p-hydroxyl phenyl)propionic acid

351

352


Table 1 (Continued ) Analyte /application Fluorescence immunoassay Gentamicin in serum


FIA. Anti-gentamicin immobilised in column containing sol-gel. Assay based on competition with fluorescein isothionatelabelled gentamicin Immunoglobulin (Ig) for FIA. Protein G irreversibly bioprocess monitoring immobilised in cartridge. Analyte binds and then is eluted by change in pH. Detection by native fluorescence Botulinum neurotoxin A SIA. Renewable microcolumn flow cell packed with functionalised beads for sandwich assay. Secondary antibody labelled with fluorescent dye. On-column detection using laser excitation Room-temperature phosphorescence Lead(II) in seawater FIA. Complex with 8hydroxy-7-quinoline sulfonic acid. Transient immobilisation on anion exchange resin within detection flow cell 1-Naphthaleneacetic acid in FIA. Analyte immobilised on apples polymeric resin within flow cell. Thallium(I) added as ‘heavy atom’. Sulfite used to deoxygenate solution

Limit of detection

Reference

0.2 mg L1

[39]

Not stated

[40]

4 mg L1a

[41]

0.1 mg L1

[23]

1.2 mg L1

[24]

CE, capillary electrophoresis; CTAB, cetyltrimethylammonium bromide; CTAC, cetyltrimethylammonium chloride; EGTA, ethylene glycol-bis(b-aminoethylether)-N,N,Nu,Nu-tetraacetic acid; FIA, flow injection analysis; LOV, lab-onvalve; NADH, nicotinamide adenine dinucleotide; OPA, o-phthaldialdehyde; SDS, sodium dodecylsulfate; SIA, sequential injection analysis. a Lower end of calibration range (limit of detection not stated).

The chemiluminescence quantum yield (the proportion of reacting molecules that lead to the emission of a photon) depends on several factors, including the efficiencies of the chemical reaction, conversion of chemical potential energy into electronic excitation and (in the case of sensitized chemiluminescence) energy

Luminescence

353

transfer and the proportion of excited molecules that emit a photon. Consequently, quantum yields for chemiluminescence are generally lower than those for photoluminescence. Certain diaryloxalates that are used as analytical reagents and in commercially available ‘glow sticks’ exhibit quantum yields of around 0.5 and enzyme-catalysed bioluminescence systems have yields as high as 0.9. However, the quantum yields of many commonly used chemiluminescence systems (such as luminol and lucigenin) are typically below 0.01. In spite of these relatively low quantum efficiencies, chemiluminescence detection can be extremely sensitive, due to the absence of an excitation light source, which provides superior signal-to-background ratios, the ability to detect light emitted from a relatively large area, and in some cases (such as the determination of transition-metal ions with luminol) the target analyte acts as a catalyst rather than reactant. It should be noted that the chemiluminescence intensity at any particular moment is a product of the number of reacting molecules, the quantum yield and the rate of the reaction.

3.2 Chemiluminescence detection in FIA The characteristics described in the previous section have several important consequences for the optimal design of chemiluminescence detectors for FIA. Perhaps most importantly, the geometry and dimensions of the flow cell should maximize the proportion of light that is emitted when the reacting mixture is in front of the detector. For relatively fast chemiluminescence reactions, this means that the reactant solutions should merge at (or as close as possible to) the point of detection. The ‘dead volume’ of the cell should be minimized, to ensure reproducible mixing and rapid washing between injections. Unlike photoluminescence, a light source is not required and wavelength discrimination usually offers no advantage (because different analytes often lead to the same emitting species) and should be avoided due to the detrimental effect on sensitivity. One design that satisfies each of the above requirements is a coil of glass or transparent polymer tubing that can be positioned against the window of a PMT (Figure 4). Solutions can be merged at T-shaped, Y-shaped or concentric connectors prior to entering the coil. Detection cells with a spiral flow design have also been etched or machined into transparent polymer blocks or chips.

confluence point reaction coil Reagent and carrier inlets

photomultiplier tube

waste solution outlet

Figure 4 A flow cell designed for chemiluminescence.

HV input and signal output

354


In an alternative design, referred to as the ‘fountain flow cell’, the reacting mixture enters the centre of an open, shallow cylindrical space and drains near the edge. Compared with a coiled tube, the fountain cell allows a greater volume of solution to be in contact with the surface facing the PMT, but this advantage may be offset by less efficient mixing and a longer period of time required to flush the cell between injections. Fluorescence detectors (with the excitation source switched off) have also been used for chemiluminescence detection. Although suitable for some applications, this approach generally provides lower sensitivity, as the volume of the reacting mixture exposed to the PMT at any particular moment is often lower than in flow cells specifically designed for chemiluminescence, and in some cases the confluence point cannot be positioned close to the point of detection, which is disadvantageous for some of the faster chemiluminescence reactions used for analysis. For chemiluminescence reactions that last longer than a few seconds, a greater proportion of the luminescence can be captured using the stopped-flow technique, but the period of time from injection until the flow is ceased must be precisely timed to ensure reproducible measurements. Emission intensity versus time profiles collected in this manner can also be used for analytical procedures based on reaction rates and can provide insight into the kinetics and mechanism of chemiluminescence reactions. Chemiluminescence detection has been successfully employed in alternative modes of flow analysis, such as ‘multisyringe’ FIA with multicommutation [46] and pulsed-flow systems [47]. Chemiluminescence is an attractive mode of detection for portable or miniaturised devices, due to the simplicity of the instrumentation and the high sensitivity, which allows the quantification of analytes in small volumes of solution.

3.3 Chemiluminescence reagents and applications Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) is one of the best known and most widely applied chemiluminescence reagents [48–50]. The oxidation of luminol in alkaline solution evokes a blue luminescence (lmax ¼ 425 nm) that emanates from electronically excited 3-aminophthalate (Equation (5)). A wide variety of oxidants, such as hydrogen peroxide, periodate, hypochlorite and permanganate, can be employed. The reaction with hydrogen peroxide is catalysed by selected transition-metal ions (e.g. Co(II), Cu(II), Cr(III), Fe(II) and Ni(II)) and complexes (including haemoglobin and peroxidases), which enables the highly sensitive detection of these species. Numerous biomolecules, such as glucose, lactate, cholesterol and choline, can be selectively determined in complex media by coupling luminol systems with H2O2-generating enzymatic reactions. Others can be determined with high specificity using immunoreactions with peroxidase- or luminol-labelled antibodies. Luminol (with various oxidants) has also been used to detect many organic molecules that enhance or inhibit the light-producing pathway [48,49].

355

Luminescence

O

O oxidation

NH

O-

NH NH2

* O-

O

(5)

NH2 O 3-aminophthalate

luminol

Lucigenin (10,10u-dimethyl-9,9u-bisacridinium nitrate) reacts with hydrogen peroxide or oxygen to produce N-methylacridone (Equation (6)), which emits blue-green light with a maximum intensity at 440 nm. The reaction can be catalysed by numerous metal ions, including several that do not catalyse the oxidation of luminol. Both luminol and lucigenin have been used to measure reactive oxygen species [51,52]. CH3 N H2O2

CH3

CH3

N

N

*

+

(6)

base O

O N N-methylacridone

CH3 lucigenin

Tris(2,2u-bipyridyl)ruthenium(III) is an effective chemiluminescence reagent for the detection of selected amines (particularly aliphatic tertiary amines), amino acids, NADH, organic acids, alkaloids and pharmaceuticals [53,54]. The reagent is only moderately stable in acidic aqueous solutions and therefore it is normally produced shortly before use by oxidising tris(2,2u-bipyridyl)ruthenium(II) (Equation (7)). Subsequent reaction of the ruthenium(III) complex with a suitable analyte elicits the characteristic orange luminescence (lmax ¼ 610–620 nm) from an excited state of tris(2,2u-bipyridyl)ruthenium(II). If the reagent can be isolated from the spent analyte solution, it can be re-oxidized to the active ruthenium(III) complex and consequently there has been a great deal of research into the development of stable immobilized forms of this reagent. 2+

N N

N

oxidant Ru(bipy)33+

Ru

N

Ru(bipy)32+

N N

analyte Ru(bipy)32+ *

Ru(bipy)32+

(7)

356


The light generated by the reaction of substituted diaryloxalates or diaryloxamides with hydrogen peroxide in the presence of a fluorophore is collectively known as peroxyoxalate chemiluminescence (Equation (8)) [55,56]. The high-energy intermediates formed in these reactions are capable of exciting a variety of fluorophores that emit light from the near-ultraviolet to the near-infrared. O

O

+ H2O2

R

R

- 2 RH

O

O

fluorophore

fluorophore *

- 2 CO2

O O

(8)

intermediate

Unlike the previously described reagents, the properties of reactivity, energy conversion and luminescence in peroxyoxalate reagents are not the province of a single molecule. Consequently, the chemiluminescence quantum yields that have been obtained by combining an oxalate or oxamide that produces relatively high yields of the key intermediate with an efficient fluorophore in organic solvents are exceeded only by enzyme-catalysed bioluminescence systems. Some of the peroxyoxalate reagents that have been employed for detection in flow analysis are shown in Figure 5(I–III). Peroxyoxalate chemiluminescence has been predominantly used to detect hydrogen peroxide, compounds that react with enzymes to produce hydrogen peroxide, and species that either exhibit native fluorescence or have been

Cl

Cl

O2N

O O

O Cl

O

Cl

NO2 O

O NO2

O Cl

O

NO2

Cl

I

II O

O H3C

3

O

O

NO2 O

O NO2

O

O

III HO3S

CF3 SO2 O N O IV

O

CH3 3

O O N

N

N

N SO2 CF3

N

O SO3H

V

Figure 5 Selected peroxyoxalate reagents: (I) bis(2,4,6-trichlorophenyl)oxalate (TCPO); (II) bis(2,4-trinitrophenyl)oxalate (DNPO); (III) bis[2-(3,6,9-trioxadecyloxycarbonyl)-4nitrophenyl]oxalate (TDPO); (IV) 4,4u-[(1,2-dioxo-1,2-ethanediyl)bis [[[(trifluoromethyl) sulfonyl] imino]methylene]] bisbenzenesulfonic acid; (V) 1,1u-oxalyldi(imidazole) (ODI).

Luminescence

357

derivatised with an efficient fluorophore. One of the main limitations of this chemistry for detection in FIA is the poor solubility and stability of many peroxyoxalate reagents in aqueous solution. There has been some success in the addition of inert polar functionality to certain oxalates and oxamides (as shown in Figure 5(IV)), but as yet these reagents have not been widely applied. Another promising reagent is 1,1-oxalyldi(imidazole) (Figure 5(V)), which has comparable quantum yields to conventional reagents, but readily dissolves in acetonitrile and does not precipitate when merged with aqueous solutions. The reaction of acidic potassium permanganate with a wide variety of organic compounds (particularly phenols, polyphenols, catechols, indoles, antioxidants and selected alkaloids and pharmaceuticals) and a few inorganic substances evokes a broadly distributed luminescence with maximum intensity at 73475 nm [57,58]. Formaldehyde or polyphosphates are commonly used to improve the sensitivity of this reagent (the presence of polyphosphates also shifts the wavelength of maximum intensity to 68975 nm). There is strong evidence to suggest that the characteristic emission from these reactions emanates from an excited manganese(II) species. Two related reagents — soluble manganese(IV) and electrogenerated manganese(III) (see Section 4.2) — have recently been examined for chemiluminescence detection [59,60]. Although new applications involving well-established reagents continue to emerge, there has been considerable growth in detection based on chemiluminescence reactions between organic analytes and oxidants such as cerium(IV), hexacyanoferrate(III), hypochlorite, N-bromosuccinimide and periodate [61–63]. Unlike most traditional chemiluminescence reagents, the emitting species in these reactions may often be derived from the analyte rather than the reagent, but in many cases the light-producing pathway is yet to be elucidated. The emission from these reactions tends to be relatively weak, but can often be significantly enhanced by adding efficient fluorophores, such as rhodamine B, fluorescein or quinine. Lanthanide ions (La(III), Tb(III) and Eu(III)) have also been used as sensitisers [64–66]. Another weak chemiluminescence reaction that has received attention is the reaction of sulfite in neutral or weakly acidic solution with oxidants such as cerium(IV), bromate and permanganate. An interesting selection of fluorescent and non-fluorescent organic compounds have been found to enhance the chemiluminescence from this particular reaction, which has been exploited for the detection of sulfite, sulfur dioxide and numerous enhancers. Bioluminescence is enzyme-catalysed chemiluminescence that has evolved in biological systems [3,62]. The enzymes and substrates in bioluminescence reactions are commonly referred to as luciferases and luciferins. The most popular bioluminescence regents employed in analytical applications are from fireflies (Photinus pyralis) and marine bacteria (Photobacterium fischeri and Vibrio harveyi). The general reactions for these systems are shown in Equations (9) and (10). Firefly luciferin/luciferase is useful for the sensitive detection of adenosine tri-phosphate (ATP) and the enzymes and substrates linked to ATP metabolism. Bacterial bioluminescence has been applied to the selective detection of numerous biomolecules by coupling the oxidoreductase–luciferase system to reactions that produce or consume NADH or nicotinamide adenosine

358


dinucleotide phosphate (NADPH). luciferase Mg(II)

ATP + reduced luciferin + O2 ⎯⎯⎯⎯→ AMP + oxyluciferine + light (562 nm) (9) NAD(P)H:FMN oxidoreductase

NAD(P)H + FMN + H+ ⎯⎯⎯⎯⎯⎯ → NAD(P)+ + FMNH2 luciferase → FMN + RCOOH + H2O + light (478-505 nm) FMNH2 + RCHO + O2 ⎯⎯⎯⎯

(10) As described above, many chemiluminescence reagents respond to classes of compounds that possess certain chemical functionality, rather than specific analytes. However, it is often the case that a reagent is far more sensitive towards certain compounds than others within the same general class and consequently there are some fortuitous situations where an analyte of interest can be selectively determined using the most simple FIA approach and without complicated sample preparation. When required, greater selectivity can be obtained by coupling chemiluminescence detection with additional operations (such as extraction, gas-diffusion or ionexchange) that can be performed on-line within the flow-analysis manifold. Examples of analytical applications involving chemiluminescence detection that illustrate the range of approaches discussed in this section are shown in Table 2.

4. ELECTROCHEMILUMINESCENCE 4.1 Introduction ECL is the emission of light from an electronically excited intermediate of a chemical reaction involving at least one species generated at an electrode surface [100–103]. The term is often (but not always [103]) used interchangeably with electrogenerated chemiluminescence. ECL can arise from organic as well as inorganic substances. It can be produced by annihilation reactions between sequentially reduced and oxidised forms of the same species or by electrolysis at a single potential in the presence of a co-reactant (a species capable of forming an energetic intermediate following its oxidation or reduction). The analytical utility of the annihilation pathway is limited because the reduced species cannot usually be effectively generated in aqueous media, but the co-reactant pathway has found growing application as the basis for analytical techniques in which either the co-reactant or the luminophore itself are quantified. A typical mechanism [104] involving the co-reactant, oxalate and tris(2,2u-bipyridyl)ruthenium(II) is shown in Equation (11). RuðbipyÞ2þ e 3

!

RuðbipyÞ3þ 3

RuðbipyÞ3þ þ C2 O2 3 4 d C O

!

RuðbipyÞ3þ 3

!

RuðbipyÞ2þ þ C2 Od 3 4 CO2 þ COd 2 RuðbipyÞ2þ þ CO2 3

!

RuðbipyÞ2þ þ light 3

2

4

þ CO2d RuðbipyÞ2þ 3

!

ð11Þ

Luminescence

Table 2

359

Selected analytical applications of chemiluminescence detection

Application

Comments

Luminol chemiluminescence Iron(II) and iron(III) in On-line mini-columns with marine waters immobilised 8-HQ for clean up and preconcentration. Fe(II) catalyses oxidation of luminol. Fe(III) reduced with sulfite prior to analysis Mercury(II) in sea and On-line gas-diffusion river waters device. Reduced Hg crosses membrane as vapour at 851C. Oxidation back to Hg(II), which catalyses luminol/H2O2 Hydrogen peroxide in Stopped-flow analysis. natural waters Reaction with luminol and cobalt(II). SDS used as an enhancer. Ion-exchange used to remove interference from metal ions Glucose in drinks and SIA with on-line sample honey dilution. Soluble oxidase enzyme. Cobalt(II) used to catalyse reaction between H2O2 and luminol Silicate in freshwaters Heteropoly acid formed with molybdate reacts with luminol. Interferences removed by on-line chelating and ionexchange columns Iodide in multivitamins On-line pervaporation device. Analyte oxidised to iodine, which permeates through the membrane and reacts with luminol Dopamine in Inhibition of pharmaceuticals chemiluminescence reaction between luminol and hexacyanoferrate(III)

Limit of detection

Reference

2 1011 M

[67]

0.8 mg L1

[68]

5 1010 M

[69]

1 106 M

[70]

0.35 mg L1

[71]

0.5 mg L1

[72]

5 mg L1

[73]

360


Table 2 (Continued ) Application Ampicillin and amoxicillin in pharmaceuticals

Comments

Enhancement of the chemiluminescence reaction between luminol and periodate Nitrite in urine Analyte binds with myoglobin, which accelerates the electron transfer of luminol Carp vitellogenin SIA. Sandwich immunoassay. Antibody immobilised on beads that could be held in detector using a magnet. Secondary antibody labelled with HRP, which reacts with luminol, H2O2 and p-iodophenol Lucigenin chemiluminescence Total antioxidant Discretely actuated solenoid capacity micropumps. Inhibition of emission from the oxidation of either lucigenin or luminol Analyte consumes Superoxide dismutase superoxide produced in activity in enzymatic reaction, which erythrocytes reduces lucigenin chemiluminescence Isoniazid in Chemiluminescence pharmaceuticals reaction with lucigenin and periodate Tris(2,2u-bipyridyl)ruthenium(III) chemiluminescence Oxalate in Bayer On-line anion exchange process samples column and step gradient Proline in wines Reverse FIA, with recirculating system to maintain oxidised form of the reagent Amiodarone in Merging zones technique. pharmaceuticals On-line photooxidation of 2 RuðbipyÞ2þ 3 (with S2O8 in solution)

Limit of detection

Reference

5 and 30 mg L1

[74]

0.02 ng L1

[75]

2 mg L1

[76]

N/A

[77]

0.1 mg L1

[78]

3 mg L1

[79]

5 106 M

[80]

1 108 M

[81]

0.3 mg L1

[82]

Luminescence

Table 2 (Continued ) Application

Comments

RuðbipyÞ2þ 3 and analyte oxidised with cerium(IV). Enhanced by silver nanoparticles Peroxyoxalate chemiluminescence Hydrogen peroxide TDPO in aqueous imidazole/acetonitrile medium. Sulforhodamine 101 fluorophore Dodecylamine in diesel On-line solid-phase fuels extraction. DNPO/H2O2 with Sulforhodamine 101 fluorophore Choline/acetylcholine ODI and TCPO. Immobilised or glucose in urine peroxide-forming enzymes. Ion-exchange added to separate choline and acetylcholine. Fluorophore immobilised in detection cell Gentamicin in Off-line derivatisation with pharmaceuticals OPA. TCPO/H2O2 with imidazole catalyst. SDS reduced reagent degradation in water Acidic potassium permanganate chemiluminescence Morphine in industrial FIA. Tetraphosphoric acid process streams used as enhancer. Sample preparation involved only filtration and dilution Antioxidant levels in FIA. Polyphosphate used as wines enhancer Anilide pesticides in Multicommutation. Analyte natural waters degraded in on-line photoreactor prior to detection Other chemiluminescence reactions Bromate in water Sulfite in an acidic solution. Enhanced by hydrocortisone Arginine in dietary Alkaline hypobromite supplements Citrate in a urinary alkaliniser

Limit of detection

Reference

4 109 M

[83]

3 109 M

[84]

0.2 mg L1

[85]

3 109 M (glucose)

[86]

1 mg L1

[87]

5 108 M

[88]

4 1010 M (quercetin) 8 mg L1 (propanil)

[89]

8 108 M

[91]

1 107 M

[92]

[90]

361

362


Table 2 (Continued ) Application

Comments

Limit of detection

Reference

Cyanocobalamin in eye lotions

Pretreatment with acid to release cobalt(II). Lophine/ H2O2 in alkaline solution enhanced by hydroxylammonium chloride SIA with flow cell in holding coil. Analyte converted to nitrite, which reacts with H2O2. Enhanced with uranine and CTAB [Eu(EDTA)] enhances chemiluminescence from NaIO4/H2O2 Gallic acid/H2O2 in alkaline solution Cerium(IV)/rhodamine B in acidic solution

5 108 M

[93]

5 108 M (nitrite)

[94]

6 108 M

[65]

4 108 M

[95]

1 108 M

[96]

Cerium(IV)/sulfite. Enhanced by terbium(III) FIA or SIA with flow cell packed with immobilised antibodies. Competitive immunoassay using antigen labelled with acridinium ester, which reacts with H2O2

0.01 mg L1

[64]

0.4 mg L1

[97]

Immobilised dehydrogenase enzyme and coimmobilized bacterial luciferase/NADH:FMN oxidoreductase. Other reagents introduced by permeable membrane reactor Firefly luciferase-luciferin. FIA with dual injection valve

1 107 M

[98]

1 1010 M

[99]

Nitrogen oxide in air

Europium(III) in rare earth oxides Formaldehyde in water Folic acid in pharmaceutical formulations Grepafloxacin in tablets and spiked urine Triiodothyronine in serum

Bioluminescence systems 3a-hydroxy bile acids in serum

ATP

CTAB, cetyltrimethylammonium bromide; DNPO, bis(2,4-trinitrophenyl)oxalate; EDTA, ethylenediaminetetraacetic acid; FIA, flow injection analysis; 8-HQ, 8-hydroxyquinoline; HRP, horseradish peroxidase; ODI, 1,1u-oxalyldi(imidazole); OPA, o-phthaldialdehyde; RuðbipyÞ2þ 3 , tris(2,2u-bipyridyl)ruthenium(II); SDS, sodium dodecylsulfate; TCPO, bis(2,4,6-trichlorophenyl)oxalate; TDPO, bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl]oxalate; TEA, triethanolamine; TMP, 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine.

Luminescence

363

For the purposes of this chapter, it is convenient to distinguish between two general approaches that we will refer to as (i) external or on-line electrochemical generation of chemiluminescence reagents and (ii) in situ ECL. In the first, the reagent is electrochemically oxidised or reduced in a flow-through electrolytic cell, prior to merging with the analyte solution to initiate a chemiluminescence reaction; the detectors discussed in Section 3.2 therefore remain appropriate options. In the second approach, the electrochemical reaction occurs within the detection cell, so both reagent and analyte may undergo electrochemical reactions. In this case, the light is emitted from a reaction zone close to the electrode surface.

4.2 Electrochemical generation of chemiluminescence reagents The on-line electrochemical generation of reagents is advantageous in cases where alternative methods of preparation are time-consuming or potentially hazardous, or the reproducibility of the procedure would otherwise be compromised by the limited stability of the reagent [59]. On-line generation of reactive oxidants such as manganese(III) cobalt(III) and silver(II) from stable precursors by constant current electrolysis has uncovered new avenues for chemiluminescence detection. Numerous applications of these nascent reagents have emerged in the open literature, but to date most involve the analysis of simple pharmaceutical formulations. As with photoluminescence and chemiluminescence, the true potential of this sensitive mode of detection will perhaps be realised through coupling with additional FIA operations that impart greater selectivity for the determination of analytes of interest in more complex sample matrices. Tris(2,2u-bipyridyl)ruthenium(III) has been successfully produced from the corresponding ruthenium(II) complex by off-line chemical oxidation (see Equation (7)), but it is difficult to maintain the reagent in this oxidation state for extended periods of time [105]. This issue has been addressed with on-line chemical or electrochemical oxidation of tris(2,2u-bipyridyl)ruthenium(II) prior to detection, but generation in the presence of the analyte within an ECL detector (as described in the following section) is far more commonly applied [106]. Some published accounts of on-line electrochemical generation of reagents for chemiluminescence detection are summarised in Table 3. Moreover, the on-line electrochemical generation of hypochlorite and hypobromite is a convenient way to prepare these oxidants for analytical applications involving luminol chemiluminescence [61]. Similarly, analytes can be electrochemically oxidised or reduced to form species that react with conventional chemiluminescence reagents. For example, vanadium(V) has been determined by on-line electrochemical reduction to vanadium(II) and subsequent chemiluminescence reaction with luminol and dissolved oxygen, with a limit of detection of 0.2 mg L1 [113].

4.3 In situ electrochemiluminescence detection In situ ECL systems exploit the inherent sensitivity, selectivity and wide linear range of chemiluminescence methods, but offer several potential advantages not

364

Table 3


External electrochemical generation of reagents for chemiluminescence detection

Reagent

Analyte

Limit of detection

Reference

Silver(II) from AgNO3 Manganese(III) from MnSO4 Cobalt(III) from CoSO4 Hypobromite from KBr RuðbipyÞ3þ 3 from Ru(bipy)3Cl2 [Cu(HIO6)2]5 from Cu(NO3)2 in aqueous KIO4-KOH medium

Captopril Isoniazid Pipemidic acid Sulfide 1,3-Cyclopentanedione Chlortetracycline

6 mg L1 30 mg L1 3 mg L1 5 108 M 50 fmol 0.5 mg L1

[107] [108] [109] [110] [111] [112]

found in traditional spectroscopic methods [114]. The main advantages lie in the electrochemical control over the chemiluminescent reaction. For example, the FIA manifold can be greatly simplified because the luminescent reagent is produced in situ from passive precursors already in the sample or carrier stream. A singleline manifold is quite feasible for ECL detection [106,115]. Furthermore, as the active form of the luminescent reagent is (in most cases) constantly regenerated by the electrode, each luminophore may emit many times, enhancing sensitivity. Background correction is also possible, because the luminescence can be switched on and off by modulating the applied potential. Precise spatial control is achieved by varying the electrode geometry or its position in relation to the detection element, since the light emission is concentrated close to the electrode surface. The main disadvantage of ECL detection is perhaps the possibility of electrode fouling, resulting in poor reproducibility. Although this may occasionally be a problem in the presence of complex sample matrices, rinse cycles and/or potential pulses are generally effective counter measures. Moreover, new electrode materials that are less subject to fouling (such as boron doped diamond [116]) have been developed, and disposable screen-printed electrodes [117] that can be discarded after use are becoming more common. ECL detection in flowing streams requires a flow cell, a means to control the applied potential (a potentiostat), suitable electrodes and a photodetection element. Flow cells for ECL are, by and large, similar to those used for conventional amperometric detection, except that provision is made to detect the light emitted from the vicinity of the working electrode. A two- or three-electrode configuration may be used. In a three-electrode setup, the reference electrode is generally placed downstream while the body of the detector itself often functions as the counter electrode. Glassy carbon, graphite, gold and platinum are common working electrode materials, but specialised materials such as boron doped diamond [116] and transparent indium-tin oxide coated glass [118] have been shown to be advantageous in some instances. Apart from instruments dedicated to immunoassays and DNA probe analysis, there is little in the way of commercially available equipment specifically designed for ECL detection, so the majority of the systems described in the literature have been either custom built or amperometric detectors adapted

Luminescence

Counter electrode connection

Working electrode

Working electrode block

365

Reference electrode

Teflon spacer

Solution outlet

Solution inlet

To PMT

Light guide

Transparent window

Figure 6 A thin layer flow cell designed for electrochemiluminescence detection.

for the purposes of ECL detection. Thin layer designs such as that shown in Figure 6 are the most common as they facilitate placement of the detection window in very close proximity to the ECL reaction. A light guide such as a fibre optic bundle or liquid light guide may be used to collect the emitted light which is detected with a conventional photodetector such as a PMT. Alternatively, a detector such as a miniature PMT or a photodiode can be placed directly in front of the detection window. Most ECL detectors will in principle also function as conventional amperometric detectors, but the potential advantages of simultaneous transduction of the dual signals of light and current have only occasionally been explored [115,119–121]. Although a wide range of reactions have been examined [101], the majority of analytical applications are based on tris(2,2u-bipyridyl)ruthenium(II) and related complexes. Early work in this area focused on the reaction with oxalate [104,122] (Equation (11)), but as with the chemically induced luminescence from tris(2,2ubipyridyl)ruthenium(III), the greatest ECL intensity is generally achieved with compounds containing amines [54,123]. In general, ECL intensity increases in the order 11 amineso21 amineso31 amines and the direct detection of compounds of pharmacological and biological importance, such as codeine, heroin, proline and oxyprenolol, is a large area of application for ECL detection. In addition to these direct detection methods, indirect methods for analytes such as glucose and ethanol, have been developed, based on enzymatic reactions that produce the tertiary amines NADH or NADPH [124,125]. Although lower detection limits have been reported in some cases, background processes, such as the reaction of tris(2,2u-bipyridyl)ruthenium(III) with hydroxide ions, generally limit the sensitivity of the co-reactant detection in aqueous media to concentrations of the order of 106–108 M. Much lower limits

366


O N

O N O

N

O N

2+ N Ru N N

H3C I H3C H2N

O N

NH NH

II

O

Figure 7 Labels for chemiluminescence or electrochemiluminescence detection: (I) RuðbipyÞ2þ 3 -NHS ester and (II) N-(aminobutyl)-N-ethylisoluminol (ABEI).

of detection are possible in applications where the luminophore itself is the species detected and the co-reactant (almost always tripropylamine) is present in large excess in the carrier stream. There has been considerable research on the synthesis and application of ECL labels based on tris(2,2u-bipyridyl)ruthenium(II), such as the N-hydroxysuccinimide ester shown in Figure 7(I). ECL analysers for immunoassay and nucleic acid probe assays have been commercially available since the mid-1990s, beginning with the ORIGEN Analyser [126,127] from IGEN International (now owned by Roche Diagnostics). This instrument and others based on ORIGEN technology are designed to detect tris(2,2u-bipyridyl)ruthenium(II) ECL labels on the surface of paramagnetic beads (used as supports for binding assays). A flow-analysis manifold within the instrument is used to present samples to the detector, where the beads are magnetically captured. The ECL reaction is initiated after the unbound labels are washed away and the tripropylamine co-reactant is introduced. This instrumental approach has found wide application in clinical diagnostics, food and water testing, and the detection of biowarfare agents [128]. The mechanism of co-reactant ECL with amines is complex and varied, depending on pH, electrode material, electrode potential and the relative concentrations of co-reactant and emitter. In cases where the co-reactant is the analyte and relatively high concentrations of tris(2,2u-bipyridyl)ruthenium(II) (mM) are employed, the generally accepted mechanism involves oxidation of the amine followed by deprotonation to form a reactive radical intermediate that reduces electrogenerated RuðbipyÞ3þ back to RuðbipyÞ2þ in an electronically 3 3 excited state. When the emitter (such as a RuðbipyÞ2þ -based label) is the species 3 being detected, sub-micromolar levels of this species are typically present with a large excess of amine co-reactant. Under these circumstances the electrooxidation of the amine becomes more important than that of the ruthenium complex and light emission is observed at less positive potentials [128]. The

Luminescence

367

oxidation rate of the amine and thus ECL efficiency may be optimized by judicious choice of electrode material and/or the use of various additives in the flowing stream [129–131]. Luminol ECL [123,132] has also been shown to be useful, particularly in the detection of hydrogen peroxide, a common by-product of enzyme substrate reactions. However, somewhat extreme conditions of pH are required and the electrochemical oxidation of luminol is irreversible, so unlike ruthenium complexes, it does not have the advantage of regeneration. The mechanism of luminol ECL is thought to involve electrochemical oxidation of both the luminol and peroxide anions and subsequent reaction of the luminol radical anion or diazaquinone with superoxide or peroxide anions, leading to the aminophthalate emitter [133]. As with the chemical oxidation of luminol, this system has been used to detect transition-metal catalysts, peroxides, the substrates of enzymatic reactions in which H2O2 is produced, and compounds that enhance or inhibit the light-producing pathway [134]. A range of luminol-based labels (such as N-(aminobutyl)-N-ethylisoluminol (ABEI), Figure 7(II)) for chemiluminescence and ECL detection have been developed. Examples of analytical applications involving electrochemiluminescence detection are shown in Table 4.

5. FUTURE DIRECTIONS The reagents used for luminescence detection impart varying degrees of selectivity towards the target analyte(s): in some cases an analyte can be accurately determined in complex matrices with minimal or no sample preparation. However, an increasing number of FIA procedures based on a small group of commonly used reagents (particularly for chemiluminescence detection) are emerging in the literature. Whilst these procedures may be suitable for the determination of analytes in simple and/or well-characterised matrices such as pharmaceutical preparations and industrial process streams (and provide reasonable percentage recoveries when spiked environmental and clinical samples are examined), the sheer number of procedures proposed for each reagent indicates their lack of selectivity in real clinical and environmental applications, where the presence of other species that also respond to the reagent is uncertain. Three areas of development will help address this issue: (1) a greater understanding of the reagents in terms of the relationship between analyte structure and luminescence intensity, and the reaction mechanism to form the luminescent species, (2) further exploitation of instrumental and chemical conditions to enhance the inherent selectivity of the reagents towards a target analyte and (3) when required, coupling luminescence detection to additional FIA operations that increase the selectivity of the overall procedure. Furthermore, as already shown by several researchers [8,147,148], FIA (and related techniques) can be used to combine sample preparation and several different analytical procedures within a single automated instrument. ECL detection for FIA is less advanced than fluorescence or chemiluminescence, but has considerable potential. Further study on the design of ECL flow

368

Table 4


Selected analytical applications of electrochemiluminescence detection

Analyte/application

Approach

Tris(2,2u-bipyridyl)ruthenium(II) electrochemiluminescence Proline and certain ECL reaction with RuðbipyÞ2þ 3 peptides using lysozyme modified electrode Heroin RuðbipyÞ2þ 3 immobilised in zeolite Y modified carbon paste electrode Thiouracil in spiked Novel flow cell design based meat on hollow platinum ring electrode b-Lactam antibiotics and Hydrolysis by b-lactamase b-lactamases in milk was signalled by an and bacterial broth increase in ECL culture Calcium in plasma Precipitation of analyte with excess oxalate. Residual oxalate measured by FIA with RuðbipyÞ2þ 3 ECL Tetracyclines in Inhibition of the ECL reaction pharmaceuticals and of RuðbipyÞ2þ 3 and honey tripropylamine. Manual extraction procedures performed prior to FIA Glucose, lactate and Dehydrogenase enzymes ethanol immobilised in polymer film adjacent to RuðbipyÞ2þ 3 loaded film on a platinum electrode. NAD(P)H from enzymatic reaction measured by ECL reaction with RuðbipyÞ2þ 3 Luminol electrochemiluminescence Chlorogenic acid in Catechols such as chlorogenic cigarettes acid inhibit luminol ECL. Sample preparation included paper chromatography to remove interferences Human immunogloblin Homogeneous immunoassay G in serum using anti-hIgG labelled with an isoluminol derivative (ABEI). FIA–ECL used to measure bound and unbound labelled-antibody

Limit of detection

Reference

2 107 M (proline)

[135]

1 106 M

[136]

5 mg kg1 (4 108 Ma)

[137]

Low mM

[138]

5 104 Mb

[139]

4 mg L1

[140]

1 108 M (NADPH)

[141]

5 109 M

[142]

80 ng L1

[143]

Luminescence

369

Table 4 (Continued ) Analyte/application Glucose and lactate in serum

Approach

Oxidase enzymes immobilised on membranes in close proximity to a glassy carbon electrode within ECL detector. H2O2 generated in the enzymatic reactions measured by ECL reaction with luminol Other electrochemiluminescence systems Cadmium Reaction with 1,10phenanthroline Calcium in milk ECL of calcein blue enhanced by analyte

Limit of detection

Reference

60 and 30 pmol

[144]

1 ppb

[145]

2 106 M

[146]

ABEI, N-(aminobutyl)-N-ethylisoluminol; ECL, electrochemiluminescence; FIA, flow injection analysis; NADH, nicotinamide adenine dinucleotide; RuðbipyÞ2þ 3 , tris(2,2u-bipyridyl)ruthenium(II). a Under ideal conditions. b Lower end of linear range (limit of detection not stated).

cells remains important. New luminophores continue to be investigated; among the most promising of these are the cyclometallated complexes of iridium with reported efficiencies in some cases far exceeding that of tris(2,2u-bipyridyl)ruthenium(II) [149,150]. New co-reactants have also been discovered. For example, tris(2,2u-bipyridyl)ruthenium(II) can be detected at an order of magnitude lower concentration using 2-(dibutylamino)ethanol, compared with tripropylamine. Moreover, it is less toxic, less volatile and easier to prepare in aqueous solution [151]. Immobilisation of the luminophore directly on the electrode surface has obvious benefits in terms of greater simplicity and conservation of the (often expensive) luminescent reagent. Approaches based on thin polymer [115,152,153] or sol-gel layers [154] or self-assembled monolayers [155] have been described which show significant promise, but as yet these systems have not found real world application.

ABBREVIATIONS 8-HQ ABEI CE CTAB CTAC DNPO ECL EDTA EGTA

8-hydroxyquinoline N-(aminobutyl)-N-ethylisoluminol capillary electrophoresis cetyltrimethylammonium bromide cetyltrimethylammonium chloride bis(2,4-trinitrophenyl)oxalate electrochemiluminescence ethylenediaminetetraacetic acid ethylene glycol-bis(b-aminoethylether)-N,N,Nu,Nu-tetraacetic acid

370


FIA HRP LOV NADH ODI OPA RuðbipyÞ2þ 3 SDS SIA TCPO TDPO TEA TMP

flow injection analysis horseradish peroxidase lab-on-valve nicotinamide adenine dinucleotide 1,1u-oxalyldi(imidazole) o-phthaldialdehyde tris(2,2u-bipyridyl)ruthenium(II) sodium dodecylsulfate sequential injection analysis bis(2,4,6-trichlorophenyl)oxalate bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl]oxalate triethanolamine 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine

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CHAPT ER

14 Atomic Spectroscopic Detection ´ Elo Harald Hansen and Manuel Miro

Contents

1. Introduction 2. Interfacing the Flow Network with Detection Devices 3. Flow Systems as Front-End Vehicles for On-Line Processing of Aqueous Samples 3.1 Solvent extraction/back extraction 3.2 Solid-phase extraction 3.3 Precipitation/coprecipitation and sorption onto knotted reactors 3.4 Gas–liquid separation: hydride generation and vapour generation 3.5 Membrane-based separations 4. Flow Systems as Front-End Vehicles for On-Line Processing of Solid Samples 5. Hyphenation with Atomic Spectroscopic Detectors Abbreviations References

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1. INTRODUCTION Next to Ultraviolet/Visible Spectrometry (UV/VIS), atomic spectroscopic techniques are the most ubiquitously employed optical instrumental detection devices used in conjunction with Flow Injection Analysis (FIA)/Sequential Injection Analysis (SIA)/Lab-on-Valve (LOV) or Multicommutated Flow Injection Analysis (MCFIA) and Multi-Syringe Flow Injection Analysis (MSFIA). By the start of 2007, these techniques accounted for close to 10% of the approximately 16,800 FIA papers then published, most of which had appeared within the last 10 years [1]. Thus, according to a scientiometric study conducted by Ro´denas-Torralba et al. [2], automation and mechanization have experienced a spectacular increase in the last decade, born particularly by FIA/SIA/LOV (96%), Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00614-4


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yet with MCFIA and MSFIA also gaining momentum. This reflects not only advances in both instrumentation and software in that period, but also the desire to miniaturize flow systems, leading to a reduction in the consumption of reagents and toxic solvents, and minimizes the generation of waste, not to mention ease of sample treatment and reducing risks to the operator. Inherently implying higher selectivity than their molecular counterparts, the first of the atomic spectroscopic detection techniques to be used in FIA was, not surprisingly, flame atomic absorption spectrometry (FAAS). This was most likely because it was readily feasible to interface continuously operating FIA systems with a continuously operating detection device. The first such paper appeared in 1979, and it was soon followed by publications describing the application of flame atomic emission spectrometry (FAES), electrothermal atomic absorption spectrometry (ETAAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS) and atomic fluorescence spectrometry (AFS), and more recently by flame furnace atomic absorption spectrometry (FF-AAS), simultaneous atomic absorption spectrometry (SIMAAS) and continuum source atomic absorption spectrometry (CSAAS). This sequence very appropriately reveals how laboratories scattered around the world gradually gained access to the various instrumental techniques as they became economically available for both industrial and academic institutions. Since the ultimate objective in any analytical chemical procedure is to obtain optimal sensitivity and selectivity, the atomic spectrometric instrumental methods are inherently very attractive. However, although they are some of the most sensitive detection devices available, they are, to some extent, prone to spectroscopic and/or non-spectroscopic interferences, especially if the sample matrix contains high levels of salts. Therefore, it is often necessary to subject the sample to appropriate pretreatment procedures, i.e., to separate the analyte species from potentially interfering matrix constituents, while at the same time performing analyte preconcentration, which indeed, might be advantageous or even necessary, if minute concentrations are to be determined. And here the FIA-based flow techniques present themselves as superb vehicles, especially because they operate under dynamic conditions. Hence, and very uniquely, it is possible to exploit the interplay between thermodynamics and kinetics of the chemical reactions involved, which in contrast to using batch conditions, opens up entirely new analytical chemical avenues. Therefore, this chapter will focus in particular on such schemes, which have blossomed over the past decade. In the early 1970s FAAS was already well established as the work horse or routine instrumental tool in many laboratories, whereas ETAAS gained momentum in the 1980s. However, in the words of Professor Z.-L. Fang [3], the combination of FIA with AAS stimulated a new era of development for AAS in the 1990s, the effects being so dramatic that it actually brought ‘‘new vitality to a technique which otherwise seemed to be confronted by a period of stagnancy’’. The reasons why FAAS, or FAES, already at an early point presented itself as a very attractive detection approach are multiple. Thus, in addition to what was mentioned above, the sample is merely exposed to the flame for a very short

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period of time (normally less than 1 s), in contrast to the conventional aspiration of sample for 10–20 s. This implies that when the flame is aspirating carrier solution, the wash-to-sample ratio is high, and therefore the risk of clogging of the burner is vastly reduced, or in fact eliminated. This has most impressively been demonstrated by Schrader et al. [4], who injected a solution of 1 mg/L Cu in 30% NaCl solution 170 times over a period of ca. 80 min without encountering any problems. Besides, the use of FIA in conjunction with FAAS not only increases the precision of measurement but also its accuracy. These features were already recognized at an early stage, and have been dealt with in detail in numerous publications and several monographs (e.g., [3,5–8]). Similarly, it was also at an early point experimentally verified, that FIA was a perfect vehicle for AAS detection based on cold vapour (CV) or hydride-generation (HG) techniques. This is because under the dynamic conditions used, the selectivity of the determination is significantly improved. As it turned out, this was in fact due to the exploitation of the interplay between thermodynamics and kinetics, or more precisely kinetic discrimination schemes. In recent years, the emphasis on determination by spectroscopic and other techniques has to a considerable extent shifted from mere assay of total concentrations to chemical species analysis. In many contexts it is of much more value to determine the concentrations of the individual species, or oxidation state of a specific element rather than its total content, because different oxidation states and chemical forms can exhibit widely different characteristics (e.g., bioavailability, toxicity). While this is true for liquid samples, it is equally valid for solid samples, such as soils or sediments. Traditionally, they have been subjected to treatment and analysis by batch procedures; this is a poor approach, because events in natura occur in a dynamic rather than in a steady-state fashion. Therefore, where possible, fractionation and speciation assays should mimic the natural conditions, i.e., a dynamic approach would be preferable. Lately, it has been shown that this is perfectly possible by use of flow systems, yielding novel, interesting and most informative results. Hence, this chapter will also deal with these procedures. It is not the aim of this chapter to explain the theory behind and the operation of the individual atomic spectroscopic techniques. Rather it is the intention to demonstrate how these detection techniques, though they superficially might seem very different, can all be exploited for on-line flow analysis provided that they are cleverly interfaced with the flow manifolds. Thus, if one wishes to get an intelligent answer from a detector, it has to be fed intelligently, i.e., the sample has to be pretreated intelligently before detection. For the very same reason, it is imperative that the flow system used is not only able to provide a suitable means of transport of the injected sample, but indeed can function as an appropriate front end for subjecting the sample to the required unit operations so that only the target analyte is exposed to the detector at a specific time. This is the nub of the matter, and this is where chemistry enters the scene. The use of an instrumental detection technique does not alleviate the use of good chemistry, rather it places demands on the operator to apply his or her knowledge skillfully. Instead of trying to be satisfied with compromises, one should aim to obtain the

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optimal solution, and this is only possible if one separates the individual tasks of the procedure, i.e., pretreat the sample using intelligent chemistry and leave the detector exclusively to perform the detection. Therefore, this chapter focuses on conceptual ways and means of exploiting flow systems as clever front ends for detection devices. The content and structure of this chapter therefore deals with: (i) interfacing the flow network with the various atomic spectroscopic detectors; (ii) flow systems as vehicles for on-line processing of liquid samples comprising solvent extraction, solid-phase extraction, bead injection, precipitation/coprecipitation, gas–liquid separation including HG and CV generation and membrane-based separation; (iii) flow systems as vehicles for on-line processing of solid samples encompassing fractionation and microwave/ultrasound extraction or digestion; and (iv) concluding remarks comprising a brief account of hyphenated techniques with atomic spectroscopic detectors. All cases are illustrated by practical examples.

2. INTERFACING THE FLOW NETWORK WITH DETECTION DEVICES The interfacing of various atomic spectroscopic detection techniques with flow systems is a function of several parameters. Thus, while some detection systems (FAAS, FAES, ICP-AES/MS, AFS) and flow systems (FIA, MCFIA) operate continuously, other detection systems (ETAAS) and flow approaches (SIA, LOV, MSFIA) inherently function discontinuously. For the very same reason SIA or SIA–LOV are ideal for ETAAS, where advantage can be taken of the fact that while running the ETAAS program, the next sample can be processed for analysis in the flow network. Some detectors can only determine one element at a time (FAAS, ETAAS, AFS), while others are multi-element devices (FAES, ICP-AES/ MS, SIMAAS). Yet, what is important in this context is that all the detectors are amenable to use with the flow systems by appropriate hyphenation. Hence, by combining SIA with FIA, or LOV with FIA or MCFIA, it is possible to gain full access to, for example FAAS, ICP-MS or AFS, as demonstrated by the examples shown herein. Some detection devices call for little or no preparation of the liquid samples, while others might place severe restrictions on the nature of the sample solution to be introduced. Thus, where possible, organic liquids should be avoided in ETAAS, due to their lower surface tension as compared with water, because they tend to distribute along the length of the platform in the graphite tube, leading to lower sensitivity of measurement. However, organic solvent matrices are beneficial in FAAS, because they result in a fine and effective nebulization, in addition to supporting the flame. For ICP-MS organic solvents should generally be eliminated because of risks for polyatomic interferences, and the sample should preferably be prepared in dilute nitric acid. The amount of dissolved salts should be kept below 0.2% (m/v), because they might result in severe signal depression. The presence of solid particles should be avoided, especially if a direct-injection high-efficiency nebulizer (DIHEN) capillary is used. In contrast, samples containing particulates (slurries) might be handled in


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Figure 1 Illustration of frequently employed FIA on-line separation and preconcentration schemes interfaced to various analytical detectors. Adapted from Ref. [23]. Copyright (1996), with permission from Elsevier B.V.

FAAS and ETAAS [9]. Furthermore, there might be other restrictions, depending on the individual samples to be analysed. Despite what is said above, there are in reality no restrictions in handling the samples with the various detectors, provided that clever designs of the flow manifolds and appropriate pretreatment schemes are used. Thus, and as illustrated in Figure 1, by incorporating various unit operations within the flow systems (e.g., solvent extraction/back extraction, sorbent extraction or gas–liquid separation), the sample can be pretreated so that it can be optimally presented to the detector, using the flow system as a front end. In doing so, one has the added advantage that all the flow systems are operating under dynamic conditions, which implies that one might be able to perform manipulations involving chemistries that are not possible in batch assays. The examples given in the following sections amply illustrate this. While selectivity for the single-element detection devices is accomplished via the applied chemistries, possibly coupled to time resolution for individual species of a particular element by chromatographic separation, the front-end chemistries for multi-element instrument might advantageously be based on group reagents, with the identification of each element being accomplished in the detection device itself.

3. FLOW SYSTEMS AS FRONT-END VEHICLES FOR ON-LINE PROCESSING OF AQUEOUS SAMPLES 3.1 Solvent extraction/back extraction Liquid–liquid extraction is among the most effective sample pretreatment techniques in the separation of interfering species and preconcentration of metal or metalloid species. Figure 2 shows the principle for separation/preconcentration

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Figure 2 The principle of preconcentration by liquid–liquid extraction. The extraction efficiency can be improved by using dual-stage extraction, where the extract from the first separation stage is mixed with more analyte complex and separated in the second-phase separator (PS2) before transport to the detector. The reaction coils are made as knotted reactors in order to increase the contact area between the two immiscible phases. Adapted from Ref. [13]. Copyright (2000), with permission from Taylor and Francis.

of metal ions by liquid–liquid sequential injection extraction, here illustrated for ETAAS detection. As the metal ions in the aqueous phase are to be transferred into an organic solvent, the analyte is initially complexed with an appropriate ligand to form a non-charged complex that can be extracted into the organic phase. The extraction coil applied is advantageously made as a knotted reactor (KR) [10], which, due to the generation of a vivid secondary, radial flow pattern, facilitates the dispersion of the two phases into each other so effectively that it is actually difficult to see them separately with the naked eye. A very large area of interface between the two phases is generated, which in turn expedites the extraction. After separation of the two phases in the phase separator PS1 (optimally a dual-conical gravitational one [11,12]), a defined zone of the organic extractant is, as illustrated by the dotted line, guided to the ETAAS instrument (max 50 mL) sandwiched by air segments to minimize dispersion during transport. To improve the extraction efficiency, a two-stage solvent extraction can also be used as shown (Figure 2), where the extract from the first separation stage is mixed with more analyte complex before the extractant is separated in a second-phase separator SP2, and directed to the detector [13]. As compared with traditional FIA extractions [10], minute, welldefined segments of organic solvents are introduced into the flow network by software-controlled flow programming [14], thus giving rise to the so-called green methods. The aforementioned two-stage solvent extraction scheme can be used for two purposes: either as demonstrated to obtain a higher extraction yield by taking advantage of the preconcentration obtained in each stage, or as a vehicle to perform extraction/back extraction. The latter approach is especially attractive


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Figure 3 Hyphenated SI/FIA manifold for on-line solvent extraction–back extraction system coupled to ICP-MS. (a) Load position (preconcentration), (b) inject position (back extraction and detection). SP1, SP2 and SP3: syringe pumps; PP: peristaltic pump; EC1 and EC2: extraction coils; PS1 and PS2: dual-conical gravitational phase separators; SV: six-port selection valve; IP: infusion pump; DIHEN: direct-injection high-efficiency nebulizer; HC: holding coil; SL: sample loop; IV: two-port injection valve; BEx: back extractant with stripping agent; CS: carrier; W: waste. Adapted from Ref. [11]. Copyright (2002), with permission from the Royal Society of Chemistry.

for ETAAS, where organic solvents should be avoided. When it comes to ICP-MS, it is implicit that organic solvents are disadvantageous, because they might give rise to the generation of interfering ions. Therefore, if an aqueous sample is to be pretreated by solvent extraction and the analyte determined by either ETAAS or ICP-MS, it is preferable, or actually necessary, to effect extraction/back extraction, where the analyte is first extracted into an organic solvent, and then back extracted into an aqueous solution, which then is introduced into the detector. The SIA system is perfectly suited for effecting such schemes, as illustrated in Figure 3. A practical example may serve to demonstrate this, that is,

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the determination of metal ions (e.g., Cu(II) or Pb(II)) by means of ICP-MS according to the following chemistries [15]: MeðIIÞaq þ APDCorg ! MeðPDCÞorg

(1)

MeðPDCÞorg þ Hþ þ PdðIIÞaq ! MeðIIÞaq þ PdðPDCÞorg

(2)

where the metal ion is first reacted with ammonium pyrrolidinedithiocarbamate (APDC) to form a non-charged chelate (Equation (1)) which thus can be extracted into an organic solvent, permitting not only separation of the analyte from the matrix constituents, but also preconcentration via judicial choice of the aqueous-toorganic solution ratio. The analyte ion is subsequently back extracted into an aqueous phase of nitric acid to which Pd(II) is added as stripping agent to facilitate and speed up the back extraction (Equation (2)). A defined volume of the analytecontaining acidic extract, as entrapped within the sample loop (SL) of the injection valve (IV), is finally introduced into the detector by means of an infusion pump (IP) and using a DIHEN, i.e., the sample being sandwiched by liquids during transport. Solvent extraction might also be exploited via the formation of micelles [16]. Thus, Nan and Yan have recently utilized on-line flow injection micelle solvent extraction preconcentration and separation procedures coupled to ETAAS for determination of Pb(II) [17] and Cr(VI) [18], where the APDC complexed metal ions are dissolved in self-aggregates of Triton X-114 or cetyltrimethylammonium entrapped on a microcolumn packed with silica gel, and subsequently eluted with acetonitrile for determination. In the context of automated micelle-mediated extraction techniques, flowinjection cloud-point extraction [19], hyphenated with FAAS and ICP-AES/ICP-MS, has attracted considerable attention for the isolation and concentration of metal ions, which are either in their native form or in organometallic chelates or ion pairs that are generated on-line under appropriate conditions [20,21]. The cloudpoint phenomenon is related to the decrease of solubility of non-ionic and zwitterionic surfactants in aqueous solutions, which, when heated above the so-called cloud-point temperature, results in phase separation and uptake of the target species within the organized entities by hydrophobic or electrostatic interactions [16]. When performed in an on-line fashion, the surfactant enriched phase is initially isolated within a microcartridge containing a filtering material, e.g., cotton, glass wool or nylon fibres, followed by elution and introduction of the eluate into the atomic spectrometer [14]. Besides heating, the addition of salting-out agents has proven effective for inducing phase separations, yet, the use of concentrated salt solutions might require careful optimization of the separation step to prevent non-spectroscopic interferences from the electrolyte medium.

3.2 Solid-phase extraction As a survey of the literature readily reveals, sample pretreatment by means of incorporated column-based solid-phase extraction is one of the most common


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and efficient, on-line approaches. The broad range of commercially available sorbent materials (normally in the form of small beads) with different surface characteristics makes this technique very attractive for on-line sample processing. Column-based solid-phase extraction has been extensively employed for on-line separation and preconcentration assays of ultra-trace levels of metals in the various generations of flow injection analysis coupled to atomic spectrometric detection [22–26]. Conventionally, the sorbent column is treated as a permanent component of the system, being used repeatedly for the sample loading/elution sequences, and it is replaced or repacked only after long-term operation. However, this approach might give rise to some problems. For instance, the performance of the procedures might often deteriorate because of the build-up of flow resistance or backpressure caused by progressively tighter packing of the sorbent material due to repetitive operations [27]. It is even worse if the sorbent beads undergo volume changes during the analytical cycle, i.e., swelling or shrinking, with the change of experimental conditions [27]; and it becomes critical if the surface properties of the column material, which are associated with the retention efficiency and the kinetics during the sorption–elution process, become irreversibly changed due to contamination, deactivation, or even loss of functional groups or active sites [28,29]. In this context it should also be borne in mind, that it is always desirable to completely elute the retained analyte from the sorbent with the minimum amount of eluent in order to get maximum enrichment factor. In practice, this is, however, not always feasible, which consequently leads to risks of carry-over between sample runs, unless special cleansing procedures are implemented. The difficulties associated with flow resistance can be alleviated to a certain extent by various approaches, including use of bidirectional flows during the sample loading and elution sequences [6]. Yet, the malfunctions of the sorbent surfaces themselves are not addressed by these means. Therefore, a superb alternative for eliminating problems associated with the changes of the surface properties of the sorbent materials and/or the creation of flow resistance in a column reactor is to employ a surface renewal scheme. That is, the contents of the packed column are simply renewed or replaced for each analytical run. Such a scheme is readily feasible in the so-called SIA–bead injection–lab-on-valve (SIA–BI–LOV) system [27,29–34]. However, in order to be operated in the SI–BI–LOV there are some stringent requirements of the solid-phase materials employed. They must be (a) perfectly spherical i.e., in the form of globe-shaped particles; (b) uniform in size distribution, falling within a range of 40–150 mm and (c) possess a density close to that of water. Basically, there are two types of beads which are applicable: hydrophobic and hydrophilic materials to which should be added materials which exhibit a combination of both characteristics. Figure 4 illustrates such an SIA–BI–LOV system, in which not only can sample be aspirated, but packed column reactors can also be generated by aspirating beads, with special surface characteristics, from a reservoir (the syringe on top). The reactors are, as shown on the right, prepared by fitting the column positions with appropriate restrictors, which will retain the beads, yet allow solutions to

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Figure 4 Diagram of an LOV system for bead injection (BI) incorporating two microcolumn positions (C1 and C2), along with a close-up of a packed renewable microcolumn. In order to withhold the aspirated beads within the column positions, they are each furnished with small PEEK rods, which allow solutions freely to flow, either along the walls or through the hole in the middle, but effectively retain the beads. Adapted from Ref. [32]. Copyright (2003), with permission from Elsevier B.V.

flow freely. Furthermore, one can take advantage of the fact that the beads are commercially available with various surface groups/properties, and can be manipulated exactly as liquids, whereby the beads can even be directed between different column positions within the LOV [31]. Appropriate eluents can be aspirated, and the eluate propelled to an external detection device (and all atomic spectrometric methods are applicable), sandwiched by air or liquid segments in order to preserve its integrity. After the assay, the beads can be reused or they can be discarded and new ones aspirated (i.e., the so-called renewable approach), depending on the circumstances. The operational principle used is illustrated in Figure 5 for the separation/ preconcentration of trace concentrations of metal ions by solid-phase extraction as facilitated via ion exchange by means of hydrophilic Sephadexs-type cation exchange beads and detection by ETAAS or ICP-MS [29]. Thus, firstly the column is packed with the column material. Then the sample is passed through the column, and the analyte is retained by the beads, while the matrix goes to waste. Thereafter, the retained analyte is eluted by a small, well-defined volume of eluent, which subsequently is transferred to the atomic spectrometer for quantification. In practice the implementation of the procedure is as follows (see Figure 4): Firstly, a fixed volume of sample solution is aspirated from port 5 and stored within the holding coil (HC), followed by aspiration of a small amount of beads suspension, usually 15–20 mL, which is initially captured in column position C1. Thereafter, the central channel is directed to communicate with column position C2, and while the syringe pump (SP) moves slowly forward, the beads are


385

Figure 5 The principle of sample pretreatment (preconcentration and separation) in the LOV approach via the use of packed column reactors for determination of metal ions. Firstly, the packed column is prepared, and then the sample solution is passed through the column. If a cation-exchanger is used, the metal ions are retained directly on the column, while if a hydrophobic column material is employed, the metal ions are initially reacted with a suitable ligand to form non-charged complexes, and the matrix goes to waste. Afterwards, the retained material is eluted by an appropriate eluent, and transferred to the detector, sandwiched by air segments for ETAAS or liquid segments for ICP-MS. Reprinted from Ref. [33]. Copyright (2004), with permission from Elsevier B.V.

transferred to C2 followed by sample solution, and separation/preconcentration occurs in this column position (if necessary, a washing step can readily be implemented). Then a fixed amount of eluent (usually 30–40 mL) is aspirated from port 1 and placed in HC, whereafter it is forwarded through C2, and the resulting eluate is eventually transported via port 4 into the detector using air-segmented flow (not shown). The reasons that the beads have to be manipulated between the two column positions, and that the eluent first has to be parked in the HC before being used to elute the packed column reactor, are that all external communications (pumping and aspiration) are effected by the SP, via the central communication line. When using ETAAS as the detection device, two different schemes for dealing with the analyte-loaded beads can be exploited [27,35]. Thus, in addition to the elution procedure just explained above, a novel and unique alternative approach, is to transport the loaded beads directly into the graphite tube, advantage being taken of the fact that the beads consist primarily of organic materials, and hence that they can be pyrolyzed, thereby allowing ETAAS quantification of the analytes. While the former approach can be used with ICP-MS as well, the latter is obviously not possible for this detection device.

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The use of hydrophobic beads as a means for separation/preconcentration of metal ions requires that the metal analyte ions be reacted with appropriate ligands to form non-charged or neutral chelates, which can be retained on hydrophobic surfaces. However, this approach entails some specific advantages, because by intelligent choice of the chelating reagent, increased selectivity can be obtained, and one can, again by playing on good chemistry, obtain higher tolerance for potentially interfering ions and eliminate inert ions in samples of high salt contents. A number of hydrophobic bead materials are applicable in LOV, including chemically modified poly(styrenedivinylbenzene) (C-18 Polysorbs) and PTFE (Teflons) . While the Teflons beads exhibit the best performance in terms of enrichment factor and retention efficiency, they are, however, somewhat difficult to manipulate because of their size, non-spherical morphology, higher density and uneven size distribution, and for the same reasons they must be aspirated as a slurry from an external, stirred reservoir [36]. As an alternative, Anthemidis et al. have successfully taken advantage of the qualities of Teflons by using turnings of the material in permanent column reactors in FIA systems [37,38]. In the on-line procedures the sample containing the target metal ion is usually mixed with the selected complexing reagent and the chelate formed is retained on the hydrophobic beads contained within a packed column reactor [33,39]. Following appropriate washing of the loaded beads, the chelate is then stripped out by a suitable eluent and the metal is determined in the attached detection device, generally FAAS, ETAAS, ICP-AES or ICP-MS. Although suitable in many instances, this approach might give rise to some problems, especially related to the kinetics of the formation of the chelate itself, of its concurrent retention on the bead surface and of its elution for final quantification. However, these problems can be solved in the SI-LOV approach by resorting to a combination of two schemes as outlined in Figure 6. Firstly, by implementing off-line pretreatment of the hydrophobic beads with the selected ligand, the conditions for the impregnation step can be readily optimized for factors such as the pH value and the time frame for strong sorption of the organic ligand onto the hydrophobic surfaces. The experimental conditions for complexation/retention of metal species by the reagent-loaded sorbent, can again be manipulated (e.g., for pH) so that they are optimal for on-line operation. The advantage is that kinetic problems associated with chelate adsorption can hereby be vastly reduced or even eliminated. Secondly, in the elution step, where the chelate is eluted from the hydrophobic surfaces, there are two further options, because it does not matter how the stripping process actually occurs. Thus, whether it involves the release of the whole complex as such, or a splitting up of the complex (where the ligand might either remain on the bead surface or dissolve into the eluent medium), or as a combination of both, is of no concern, because in the LOV configuration the beads can readily be renewed for each measurement cycle. This is why this pretreatment scheme using beads has also been termed the universal approach, because whatever the ligand is that can be adsorbed, it is applicable, irrespective of the kinetics involved and the elution protocol needed for the final quantification.


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Figure 6 The concept of the universal approach, where the hydrophobic beads initially are preimpregnated off-line with the selected ligand, advantage being taken of operating under optimal reaction conditions to affix the ligand. These pretreated beads are then used for on-line retention of the analyte metal species, the elution and subsequent determination of the metal being unaffected of the mechanisms involved in the liberation of the retained chelate, because the beads are renewed for each sample cycle. Reprinted from Ref. [34]. Copyright (2007), with permission from Elsevier B.V.

A very good example to demonstrate this approach is the determination of Cr(VI) using spherical, hydrophobic beads consisting of poly(styrenedivinylbenzene) containing pendant octadecyl moieties (C18-PS/DVB) pre-impregnated off-line with 1,5-diphenylcarbazide (DPC) [40]. Although the determination of Cr(VI) with DPC is a well-known and a widely used procedure in batch assays, it was found virtually impossible to implement the chemistry on-line with the naked hydrophobic beads. This is because the rate-limiting step is actually the adsorption of the ligand onto the bead surface, which is a very slow process. Experimentally, it was thus observed that it takes ca. 30 min in a 5% (v/v) methanol/water medium to be accomplished, as revealed by following the progressively more intense reddish colour attained by the beads. Therefore, it is evident that the use of pre-impregnated beads in the LOV microconduits is particularly advantageous for this application. The reaction between Cr(VI) and DPC is actually rather complex as shown below:

2CrO42- + 3H4L + 8H+

2[Cr(VI)-H4L]

(beads)

(beads)

(3)

]+

[Cr(HL)2 (beads)

+

Cr3+

+ H2L + 8H2O

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Figure 7 LOV manifold used for determination of Cr(VI) by solid-phase extraction of Cr(VI) via reaction with diphenylcarbazide (DPC) using hydrophobic DPC-loaded C18 beads. For explanatory details, see text. The figure shows that step where sample solution (propelled by syringe pump SP1) and pH-adjustment reagent (propelled by syringe pump SP2) are merged and transported to column position C2, where the target species is retained by the DPC immobilized on the contained beads. HC, holding coil; PP, peristaltic pump. Adapted from Ref. [40]. Copyright (2005), with permission from the American Chemical Society.

First a complex is formed between the Cr(VI) and the carbazide (H4L) affixed on the pre-impregnated beads, in which the Cr(VI) oxidizes the carbazide to carbazone (H2L), which in turn results in half of the generated Cr(III) being complexed by the immobilized carbazone, and hence retained on the beads, while the other half is wasted. Figure 7 shows the LOV manifold used for the actual analytical procedure. In this particular application, the beads are loaded with a merged stream consisting of the sample and of a pHadjustment reagent (1 M HNO3) in order to facilitate the reaction, which must take place in acid solution. However, the pH should not be excessive, because this will influence the quantitative binding ability of the beads for the target metal species. Besides, too high an acidity might also cause interconversion of Cr(VI) to Cr(III) in the presence of dissolved organic matter [40]. As explained above, the loaded beads are eventually transported to column position C2, allowing subsequent dissolution of the retained material with a defined volume of eluent. Regarding the separation and preconcentration of trace levels of metal ions by adsorption on suitable sorbent materials following on-line dynamic derivatization, it has been found that in many instances a continuous forward-flow system is not appropriate because a certain delay time has to be implemented, allowing the reaction sufficient time to generate the complex, which in turn, is adsorbed on the solid-phase bead material [41]. This is for instance the case in the determination of trace levels of Ni in brines with dimethylglyoxime using a hydrophobic/hydrophilic (ca. 50/50) copolymeric sorbent (poly(divinylbenzene-co-N-vinylpyrrolidone)).


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Figure 8 Schematic diagram of the SI-LOV-ETAAS system for on-line determination of Ni(II) via complexation with DMG and preconcentration on N-vinylpyrrolidone-divinylbenzene beads. Carrier, 0.2 mol L1 ammonium citrate buffer (pH 9.0); DMG, 1.2% (w/v) dimethylglyoxime in ethanol; Eluent, methanol; SP1 and SP2, Syringe pumps 1 and 2; C1 and C2, LOV micro-column positions; HC, Holding coil; RC, reaction coil; Pump, Peristaltic pump. Reproduced from Ref. [41]. Copyright (2006), with permission from Springer-Verlag.

For the very same reason, an LOV manifold such as the one shown in Figure 8 is used, where an external reaction coil (RC) is attached to one of the peripheral ports of the valve, and flow programming is exploited for accommodation of stopped-flow approaches. In fact, not only of the combined sample and reagent within the RC to promote the complex formation, but also during elution of the Ni(DMG)2-loaded beads entrapped within C2 in order to obtain a quantitative yield. The use of an external RC for conducting a necessary chemical operation has also been reported for the fully automated speciation analysis of Cr(III) and Cr(VI) at trace levels using a single hydrophilic microcolumn, namely, a polysaccharide material with covalently immobilized iminodiacetate moieties, that can complex and retain Cr(III) ions. This approach involves, as shown in Figure 9, the direct determination of Cr(III), with the total concentration of Cr(III) and Cr(VI) being subsequently quantified via on-line reduction of Cr(VI) to Cr(III) [42]. The on-line reduction is effected by on-line merging of the sample zone with hydroxylamine, yet although this was the optimal reagent of a series of reductants assessed (e.g., ascorbic acid and hydrogen sulfite), it reacted rather slowly, requiring approximately 4 min for accomplishment of an acceptable reduction yield. However, as the detector used is ETAAS, this delay time does not impair the sample throughput, because while the Cr(VI) contained in an aspirated aliquot of sample is reduced to Cr(III) in the external RC under

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Figure 9 Flow chart of the LOV procedure to which the Cr(III) and the Cr(VI) species, present in the original sample solution, are subjected. While the Cr(III) ions are separated/ preconcentrated on the chelating Sepharose beads and subsequently eluted and quantified by ETAAS, the Cr(VI) ions are reduced to Cr(III) by hydroxylamine (in an open reaction coil, as shown in Figure 8), and afterwards treated as the native Cr(III) ions. Reprinted from Ref. [42]. Copyright (2005), with permission from the Royal Society of Chemistry.

stopped-flow conditions, the indigenous Cr(III) can, after preconcentration on the beads and separation from the matrix constituents and subsequent elution, be determined through the ca. 4 min long temperature program of the graphite atomiser. When the measurement is completed, the reduced sample is ready to be subjected to the same treatment, and the total Cr-content quantified. Again, by playing on the proper timing, all reactions can be individually optimized, and the analytical cycle be greatly accelerated, regardless of the type of reagent-based assay. Alternatively, and taking into account the different nature of both oxidation states, selective sorptive preconcentration of both Cr(III) and Cr(VI) might be accomplished by using chelating and anion-exchange resins, respectively [43,44]. A rather unique on-line separation/preconcentration approach is possible when using ETAAS, namely to place a microcolumn packed with solid-phase material within or at the tip of the robotic arm of the autosampling device [45,46]. Since the column is stationary, it however suffers the shortcomings of conventional packed columns as outlined above.


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3.3 Precipitation/coprecipitation and sorption onto knotted reactors Since they were first employed in FIA for on-line collection of coprecipitate without filtration [47] and on-line sorption of neutral metal complexes [48], KRs have been widely recognized as trouble-free and very efficient collecting media. On-line collection is facilitated by the secondary flow patterns created in the KR [49]. If the precipitate is hydrophilic, the KR is made of a hydrophilic material (such as ethylvinylacetate or Nylon), whereas if the generated precipitate is noncharged, a hydrophobic KR material is employed (usually Teflons). A general review of the combination of FIA and KRs in conjunction with atomic spectrometric techniques has been published by Cerutti et al. [50]. An example of employing a hydrophilic KR is presented in the section focused on HG, while the use of a hydrophobic KR, e.g. with detection by ICP-MS, is more closely examined here. There are two main approaches to execute on-line KR sorption/retention preconcentration followed by ICP-MS detection. In most cases, the preconcentration is achieved through on-line merging of the sample solution and a complexing, precipitating or coprecipitating reagent, followed by the sorption/ retention of the neutral metal chelates or the precipitate/coprecipitate on the interior surface of the KR. After a washing step, the accumulated material is eluted or dissolved with an appropriate eluent. By using this approach, APDC [51–53] has been used for on-line flow injection formation and sorption of neutral metal complexes, while diethyldithiocarbamate (DDTC) [54,55] and ammonia buffer [56] have been employed as reagents for collecting precipitates of transition metals and rare earth elements. Another approach, which in practice is only effective for on-line sorption of metal complexes, is to precoat the complexing reagent directly onto the interior surface of the KR, followed by sample loading and thus executing the separation/preconcentration process. After a washing step, the analyte is eluted and ultimately transported into the ICP-MS. This approach has so far only been employed for on-line flow injection separation and preconcentration of rare earth elements with precoated 1-phenyl-3-methyl-4-benzoylpyrazol-5-one (PMBP) [57], but the figures of merits reported appear to indicate an improved sensitivity and overall efficiency compared to that of on-line merging of the sample and the reagent solutions. As to the molecular sorption schemes, the neutral metal complexes adsorbed onto the interior surface of the KRs are often eluted with organic solvents, such as IBMK, ethanol and methanol. To eliminate the adverse effects of organic solvents on the ICP-MS measurements, it is therefore compulsory either to employ an ultrasonic desolvation nebulization system [51,57], or, to use mineral acid, preferentially nitric acid, as eluent [52,57]. For neutral metal complexes adsorbed onto the PTFE surface, the last option is, in many cases, fully satisfactory [58]. However, with on-line precipitation or coprecipitation systems, the retained precipitates/coprecipitates on the interior surface of the KRs are generally dissolved with nitric acid of appropriate concentrations [54–56], which can

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afterwards be introduced directly into the ICP-MS instrument for quantification. This approach has, however, so far not been much employed for coupling to ICP-MS. The open-ended KRs possess the clear advantage of low hydrodynamic impedance, and thus allow high sample loading flow rates for obtaining better enrichment factors [49]. A limiting factor in using KRs as collecting media are their low retention efficiencies for most metal complexes, which restricts the actual preconcentration capability. This drawback can be eliminated by employing a bead-packed column [58] as a sorption medium instead of using a KR, as discussed in the section above. To this end, Wang et al. [59] used octadecyl chemically modified beads as collecting media for determination of ultra-trace levels of Cd(II) by adsorption of precipitated cadmium hydroxide with further elution by an acidic eluent solution.

3.4 Gas–liquid separation: hydride generation and vapour generation Several elements, such as As, Sb, Bi, Se, Te, Pb and Ge can, by reaction with a strongly reducing agent, such as sodium tetrahydroborate, become chemically converted to their hydrides, as shown by Equation (4). Gaseous hydrides can be readily separated from the sample matrix and guided to a heated quartz flow-through cell of an AAS instrument, where they are atomized by heating and excited by radiation, so that the elements of interest can be selectively quantified (Equation (5)). Originally, the HG technique was introduced as a batch procedure, but this involved several problems, as illustrated in Equations (6)–(8). The conversion of the analyte itself must necessarily take place in acidic media (Equation (6)). However, there are possibilities for side reactions and interferences (Equations (6)–(8)). The tetrahydroborate itself can react with acid and form hydrogen (Equation (6)), whereby the reagent is wasted for the hydride formation. Therefore, the tetrahydroborate must be prepared in a weakly alkaline medium and mixed with the sample and the acid precisely when it is required, and under very controlled conditions. A serious possibility for interference is the presence of free metals, particularly of Ni, Cu and Co. Hence, if ionic species of these metal constituents are present in the sample, they become reduced by the tetrahydroborate, giving rise to the formation of colloidal free metals (Equation (7)), which have been shown to act as superb catalysts for degrading the hydrides before they can reach the measuring cell (Equation (8)).

Hydride generation/atomization − BH4 ↑ ↑ ↑ As3+, Sb3+, Sn4+ → AsH3 , SbH3 , SnH4

(4)

Acid(HX)

Δ AsH3, SbH3, SnH4 ⎯⎯ → As, Sb, Sn + nH2

(5)


Side reactions/interferences BH4- + 3HX + H+ → BX3 + 4 H2

↑

393

(6)

−

Me2+

(Ni, Cu, Co)

BH4

→

Me0(slower) Acid(HX) 0

AsH3, SbH3, SnH4 ⎯Me ⎯ ⎯→ As, Sb, Sn + nH2

(7)

(8)

However, because of the dynamic conditions prevailing in the flow system, and because of the inherently short residence time of the sample in the manifold, these side reactions can to a large extent be eliminated or kinetically discriminated against at the expense of the main reaction. If side reactions do occur, the precise timing of the flow system ensures that they take place to exactly the same extent for all samples introduced [60]. A concrete example will readily ˚ stro¨m [61] found that it was totally impossible to illustrate this: In his work, A determine minute quantities of Bi(III) (25 mg L1) in the presence of 100 mg L1 Cu(II) in a batch system, because the hydride formed was degraded before it could reach the detector. However, when implementing the very same analytical procedure in an FIA system it was perfectly feasible. In fact, it yielded close to 100% response, i.e., the interference due to Cu was practically eliminated. Similarly, it has been shown that As could readily be determined in extracts from polluted soils containing As, Cr and Cu (remnants from facilities impregnating wooden materials) without any problems even in the presence of the elevated concentrations of Cu. There are two main approaches for obtaining the hydride on-line. The most widely employed one is the chemical HG scheme by using a suitable reductant such as sodium tetrahydroborate in an acidic medium. This approach has, so far, been applied extensively for on-line flow injection separation/preconcentration of hydride-forming elements in various sample matrices [62–64]. A second means is the on-line electrochemical hydride generation (EcHG) technique, whereby the hydride is generated on-line at an electrode by application of an appropriate electrolysis current [65]. Both schemes can completely eliminate the matrix components, and they are thus most favourable for the analyses of hydrideforming elements in biological samples, where serious matrix effects and consequently signal suppression very often are encountered. Some elements possess at room or at elevated temperatures sufficiently high vapour pressure to be determined by AAS, AES or AFS. The most notable is Hg, which has given its name to a specific procedure, viz.; cold vapour AAS (CVAAS), where the higher oxidation states of the element are reduced by strong reductants such as tetrahydroborate or Sn(II), followed by sweeping the released Hg into the AAS by an inert carrier gas such as Ar or N2. Preconcentration may be accomplished by incorporating a trap of gold-wire, which will retain the Hg as amalgam [66]. When heated after an appropriate sample loading time, the Hg is released. Cadmium, zinc, gold or volatile organometallic species may also be

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quantified by vapour generation (VG) following appropriate sample processing (e.g., chemical reduction) [25,63]. In these instances, the formed gases are directed to continuously operating detectors such as AFS or quartz-tube AAS [67] or alternatively, trapped-in-analyser in a graphite tube of an ETAAS and followed by atomization [68]. The successive sequestration of vapours in the graphite tube provides not only an efficient means to preconcentrate the analyte, but also an elegant way to eliminate the matrix components. The very same approach of sequestration can be used for hydrides as well, by adjusting the temperature so that it is high enough for the decomposition of the hydride, but not sufficient to atomize the analyte. In addition, an appropriate coating on the interior surface of the graphite tube can offer much improved performance [69,70]. Various materials have been investigated to precoat the atomiser, including Pd, Zr, Ir, Au and Nb–Ta–W–Ir/Mg–Pd/Ir. A precoated graphite tube can be used for several hundreds of trapping-atomization cycles [71], with conditions varying according to the trapping temperature and the sequestrating time. However, this approach of repeated in-atomiser trapping is inherently very time-consuming. The interfacing of a flow system with an on-line hydride/vapour generation system to ICP-MS is readily feasible. The compatibility between the two set-ups allows the hydride/vapour to be introduced directly into the ICP by using an argon flow [63,72]. In many cases, however, the sensitivity achieved by direct introduction of the hydride/vapour into the ICP is not sufficient. This can be overcome by employing electrothermal vapourization (ETV) after on-line hydride/vapour generation, i.e., the hydride/vapour is successively sequestrated by in-atomiser trapping in a precoated graphite tube as in the case of ETAAS determination [73] in order to reach a certain preconcentration level, whereupon the trapped analytes are atomized and introduced into the ICP for quantification. It should, however, be mentioned that although the in-atomiser sequestration procedure might appear attractive, it is, from an operational point of view, more preferable to effect all the manipulations on-line in a single cycle using solidphase extraction protocols, but to separate the preconcentration part of the procedure from that of the hydride/vapour generation part [74,75]. Being readily feasible using an FIA system, this approach renders an extra degree of freedom, because it allows not only selection of the preconcentration chemistry completely independently of the HG/VP scheme used, but also the attainment of much higher enrichment factors using considerably shorter sampling times. An example of such a system is depicted in Figure 10, which was used for the determination of ultra-trace levels of selenium(IV) [76], where the analyte was preconcentrated via coprecipitation with lanthanum hydroxide. Thus in (a), which shows the system in the ‘‘fill’’ position, the sample is aspirated by pump P1 and mixed on-line with buffer and La(III) coprecipitating agent. The coprecipitate generated is entrapped in the KR. In (b), where the system is in the ‘‘inject’’ position, valve V is switched over, permitting the eluent, HCl, to pass through the KR to dissolve and elute the precipitate and forward it to mixing with the reductant, NaBH4, which leads to the formation of the hydride. The hydride is separated from the liquid matrix in the gas–liquid separator (SP) and


395

FILL-POSITION QTA (900°C)

Argon

SP

RC W1 NaBH4

W2

KR MC

HCI S

ON P1

W3 V

La Buffer

ON P2

(a)

INJECT-POSITION QTA (900°C)

Argon

SP

RC W1 NaBH4

KR MC

W2

HCI S

ON

W3

P1 V La Buffer (b)

OFF P2

Figure 10 Schematic diagram of the flow injection–hydride generation–atomic absorption spectrometry (FIA–HG–AAS) system for on-line coprecipitation–dissolution of selenium or arsenic. (a) Coprecipitation sequence; and (b) dissolution and detection sequence. For details, see text. Reprinted from Ref. [76]. Copyright (1996), with permission from the Royal Society of Chemistry.

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subsequently by means of an auxiliary stream of argon gas guided to the heated quartz tube atomization cell (QTA) of the AAS instrument. An interesting combination of MSFIA, LOV and HG coupled to an AFS detector has been described by Long et al. [77]. This work exploited the LOV for the separation and preconcentration of As, and an MSFIA flowing stream network for on-line post-column derivatization of the eluate from the LOV and for communication to the detector. Thus, by employing quantitative preoxidation of As(III) to As(V) in the samples by means of permanganate, the method involved the preconcentration of arsenate at pH 10 on a renewable anion exchanger, namely Q-Sepharose, packed in an LOV microcolumn. The analyte species was afterwards stripped out, and concurrently prereduced by an eluent plug containing 6 mol L1 HCl and 10% KI. The eluate was merged downstream with a metered volume of sodium tetrahydroborate for generation of arsine, which was subsequently quantified by AFS. The flow system facilitated oncolumn reduction of the retained arsenic with no need for application of programmable stopped-flow. However, the high concentration of reductant and extreme pH conditions required for elution limited the reuse of the sorbent because of the gradual deactivation of the functional moieties, and therefore the maximum benefit could be derived from the application of the bead disposal/ renewable strategy. The proposed procedure is characterized by a high tolerance to metal species and interfering hydride-forming elements. In fact, ratios of Se(IV) to As r5,000 and Sb(V) to As r500 are tolerated at the 10% interference level.

3.5 Membrane-based separations The coupling of flow-through membrane-based separation processes, including dialysis, gas-diffusion and pervaporation with atomic spectrometric detection has been utilized for on-line isolation of low-molecular-weight compounds and volatile species from interfering macromolecules, colloidal matter and suspended particles. Thus, both solid substrates and slurries can be directly processed in flow systems with no further hindrance. Sandwich-type units involving the incorporation of a flat sheet membrane between two machined plates are the preferred configuration for implementation of the unit operations in flow systems. Concentric or linear arrangements with tubular membranes have been also employed, especially for construction of dynamic headspace devices and microdialysis probes [78]. Strict kinetic control of the transfer process in flow systems allows operation under non-steady-state conditions, thus implying that a minor, but reproducible, fraction of the analyte is transferred across the membrane [79]. Flow-through passive dialysis has actually been exploited as an on-line sample dilution technique for monitoring alkaline, alkaline-earth and transition metals in industrial effluents [80,81] and plant digests [82] with detection by FES and FAAS. Depending on the intended application, flow dialysis can be tuned not only to obtain a high degree of sample dilution but to give almost complete recovery of the analyte or significant removal of interfering species by application of sample


397

recirculation strategies and trapping of diffused analytes onto ion-exchange resins. This was demonstrated by the determination of trace elements in serum samples using isotope-dilution ICP-MS [83]. In contrast to passive dialysis, Donnan or active dialysis capitalizes on the use of permanently charged membranes. As a result of the electrostatic interactions between the sample components and the ionogenic moieties of the membrane [84], it offers analyte preconcentration capabilities and improved selectivity for ionic species. Typical applications are trace metal enrichment followed by FAAS and ICP-AES detection [84,85]. Microdialysis is a specialized application of dialysis used traditionally in neurochemistry and pharmacokinetic studies for dynamic monitoring of extracellular chemical events in living tissues. On-line microdialysis sampling coupled with ETAAS or ETV-ICP-AES has been recently reported for in vivo monitoring of trace metal ions in brain extracellular fluid [86] and cell suspensions [87]. The (micro)dialysis concept can be expanded beyond its current applications, since (micro)dialysers have recently been employed as analytical tools for automatic microsampling and monitoring of the bioavailability of transition metals from soils and foodstuffs using fractionation schemes or simulated gastrointestinal digestion [88,89]. Gas-diffusion/pervaporation approaches are based on diffusion of volatile species across a microporous, hydrophobic membrane. Since only relatively few compounds are sufficiently volatile at room/mild temperatures, these techniques are associated with a high degree of selectivity enhancement [90], which has been exploited for the speciation analysis of inorganic and organic mercury compounds with no need for chromatographic separations [67]. Coupling of pervaporation with on-line chemical derivatization and detection by AFS and AAS has also proven effective for speciation analysis of inorganic arsenic [91] and mercury species [92] in particle containing samples and environmental solids.

4. FLOW SYSTEMS AS FRONT-END VEHICLES FOR ON-LINE PROCESSING OF SOLID SAMPLES Though originally conceived for wet chemical analysis, flow-based systems have proven themselves to constitute excellent vehicles for automated pretreatment of solid samples of diverse origin, namely: soils, sediments, sludges and foodstuffs. These materials can be contained in dedicated microcartridges and hyphenated to AAS or ICP-AES/MS detectors. Several authors have exploited on-line leaching schemes aided by auxiliary energy devices, such as microwaves [93] or ultrasound [94], aimed at expediting the complete dissolution of the solid substrate and determining the total concentration of a given target metal species [95]. In the environmental field, however, the impact of metal ions on biota cannot be evaluated by measuring merely the total concentration of individual species, because the mobility, bioavailability and the eventual impact of anthropogenic metal ions on ecological systems largely depends on their

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chemical forms and types of binding [96]. Accordingly, analytical attempts to support this research field have undergone considerable changes during the past decades, and a multitude of novel procedures and instrumentation have been developed to determine specific chemical forms of elements via application of sequential extraction (fractionation) protocols. In fractionation studies, the environmental solid is sequentially subjected to various leaching solutions, which because of their composition, provide information on the potential bioavailability of the analytes present in the sample. The analytes might typically be microconstituents (such as metals or metalloids) or macronutrients. Thus, by using operationally defined and internationally accepted leaching agents of increasing aggressiveness, it is possible to distinguish between fractions such as the ‘‘exchangeable’’, ‘‘acid-soluble’’, ‘‘reducible’’ and ‘‘oxidizable’’ metal species, and thus assess the potential bioavailability and toxicity of anthropogenic agents. Traditionally such extraction schemes have been implemented by batch procedures, which are very tedious, but also do not mimic the dynamic conditions in the environment. All these manual procedures are based on the establishment of equilibrium between the solid and the liquid phases, but suffer from the inherent drawbacks of element readsorption and redistribution between phases during extraction. More importantly, these schemes provide information solely on the averaged concentration of leachable metals in each extractant. Hence, knowledge is lost as to the kinetics of metal release, which otherwise could assist in the interpretation of elemental associations in the various soil compartments [97]. Therefore, it is preferable to effect such schemes in a continuous-flow/flowinjection fashion, by incorporation of a solid sample-containing cylindrical microcolumn into the flow network [98–102]. Not only does this approach more realistically imitate how the leaching occurs in natura, but it also yields a detailed insight into the extraction processes through the recording of the extractograms, i.e., the representation of the amount of extracted trace elements versus time or leachant volume [97]. In this context, the sequential injection approach presents itself as a very attractive alternative to FIA set-ups for on-line processing of solid substrates prior to presentation of the analyte species to the detector [103]. Actually, by clustering the sample container at a peripheral port of the multiposition selection valve and exploiting programmable flow, it is feasible to conduct uni-, bi-, multi-bi-directional and even stopped flow-based extraction protocols with the subsequent mitigation of backpressure or clogging effects due to solid-phase compaction [104], which are frequently encountered in continuous-flow or uni-directional flow-injection fractionation manifolds. One of the major limitations of miniaturizing these fractionation schemes is the risk that the extracted samples may be poorly representative of the bulk sample. To tackle this shortcoming, a specifically designed and dedicated dual-conical microcolumn has been recently devised [103]. In contrast to cylindrical containers which solely admit small samples, typically o50 mg [98–100], the dual-conically shaped column is able to accommodate substrate amounts up to 300 mg without undue pressure increase, and additionally features fluidized bed-like conditions that result in appropriate mixing between sample and extractant [102,105].


399

Direct coupling of flow-through microcolumn approaches with FAAS, AFS, ETAAS or ICP-AES/MS for assessment of the most ecotoxicologically significant forms of trace metals, the so-called water-soluble, exchangeable and mild acidsoluble fractions, has two major limitations: (i) it is merely applicable to highly contaminated substrates because the concentration of such metal forms in moderately polluted solids is mostly below the detection limit of the spectrometer; and (ii) the reliability and accuracy of the determinations are strongly dependent on the magnitude of the spectral and non-spectral interfering effects caused by the sample matrix itself and/or the high electrolyte content of the extracting reagent. To circumvent the above drawbacks, appropriate on-line treatment of the sample extracts prior to detection is therefore called for, and maximum benefit can be taken from the concept of SIA-LOV with renewable solid-phase extraction in the bead-injection fashion. Thus, the third generation of flow injection, that is, LOV, can be regarded as a promising tool for on-line soil/ sediment fractionation with automated matrix isolation and concomitant analyte preconcentration [106], as exemplified here by the accurate monitoring of easily mobilized hexavalent chromium in soil environments at the sub-low parts-permillion level. The microflow arrangement, which is shown schematically in Figure 11, integrates dynamic leaching of Cr(VI) using deionized water or artificial acid rain as single extractants; on-line pH adjustment of the extract to

Figure 11 Schematic diagram of the SI–BI–LOV–ETAAS system for dynamic fractionation of Cr(VI) in environmental solids. The Cr(VI) released from the soil is aspirated into the holding coil (HC) and subsequently mixed with the pH-adjustment reagent (Tris-HNO3 buffer at pH 8.0) and transported with the previously aspirated beads (Q-Sepharose; strong anionexchanger) into column position C2, where preconcentration/separation takes place. Afterwards the beads are eluted by 40 mL of 0.5 mol L1 NH4NO3/NH4OH buffer (pH 8.0) and the eluate, via air-segmentation, is transported to the detector (ETAAS). CC, central communication channel. Reprinted from Ref. [106]. Copyright (2006), with permission from the Royal Society of Chemistry.

400


minimize undesired Cr(VI)/Cr(III) interconversions in the slightly acidic medium of the aqueous extractants; isolation and preconcentration of the chromate leached from the matrix constituents and reagent medium onto strong anion-exchange beads (Q-Sepharose) freshly packed into the microconduits of the LOV assembly; air-segmented elution of the sorbed species which are detected by ETAAS and finally withdrawal of the used beads for each step of the multiple extraction protocol to overcome progressive sorbent deterioration and the influence of irreversible interferences from the soil matrix. In this configuration, the upright disposition of the microcolumn is intended to locate the entire substrate in the lower conical cavity of the container for facilitating fluidized bed-mixing conditions during the progressive outward pumping of the leaching reagent through the packed column as well as to strip quantitatively the extractant out of the moistened solid whenever the solution is pulled back toward the valve by the reverse motion of the SP. In Figure 12, an example of a multiple-step dynamic extraction profile is depicted for the assessment of the readily bioavailable content of Cr(VI) in soils as obtained by extracting a moderately polluted soil material (SRM 2709) and

Figure 12 Extraction patterns of readily bioavailable Cr(VI) in San Joaquin Soil (SRM 2709, Baseline Trace Element Concentrations) and spiked soil samples as obtained from the SI–BI– LOV microcolumn system using mild extractants, namely deionized water and simulated acid rain. Soil amount, 100 mg; subfraction volume, 500 mL; Spike 1, 5.0 ng g1 Cr(VI); Spike 2, 8.0 ng g1 Cr(VI). Reprinted from Ref. [106]. Copyright (2006), with permission from the Royal Society of Chemistry.


401

various spiked batches with mild extractants. For this particular case, the progressive acidification of the extraction media did not increase the leachability of soluble Cr(VI) from the sample, which is attributed to the efficiency of distilled water for quantitative removal of soluble (surface-bound) chromate in an on-line dynamic mode, and the ability of the soil material to raise the pH of the applied extractant, thereby precluding the additional release of sparingly soluble forms of Cr(VI). The potential extension of the SI analyser for speciation/fractionation of Cr(VI) and other anthropogenic metals in highly polluted samples has also been assessed by using the miniaturized unit as a front end to FAAS rather than ETAAS. Despite the continuously operating nature of the detection instrument and the discontinuous flow inherent in SIA operation, hyphenation between both set-ups can be easily realized by interfacing a rotary injection valve for continuous injection of the extracts into the FAAS nebulizer stream [105]. As a result of the flexibility of the SIA–LOV–AAS coupling, environmental solids with variable amounts of available trace elements ranging from the sub-mg kg1 to the mg kg1 level, i.e., above the maximum permissible concentrations for agricultural use, may be automatically treated and further analysed in the fully enclosed flow assembly.

5. HYPHENATION WITH ATOMIC SPECTROSCOPIC DETECTORS Hyphenation in chemical analysis is a colloquial term describing the combination of different techniques or approaches used for front-end sample processing. Hyphenation is required in order to subject the sample to appropriate pretreatments so that the detector(s) used can identify the individual analyte species with suitable selectivity and sensitivity. As atomic spectroscopic detection devices only can discriminate elemental rather than molecular species, such pretreatment schemes typically entail physical/temporal separation of individual compounds via chromatographic techniques for speciation purposes as demanded by current metabolomic research. This might also involve the separation of the analytes from potentially interfering matrix constituents, the execution of sample preconcentration protocols or a combination of all strategies. And here the various generations of the FIA systems present themselves as superb vehicles to implement these procedures on-line [7,107], not the least because they are totally automated and fully computer-controlled. This is exactly what the authors of this chapter have attempted to illustrate with the examples given. Thus, with the SIA or LOV systems one has the advantage of being able to attach external units to facilitate individual operations, such as exploiting an extraction microcolumn for fractionation/extraction of target metals from soils, sediments or edible matrices, or a chromatographic column for spatial separation of individual components in a given sample, or for applying external energy sources such as UV light, or ultrasound or microwave radiation in order to effect desired reactions. But most importantly, a proper hyphenation will allow the implementation of intelligent chemistries, where

402


advantage can be taken by the fact that the on-line systems operate under dynamic conditions so that one can exploit the interplay between thermodynamics and kinetics. The kinetic discrimination schemes are superb examples of this, as used for instance in the HG procedures. So too, is the use of the stopped-flow approach in order to gain sufficient reaction/delay time or to obtain quantitative extractive yield of the analyte retained on incorporated column reactors (see Section 3.2). The various combinations to be applied are numerous, and the road for potential usage is wide open. Thus, what is needed in future applications is a realization of what this interplay between thermodynamics and kinetics implies, particularly in terms of facilitating determination of individual chemical species, because this is exactly what differentiates the on-line systems from their batchwise counterparts. Therefore, the authors of this chapter are convinced that the future will bring novel and interesting applications, similar for instance to the one mentioned in Section 3.4, where LOV and MSFIA were combined, not only to facilitate the chemistries necessary, but to lower the amount of chemicals required, thus generating ‘‘green’’ chemical approaches. However, these requirements of modern analytical chemical procedures present challenges to chemists to demonstrate ingenuity and apply their chemical knowledge in an intelligent way, and not merely resort to looking at the FIA systems as means for convenient transport and the atomic spectroscopic instruments as simple detection devices. And that will be the real quest for the chemists in the future.

ABBREVIATIONS AFS APDC BI CSAAS CVAAS DDTC DIHEN EcHG ETAAS ETV FAAS FAES FF-AAS FIA HC HG IBMK ICP-AES ICP-MS IP

Atomic fluorescence spectrometry Ammonium pyrrolidinedithiocarbamate Bead injection Continuum source atomic absorption spectrometry Cold vapour AAS Diethyldithiocarbamate Direct-injection high-efficiency nebulizer Electrochemical hydride generation Electrothermal atomic absorption spectrometry Electrothermal vapourization Flame atomic absorption spectrometry Flame atomic emission spectrometry Flame furnace atomic absorption spectrometry Flow injection analysis Holding coil Hydride generation Isobutylmethylketone Inductively coupled plasma atomic emission spectrometry Inductively coupled plasma mass spectrometry Infusion pump


IV KR LOV MCFIA MSFIA PMBP QTA RC SIA SIMAAS SL UV/VIS VG

403

Injection valve Knotted reactor Lab-on-valve Multicommutated flow injection analysis Multi-syringe flow injection analysis 1-Phenyl-3-methyl-4-benzoylpyrazol-5-one Quartz-tube atomization cell Reaction coil Sequential injection analysis Simultaneous atomic absorption spectrometry Sample loop Ultraviolet/Visible spectrometry Vapour generation

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CHAPT ER

15 Vibrational Spectrometry Sergio Armenta, Salvador Garrigues and Miguel de la Guardia

Contents

1. A Short Note on the Evolution of Flow Injection Analysis in Recent Years 2. Vibrational Techniques as Detectors in Flow Injection Analysis 3. Scientometric Evolution of Vibrational Spectrometry in Flow Injection Analysis 4. Objectives 5. Infrared Spectrometry 5.1 Mid-IR 5.2 Near-IR 6. Raman Spectrometry 7. Concluding Remarks and Outlook Abbreviations Acknowledgments References

407 408 408 409 410 410 426 430 435 435 436 436

1. A SHORT NOTE ON THE EVOLUTION OF FLOW INJECTION ANALYSIS IN RECENT YEARS From the pioneering study of Ruzicka and Hansen in 1975 the interest in flow injection analysis (FIA) techniques has been exponentially increasing because of the advantages offered in automated sample processing, high repeatability, adaptability to miniaturization, containment of chemicals, waste reduction and reagent economy in systems that operate at microlitre levels. Additionally, by controlling the timing FIA allows exploitation of the kinetic aspects of chemical reactions in order to increase the selectivity of the assay. As a result of growing environmental demands for reduced consumption of samples and reagent solutions, in 1990 the first generation of FIA was supplemented by the second generation, called sequential injection analysis Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00615-6


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(SIA) [1] in which precisely metered zones of samples and reagents are aspirated by means of a syringe pump and using a multiposition valve, stacked in a holding coil, and then finally forwarded to a suitable detector. The advantage of SIA over traditional FIA is that it strongly reduces the consumption of reagents and in consequence minimizes the wastes, but one of its disadvantages is that it tends to run slower than FIA. The technique known mainly as multicommutation is an emerging approach based entirely on the manipulation of solutions by solenoid valves [2]. It is based on the alternate introduction of small solution segments by controlling (through a computer) the opening and closing times involved in the passage of the samples or reagents. Other approaches like the so-called lab-on-valve or bead injection have been introduced [3], allowing a downscaling of operations and the on-line incorporation of a number of solid-phase sample pretreatments. New developments based on the use of pinch valves [4] or minipumps [5] have also contributed to the continuous enhancement of flow analysis procedures. So, nowadays laboratories can use a large number of different flow approaches to automate or to mechanize their analytical methodologies.

2. VIBRATIONAL TECHNIQUES AS DETECTORS IN FLOW INJECTION ANALYSIS Vibrational techniques, especially those based on the use of Fourier transform, can be used successfully for detection in flow systems because they offer: (i) fast monitoring of the whole spectrum; (ii) high resolution and a wide wavenumber working range; (iii) many bands which can be employed for the determination of a single compound; (iv) simultaneous determination of several compounds in the same sample and (v) possibility for elimination of band overlapping through the use of simple strategies like derivative spectrometry or the modelling of the whole signal by multivariate calibration. In spite of the aforementioned advantages, it must be recognized that nowadays flow analysis–vibrational spectrometry is a mature analytical technique but the available applications are far from the level that it merits.

3. SCIENTOMETRIC EVOLUTION OF VIBRATIONAL SPECTROMETRY IN FLOW INJECTION ANALYSIS The evolution of flow methods involving vibrational spectrometry based detectors (Figure 1) was initially very slow, with only 20 papers published in the 1980s. However, from 1990 the number of publications has been increasing every year. Vibrational spectrometric FIA procedures can be classified in three different groups based on the type of the specific detection technique, i.e., mid infrared (MIR) (using different sampling techniques), near infrared (NIR) or Raman spectrometry. As can be seen in the inset of Figure 1, transmittance

Vibrational Spectrometry

Cumulated number of published papers

Transmittance mid-IR 46 %

Raman 18 %

160

409

140 120 100 80

ATR mid-IR 17 %

Near-IR 19 %

60 40 20

05 20

03 20

99

01 20

97

19

95

19

91

93

19

19

89

19

87

19

85

83

19

19

81

19

19

19

79

0

Year

Figure 1 Evolution of the literature on FIA with vibrational spectrometry. Inset: Distribution of the literature on FIA-vibrational spectrometry as a function of the detection techniques employed.

measurements in the MIR region are most commonly used in combination with FIA, followed by NIR, attenuated total reflectance (ATR) in the MIR region and Raman based procedures. The published literature on this topic is summarized in Figures 2 and 3, which list the journals in which papers on flow analysis and vibrational spectrometry have been published and the productivity of the main authors in this field. The names of the journals, in which at least two articles on vibrational spectrometry and FIA have been published, are listed in Figure 2 in descending order with respect to the number of articles published on this topic. As can be seen, 10 or more articles have been published in only 5 among these journals (Applied Spectroscopy, Analytica Chimica Acta, The Analyst, Analytical Chemistry and Talanta). Figure 3 lists most of the authors identified in the Analytical Abstracts database as the main contributors to research on vibrational spectrometry in FIA. This figure identifies two highly productive groups in this area of research, one in Spain and another one in Austria.

4. OBJECTIVES The main objective of this chapter is to outline the advantages, the state-of-the-art and scope of on-line vibrational spectrometry detection strategies in FIA. The articles discussed in this chapter have contributed fundamentally to the development and evolution of the flow analysis-vibrational spectrometric techniques. These techniques together with their modes of measurement are

de Ga la rrig G ue ua s rd L e ia K n G ell dl al ne l Sc igna r hi ni nd l F er Ar r a n m k en M ta i B lle Bo ae r n H uhs a ab a er in La kor s n W ern or a sf Bu Be old r rt Bu gue hod Ay rg ra or ue M a- ra C J a L C ñad hr a is t C ia D ur n an ra ie n ls o Fo n rc e J Ka un t o La n M or M ure al e ll es ye -R rs Q ubio ui R nt a uz s ic S ka Se ear dm e a T n Ve ra n n W V tu W anz on ra ei en ac ss b h en oe ba ck ch W e in W r ef e l or ls dn er

Number of published papers Number of published papers

. C ct ro hi sc . m Th . A ct e a A An na Fr al lys es .C t en he iu m s J. Ta . An lan ta a Vi br l. C .S h e pe m . c An tros M c a . l. ik J. Ag roc Pro ric him c. .F . J J. oo Ac Au . Ne t M dC a ar to ic m he I r n . M fr oc m et are he . m ho d . ds Sp J M ec . an tro ag sc .C . he Se Qu i m m ns .A .A . n ct al Sp uato . ec rs tro B s Sc An cop i.T al y . ot al Le E n tt. vi ro n. JA LA

pe

.S

pl

al

An

Ap

410 Sergio Armenta et al.

25

20

15

10

5

0

Figure 2 Journals in which flow analysis articles involving vibrational spectrometry have been published. Journal

30

25

20

15

10

5

0

Author

Figure 3 Authors who have published more than three articles on FIA with vibrational spectrometry.

outlined in the subsequent sections. Special attention has been paid to emerging flow strategies like SIA or multicommutation.

5. INFRARED SPECTROMETRY

5.1 Mid-IR

The synergistic combination between FIA and Fourier Transform InfraRed (FTIR) spectrometry developed in the last 15 years provides: (i) a simple, fast and


411

reproducible way for loading the IR flow cells (transmittance or ATR cells); (ii) an enhancement of the repeatability and accuracy because of the lack of sample handling; and (iii) an important reduction of reagent consumption and time of analysis [6]. Additionally, the major merits of IR detection in flow analysis systems include: (i) easy way of operating; (ii) real-time detection; and (iii) low maintenance. Usually, FIA–FTIR is applied for single-component analysis, but multicomponent analysis can also be carried out. The absence of a separation step does not preclude identification and quantification of several analytes in the same sample. For instance, the introduction of multivariate regression methods has enabled the simultaneous quantification of several compounds even in cases of strong band overlapping.

5.1.1 Transmittance measurements Since Curran and Collier made the first study focused on the analysis of synthetic samples in 1988 [7], in which phenyl isocyanate was analysed using a dispersive IR spectrometer, the FIA–FTIR technique has been successively applied to the determination of diverse analytes in several matrices, using different flowthrough cells and FIA approaches, as can be seen in Table 1. The main applications of the methodologies involving FIA–FTIR transmittance measurements are in the analysis of organic solvents, gasoline, pharmaceutical products, pesticides, food and beverages and environmental samples. Additionally, this technique has been used for reaction monitoring. The analytical features of the corresponding procedures are summarized in Table 1. Classical FIA is the most common flow approach used. A single application of multicommutation evidences the lack of development of this novel flow approach (see Figure 4 for schematics of FIA, SIA and multicommutation manifolds). The material employed for the flow through cell windows has been KBr only in the case of extremely non polar media. Otherwise CaF2, ZnSe or the combination of both materials have been used. The use of mixtures of ZnSe and CaF2 reduces problems related to the high diffraction index of ZnSe, thus improving the limit of detection by reducing the contribution of interference bands as illustrated in Figure 5. Limit of detection values (LOD) reported in FIA–FTIR procedures based on transmittance measurements vary as a function of the analyte and the medium of measurement. Typical LOD values of the order of 0.01% (w/w) for the original samples have been obtained except in cases involving preconcentration of the analytes [13]. Regarding the sample throughput, earlier flow systems allowed only a few measurements per hour, but nowadays, fast computer-controlled FTIR instruments allow 2 scans per second thus resulting in sampling frequencies of the order of 120 h1. However, methods that incorporate on-line sample pretreatment only permit analysis rates of between 5 and 10 h1. Production of waste in vibrational spectrometry is a serious matter, especially when using organic solvents and in particular chlorinated organic solvents.

Flow technique

Analyte

Sample

Sample throughput (h1)

LOD

Waste (mL)

Reference

ZnSe–CaF2 ZnSe–CaF2 ZnSe–CaF2

FIA/stopped flow FIA FIA

Pesticides Pesticides Pesticides

30 30 60

0.7–0.8% 12 mg mL1 16–17 mg g1

9.3 2 2.7

[8] [9] [10]

ZnSe–CaF2 ZnSe ZnSe KBr ZnSe–CaF2

FIA FIA FIA FIA FIA/stopped flow

Pesticides Pesticides Pesticides Pesticides Food

6 11–14 – 42 7.5

20 mg mL1 400–785 mg 15 mg L1 1.6 mg mL1 0.1–0.9%

3 – – – 4

[11] [12] [13] [14] [15]

CsI CaF2

FIA FIA

Food Food

24 –

– –

– –

[16] [17]

CaF2 ZnSe AgClBr (fiber optic) CaF2 ZnSe ZnSe –

FIA FIA FIA

Diuron Malathion Folpet and Metalaxyl Buprofezin Ziram thiram Carbaryl Carbaryl Aspartame and acesulfame Peroxide value Citric, malic and tartaric acids Phosphate Caffeine Sugar

Food Food Food

– 6 –

– 9 mg L1 –

– 30 –

[18] [19] [20]

Food Food Food Food

60 30 25 60

– 10 mg mL1 0.03% –

– – 20 ml h1 –

[21] [22] [23] [24]

– CaF2 – CaF2 – –

FIA FIA FIA FIA FIA FIA

Food Food Food Food Food Food

40 – – – – 45

– 39 mg L1 53 pg – – –

– – – – – –

[25] [26] [27] [28] [29] [30]

FIA FIA FIA FIA

Phosphate Caffeine Ethanol Fatty acids and moisture Fatty acids CO2 Sugars Glucose Sucrose Sucrose


Cell

412

Table 1 Recent articles published on the combination of FIA systems with mid infrared spectrometry using transmittance measurements

FIA

Sucrose

Food

–

–

–

[31]

FIA FIA

Food Food

– –

– –

– –

[32] [33]

CaF2 CaF2

FIA FIA

Food Clinical and food

– –

– –

– –

[34] [35]

ZnSe–CaF2

FIA

Reaction monitoring

–

–

–

[36]

ZnSe–CaF2

FIA

Reaction monitoring

–

–

–

[37]

CaF2

FIA

–

–

[38]

FIA FIA

Reaction monitoring Environmental Environmental

–

Quartz AgClBr (fiber optic) Quartz

15 –

0.46 mg mL1 –

10 –

[39] [40]

FIA

Carbohydrates Organic acids and sugars Organic acids Glucose and urea Amylase and amyloglucosidase activities Amyloglucosidase activities Amylase activity Oil Pesticides and solvents Oil and greases

Environmental

60

1 mg mL1

[41]

– Fiber optic

FIA FIA

Solvent recycling – –

KBr

– –

– –

Pharmaceutical

42

0.04 mg mL1

KBr

FIA

Paracetamol

Pharmaceutical

KBr

FIA

Pharmaceutical

120

0.09 mg mL1

–

FIA

Propyphenazone and caffeine Dimenhydrinate

Pharmaceutical

–

25 mg mL1

Solvent recycling Solvent recycling Solvent recycling –

[42] [43] [44] [45] [46]

[47]

413

Water Water

FIA

Metal ions Thrichloroethylene Ketoprofen


AgClBr (fiber optic) CaF2 CaF2

414

Table 1 (Continued ) Flow technique

Analyte

Sample


LOD

Waste (mL)

Reference

–

FIA

Pharmaceutical

–

–

–

[48]

–

FIA

Pharmaceutical

–

–

–

[49]

– NaCl KBr

FIA Multicommutation FIA

Pharmaceutical Gasoline Gasoline

20 81 –

0.08 mg mL1 0.004% 0.005–0.0035%

– 1.2 –

[50] [51] [52]

– KBr – KBr Si –


Gasoline Gasoline Gasoline Solvents Solvents Solvents

– – – 20 – –

0.01% 0.04% 0.02% 0.03% – –

– – – – – –

[53] [54] [55] [56] [57] [58]

KBr

FIA FIA

Solvents Solvents

– –

0.01% –

– –

[59] [60]

ZnSe –

FIA FIA

Acetaminophen Acetylsalicilic and caffeine Ibuprofen Benzene Benzene, toluene and MTBE Toluene MTBE Benzene Xylene IBMK Acetone, ethanol and THF Xylene Xylene, toluene, ehylbenzene Nicotine Phenyl isocyanate

Tobacco –

6 –

0.1 mg L1 4 mg mL1

– –

[61] [7]

Note: IBMK, isobutyl methyl ketone; MTBE, methyl tert-butyl ether; THF, tetrahydrofuran.


Cell

Syringe pump

Holding coil Detector

B) C) Carrier

Standard

Solenoid valve

Waste Detector

Solenoid valve

Waste

Mixing coil Carrier Pump

Solenoid valve

Sample

Sample Carrier Reagent 1

Reagent 2

Waste

A) Sample

Waste Pump

Mixing coil

Reagent 1 Reagent 2


Detector Carrier

Figure 4 Schematic of flow analysis systems with vibrational spectrometric detection ((A) classic FIA, (B) SIA and (C) multicommutation).

415

416


0.8 0.7

a)

0.6

Absorbance

0.5 0.4 0.3 0.2 0.1

b)

-0.0 -0.1 -0.2 c) -0.3 -0.4 2000

1800

1600 1400 Wavenumbers (cm-1)

1200

1000

Figure 5 Bensulfuron standard spectra obtained in chloroform with a micro flow through cell of 0.11 mm with (a) ZnSe windows, (b) ZnSe and BaF2 windows and (c) BaF2 windows. Note: in all the cases the spectrum of pure chloroform was also included.

For this reason, efforts towards miniaturization of the flow systems or the development of closed flow systems [5] have dramatically reduced reagent consumption and waste generation, thus providing environmentally friendly alternatives. In cases where highly polluting solvents like CCl4 or CHCl3 are used, on-line solvent recycling [44] is a major advantage of the FIA–FTIR technique. Classical FIA systems have been employed in the determination of many analytes in a great variety of matrices. Representative examples of the FIA–FTIR approach, where a sample is mixed with a reagent and the product is monitored, are the determination of different oil quality parameters (peroxide value (PV) [16] and free fatty acid content [25]). In the analysis of the first parameter, the sample stream was mixed with a solvent mixture consisting of 25% (v/v) toluene in hexanol, which contained triphenylphosphine (TPP). The hydroperoxides present in the sample reacted stoichiometrically with TPP to give triphenylphosphine oxide (TPPO). By using t-butyl hydroperoxide spiked oil standards and evaluating the band formed at 542 cm1, a linear calibration graph covering the range 1–100 PV (meq O2 kg1 oil) was obtained. The relative standard deviation (RSD) was 0.23% (n ¼ 11) and the sample throughput was 24 h1. In the second application, the sample was merged with a carrier stream of 25% toluene in propan-2-ol (solvent A), which was then merged with a stream of 0.3 M KOH in solvent A. After a short reaction time, the intensity of the FTIR peak at 1570 cm1 was measured. For oleic acid, the calibration graph was linear up to 2.5%, the RSD was 3.3% and the sample throughput was 40 h1.


417

Phosphate in soft drink samples was determined by means of pH modulation [21]. Each sample was injected into a carrier stream and mixed with an acetate buffer to adjust the pH to 5 (reference spectra). A second portion of the sample was then injected into the carrier stream and mixed with 100 mL of either carbonate buffer (method A) or NaOH solution (method B) to adjust the pH to 10 or higher than 13, respectively. Phosphate was quantified from the difference between the reference and alkaline spectra, using the peaks at 1,085–1,095 and 999–1,009 cm1 for method A and B, respectively. The calibration graph was linear from 0.1 to 1 g L1 phosphate and the sample throughput was 60 h1. This method was improved by the use of a Fabry-Perot quantum cascade lasers (QCL) as a powerful light source for MIR detection in FIA [18]. The use of QCL instead of an interferometer results in real-time spectral data acquisition since there is no need for mathematical calculations. Compared with a Fourier transform spectrometer, the signal-to-noise ratio improves by a factor of 50 when a QCL is used. Additionally, by using a QCL as the light source, optical path lengths of more than 100 mm can be used, even in aqueous matrices, which reduce the risks of cell clogging. A room-temperature MIR QCL was also successfully applied to the direct determination of carbon dioxide in aqueous solutions [26]. Aqueous carbon dioxide standards were prepared by feeding different mixtures of gaseous N2 and CO2 through wash bottles at controlled temperature. The carbon dioxide standards were connected via a selection valve to a peristaltic pump for subsequent automated measurement in the flow-through cell. A calibration curve for CO2 was obtained in the range 0.338–1.350 g L1 with a standard deviation of 19.4 mg L1 and a limit of detection of 39 mg L1. Other environmental applications using flow systems with FTIR detection, such as the determination of oil and greases in water [41], were developed. The manifolds employed in this procedure are depicted in Figure 6 and they were applied to the determination of oil in aqueous solutions after microwaveassisted extraction. A portion of the extract was injected into a carrier stream of CCl4 at a flow rate of 1.5 mL min1 and the area from 3058 to 2780 cm1, corrected for the baseline established between 3200 and 2700 cm1, was used for quantification. The detection limit was 0.6–1.1 mg mL1 of oil and greases; RSD was between 1.4 and 4.1% (n ¼ 10) and the sample throughput was 60 h1. The method was improved using an on-line system for oil extraction based on a polytetrafluoroethylene (PTFE) membrane phase separator [39]. The detection limit of oil in water was 0.46 mg mL1 and the RSD was 1.1% using carbon tetrachloride as the extraction solvent. The detection limit was improved using 1,2,3,4-tetrachloro-1,1,2,3,4,4-hexafluorobutane instead of carbon tetrachloride (0.38 mg mL1). The calibration graph was linear up to 80 mg mL1 and the RSD was 2.5%. The determination of aromatic hydrocarbons (e.g., benzene and toluene) and additives, such as methyl tert-butyl ether (MTBE), is necessary in several areas of petroleum, petrochemical and related industries. A simple way for the simultaneous determination of benzene, toluene and MTBE [52] using flow systems has been proposed. In this study, petrol was diluted with hexane and a

418

300 μL

CCl4

Pump

Waste

TRANSPORT

Sample

Water Pump

6.5 mL

Phase separator

Water

Waste

FTIR detector

CCl4

Waste

Extraction coil

CCl 4

Pump

Standard

FTIR detector

TRANSPORT + EXTRACTION

1.3 mL

Figure 6 Manifolds employed for the determination of oil and greases in water.


Sample extract (off-line)


419

portion of the solution was injected into a carrier stream of hexane. Benzene, toluene and MTBE were determined by measuring the first derivative values from 678–672, 731–725 and 1,209—1,201 cm1, respectively. The corresponding detection limits were 0.005, 0.01 and 0.035% with dynamic ranges from the detection limit up to 0.8%, 2.0% and 2.0%. The RSD values were 1.0%, 0.9% and 0.6% for benzene, toluene and MTBE, respectively, and the sample throughput was 25 h1. FIA–FTIR was also employed for the determination of o-xylene, toluene and ethylbenzene in n-hexane [60]. The results were processed using a multivariate partial least squares (PLS) PLS1 algorithm, giving relative root-mean-square standard errors of cross-validation of less than 7% when using mean-centred data and the first derivative spectra for o-xylene. An FIA system was used for sensing of persistent organic pollutants in water [40]. The manifold (Figure 7) is composed of an FTIR spectrometer coupled via an AgClxBr1x optic fibre to a polymer coated sensor cell, where the analyte is preconcentrated thus minimizing water interferences. For trichloroethylene detection, the fibre was coated with poly(isobutylene) whereas for alachlor detection, a coating of PVC containing 5% chloroparaffin was used. The detection limits were 3 mg L1 for trichloroethylene and 5 mg L1 for alachlor. The applicability of this technique to multi-analyte determination in water was also demonstrated. Modern alternatives to FIA methods, such as SIA methodologies have been successfully applied not only for the simultaneous determination of different analytes in the same matrix (glucose, fructose and sucrose in soft drinks) but also for the automated preparation of a multi-component calibration solution [31].

FTIR spectrometer and IR source

Flow in Input fibre Flow-through cell Optic coupler

Optic coupler

Sensing fibre Output fibre Flow out

MCT detector

Figure 7 Setup employed for sensing of organic pollutants in waters. Reproduced from Ref. 40. Copyright (1996), with permission from the Royal Society of Chemistry.

420


In this paper, the spectra were recorded using a homemade fibre optic flow cell, in which two AgClxBr1x fibres with plane-parallel faces were assembled in a PTFE block coaxially to each other with a gap of 30 mm between the fibre tips. Multicommutation has been successfully coupled with mid range FTIR and this has enhanced on-line sample dilution, external calibration and standard addition processes [51]. This method has permitted the direct determination of benzene in gasoline without any sample pretreatment and with a limit of detection of 0.004% (v/v), a variation coefficient of 1.2%, a solvent consumption of 1.2 mL per determination, and a sample throughput of 81 h1.

5.1.2 Attenuated total reflectance (ATR) The use of ATR flow cells is well known since their introduction in 1985 by Kennedy, White and Browne [62]. Thus, from this first application of ATR-based monitoring, in which quality parameters of milk powder were determined in a continuous-process stream analyser, to applications involving modern flow techniques such as SIA [63] and multicommutation [64] for the analysis of surfactants, a number of other applications based on continuous ATR sensing have been reported. They have involved the use of ATR– FTIR as the detection technique in flow systems for the analysis of a great variety of analytes in different sample matrices, as shown in Table 2. A micro-circle flow-through cell was used for a reagent-free analysis of urine by ATR spectrometry combined with multivariate calibration [67]. Multivariate models enabled quantification of urea, creatinine, uric acid, phosphate and sulfate in the same sample. PLS-micro circle ATR was also used for the determination of different sulfur oxygen (e.g., hydrogensulfite, thiosulfate, sulfate and sulfite) and nitrate anions in water [70]. The method was used for kinetic analysis of the decomposition products of the dithionite ion in an aerobic aqueous environment at 551C over the concentration range 0.5–32.3 mM. The root-mean-square error found was 0.2 mM. Qualitative and quantitative FTIR analyses of different warfare agents (i.e., tabun, sarin and soman) were performed with a circle cell accessory fitted with a modified high-pressure micro flow-through sampling cell [71]. The detection limits were 0.3 ng mL1 for tabun and 0.2 mg mL1 for sarin and soman, and the intra- and inter-day coefficients of variation were 3.0% and 6.0%, respectively. Reaction processes could be monitored by ATR–FTIR. The method has been used to predict the concentrations of glucose and ethanol during baker’s yeast fermentation [76]. A completely automated flow system was employed as an interface between the bioprocess under study and the FTIR spectrometer. By using an automated flow system, experimental problems related to adherence of CO2 bubbles to the ATR surface, as well as formation of biofilms on the ATR surface, could be efficiently eliminated. The recorded data from different fermentations were modelled by PLS regression comparing two different strategies for the calibration. On the one hand, calibration sets were constructed from spectra recorded from either synthetic standards or from samples drawn during fermentation, while on the other hand, spectra from fermentation samples and synthetic standards were combined to form a calibration set. The optimal

Table 2 Recent articles on the use of flow systems and attenuated total reflectance mid infrared spectrometry Analyte

Sample group

LOD

Reference

Single bounce ATR ATR ATR Micro circle ATR ATR ATR Micro circle ATR Circle cell Circle cell Membrane ATR ATR/trans Circle cell/trans Circle cell Membrane ATR ATR ATR Micro circle ATR Circle cell ATR ATR Circle cell ATR

Quality parameters Quality parameters Quality parameters Multi analyte Glucose Multi analytes Sulfur, oxygen, anions and nitrate Chemical warfare agents Chemical warfare agents Ethyl acetate Xylene, toluene and ethylbenzene Acetone, ethanol and THF pH sensor Surfactants and oils Fermentation process Fermentation process Choline Choline Acetaminophen Surfactants Surfactants Surfactants

Food Food Food Clinical Clinical Clinical Water Water Water Solvents Solvents Solvents – – Reaction monitoring Reaction monitoring Pharmaceutical Pharmaceutical Pharmaceuticals Liquid formulations Formulations Formulations

– – – – – – – 0.3–0.2 ng mL1 0.1 mg mL1 – – – – – – – 0.30% 0.5–0.2 ng mL1 – – 0.77 mg –

[65] [62] [66] [67] [68] [69] [70] [71] [72] [73] [60] [58] [74] [63] [75] [76] [77] [78] [79] [64] [80] [81]


Measurement mode

421

422


PLS regression method was obtained using the mixed calibration set of samples from fermentations and synthetic standards. The root-mean-square errors in this case were 0.267 and 0.336 g L1 for glucose and ethanol concentrations, respectively. Another type of sampling system for ATR in flow analysis is based on the adsorption of the analyte on a film deposited onto the surface of the internal reflection element (IRE) [73]. Such a systems was used to study the adsorption of ethyl acetate in n-heptane. The silica-coated ZnSe IRE was mounted in a flow-through tunnel cell and a series of solutions of ethyl acetate in n-heptane (5.1–77.0 mM) were pumped through the IRE for 5 min at a flow rate of 1 mL min1. After exposure to each solution, ATR–FTIR spectra were acquired at the solid/liquid interface and the characteristics of adsorption of ethyl acetate from n-heptane onto silica were discussed. Modern SIA approaches have been successfully used in monitoring reactions, for example in the exhaustion of alkaline degreasing baths [63] as illustrated in Figure 8. In this study, an ATR–FTIR membrane-based sensor was integrated into an SIA manifold for the determination of the parameters of interest. The system was based on m-liquid–liquid extraction of the analytes through a polymeric membrane from the aqueous to the organic solvent layer which was in close contact with the IRE and was continuously monitored. The signals obtained were processed by a multivariate calibration technique. The mechanization of ATR measurements in FTIR spectrometry through the use of multicommutation was evaluated in order to reduce sample consumption and waste generation as well as to minimize the risks of cell breaking [64]. The procedure was proposed for the determination of sodium alpha-olefin sulfonate (AOS) in liquid detergent formulations. The main advantages of the method are the low sample consumption (96 mL per 100 determinations) and a sample throughput (23 vs 15 h1 compared to the manual mode). Results obtained for

Holding coil Waste SIA valve

Water

ATR-FTIR CCl4 Outlet

Syringe pump

Inlet W S

Polymeric membrane

Sample

I

G S

W G W S

G S

S

W S I I G S GS I W S G G IRE

G S

W I

GS

SG G

External chamber OSL

Figure 8 SIA–FTIR setup used to monitor the exhaustion of alkaline degreasing baths.


423

reference samples containing 7.52–9.52% (w/w) AOS were in the 0.5% (w/w) accuracy error range, with a mean precision of 0.3% RSD. Recovery studies demonstrated the accuracy of the method, with average recovery values of around 100%. The possible use of ATR–FTIR in the analysis of different types of liquid samples, such as beer, orange juice, milk and olive oil, was evaluated, showing the great versatility of this approach to improve sample introduction and cell cleaning.

5.1.3 Vapour phase FTIR Problems arising from the poor transparency of water to MIR radiation, and from the toxicity and side effects of chlorinated hydrocarbons (discussed above) could be avoided by obtaining the FTIR spectra of analytes in gas phase in a nitrogen atmosphere. This would provide transparent and unreactive medium thus offering the possibility to work with long-path cells. However, use of gases to obtain IR spectra has serious limitations, such as (i) difficulties associated with accurate calibration, caused by the strong dependence of the analyte concentration on the working pressure; (ii) the need for strict control of leaks required for gas handling; and (iii) reduced number of compounds that are gases at room temperature [6]. The development of an attractive analytical technique called vapour generation, based on FIA–FTIR in a N2 carrier opened new possibilities in gas analysis by FTIR [6]. The technique relies on the injection of discrete liquid sample volumes into an electrically heated Pyrex glass reactor in which the compounds to be determined are volatilized. The vapours generated are transported by the nitrogen carrier flow into an IR gas cell and the corresponding FIA recording is registered as a function of time (Figure 9). In addition to the inherent advantages of measurements in the gas phase, this technique allows efficient matrix removal, thus reducing spectral interferences. The principles of operation of vapour generation extend the application of FTIR to those compounds that are easily volatilized in N2 flow at temperatures lower than 2001C. The first studies on vapour generation FTIR were based on direct determination of different analytes in several matrices (Table 3). One of those studies involved the determination of ethanol in blood [101]. This method was based on the injection of a discrete sample volume into a Pyrex glass reactor electrically heated at 901C. The ethanol was volatilized and introduced by means of a N2 carrier flow inside a long-path infrared gas cell and the corresponding flow analysis recording registered as a function of time. The limit of detection of the method was 0.020 g L–1, the RSD value varied between 0.3% and 1.9% and the sampling frequency was 40 h–1. The use of vapour generation FTIR for ethanol analysis in blood samples not only minimizes the problems related to water interference but also provides an excellent means for the on-line removal of proteins, which are thermally degraded without volatilization. A different approach to converting the analyte into the vapour phase is by means of chemical reactions (Figure 9), such as those based on the reaction of analytes with acids or bases. These types of procedures have been developed for

424 Sergio Armenta et al.

Figure 9 Different configurations used for the generation of the vapour phases in FTIR spectrometry.

Table 3 Recent articles on the use of vapour phase generation and mid infrared spectrometry LOD

RSD (%)


Reference

Nitrite Antimony Antimony, arsenic, tin Lead Nitrogen Carbonate Carbonate CO2 Organic and inorganic carbon VOC Organic pollutants Contaminants Contaminants Contaminants Butyl glycol Butyl acetate, toluene and MEK Acetone and isopropanol Paint solvents Ethanol Ethanol Thiourea Ziram Methanol and ethanol Methanol and ethanol Trimethylamine

Food Pharmaceuticals Synthetic samples Reference and soil samples Hydrolyzed proteins Sediments Waters Natural water Water Aqueous solutions Water Waste water Waste water Waste water Paint solvents Paint solvents Nitrocellulose paints Paint Solvents Blood Film developing solutions Pesticide formulations Beverages and cosmetics Beverages and cosmetics Seafood

0.3 mg NO2 mL–1 0.9 mg L1 0.25, 0.3, 1.2 mg L1 0.28 mg L1 1.4 mg L1 0.2 mg 4.6 mg L1 – 1.5 mg L1 100 ng mL1 – – – – 2.7–4.5 mg L1 1.4–3 mg 0.5–3.9 mg 1–4 mg 0.02% v/v 0.020 g L1 10 mg L1 0.055 mg – 0.21–0.04% 0.6 mg L–1

0.7–2.8 1 0.3 2 3.0 2 1.3 – – – – – – – 1 0.5–1 0.8–0.9 0.4–2 – 0.3–1.9 1.1 6 2.3–2.7 0.5–3.5 0.6

40 28 – 60 60 15–20 30 30 – – – – – – 65 – 50 – – 40 14 17 – – 30

[82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106]

Note: MEK, methyl ethyl ketone.

425

Sample group


Analyte

426


the determination of carbonates in water [88] and sediments [87], nitrogen in hydrolyzed proteins [86] and trimethylamine (TMA) in seafood [106]. The typical manifold required for this kind of determinations is based on continuous mixing of the sample or standard carrier stream with an acid or alkaline reagent stream, or on the use of removable reactors (Figure 9). In the latter approach, solid samples are weighed, inserted into a setup in which the acid or base is injected, and the vapours generated are transported to the gas cell. The method proposed for the determination of total carbonate in waters is based on the simultaneous injection of a discrete volume of sample with 300 mL nitric acid in a two-channel manifold with a merging zone. A coil located inside a microwave oven enhances the evaporation of CO2 which is separated from the distilled water employed as carrier by means of a gas–liquid separator and is introduced by a nitrogen carrier flow into a long-path infrared absorption gas cell. The corresponding flow analysis recording is registered as a function of time. This procedure has a limit of detection of 15 mg HCO–3 L–1 and a variation coefficient of 1.3%. A recently developed method for the determination of TMA in fish and cephalopod samples was based on vapour phase generation FTIR spectrometry. Samples extracted with trichloroacetic acid (TCA) were filtered and aspirated into a two-channel manifold. The extract was alkalinized with 2.0 M NaOH in an on-line system and TMA generated was separated from the solution in a gas-liquid separator, after which it was transported by means of a nitrogen carrier into a homemade IR gas cell. The method was applied to the determination of TMA in natural samples providing concentration values statistically comparable to those obtained by head space gas chromatography used as the reference procedure. Vapour generation FTIR was applied by the authors to the determination of dithiocarbamate pesticides in agricultural fungicides [103]. This method was based on the decomposition of dithiocarbamates on heating the sample in an acid medium and the continuous FTIR measurement of the CS2 evolved. Recently different research groups have developed methods for the determination of metalloids and metals such as Sb, As, Sn [84] and Pb [85] by means of hydride generation coupled to vapour phase FTIR measurement thus improving previous methods based on vapour phase UV spectrometry [107]. In summary, vapour generation FTIR has become a mature analytical technique, useful for the quantitative analysis of both, organic and inorganic samples in the solid and liquid phases. It offers a sustainable alternative to the use of toxic solvents without sacrificing the advantages of the FTIR method.

5.2 Near-IR Near-IR (NIR) spectrometry could be used as a universal detector for FIA systems. In fact, the NIR technique provides a number of attractive advantages, namely: (i) direct recording of spectra for solid [108] and liquid [109] samples with little or no sample pretreatment; (ii) provision of chemical and physical information on samples such as viscosity, moisture content or polymorphism [110]; and (iii) multi parameter determinations based on a single spectrum [111].


427

On the other hand, NIR spectrometry has the following major disadvantages: (i) NIR spectra exhibit strong band overlap, which requires the use of multivariate chemometric techniques in both qualitative and quantitative analytical applications [112]; and (ii) the low sensitivity of the technique restricts its scope to major components and a few minor components. Table 4 summarizes the articles published on this topic that have been found by the authors. It can be seen that NIR measurements are usually conducted in transmittance or fluorescence mode using diffuse reflectance or fibre optics. Analytes determined by FIA–NIR include heavy metals [131], Al [114], anions [132], water [130], organic compounds [120] and NaOH [116] in industrial, environmental, clinical and food samples which vary from water to solvents and supercritical CO2. Regarding the flow techniques used, most of the applications involve classical FIA and there are single applications based on the use of multicommutation [113], stopped-flow [122] and on-line measurements [126]. An example of a classical FIA application is the determination of ethanol as stabilizer of CHCl3 [129], which was based on the direct measurement of the stabilizer using a dry chloroform carrier and a homemade flow through cell (1 mm path length) sitting in an NIR spectrophotometer for continuous monitoring of absorbance. The procedure was validated by comparing the results with a reference method based on gas chromatography. A sample throughput of 240 h1 was obtained. In the same way, dichloromethane and isobutylmethylketone were used as test systems for the determination of water in organic solvents by FIA–NIR using a setup which avoided the influence of the ambient moisture [130]. A study was carried out on the effect of the flow variables on the sensitivity and speed of the determination. For dichloromethane and isobutylmethylketone, detection limits of H2O were 0.01% and 0.005% v/v, respectively, and the corresponding calibration graphs were linear up to 0.2% and 0.5%. The results obtained compared well with those obtained by FIA with spectrophotometric detection. In order to reduce the acquisition cost of the instrumentation, a compact photometer based on up to seven different light-emitting diodes (LEDs), from blue to near-IR, was applied to simultaneous determination of several model metal ion mixtures in combination with several photometric reagents in an FIA system using rapid computer-controlled switching of the LEDs [127]. The introduction of a solid-state acousto-optic tunable filter (AOTF) in NIR spectrometers provides a high scanning speed and wavelength accuracy. Such an instrument was applied to the determination of traces of H2O in CHCl3 and benzene and H2O in ethanol [128]. Diffuse reflectance (DR) NIR spectrometry has also been used for detection in flow systems. The method is based on the retention of metal ions in flow cells containing fibrous material or fabric discs with immobilized organic reagents. Two types of sensors were proposed; such as carrier discs with a previously immobilized reagent (coloured discs); and white carrier discs with on-line immobilization of the reagent. The aforementioned sensor systems were used to determine Co, U, Pd, Ni, and Cr in fresh water [131].

428

Measurement cell

Technique

Flow technique

Analyte

Sample


LOD

Reference

Quartz Immobilized eriochrome cyanine –

NIR transmittance DR NIR

Multicommutation FIA

Hexythiazox Al (III)

Pesticides Environmental

52 20

0.1 mg mL1 0.34 mg mL1

[113] [114]

NIR fluorescence NIR transmittance NIR transmittance NIR spectrofluorimetric

FIA

Environmental –

0.006%

[115]

Environmental –

–

[108]

FIA

Na dodecyl sulfate Commercial surfactants NaOH

Environmental –

–

[116]

FIA

H2O2

5.58 108 mol L1

[117]

Sapphire

NIR transmittance

FIA

Urea

–

–

[118]

Quartz Quartz

NIR fluorescence NIR transmittance

FIA FIA

Nile red Urea, creatinine, glucose, protein, ketone

Clinical and environmental (rainwater, serum and plant) Clinical (effluent dialysate) Clinical Clinical

– –

1 mM –

[119] [120]

– – Quartz

FIA


Table 4 Recent articles published on the use of FIA–NIR

Custom made flow through cell – Glass – – Transmission probe Transmission probe Quartz Quartz Quartz Discs Quartz – CaF2 –

FIA

Multicomponent

Clinical

–

–

[121]

Fibre optic CCD NIR NIR transmittance NIR transflectance NIR transmittance NIR transmittance

Stopped flow FIA FIA FIA On-line

Actinide Ethanol Quality parameters Quality parameters Esters

Monitoring Food Food Food –

– 240 4.2 – –

– – – – –

[122] [123] [124] [125] [126]

NIR transmitting LED AOTF NIR NIR transmittance NIR transmittance DR NIR NIR transmittance NIR transmittance NIR transmittance NIR fluorescence

FIA

Metal ion mixtures –

–

–

[127]

FIA FIA FIA FIA FIA FIA FIA FIA

Solvents Solvents Solvents Water Water Gasoline Hydrocarbons –

– – – – – – – –

10–80 mg mL1 0.0045% 0.01–0.005% – 1.5 mg L1 – – –

[128] [129] [130] [131] [132] [133] [134] [135]

Fibre optic NIR

FIA

H2O Ethanol H2O Heavy metals Nitrate Octane number Physical properties Warfarin and flufenamic acid Squalana

Supercritical CO2

–

–

[136]

Note: CCD NIR, Charge coupled device near infrared.


High-pressure optical cell

NIR transmittance

429

430


Standard Solenoid valve

NIR flow cell

Sample Waste

Mixing coil Pump Carrier

Solenoid valve

Figure 10 Multicommutated flow system used to determine hexythiazox in pesticide formulations.

Other interesting applications of the use of NIR spectrometry as a detection technique in FIA include the determination of the octane number in gasoline [133] and different properties such as heat of formation, mean molecular weight and number of methyl groups per molecule in hydrocarbons [134]. An FIA liquid–liquid extraction procedure using NIR fluorescence detection was applied to the determination of warfarin and flufenamic acid using methylene blue as the fluorophore [135]. The method used CHCl3 as the extraction solvent and the limits of detection obtained were equal or lower than 1 and 2 mM for warfarin and flufenamic acid, respectively. Some advantages of NIR detection systems are the possibility to carry out multi-component analysis at high speed and with no reagent or sample preparation required. Thus urea, creatinine, glucose, ketone and protein were analysed by this approach in urine samples by means of PLS regression [120]. Multicommutated FIA systems have been also successfully coupled to NIR spectrometers. Figure 10 shows a multicommutated FIA system, which was designed to allow high sample throughput and to control the thermal effects on the NIR spectra [113]. It was used for the determination of hexythiazox in pesticide formulations. An on-line standard addition procedure was carried out showing the versatility and repeatability of multicommutation for the on-line mixing and dilution of solutions. Results obtained for commercial samples were statistically comparable to those obtained by a high performance liquid chromatography (HPLC) reference method. This multicommutation FIA–NIR system provided a sample throughput of up to 52 h1 compared to 30 h1 offered by a NIR-batch procedure and 7 h1 in the case of the HPLC reference method.

6. RAMAN SPECTROMETRY The most significant drawback of Raman spectrometry is its inherently poor sensitivity, which makes it unsuitable for trace analysis. Because of that, the use of FIA with Fourier transform Raman spectrometry as the detection technique has been reported once only [137]. The molecular and structural information contained in Raman spectra together with selective retention of the species of interest on a sorbent makes the proposed methodology highly selective. A flow-through sensor


431

of this type allowed the direct quantitative determination of sulfathiazole and sulfamethoxazole in the presence of other species, normally encountered with these analytes. The corresponding FIA system used Sephadexs QAE A-25 resin as packing material for the flow-through cell on which sulfonamides were temporarily retained. Samples were transported by a carrier solution of 0.01 M NaOH (pH ¼ 12) and 2 mL of a solution containing 0.10 M NaCl and 0.01 M NaOH were employed as the eluent. The analytical signal was linear in the range 0.5–7 and 0.5–10 g L1, for sulfathiazole and sulfamethoxazole, respectively. RSDs lower than 4% were obtained for both analytes. This method was satisfactorily applied to several commercial pharmaceutical preparations for humans and animals in different physical presentations. The discovery of the surface enhanced Raman scattering (SERS) effect in the 1970s offered exciting possibilities for overcoming the lack of sensitivity of traditional Raman spectrometry. The SERS enhancement is due to a combination of chemical and electronic effects [138,139]. Coupling the high sensitivity of SERS with the instrumental benefits of Raman spectrometry, including the sensitivity to small structural changes, non-invasive sampling capability, minimal sample preparation and high spatial resolution, yields a methodology that can provide highly specific molecular information on microscopic scale. The three most common types of substrates used in SERS include electrodes, island films prepared by vacuum deposition [140] and colloidal sols [141]. The last type has the following advantages: (i) they are easily and inexpensively prepared by chemical reduction techniques, (ii) silver colloidal hydrosols are easily characterized by their absorption spectra and (iii) they provide the possibility of continuous renewal of the sample by flow, which is thus potentially useful in FIA and liquid chromatography (LC). The disadvantage of such colloidal systems is their tendency to flocculate and there are also a large number of experimental parameters that are adsorbate- and substrate- dependent and contribute to the poor reproducibility of SERS on colloidal dispersions. To overcome those problems several researchers have adapted colloidal SERS systems for detection in flowing streams (FIA and LC) [142] and it has been demonstrated that flow techniques yield the most reproducible results. FIA systems with SERS using silver colloids [143,144] were optimized with respect to pH, sol preparation and detector response and were reported to exhibit an RSD of 3.2% for successive injections of p-aminobenzoic acid and a detection LOD of 30 ng [145,146]. Freeman et al. were the first to report SERS on silver sols that were applied to the determination of pararosaniline [147]. These experiments showed reproducibility of 1% for pararosaniline hydrochloride, a linear response from 0.1 to 50 pg mL1 and a dynamic range of over three orders of magnitude. Under continuous flow conditions, a major difficulty encountered is the maintenance of an active and clean SERS substrate, especially when several compounds are subsequently introduced into the FIA system. By using a short electrochemical roughening procedure, Force [148] was able to regenerate an SERS-active Ag electrode surface under flowing conditions. However, only pyridine was tested and the method required a reproducible roughening

432


Rinsing solution

Sample SERS flow cell Waste

Pump

Raman spectrometer

Glass window Metal screw

Brass cylinder

Collection optic Prism

SERS substrate (brass cylinder)

Teflon block Laser beam

From the FIA manifold

Figure 11 Setup for SERS determination of nicotinic acid in aqueous solutions.

procedure, as well as a potential step to remove the adsorbate resulting in a cumbersome procedure. The capabilities of surface enhanced Raman spectrometry coupled with FIA for monitoring a fermentation process have been demonstrated [149]. The detection limits for ethanol, methanol and acetone were 0.04%, 0.02% and 0.09%, respectively. SERS has been used in stopped-flow mode for the determination of nicotinic acid using a flow-through cell containing a circular brass plate which was electrochemically roughened (Figure 11). Multiple SERS measurements on the same substrate were achieved by rinsing the substrate with 3 M KCl or 0.1 M NaOH between measurements. A linear calibration graph was obtained up to 100 mM nicotinic acid and the detection limit was 1.7 mM [159]. A flow-through cell for detection in HPLC and FIA has been described that incorporated a cascade geometry capable of accepting modified SERS substrates. In the FIA–SERS of BTEX (benzene, toluene, ethylbenzene, xylenes) the detection limits were equal or lower than 190 mg L1 [150]. In order to overcome problems related to strong memory effects of colloidal sols, tailing of the peaks, baseline drift, contamination problems and overlapping of the spectra of the previously separated compounds, a windowless flow cell has been proposed [151]. SERS has been successfully interfaced to an FIA system to detect ribonucleic acid (RNA) bases in real time [152]. Four of the major bases of RNA (i.e., uracil, cytosine, adenine and guanine) were introduced into the FIA system and were mixed with an Ag sol prior to the SERS measurements. A recently published study involving SERS is of particular interest because it reports on a new strategy for on-line monitoring of chemical reactions

433


Raman excitation laser

Levitated drop Ultrasonic levitator transmitter

Ultrasonic levitator reflector Automated flow system Waste Holding coil

Waste

Flow-through microdispenser

SIA valve

Water

Syringe pump Reagents / Samples

Figure 12 An SIA setup for on-line monitoring of chemical reactions in ultrasound levitated droplets.

in ultrasonically levitated, nanolitre-sized droplets by Raman spectrometry (Figure 12). A well-defined sequence of reagents were injected via the microdispenser into the levitated droplet placed in the focus of the collection optics of the Fourier transform Raman spectrometer. In that way, chemical reactions could be carried out and monitored on-line. The proposed system was used for fast, reproducible in situ synthesis of a highly active SERS sol resulting from the reduction of silver nitrate with hydroxylamine hydrochloride under basic conditions. The silver sol, prepared in this way, was used for trace analysis of several organic test molecules that were injected into the levitated SERS-active droplet using the same microdispenser [153]. Other interesting applications of SERS as a detection technique in FIA systems, published in the literature, are listed in Table 5. Surface enhanced resonance Raman scattering (SERRS) has also been successfully coupled to FIA. SERRS only occurs with analytes that contain a chromophore with an absorption that matches the wavelength of the excitation source. The use of FIA systems yields the most reproducible results in SERRS analysis and avoids problems of localized heating, photo-dissociation, variable mixing times and scattering geometry. Analyte solutions and silver sol are pumped at equal rates and Raman spectra are obtained as the mixture moves through the laser beam in a capillary tube or open stream under a 16 objective lens. Such a system was used to obtain the spectra of 1 pM crystal violet solution with a coefficient of variation of around 2.4% [163]. This technique was also used for the determination of Fe(II) as its tris(bipyridyl) complex [164], which exhibits both a resonance Raman effect when excited with Argon laser (514.5 nm

434

Table 5

Articles published in recent years and reporting on the combination of FIA systems with Raman spectrometry Analyte

Samples

LOD

Reference

SERS nanodroplets

[153]

0.02–0.09%

[149]

SERS

Haemoglobin and NAD+

–

[154]

SERS liquid and vapour

Organic compounds

Monitoring chemical reactions Monitoring chemical reactions Monitoring chemical reactions Air and water

–

Raman

6-mercaptopurine, thiamine and acridine Fermentation products

[155]

SERS SERS SERS SERS SERRS Raman waveguide SERS SERS SERRS SERS SERS SERS SERS SERRS SERS SERS SERS

Pesticides Cyanogenic glucosides Nicotinic acid Pararosaniline HCl Nicotinic acid Solvents BTEX Drugs Fe (II) Pyridine adenosine amp RNA bases RNA bases Pyridine Crystal violet Aminobenzoic acid acridin-9-amine Aminobenzoic acid Aminobenzoic acid acridin-9-amine

Environmental Biological matrices Aqueous solutions Aqueous solutions Aqueous solutions – – – – Clinical Clinical Clinical Clinical – 40% ethanol/ethanol 40% ethanol –

15 ng mL1 (nicotine) – – – 2 ng 1.7 mM – 190 mg L1 – 1 nM 800 pmol – 175–233 pmol 250 nM 1 pM – – –

[156] [157] [158] [147] [159] [160] [150] [151] [161] [162] [152] [163] [148] [164] [143] [144] [146]


Technique


435

radiation) and surface enhanced Raman scattering when adsorbed on Ag. The calibration graph for Fe(II) was sigmoidal in shape with a working range between 10 and 10 mM and the detection limit was 1 nM, which represented detection of Fe(II) at the femtomol level.

7. CONCLUDING REMARKS AND OUTLOOK Among the different vibrational techniques coupled with FIA systems MIR spectrometry is most commonly used, both in transmittance and ATR measurements. This is due to its inherent advantages such as the presence of many bands to be employed for the determination of a single compound and the possibility of simultaneous detection of several sample components at a relatively low cost and with an easily available instrumentation. Concerning the different flow approaches, it can be concluded that classical FIA, in which a volume of sample is injected into a carrier stream, is the flow configuration most commonly employed. It has the advantage of simplicity but consumes higher amounts of reagents than more advanced flow approaches such as SIA and multicommutation. However, these approaches have been scarcely combined with vibrational spectrometry based detectors. They would enable a considerable reduction in reagents and solvents consumption and in waste generation to be achieved. In the case of multicommutation, this could be achieved without any decrease in the main analytical parameters. The relatively low sensitivity achieved by vibrational spectrometric techniques could be a possible explanation for the absence of attempts at miniaturization. In this sense, the incorporation of different technical advances to the instrumentation, such as QCL and acousto-optic tunable filters, has overcome partially this disadvantage. Therefore, it can be concluded that, despite the importance of vibrational based detection in FIA, the development of modern technical advances in both flow and vibrational techniques will be crucial for increasing the number of vibrational FIA applications in the next years.

ABBREVIATIONS AOS AOTF APTF ATR BTEX CCD NIR DR FTIR HPLC IBKM IRE

Alpha-olefin sulfonate Methyl tert-butyl ether Acousto-optic tunable filter Attenuated total reflectance Benzene, toluene, ethylbenzene, xylenes Charge coupled device near infrared Diffuse reflectance Fourier Transform Infrared High performance liquid chromatography Iso butyl methyl ketone Internal reflection element

436

LC LED LOD MEK MIR MTBE NIR PLS PTFE QCL RNA SERRSS SERS TCA THF TMA TPP TPPO


Liquid chromatography Light emitting diode Limit of detection values Methyl ethyl ketone Mid infrared Methyl tert-butyl ether Near infrared Partial least squares Polytetrafluoroethylene Quantum cascade lasers Ribonucleic acid Surface enhanced resonance Raman scattering Surface enhanced Raman scattering Trichloroacetic acid Tetrahydrofuran Trimethylamine Triphenylphosphine Triphenylphosphine oxide

ACKNOWLEDGMENTS The authors acknowledge the financial support of the Ministerio de Educacioń y Ciencia (Project CTQ2005-05604, FEDER) and Direccio´ General d’Investigacio´ i Transfere`ncia Tecnolo`gica de la Generalitat Valenciana (Project ACOMP06-161). S. Armenta also acknowledges the FPU grant (Ministerio de Educacioń y Ciencia (Ref. AP2002-1874)).

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J.J. Laserna, A. Berthod and J.D. Winefordner, Microchem. J., 38 (1988) 125–136. R.D. Freeman, R.M. Hammaker, C.E. Meloan and W.G. Fateley, Appl. Spectrosc., 42 (1988) 456–460. R.K. Force, Anal. Chem., 60 (1988) 1987–1989. T.B. Shope, T.J. Vickers and C.K. Mann, Appl. Spectrosc., 41 (1987) 908–912. B.J. Kennedy, R. Milofsky and K.T. Carron, Anal. Chem., 69 (1997) 4708–4715. L.M. Cabalin, A. Ruperez and J.J. Laserna, Anal. Chim. Acta, 318 (1996) 203–210. F. Ni, R. Sheng and T.M. Cotton, Anal. Chem., 62 (1990) 1958–1963. N. Leopold, M. Haberkorn, T. Laurell, J. Nilsson, J.R. Baena, J. Frank and B. Lendl, Anal. Chem., 75 (2003) 2166–2171. H.Y. Chen and Y.T. Long, Anal. Chim. Acta, 382 (1999) 171–177. J.M.E. Storey, T.E. Barber, R.D. Shelton, E.A. Wachter, K.T. Carron and Y. Jiang, Spectroscopy, 10 (1995) 20–25. R. Kellner, B. Mizaikoff, M. Jakusch, H.D. Wanzenboeck and N. Weissenbacher, Appl. Spectrosc., 51 (1997) 495–503. L.G. Thygesen, K. Joergensen, B.L. Moeller and S.B. Engelsen, Appl. Spectrosc., 58 (2004) 212–217. B. Lendl, H. Ehmoser, J. Frank and R. Schindler, Appl. Spectrosc., 54 (2000) 1012–1018. N. Weissenbacher, B. Lendl, J. Frank, H.D. Wanzenboeck and R. Kellner, Analyst, 123 (1998) 1057–1060. M. Holtz, P.K. Dasgupta and G.F. Zhang, Anal. Chem., 71 (1999) 2934–2938. N.J. Pothier and R.K. Force, Appl. Spectrosc., 48 (1994) 421–425. N.J. Pothier and R.K. Force, Anal. Chem., 62 (1990) 678–680. G.T. Taylor, S.K. Sharma and K. Mohanan, Appl. Spectrosc., 44 (1990) 635–640. V.J.P. Gouveia, I.G. Gutz and J.C. Rubim, J. Electroanal. Chem., 371 (1994) 37–42.

CHAPT ER

16 Electrochemical Detection Ari Ivaska

Contents

1. 2. 3. 4.

Introduction Detector Design Conductometric Measurements Potentiometric Measurements 4.1 General 4.2 Applications 5. Voltammetric and Amperometric Measurements 5.1 General 5.2 Stripping techniques 5.3 Bead injection 6. Coulometric Measurements 7. Conclusions Acknowledgments References

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1. INTRODUCTION Many different analytical methods are used as detection techniques in flowinjection analysis (FIA). The spectrophotometric methods are the most frequently used. This is mainly due to the simple construction of a spectrophotometric flowthrough cell and that, over the years, many spectrophotometric methods have been developed for batch analytical determinations of a large variety of different ions and compounds. In a spectrophotometric determination the light has to pass through the sample plug and the signal is generated as a result of the interaction between electromagnetic radiation and matter. The electrochemical methods of analysis, however, are based on the interaction of electrical energy and matter. One of the advantages of electrochemical methods is that the signal generated is an electrical signal that can directly be used in data processing and no further transformation processes are required. The measurement in flow analysis is done Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00616-8


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in a flow-through electrochemical cell containing at least two electrodes. The carrier flow is either continuously propelled through the cell and when the analyte passes through the cell a transient signal is recorded. The flow may also be stopped in the cell when the entire analyte or a certain part of it is in the cell. The possibility for flow manipulation increases the versatility of the flow analytical methods especially when the sequential injection analysis (SIA) technique is used. In most of the electroanalytical methods the analyte has to be in physical contact with the electrodes bridging the electrodes together in order to form a closed electrical circuit and in many methods there is a charge transfer reaction taking place at the surface of the electrode. There are also methods where the electrodes are located outside the cell and are not in physical contact with the analyte. In those methods the electrolyte ( ¼ analyte) participates in the measurement procedure as signal conveying part and the magnitude of the signal will depend on the concentration of the analyte. In all the cases of electrochemical detection the magnitude of the measured electrical signal is in a specific way depending on the properties of the electrolyte. In most of the methods, a concentration-dependent parameter, such as voltage, current, resistance or charge, is measured while the other parameters are kept constant or manipulated to receive the desired signal correlating with the sample composition. The electroanalytical techniques can be divided into different groups depending on the electrical signal that is measured. A schematic presentation of the techniques is presented in Figure 1. The scheme is based on the recommendation of the International Union of Pure and Applied Chemistry

Electroanalytical methods

No electrode reaction is considered: measurement of bulk property -conductometry, G ∝ C

Electrode reaction is at equilibrium: measurement of potential at zero current -potentiometry, E ∝ lgC

Measurement of current - voltammetry - amperometry, at constant potential i∝C

Figure 1 Classification of electroanalytical techniques.

Electrode reaction is considered

Electrode reaction is not at equilibrium: measurement at polarized electrode

Measurement of current and time -coulometry amount of substance ∝ ∫ i dt t

Electrochemical Detection

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(IUPAC) [1]. The methods are divided between those where the measured signal depends either on an electrical property of the bulk of the sample or on an electrochemical reaction at the working/indicator electrode. Measurement of electrical capacity of the cell and conductivity of the sample belong to the first category where only bulk properties are considered. In the conductivity measurements the bulk ohmic impedance of the cell is measured. All ions, anions and cations contribute to the conductance and therefore the conductivity method is not specific. Measurement of the electrical capacitance of the cell is also an impedance measurement and the response is not specific either, but is related to the dielectric constant of the sample. The methods where electrode reactions are considered can be divided into two groups: measurement at a non-polarized electrode where the reaction is at equilibrium or at a polarized electrode where the electrode reaction is forced to take place by applying an external voltage to the electrode. In a measurement with a non-polarized electrode, potentiometric measurement, a thermodynamic property of the cell is considered, i.e. the equilibrium potential of the cell. The activity of the analyte can be related to the equilibrium potential by the Nernst equation giving a logarithmic relationship between them. This method can be regarded rather specific because sensors selective to certain ions can be used in the flow cell. There are numerous techniques where polarized electrodes are used. Only the most common ones, voltammetry and coulometry are shown in Figure 1. In voltammetry the electrical current passing through the cell is measured as a function of the applied potential and is linearly dependent on the concentration of the analyte. In amperometry the measurement is done at a constant potential and the method is a subgroup of voltammetry. In coulometry the current is integrated over a period of time giving the charge as the parameter measured. The amount of substance, number of moles, of the analyte is directly related to the charge consumed during the electrode reaction according to Faraday’s law. Coulometric measurements may be performed either in constant current or constant potential mode. Voltammetric and coulometric methods are not as selective as potentiometric methods because all the substances that can undergo an electrochemical reaction at the applied potential and are present in the sample will contribute to the measured signal. In conductometric as well as in potentiometric and voltammetric measurements the response is related to the concentration or activity of the analyte and can be evaluated by using calibration curves. In coulometry, however, the measured charge gives directly the amount of substance and therefore no calibration curves are needed. However, in coulometry the analyte is consumed during the electrode reaction and in order to be a reliable method 100% current efficiency is required. Conductometry and potentiometry are sample nonconsuming methods. In voltammetry only an insignificant amount of the analyte is consumed and therefore several measurements can be done on the same sample. Only in voltammetric stripping methods involving very low concentrations of the analyte, the amount consumed during the electrode reaction has to be considered if several measurements are to be done on the same sample.

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A number of reviews have been published dealing with different aspects of electrochemical detection in flow analysis. Fernandez-Abedul et al. have covered the coupling of FIA to immunoassays [2]. In their review Pe´rez-Olmos et al. have discussed electrochemical detection in SIA mainly from the application point of view [3]. Trojanowicz has in his review described the achievements in the application of different electrochemical detection methods, e.g., in FIA [4].

2. DETECTOR DESIGN In electrochemical detection in flow analysis the carrier stream is in physical contact with the electrodes except in contactless conductivity measurements. The flow pattern is preferably laminar in order to make reproducible measurements. In general, three different flow configurations are used in electrochemical detection in flow analysis. Schematic diagrams of these configurations are shown in Figure 2. In Figure 2a, a laminar flow passes a planar electrode. This kind of configuration is used in cases where, e.g., a planar ion-selective or voltammetric electrode is immersed in the flow channel as in some commercially available thin-layer cells. A tubular electrode, Figure 2b, can be used in conductometric measurements but also in potentiometric redox measurements and in voltammetric detection as the working or the counter electrode. When using the wall-jet configuration, Figure 2c, the solution is impinged against the electrode surface from which it is then radially dispersed. The wall-jet electrode is mainly used in amperometric detection where the mass transfer is of importance and is considerably improved by forced convection. The current response depends on the ratio between the radii r1 and r2. Potentiometric measurements are also done in wall-jet cells in order to improve the response time of the flow-through system. Equations describing the electrical currents in amperometric measurements with the three measurement configurations shown in Figure 2 are given in a paper by To´th et al. [5].

3. CONDUCTOMETRIC MEASUREMENTS The electrical current passing through the solution is accomplished by movement of ions. The electrical conductivity of a solution is reciprocal of the bulk ohmic resistance and is due to migration of ions through the solution. The concentration of all ions and their mobilities determine the electrical conductivity of a solution. Mobility depends on the charge and size of the ions, temperature of the solution, and the bulk properties of the solvent, such as dielectric constant and viscosity. Measurement of electrical conductivity of a solution is normally done in two different ways depending on the application: with contact electrodes or with contactless (inductive measurement) electrodes. In the contact electrode measurement two similar electrodes, normally of platinum and coated with platinum black, are immersed in the sample solution. In the flow analytical measurements these electrodes are normally embedded in series in the flow


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(a)

Electrode

Flow

(b)

Electrode

Flow

Flow

(c)

Tubing r2 r1

Electrode

Figure 2 Laminar flow passes a planar electrode (a), laminar flow through a tubular electrode (b) and wall-jet electrode (c).

channel forming the conductivity cell. A schematic diagram is presented in Figure 3a. In the electrical measurement procedure this cell is a branch of a Wheatstone bridge. To avoid polarization of the electrodes an alternating potential with frequency o1 kHz is applied between the electrodes. The solution resistance is measured with the bridge. The measured conductance, G, can be expressed by the following equation: A (1) l where k is the conductivity, A the area of the electrode and l the distance between the electrodes. The unit of conductance is siemens, S, that is the reciprocal of ohm, O. The common unit of k is S cm1. G¼k

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(a)

(b)

Figure 3 Conductivity measurement in a flowing stream with contact electrodes (a) and in contactless mode (inductive coupling) (b).

Conductometric detection offers a simple way to follow changes in electrolyte concentrations and can be used for analytical determinations in cases where the analyte itself is the only ionic compound present or the ion concentration of the sample depends on the analytical reaction. Urea was determined by enzyme hydrolysis in measuring the conductivity before and after hydrolysis with an FIA instrument [6]. FIA with conductivity detection was used in the determination of lead by hydride generation reaction [7] and ammonium ion in an SIA system connected to a gas permeable membrane [8]. The contactless or inductive conductivity measurement in its essence measures the resistance of a closed loop of solution by the extent to which the loop couples two transformer coils. A schematic diagram of the measuring system is shown in Figure 3b. The primary coil is connected to an oscillator that supplies an alternating voltage. Through the solution another alternating voltage is induced in the secondary coil and is transmitted to the detector part of the measuring system. With constant input excitation signal, the induced response will depend on the ionic composition of the sample in the flow cell. Contactless measurement can also be done in capacitive way. Pungor et al. have designed an oscillometric, i.e., working in capacitive mode, flow cell for measurement of conductivity and permittivity. The cell was used in the determination of glucose by FIA [9]. Hauser has exploited this detection method in capillary electrophoresis where a flow analysis manifold was coupled to the capillary system [10,11].


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Hoherca´kova´ et al. have used flow-through cells with capacitive contactless detection in FIA [12,13] and Xuan et al. in capillary flow injection and capillary electrophoresis when studying iron species as plant metabolites [14].

4. POTENTIOMETRIC MEASUREMENTS 4.1 General In potentiometric measurements the potential of an electrode is measured when no current is drawn through the electrode, i.e., the electrode reaction is at equilibrium. But in reality a small current has always to be drawn through the measuring system in order to be able to measure the equilibrium potential. This current, however, is negligible and does not disturb the equilibrium at the electrode surface. Let us consider a redox reaction at the electrode surface (Equation (2)): Ox þ ne $ Red

(2)

where Ox and Red denote the oxidized and reduced forms of a compound, respectively. The number of electrons involved in the reaction is n. The potential of the electrode, E, follows the Nernst equation: RT aOx ln E ¼ E0 þ (3) nF aRed where E0 is the standard electrode potential for the electrode reaction. R is the gas constant, T – the absolute temperature and F – the Faraday constant. The activities of the species Ox and Red are denoted by aOx and aRed, respectively. Equation (3) is valid for redox measurements where the indicator electrode is of platinum, gold, glassy carbon or any other material that does not participate in the electrode reaction in any other way than by conveying electrons between the species in the solution and the solid electrode substrate. It is also valid for a metal electrode in contact with its own ions. In that case aRed is the activity of the solid metal phase and is by definition 1. If ion-selective electrodes are used where the electrode membrane contains an ionophore or a compound with which the analyte ion selectively reacts leading to partitioning of the ion between the solution phase and the membrane according to the following reaction: Ion ðsolutionÞ $ Ion ðmembraneÞ the potential of the electrode is described by the following equation: RT ln aion E ¼ E0 zF

(4)

(5)

The charge of the ion is denoted with z and the sign is + for cations and – for anions. The activity of the ion is denoted by aion. It should be noted that ionselective electrodes respond to activities rather than to concentrations of free ions and not to those in any complex form. This is an advantage of potentiometry with ion-selective electrodes because in many cases activities of free ions are of interest, e.g., in clinical determinations of ionic species in body fluids.

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4.2 Applications Applications of potentiometric sensors in flowing solutions have been discussed earlier in the literature [15,16]. The potential of a single electrode cannot be measured but rather the potential difference between two electrodes. Therefore, a reference electrode has to be used in all potentiometric measurements. It is constructed in such a way that it has a constant potential. The most common reference electrodes are the silver–silver chloride and calomel electrodes. A schematic diagram illustrating the principles of potentiometric measurements in flowing streams is shown in Figure 4. Both the indicator electrode, i.e., an ionselective, and a reference electrode are immersed in the flow. The reference electrode has to be placed downstream of the indicator electrode in order to avoid interferences at the indicator electrode due to slow diffusion of the internal electrolyte from the reference electrode. One advantage in the use of a reference electrode in flowing solutions is that the liquid junction potential is kept constant due to the continuous flow maintaining constant solution composition at the tip of the electrode. In designing the flow cell, it should also be considered that the hydrostatic pressure in the reference electrode should always be higher than the pressure in the flowing solution in order to avoid reverse flow of ions and contamination of the internal solution from the flowing solution transporting the sample. There are several problems connected with potentiometric measurements in flow analysis. Gas bubbles originating either from leakage at joints or from dissolved gases are one of the main problems in FIA in general and in electrochemical measurements in particular because they create disconnection in the electrical measuring circuit. Especially in voltammetric and amperometric measurements it may take a rather long time before the measuring system can recover from the open circuit situation created by bubbles. Formation of static electricity in the flow cell may also give extra noise in the signal. Streaming potential is another type of disturbance that should be considered in flow analysis. It is usually created when high flow rates and narrow channels are used in cases where the flowing solution has low electrical conductivity. Such potentials are formed in continuously working flow-through cells and give extra

Figure 4 Schematic diagram of a potentiometric measurement in a flowing stream.


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Figure 5 Experimental setup of the SIA–LoV (lab-on-valve) instrument for potentiometric experiments with a solid state ion-selective electrode. Reprinted from Ref. [24]. Copyright (2007), with permission from Elsevier B.V.

disturbances in the form of potential fluctuations when the flow rate varies [17]. By increasing the conductivity of the flowing medium, e.g., by adding a neutral electrolyte to the stream, the effect of streaming potential can be reduced. A practical way to minimize the streaming potential is to connect the inlet and outlet of the flow cell electrically and ground this connection as shown in Figure 5. The signal in potentiometric detection, in theory, is independent on the flow rate. The detection limit of potentiometric sensors, however, is improved in flowing streams [18]. This is obviously due to the fact that in static measurements the detection limit will partially be determined by the diffusion of the active components from the sensing membrane. In flowing streams, however, these components are flushed off from the membrane surface keeping it in a more pristine state and the concentration at the surface is the same as or close to the concentration in the bulk. Due to the short contact time between the sample and the electrode in flow analytical methods the electrode itself cannot influence the sample solution. The response time is also improved in FIA methods where the diffusion layer at the electrode surface is rather thin. The response time of the electrode should be short in order to respond to the rapid changes in the analyte concentration when the sample passes the electrode. The potential–time curves obtained in a flow-through detector as a result of a single sample injection were already examined in the early days of FIA [19]. There is always a drift in the electrodes, especially in continuous use, and in order to obtain reliable readings they should be frequently calibrated. A special calibration sequence should be introduced between the measurements where standard solutions are injected in the carrier solution and the calibration curve is refreshed.

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In one of the first applications of potentiometric detection in FIA a conventional ion-selective electrode was used [20]. The carrier solution was flushed against the electrode membrane and then let freely to flow to a beaker where the reference electrode was placed. Due to the flowing solution both electrodes were all the time in electrical contact with each other through the carrier solution and the small flow of electrolyte from the reference electrode did not affect the measurement. Even disturbances from air bubbles could be reduced by this simple construction. The cell design could accommodate most of the conventional ion-selective electrodes. Ion-selective field effect transistors have also been used in potentiometric detection in FIA [21]. Potentiometric detection was also used in the microconduits introduced by Ruzicka and Hansen [22]. A silver or platinum rod was introduced in the flow channel and their surfaces were modified in order to make them selective electrodes. The concept of solid-contact ion-selective electrodes can with great advantage be used in similar cases. In that concept a layer of conducting polymer is introduced between the solid substrate, platinum or glassy carbon, and the ionselective membrane [23]. The solid-contact ion-selective electrodes are robust and can be miniaturized and are therefore very suitable to be used in different flow channels and in the lab-on-valve (LoV) concept as well. The experimental setup for an LoV experiment with a calcium ionophore-based solid-contact electrode and a polyaniline-based pH electrode is shown in Figure 5 [24].

5. VOLTAMMETRIC AND AMPEROMETRIC MEASUREMENTS 5.1 General The electrochemical cell in voltammetric experiments consists of three electrodes: the working electrode, the reference electrode and the counter or auxiliary electrode. The electrochemical reaction takes place at the surface of the working electrode and the electrical current in the cell flows between the working and the auxiliary electrodes. There are also applications where only two electrodes are used. The most common materials for working electrode in flow analysis are platinum, gold, glassy carbon and a thin film of mercury. Boron-doped diamond has also been used. Voltammetric measurements are done by applying a potential scan to the working electrode and the current is measured. It is produced in the electrochemical reaction (Equation (2)) that is forced to take place in one of the directions by applying an overpotential from an external potential source. The current is normally proportional to the concentration of the electroactive compounds in the solution. When the current is measured at a constant potential the technique is called amperometry. In voltammetric measurements the current measured consists of two components: the Faradaic current and the charging current. The Faradaic current originates from the electrochemical reaction and is proportional to the concentration of the analyte. The charging current, however, is not an analytical signal but rather interference or noise because it is formed


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when the double layer at the working electrode is either charged or discharged when the potential of the electrode is changed, e.g., by scanning. Different voltammetric techniques have been developed in order to eliminate the charging current and to enhance the Faradaic current. In amperometry the charging current is practically zero and the current response is a direct measure of the analyte concentration and therefore most of the voltammetric detection methods used in flow analysis are based on amperometric detection due to its simplicity. The coupling of FIA with voltammetry has been discussed by Janata and Ruzicka [25]. Two different designs of voltammetric flow-through cells are shown in Figure 6. The cell in Figure 6a has the wall-jet configuration where the carrier stream is impinged against the working electrode. The reference electrode is placed at the outlet of the cell which is a platinum tube functioning simultaneously as the auxiliary electrode. In some designs the inlet tube is used as the auxiliary electrode. A thin-layer cell construction is shown in Figure 6b where the flow passes the planar working electrode immersed in the flow channel. The auxiliary electrode can be placed adjacent to the working electrode in the channel as shown in Figure 6b or it can be an outlet platinum tube. The reference electrode in this design is normally placed somewhere outside the cell. Because the electrical current in voltammetric measurements flows between the working and the auxiliary electrode they should be rather close to each other in order to minimize the potential drop due to solution resistance in the electrical measurement circuit. As discussed in the potentiometric section, the presence of air bubbles in the carrier stream often results in disconnecting the electrical circuit and it may take a while before the signal is stabilized. In a recent work all the three electrodes (working, reference and auxiliary) were placed close to each others in a flow channel and were covered with a thin film of the ionic liquid: imidazolium salt-functionalized polyelectrolyte that is only sparely soluble in water [26]. In this configuration the electrodes were all the time in electrolytic contact with each other irrespective of the presence of air bubbles in the carrier flow. However, the electroactive compounds to be determined had to diffuse from the carrier stream into the thin layer of the ionic liquid and to the surface of the working electrode. This could result in a short lag time in the detector signal but in most cases this delay should be negligible. A two-channel fountain cell has also been used in voltammetric detection in flow analysis [27]. The design of the cell is shown in Figure 7. Two separate flows enter the cell in the middle. One of the flows is 0.1 M KCl and the other one is the carrier solution. How the two flows are divided in the fountain cell depends on the individual flow rates: the flow with higher flow rate occupies a larger sector of the cell and equal flow rates result in equal division of the cell volume. The broader line between the sectors is very sharp and practically no dispersion takes place between the flows in the cell. A planar silver electrode is embedded in the bottom of the fountain cell in the sector occupied by the flow of the KCl solution. This part of the cell functions as the reference electrode. Planar gold and glassy carbon electrodes, the working and the auxiliary electrodes, are embedded in the part of the fountain cell occupied by the carrier stream. The performance of the cell was tested in flow measurement of the

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Figure 6 Electrochemical flow-through cells used in voltammetric detection with wall-jet (a) or thin layer (b) design.

Fe(III)/Fe(II) redox couple. Use of the fountain cell in potentiometric detection has also been demonstrated in the same work. The most common amperometric biosensors contain a layer of immobilized enzyme on a platinum-working electrode. When the sample zone enters the flow


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Figure 7 Schematic diagram of the fountain cell in assembled form from the side (C) and separated into parts: (A) inlet block; (B) cell top; (D) gasket/spacer; (E) electrode block. Reprinted from Ref. [27]. Copyright (1996), with permission from Elsevier B.V.

cell a specific enzymatic reaction takes place producing hydrogen peroxide which is detected through an oxidation reaction at the electrode. The biosensor layer may also contain a mediator that is reduced in the enzymatic reaction and is reoxidized at the working electrode. Electronically conducting polymers incorporating the enzyme can also be used as the sensing layer on the working electrode. In such cases the electric signal formed in the enzymatic reaction is transmitted through the polymer network to the electrode. The current peak produced is the analytical signal. Stop-flow technique can be

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used in cases where the enzymatic reaction is slow. Several review papers have been published dealing with biosensors with amperometric detection in FIA [28–30].

5.2 Stripping techniques Electrochemical stripping analysis is mainly used in the determination of amalgam forming metal ions. The analytical procedure consists of several steps. The analyte is first preconcentrated on the surface of the working electrode that most often is a thin layer of mercury. Depending on the electrochemical procedure used in the determination, several techniques are applied: potentiometric, anodic, cathodic and adsorptive stripping. Both thin-layer and wall-jet cells have been used in flow analysis. In the potentiometric stripping technique the metal ions are reduced at the mercury film electrode by applying a negative potential at which the ions of interest are reduced and form amalgam and are dissolved in the mercury film. In the anodic-stripping technique the preconcentration step is the same. In potentiometric stripping the electrolyte in the cell is changed to a solution containing an oxidizing agent that chemically oxidizes the metals dissolved in the mercury film. A carrier stream containing dissolved oxygen has even been used in some cases. During the stripping step the flow is stopped. The potential of the working electrode is then monitored. The metals are oxidized at different potentials giving qualitative information of the sample composition. The technique has therefore also been called as chemical stripping. The quantitative information is based on the time the working electrode spends at a particular potential which corresponds to the stripping of the metal of interest. In anodic stripping, the oxidation of the amalgamated metals is accomplished by applying a potential scan in anodic direction from the preconcentration potential. Quantitative information is derived from the oxidation current at different potentials. In the cathodic-stripping technique the analyte forms a chemical compound with mercury and is mainly used for the determination of halides, sulfides and selenides. The preconcentrated analytes are then stripped off from the mercury surface by applying a cathodic scan. In adsorptive-stripping voltammetry the analyte is adsorbed on the electrode surface or forms a complex that is adsorbed. The technique is mainly used for the determination of organic substances and metal ions that do not form amalgams. Due to the versatility of FIA, the stripping techniques are rather suitable detection methods for the determination of many different types of compounds, though these are mainly metal ions. Potentiometric stripping is a rather simple technique and therefore is often used [31–33]. Anodic stripping is a good method for the determination of metal ions at low concentrations and has also been used in flow analysis [34,35]. To improve the accumulation of metal ions in the preconcentration step in anodic-stripping analysis, Wang et al. introduced the concept where the flow was reversed several times


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when the analyte zone was in the flow cell [36]. In this way the residence time of the analyte in the cell was increased. Adsorptive-stripping detection in FIA has been used both for the determination of heavy metals [37,38] and pharmaceutical compounds [39,40]. Speciation of selenium has been studied by flow-injection cathodic-stripping technique [41]. The introduction of SIA has revolutionized the sample handling in flow analytical methods [42]. By that technique it is a rather simple matter, by modifying the software, to manipulate the flows in the instrument. That was demonstrated in anodic-stripping analysis of heavy metals [43]. The whole stripping procedure with plating of the thin mercury film, accumulation step, exchange of the stripping medium and removal of the used film was easily controlled by the software. Even complex mixtures of metals with overlapping stripping peaks could be determined simultaneously by changing the stripping medium. The versatility of the sequential-injection technique was further demonstrated in the adsorptive-stripping mode [44]. Both FIA and SIA techniques have been used in the simultaneous determination of some heavy metals in wastewater samples [45].

5.3 Bead injection Electrochemical detection can also be implemented in the bead-injection technique. A jet-ring cell was used in the amperometric detection of glucose in an SIA system [46]. Agarose beads with immobilized glucose oxidase were injected in the cell where they were captured at the working electrode. When the injected sample containing glucose infused the bead layer, hydrogen peroxide was released in the enzymatic reaction and was oxidized at the glassy carbon-working electrode. For the next determination the used beads were discarded and a new layer of beads was injected allowing determination on a renewed reactant surface. Mayer and Ruzicka developed a bead-injection system where electrically conducting glassy carbon beads and non-conducting beads with different immobilized enzymes were injected in the flow cell capturing the beads [47]. The procedure and the cell are shown in Figure 8. They applied the system to the amperometric determination of alcohol, galactose, glucose and lactate. The concept of conducting beads was tested in the determination of hexacyanoferrate, demonstrating that the system can be used in electrochemical experiments with renewable electrode surface. After each determination a new injection of beads was done and the problem of working electrode fouling was avoided.

6. COULOMETRIC MEASUREMENTS In coulometric methods the amount of charge consumed in the electrochemical reaction is measured. The reaction must be brought to completion. The charge is a

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Figure 8 Electrochemical jet-ring sensor. (A) Beads are introduced into the cell; (B) beads are trapped and accumulated on the sensor surface; (C) sample (dark shading) is perfused over the beads, and current is continuously monitored; (D) the outlet gap is opened and the beads are discarded. CE, counter electrode; TT, Teflon tube; RE, reference electrode; WE, working electrode. Reprinted from Ref. [47]. Copyright (1996), with permission from the American Chemical Society.

measure of the amount of substance consumed in the reaction or reagent produced for the complete reaction with the analyte. Coulometric methods relay on Faraday’s law: Q (6) N¼ nF where N is the amount of substance consumed or produced by the charge Q. The fundamental requirement of coulometric analysis is that only a single reaction takes place at the working electrode and that the reaction proceeds with


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100% current efficiency. In a coulometric experiment either the potential or the current are held constant or in general the current, i, is integrated over the time of the experiment, t: Q¼

Z

t

idt

(7)

t¼0

Coulometric detection was used already in the early days of FIA, despite the many problems connected with that mode [48]. A reticulated vitreous carbon flow-through electrode was used in both an amperometric and coulometric mode of operation. Different designs of flow-through coulometric cells have been devised over the years improving the current efficiency [49–51]. Carbohydrates have been determined by pulsed coulometric detection at a constant detection potential [52]. The authors improved their method later by cycling the potential over a specified range in order to eliminate the baseline drift caused by surface roughening and changes in pH [53]. Coulometric titrations have also been performed by the flow analysis methodology. Redox and acid–base titrations where the reagent is generated coulometrically have been done in flow-through reactors with different designs and mode of detection [54–56].

7. CONCLUSIONS Electrochemical detection in flow analysis has several advantages but disadvantages as well. The detector design with the electronics can be simple and compact allowing the construction of portable instruments especially when conductometric, potentiometric and amperometric detection is used. The detection limit in potentiometric measurements is lower than in batch methods. Air bubbles in the carrier stream and electrode fouling, requiring periodical calibration and renewing of the surface, create problems. SIA methodology offers unique possibilities of liquid handling for both the sample and reagents enabling complicated assays. The possibilities electrochemical detection can offer to the bead-injection technique have not yet been fully exploited.

ACKNOWLEDGMENTS ˚ bo Akademi Process Chemistry Centre nominated as the This work is part of the activities of the A National Centre of Excellence in research by the Academy of Finland for 2000–2011. The author acknowledges Fredrik Sundfors and Kim Granholm for the drawings in this chapter.

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CHAPT ER

17 Miscellaneous Detection Systems Kate Grudpan and Jaroon Jakmunee

Contents

1. Introduction 2. Conductometric Detectors 3. Miscellaneous Non-Spectrophotometric, Optical Detection Systems 3.1 Visual optical detection 3.2 Refractometry 3.3 Turbidimetry, nephelometry, and light scattering 3.4 Cytometry and microscopy 3.5 Optrode devices 4. Radiometric Detection 5. Thermometric and Enthalpimetric Detection 6. Dynamic Surface Tension Detector 7. Mass Spectrometry 8. Nuclear Magnetic Resonance (NMR) 9. Piezoelectric Detection 10. X-Ray Fluorescence 11. Conclusion Abbreviations References

461 463 464 464 465 467 470 472 474 476 477 479 480 481 485 505 505 506

1. INTRODUCTION Flow injection analysis (FIA) was originally conceived as a means of automating serial assays. In this approach a sample is injected as a discrete portion into a stream of reagent or carrier flowing in a narrow-bore tube, and the concentration gradient of analyte or reaction product is continuously followed with a flowthrough detector. The resulting detector response is related either directly or indirectly to the concentration of analyte. FIA may be considered as a technique for information gathering from the concentration gradient formed by an injected, well-defined zone of a fluid dispersed into a continuous unsegmented stream of a carrier. Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00617-X


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FIA may be used:

for serial assay, for continuous monitoring and process control, for linking chemistry to instrumentation, for enhancing detector performance, for hyphenation to yield new information via multidimensional readout, as an impulse-response technique, and for miniaturization and integration with other detection techniques.

The development of various detector systems has been a major emphasis in the evolution of FIA and successive generations of sequential (SI) and bead injection (BI) analysis. Detection methods applied to FIA and related techniques may include those based on optical (atomic absorption, chemiluminescence, fluorometry, phosphorimetry, flame emission, inductively coupled plasma-atomic emission spectrometry (ICP-AES), inductively coupled plasma-mass spectrometry (ICP-MS), molecular emission cavity analysis (MECA), UV-VIS, turbidity, nephelometry, IR, Raman, refractrometry), electrochemical (amperometry, conductometry, polarography, potentiometry, potentiometric stripping analysis, voltammetry, chemfets, coulometry), and other phenomena (thermochemistry, viscosity, surface tension, radiochemistry). Detection systems for FIA and related techniques may involve measuring either the properties of species originally present in the injected sample using selective sensing devices (e.g. by atomic spectroscopic or potentiometric methods), or selectivity may be achieved by monitoring the properties of products resulting from sample modification or chemical manipulation. In conventional FIA systems involving injection of sample into the carrier stream, analyte detection may rely on (i) the direct measurement of analyte species which are inactive to the detector in their native state, but which yield active, measurable products following reaction, or (ii) indirectly when the analyte detector response is either less than that for the carrier, or the analyte reacts to produce species that are not sensed by the detector. Conversely, in a reverse FIA system, reagent is injected into a sample stream, and the analyte is sensed (i) directly, by following the formation of the product of reaction between analyte and reagent, or (ii) indirectly by measuring the disappearance of the reagent as it reacts with the analyte. Detectors for FIA, similar to those for liquid chromatography (LC), should have high sensitivity, low noise, rapid response, and a minimum contribution to mixing/dispersion processes in the flowing stream. The first two factors influence the detection limit of an analytical method, while the second two affect the separation between successive peaks in the detector response versus time output. The quality of electronic and transducing devices influences the signal to noise ratio of the detector output, and hence also affects the ultimate sensitivity of a particular detector assemblage. Peak broadening is affected by the internal geometry of the flow-through detector and the dimensions of the tubes connecting the detector to the manifold, which also serve as reactors for the detection chemical reactions. The flow patterns in the tubing as well as in the

Miscellaneous Detection Systems

463

flow-through cell critically influence mixing and dispersion processes and can have a major effect on analytical sensitivity. Apart from these factors, a detector as a chemical sensor should have characteristics of sensitivity (enabling detection of low analyte concentrations), selectivity, baseline and calibration stability, flexibility (for various applications, either in laboratory or in the field), longevity, simplicity, and cost-effectiveness.

2. CONDUCTOMETRIC DETECTORS Conductivity measurement, although not selective, is a simple, convenient means of electrochemical detection that is still a versatile and interesting choice of detection [1]. Apart from its popular use as a detector in ion-chromatography, conductometric detection has been applied to FIA by incorporation of separation techniques such as gas diffusion for improvement of selectivity. Applications for determination of ammonium [2], carbonate [3], and arsenic [4] have been developed employing detection of diffused gaseous species in gas-diffusion FIA. Ascorbic acid [5], acetic acid [6], volatile acidity [7], acidity in hydrated ethanol [8], urea in serum and urine [9], and ammonium in Kjeldahl digests [10] have all been determined by using reactions to produce the above related gaseous species. Typically, only 10–15% of the analyte is transferred through the gas diffusion membrane, resulting in limited sensitivity (see Chapter 8). A membraneless gas diffusion has been introduced to tackle this limitation [11]. The detection cell typically consists of two inert electrodes placed opposite to each other in the flow channel (Figure 1 [8]) or wires placed either across the flow path, parallel to it, or tubular electrodes (Figure 2 [12]). The conductance change due to passing of the diffused ions to the electrode is directly proportional to the analyte concentration in the injected sample. Recently, a capacitively coupled contactless conductometric detection (C4D) has been developed for flow techniques, such as capillary electrophoresis [13–14], anion exchange

Figure 1 PTFE tubular conductometric flow cell: A, stainless steel screws with platinized tips; I, inlet and O, outlet solution; B, epoxy resin. Reprinted from Ref. [8]. Copyright (1998), with permission from Elsevier B.V.

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l

g

2

g

1 l

4

d (A)

(B)

g

l

3 d

1

(C)

Figure 2 Conductometric cells with wire electrodes oriented across the PTFE tube (A), along the tube axis (B), and with tubular electrodes (C): (1) PTFE tube, (2) wire electrode, (3) tubular electrode, and (4) hot-melt glue. d, diameter; l, electrode length; g, gap between electrodes. Reprinted from Ref. [12]. Copyright (2005), with permission from Elsevier B.V.

chromatography [15], and FIA [12]. This approach provides a number of advantages, viz, avoidance of electrode passivation, greater stability, and cleaner flow cell design (simply a tube) with less potential for mixing and dispersion. Surfactant bubbles have a high surface area to liquid volume ratio, and have been exploited as effective atmospheric gas sampling devices, with the electrical conductivity of the bubble being used as the basis of analyte quantification [16].

3. MISCELLANEOUS NON-SPECTROPHOTOMETRIC, OPTICAL DETECTION SYSTEMS 3.1 Visual optical detection Use of visible optical detection has been applied in FIA, e.g. for the assay of acetic acid in vinegar by FIA titration. In this system, an observer simply notes the time between indicator colour changes, which equate to the peak width measurement in an FIA titration, which in term is related by calibration to concentration. Despite the simplicity of the approach and the equipment, the accuracy and precision are quite acceptable [17]. Components of the set-up were placed on a piece of clear acrylic sheet (30 30 0.2 cm), as illustrated in Figure 3. The detection point, a point for observation for visual discrimination of coloured products or for timing period of a


465

Figure 3 Arrangement of the FIA system on an acrylic sheet (A) for placing on an overhead projector or white background: R, reagent/carrier bottle; T, polyethylene tubing; C, former made of clear plastic; I, injection port; M1, mixing coil; M2, mixing chamber; D, detection point; P, receiver. Reprinted from Ref. [17]. Copyright (1993), with permission from the Royal Society of Chemistry.

colour change, is a chamber of similar design to a mixing chamber which can be fabricated as depicted in Figure 4. Siphon action together with gravity is applied as a means of regulating the flow. The system may also be used without an overhead projector but placed on a white background for visualisation. Despite the simplicity of the system and the use of visual detection, the experimental results obtained for a flow injection titration of acetic acid in vinegar are quite acceptable (Table 1). A sodium hydroxide/bromothymol blue solution is used as a carrier. A volume of acid is injected into the moving blue coloured stream, which changes to a yellow plug, which can be observed as it passes through the mixing, coil, and at the detection point. An elapsed time, Dt, between the colour change, from blue to yellow and back to blue, should correspond to the peak width at half height: V C0 SV Dt ¼ ln 10 log (1) Q CNaOH V where V is the volume of the mixing chamber, Q the flow rate, C0 the concentration of the standard or sample injected, Sv the volume injected, and CNaOH the concentration of sodium in the hydroxide carrier stream. Thus a plot of Dt versus log C0 for a set of standards should give a straight line calibration graph, as illustrated in Figure 5, from which the unknown sample concentration can be determined.

3.2 Refractometry Photometric cell systems may be utilized as refractometric detectors in FIA. The sensitivity to refractive index (RI) arises because of differences between the RI

466


Figure 4 Mixing chamber: A, acrylic sheet (3.5 3.5 0.2 cm); H, hole (0.8 cm diameter); T, inlet and outlet polyethylene tubing, fixed to A with epoxy glue; C, cover glass; OT, opaque tape. Reprinted from Ref. [17]. Copyright (1993), with permission from the Royal Society for Chemistry.

values of the injected zone and the adjacent carrier. For example, a sample solution with a high salt concentration injected into a stream of distilled water would quickly adopt a parabolic shape under the prevailing laminar flow conditions, and this would give rise to a lensing effect in the flow cell that focuses or disperses light on or away from the optical sensing element [18] (Figure 6 [19]). The magnitude of this schlieren response provides a measure of the original RI of the sample. Figure 7 [19] shows peak profiles obtained from a flow set-up using LED for chloride solutions with different concentrations. It has been reported that the device functions as a differential refractometer as well as a photometric detector. It is capable of better discrimination than Abbe refractometer and is almost as sensitive as a conventional differential

467


Table 1 Determination of acetic acid in vinegar: a comparison of flow injection with titrimetry. Modified from Ref. [17] Elapsed times (s) Nominal

Observed

Average

%(w/v)

a

Acetic acid concentrations

Standards 0.49 M 0.58 M 0.78 M 0.98 M 1.17 M

15.4, 16.6, 18.4, 19.5, 21.1,

15.5, 16.2, 18.3, 19.5, 21.0,

15.5 16.6 18.3 19.7 21.0

15.5 16.5 18.3 19.6 21.0

Samplesa 1 4 2 5 3 5 4 5 5 —

17.1, 19.8, 19.2, 19.0, 16.9,

17.3, 19.7, 19.3, 19.1, 16.3,

17.3 20.6 19.3 19.0 16.3

17.2 20.0 19.3 19.0 16.5

FIA

Normal titration

M

% (w/v) M

% (w/v)

0.655 0.930 0.909 0.880 0.550

3.93 5.58 5.40 5.28 3.30

4.26 5.28 5.00 5.62 3.48

0.710 0.883 0.833 0.936 0.581

Different commercial vinegars.

refractometer. The measurements made are thus relative, requiring a prior calibration step. Differential refractometric techniques developed in this manner afford a resolving power of 0.1 g L1, regardless of the concentration range. Problems can arise in absorptiometric determinations owing to the sensitivity of the transducer to differences in RI between the sample and the carrier stream. This presents no problem if the samples are all of similar refractometric response. If, however, large variations in the sample RI do occur, there will be a detection limit set at which point the refractometric response becomes significant in the absorptiometric signal. One of the earliest applications of FIA to viscosity measurement was to the determination of glycerol [20]. The extent of sample plug–carrier mixing was measured as a function of the sample viscosity. The use of a coloured carrier permitted detection of the sample plug by means of the change in absorbance. An automated viscometer has been developed for use in FIA. This is a modified Ostwald viscometer in which the sample to be determined is injected into a suitable carrier moving along a length of narrow-bore tubing. The time taken by the sample to travel a given distance is indicative of its viscosity [21].

3.3 Turbidimetry, nephelometry, and light scattering Light scattering phenomena can be followed in the modes of turbidimetry, nephelometry, or light scattering by making use of a simple colorimeter or

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20

B

Et/s

15

A

10

C

5

D

0 10-1

2

3 4 5 6 10-0 Acid concentration/ mol-1

10-2

2

3

4 5 6 7 8 9 10-1 Injection volume/ml

2

3

2

3

Figure 5 Elapsed time (Dt) between colour changes as a function of log(HCl injection volumes) (curve A) or a log(acid concentration) (curves B, C, and D); height reagent bottle, 50 cm; flow rate 4.8 mL min1. (A) 0.6 M HCl vs 0.02 M NaOH; (B) 0.10 mL HCl standard vs 0.02 M NaOH; (C) 0.15 mL HCl standard vs 0.1 M NaOH; (D) 0.15 mL CH3COOH standard vs 0.1 M NaOH. Reprinted from Ref. [17]. Copyright (1993), with permission from Royal Society of Chemistry.

spectrophotometer. In some reports, a fluorometer has also been employed. The popular applications of these modes of detection involve the determination of sulfate using barium chloride [22–24]. The barium sulfate precipitate/colloid that is formed in-line can be stabilized by using a surfactant or high viscosity reagent, e.g. Tweens, glycerol, or poly(vinyl) alcohol (PVA) in order to obtain reproducible output signals due to light scattering of the colloid. Use of a colloid stabilizer also helps reduce adsorption of the precipitate to the tube wall or the windows of the flow cell. Alternately, a dissolving agent, e.g. alkaline EDTA, can be used for washing the flow system. A windowless optical cell based on a liquid drop was designed to avoid this problem [25]. Sensitivity of light scattering detection depends on several factors such as the ratio of particle diameter to the wavelength of the light, polarization, angle and coherence of the incident light, shape and stability of particle. A wavelength of 410 nm is widely used as it is well suited for colloid particle size. Formation of highly insoluble colloids of narrow size range in stable suspension gives improved sensitivity and reproducibility of the measurement. Selection of a suitable solvent or carrier can reduce solubility of the colloid, and hence improve sensitivity [26]. Crystal seeding may increase reaction rate and stability of the precipitate [27]. Measurement of light intensity at 1801 (turbidimetry) or 901 (nephelometry) to


(a)

469

Normal

Light Detector

η'

LED

η Isohaline

η < η' Light focused Direction of flow (b)

Normal

Light detector

η'

LED

η η > η' Light diverged Direction of flow Normal

(c)

Light Detector

η

η'

LED

η > η' Light focused Direction of flow

Figure 6 Refraction by isohalines in transducer cell: (a) leading interface for sample of higher refractive index than stream; (b) training interface for sample of higher refractive index than stream; and (c) leading interface for sample of higher refractive index than stream with direction of flow through cell reversed; Z, refractive index. Reprinted from Ref. [19]. Copyright (1978), with permission Royal Society of Chemistry.

the light source is usually performed in FIA due to simplicity of the detector. Measurement of light scattering at different angles could also be employed for determination of particle size, which has been widely applied in field flow fractionation (FFF) [28,29]. A more sophisticated evaporative light scattering detector (ELSD), which is a general non-selective but sensitive detector usually used for high-performance liquid chromatography (HPLC) was also applied in FIA [30]. This detection technique is based on evaporation of solvent to produce a

470


Figure 7 Typical peak profiles for refractive-index responses for sodium chloride solutions: (A) 1; (B) 2; (C) 3; (D) 4; and (E) 5 g L1. Carrier stream was distilled water. Reprinted from Ref. [19]. Copyright (1978), with permission from the Royal Society of Chemistry.

greater concentration of solute or crystal, which can cause refraction, or scattering of light. Apart from sulfate, light scattering detection has also been applied in different flow-based analysis systems for chloride [31], total organic carbon [32], anionic surfactants [33], several drugs [34,35], and proteins [36–38].

3.4 Cytometry and microscopy Cytometry and microscopy used in conjunction with FIA and related techniques, especially SIA and BI, have been aimed at clinical and drug discovery applications. Flow cytometry is a valuable tool for studying the kinetics of cell activation and response, ligand binding and macromolecular assembly. The flow injection approach offers a flexible framework for facilitating different flow patterns. Use of flow injection with cytometric detection allows physical dispersion and the cellular reactions that occur along the chemical gradient thus formed to be followed. The FIA system can be coupled to a flow cytometer via a 6-port injection valve to manipulate the flow pattern without affecting the fluidics in cytometer, facilitating injection of accurate volumes of sample and reagent, and ensuring reproducible timing and controlled mixing conditions not achievable by manual manipulation. Figure 8 illustrates a flow injection cytometric set-up for a model experiment involving on-line staining of trout erythrocytes with 4u,6diamidino-2-phenylindone and prodidium [39]. Flow injection microscopy also provides a means of performing rapid monitoring of the initial kinetics of cellular responses for biologically active ligands. This is very useful for fast drug screening in drug discovery, and simplifies the process of obtaining dose–response relationships, because a


CF

TL

471

HC SV

SF P

D IV W

W AW

S

R

Figure 8 Flow injection cytometry set-up. P, peristaltic pump; SV, 10-port selection valve; IV, 6-port injection valve; HC, holding coil; S, sample aspiration port; R, reagent aspiration port; AW, auxiliary waste; TL, transfer line; W, waste line; CF, core flow; SF, sheath flow; D, detector. Adapted from Ref. [39].

Figure 9 (A) Cutaway view of the radial flow chamber showing the bottom formed by NUNC cell in which cells are shown attached to the cover slip. (B) Overhead view showing radially symmetric outflow into the chamber and the concentration gradient of stimulant that forms in the gap between the end of the outlet tube and the NUNC chamber. (C) Enlarged cutaway view of the gap between the inlet tube and the chamber bottom, showing the shape of the injected bolus as it broadens towards the chamber circumference. Reprinted from Ref. [40]. Copyright (1996), with permission from the Royal Society of Chemistry.

complete dose–response curve can be constructed from a distinct set of cells. Figure 9 depicts a flow injection microscopy set-up for a study of the responses of live cells to agonist, antagonists, and other physical stimuli, using an inverted radial flow chamber [40].

472


Top plate Syringes Screws

O-ring Sample

Bottom plate (a)

F1

P

M

FC

B

(b)

R

W

Figure 10 (a) Side view of the all-metal flow cell. (b) Schematic diagram of the FIA system. B, scanning tunnelling microscope; FC, flow cell; FI, HPLC injection valve; P, pressure equalizer; M, peristaltic pump; R, solvent reservoir; W, waste container. Reprinted from Ref. [41]. Copyright (1995), with permission from the American Institute of Physics.

Figure 10 illustrates a system that combines a simple FIA system with scanning tunnelling microscopy for a study of surface chemical processes [41,42].

3.5 Optrode devices An optrode or optode is an optical sensor device that selectively responds to an analyte in a manner analogous to an ion selective electrode. It is composed of a chemical transducer, usually immobilized on a polymer, and instrumentation (optical fibre, light source, light sensor, and other electronics). Optrodes can apply various optical phenomena such as absorption, reflection, fluorescence, and chemiluminescence, and in different configurations for measurement.


From light source

473

To detector

Bifurcated optical fiber 3 cm 0.6 cm 1.2 cm Window PTFE spacer

Anionexchange disk Frit Flow in

Flow out

Figure 11 Design of the flow cell employed for use of optrodes in FIA mode. Reprinted from Ref. [43]. Copyright (2005), with permission from Elsevier B.V.

The FIA system is employed as a solution-handling device for reaction, concentration, and washing. For example, a reflectance optrode is depicted in Figure 11 that is used for the determination of Fe(III) [43]. An anion exchange disk selectively sorbs FeðSCNÞ3 that is formed by on-line reaction between 6 Fe(III) and SCN, and this is detected by reflectance spectrometry at 480 nm using a bifurcated optical fibre. The selectivity and sensitivity of the optrode are determined by the nature of its chemical transducer and the detection method used. Because of their relative simplicity, optrodes can be made compact and physically robust, making them more convenient for use in the field. When used in conjunction with FIA, optrode performance (sensitivity, selectivity) can be improved because of the ability to perform complex solution handling including the treatment of potential interferences on-line. Flow-based systems for the determination of iron [43], copper [44], selenium [45], sulfide [46], nitrite [47,48], and pesticide [49] are good examples of the use of optrodes in this mode.

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4. RADIOMETRIC DETECTION Use of radiometric detection in FIA was first demonstrated using very simple flow-through detectors to monitor radioactivity [50–51], In addition to simplicity, there are several other advantages associated with use of FIA and related techniques in this context, such as the ability to perform safe handling and closed system manipulation (dilution, separation, extraction) of radioactive materials.

(a)

side

top

(b)

(c)

plastic sheet side

top

(d)

Count rate

(i)

(ii)

(a) (b) (c) (d)

2 min Scan

Figure 12 (i) Radiometric flow through cells for FIA: configurations of 40 cm lengths of 1 mm i.d. tubing: (a) and (b) end window and GM counters, respectively; (c) and (d) cylindrical and well-type NaI(Tl) scintillation counters, respectively. (ii) Effect of flow rate on FIA peaks obtained with well-type NaI(Tl) scintillation detector (a) 2.5; (b) 3.3; (c) 5.0; (d) 7.5 mL min1. Reprinted from Ref. [50]. Copyright (1991), with permission from Elsevier B.V.

475


R C B

I

D SH

SH

P

W SH

Figure 13 Schematic diagram of a flow injection radio release system for determination of vanadium. B, acetate reservoir buffer (0.1 M, pH 3); I, injection valve with 0.5 or 0.7 mL sample loop; C, microcolumn; D, scintillation detector (NE7D8); P, peristaltic pump (1.2 mL min1); W, waste container; SH, lead shielding; R, chart recorder. Reprinted from Ref. [51]. Copyright (1991), with permission from Elsevier B.V.

Four types of flow-through radiometric cells have been applied in FIA. The cells are assembled by using pieces of tubing (40 cm lengths and 1 mm inner diameter) for end-window and liquid Geiger–Mu¨ller (GM) counters and for cylindrical and well-type NaI/TI scintillation counters (Figure 12(i)) [50]. It has been observed that the FIA peak-shape depends on flow rate and the effective volume of the detector, i.e. residence time (Figure 12(ii)), and detector responses have been optimized by adjustment of flow rate. The sensitivity of this technique could be improved by use of commercially available radioactive flow detectors designed for HPLC [50]. These inexpensive flow cells could be useful for automatic radioactivity measurement of highly radioactive liquid samples after on-line dilution using an FIA manifold, and offer a viable alternative to the liquid type GM counter, which is no longer commercially available [50]. The cylindrical NaI/TI flow detector was demonstrated in application of measurement of radioactive silver (Ag-110m), released from the reaction with vanadate in a FIA radio release analysis for vanadium (Figure 13) [51] and in flow injection neutron activation analysis for silver [52–53]. Recent radiometric applications of FIA and its subsequent generations (e.g. SIA) have proven to be most beneficial. A SIA system (Figure 14) [54] was proposed for automated fast determination of Sr-90 in nuclear waste with online rapid separation of Sr-90 from Y-90, using an adsorbent extraction minicolumn packed with EIchrom Sr-Specs resin. Eluted Sr-90 was merged with a liquid scintillation cocktail and continuously detected by a flow-through commercial liquid scintillation counter. A SIA system with a renewable separation column that did not require elution [55] has also been described for automatic separation and determination of Sr-90, Am-241, and Tc-99 in nuclear waste, also using on-line flow-through liquid scintillation detection (Figure 15a and b). Multi-syringe flow injection systems for on-line separation of radionuclides with off-line a and b detection have also been investigated, as shown by the exemplar in Figure 16 [56].

476


MPV

HC

C

R PP

W

SC

S

LSCP DV LSC

W MC Flow detector

DC

W

Figure 14 Schematic diagram of the SIA used for determination of Sr-90 with on-line separation Y-90 in nuclear waste. C, carrier; PP, peristaltic pump; HC, holding coil; R, reagent line (8 M HNO3); S, sample; W, waste; MPV, multiposition valve; SC, sorbent column containing Sr-Spec; DV, diverter valve; LSC, liquid scintillation cocktail; LCSP, LSC pump; MC, mixing coil; DC, detector flow cell. Reprinted from Ref. [54]. Copyright (1996), with permission from the American Chemical Society.

5. THERMOMETRIC AND ENTHALPIMETRIC DETECTION Thermometric and enthalpimetric detection have been employed in FIA for a long time. It is well known that some reactions involve significant absorption or evolution of heat. A thermistor is usually employed as the sensing element in such a system due to its compact size and good sensitivity. Actually, thermistor responses to temperature change, so the sensitivity of this detection depends on molar enthalpy of the involved reaction and heat capacity of the system, including the solvent. Organic solvents, which generally have heat capacities two or three times lower than water, can be employed to enhance the sensitivity. Unlike many optical or electrochemical sensors, a thermometric sensor is not selective, and thus selectivity of the system is dependant on the selectivity of the reaction alone, and this is especially the case for those involving enzymes or other biochemical reagents. Use of enzyme thermistor sensors is well established in bioprocess control. Thermometric and enthalpimetric detection systems have also been developed for enzymatic FIA/SIA, where the flow technique provides the fluid handling capability of the system, and the detector exhibits good stability and sensitivity under thermostat control [57]. Applications include monitoring of the bioconversion of glycerol to dihydroxyacetone (Figure 17) [58], determination of glucose in drinks and serum [59], and fluoride in cosmetics [60]. Thermometric detection has also been applied in gas phase FIA for determination of carbon dioxide by reaction with ammonia in the presence of water vapour [61].


477

TL W

MPV 2

HC

3

SDL C 1

TPV A 4

SP

CB

W RS, R, S FR

2 3

D

TPV B W

1

(a)

4

A) B) CB

FR TPV

(b)

Figure 15 (a) Schematic representation of the SI-RSC instrument for separation of Sr-90, Am-241, and Tc-99. C, carrier; SP, syringe pump; HC, holding coil; MPV, multiposition valve; W, waste lines; RS, R, S, reagents, sorbent slurries, and samples, respectively; SDL, slurry delivery line; TL, transport line; TPV A, four-port, two-position valve A; CB, renewable separation column body; TPV B, four-port, two-position valve B; FR, frit restriction; D, detector or fraction collector. (b) renewable separation column configuration using a two-position valve, TPV, modified with a frit restriction, FR, connected at the bottom of the column body, CB. (A) Valve position during column packing and (B) during disposal of separation material. Reprinted from Ref. [55]. Copyright (1999), with permission from the American Chemical Society.

6. DYNAMIC SURFACE TENSION DETECTOR A dynamic surface tension detector (DSTD) is a sensor for the non-equilibrium interface tension, which is known as ‘‘dynamic interface tension’’. The DSTD is based on drop pressure measurement during growth of a drop. The detector consists of a capillary sensing tip connecting to a main flow and a pressure sensor attached to a side arm of the main flow. As illustrated in Figure 18 [62],

478


M1

HNO32M Autosampler M2

TRU-resin

Sample

T1

T2

M3

on

on

HCI 4M / TiCI3 0.02M off

H2O

HCI 4M V 1 off

V2

Multipump system (MPFS)

H.C1

H.C2 PC off on off

on

off

on off

on

S1

S1

S1

S1

10 ml

10 ml

10 ml

1 ml

Multisyringe module (MSFIA)

H2O H2O

NaNO20.25M

HNO3 0.05M

Figure 16 MSFIA–MPFS system for the separation and preconcentration of Am and Pu. Reprinted from Ref. [56]. Copyright (2008), with permission from the American Chemical Society.

the pressure sensor monitors the internal pressure of growing drop in relation to the atmospheric pressure, during pneumatic detachment, drops are blown off the sensor tip at a preset time, well before the drops would fall due to gravity. The data can be processed into a usable form as dynamic surface pressure or surface tension (Figure 19) [63] providing real-time information on the surface activity of the analyte of interest [64]. Figure 20 shows a SIA system configured for differential surface tension detection [65]. Measurements of differential pressure as a function of time across the liquid– liquid interface of organic liquid drops can be made by a dynamic interfacial pressure detector (DIPD) based on a similar principle to DSTD [66]. Although DSTD is not a selective detector, it has a high sample throughput compared with manual methods, and it is typically employed in LC, FIA, and SIA for a wide variety of samples including surfactants, polymers, and proteins. Cations and anions can affect the kinetic dynamic pressure of SDS. By introducing SDS to water sample using a SIA system, the DSTD signal pattern could be used to identify the quality of the water, and thus used as a very fast alternative screening method for water quality in the electronics industry [64].


Bridge/ Amplifier

479

Computer

Sample

Outlet

Buffer

Thermistor Heat exchanger

Polyurethane-foam insulation

Enzyme column

Aluminium block

Figure 17 Schematic representation of an enzymatic thermistor system operated in FIA mode. Reprinted from Ref. [58]. Copyright (2001), with permission from Elsevier B.V.

7. MASS SPECTROMETRY FIA and SIA have been used in hyphenated mode with mass spectrometry for sample introduction and sample handling, and this approach has proven especially useful in the area of bio-medical analysis. FIA was first interfaced to a quadrupole mass spectrometer for fast mass spectrometry (MS) characterization [67]. By use of FIA fluid handling, the total analysis time for samples in a 96-well microtitre plate was reduced to 12 min using a Gilson 215 eight probe autosampler (Figure 21), and compared very favourably with the 48 min required for a single probe autosampler. Reducing the injection volume and the time between each injector rotation could further improve the speed of analysis. Such improvement could be achieved by sequencing column switching valves to ensure that one is always active while other seven are off-line, as illustrated in Figure 21 [68]. Using this approach, the analysis time for a 96-well microtitre plate could be reduced to as little as 5 min. FIA-MS used in this mode has been usefully employed in the analysis of the products of combinatorial synthesis, and for high-speed analysis of chemical repository sample stability, and in biological screening. SIA-LOV (Figure 22) [69] with BI and coupled to a UV/VIS spectrometer and electrospray ionisation-mass spectrometry (ESI-MS) has been used for automated in situ monitoring of affinity capture and release of biotin-containing conjugates immobilized on streptavidin. UV/VIS spectrometry is useful for determining the dissociation rate constants for release from streptavidin of chromophore-tagged biotin conjugates, while ESI-MS simultaneously monitors dissociation the

480


A

Flow

Load sample Injection Valve

Solvent Pump

Capillary for Pressure Measurement

Waste

Pressure Sensor Drop

PC

Waste Collection B

Flow Pressure Sensor Dc

Open to Atmosphere 1.0 cm Membrane D 0.4 cm R' r

Figure 18 Diagram of a dynamic surface tension detection system (DSTD) showing (A) instrument design. Sample is injected into the flow, and passes through the capillary forming a repeating drop at the capillary tip. The pressure is measured as a function of time. (B) Detailed view of the stainless steel capillary and sensor. Deformation of the sensor membrane is converted into an electrical signal. Reprinted from Ref. [62]. Copyright (1997), with permission from the American Chemical Society.

dissociation products. A short analysis time (4.5 min/full cycle) with robust operation has been reported. The instrument can also be applied to repetitive assays of lysosomal b-galactosidase in human cell homogenates [69]. A similar set-up has been employed for simultaneous measurements of multiple ligand affinities to proteins immobilized on beads. The instrument offers fast determination of Kd (10–0.1 mM) for sample mixtures, and is useful for screening a large number of compounds for multiple proteins [70].

8. NUCLEAR MAGNETIC RESONANCE (NMR) The increasing demand for fast methods for high throughput screening of active compounds from natural product or combinatorial synthesis libraries, and the


A

481

Sample Introduction

Air Pressure Sensor Sensor Tip Capillary

Pneumatic Detachment Capillary

P(t)

B

60

Pressure Signal, P(t)

58

0

30 60 90 Time, seconds

120

Figure 19 (A) Schematic of DSTD capillary sensing tip. Sample from the FIA system forms a drop at the capillary sensor tip, and the internal drop pressure is monitored via a pressure sensor. Computer controlled pulses of air from a small capillary are used to detach the drop from the sensor capillary. (B) Raw pressure data obtained from a DSTD using pneumatic detachment for a sample of 5% acetic acid. Reprinted from Ref. [63]. Copyright (2001), with permission from Elsevier B.V.

advancement of NMR techniques, such as high field NMR, miniaturization of NMR probe heads, and the advent of powerful solvent suppression schemes, has lead to the development of flow-based NMR, e.g. HPLC-NMR and FIA-NMR. There have been a number of applications employing FIA-NMR that use FIA for sample handling [71–74], which provides high sample throughput and requires minimal spectrometer optimization. In combination with multiple on-line spectroscopic analysis techniques (UV, IR, and MS), almost complete structural characterization is achievable [72,73]. Chemometric techniques, such as pattern recognition methods, are required for evaluation of the spectra [74].

9. PIEZOELECTRIC DETECTION The piezoelectric properties of some materials enable the construction of devices such as quartz crystal microbalances (QCM), which have an important role in

Computer

Mixing Coil 70 cm.

Mixing Coil TeeConnector 40 cm. Solenoid Valve

Holding Coil 330 cm. Injection Valve Sl Valve Pressure Sensor

Air Burst Capillary Capillary Sensing Tip and Drop

Sl Syringe Pump

HPLC Syringe Pump

Figure 20 Schematic diagram of a SIA system incorporating a dynamic surface tension detector. Reprinted from Ref. [65]. Copyright (2003), with permission from Elsevier B.V.

HPLC Pumping system

I

II

Autosampler

Mass Spectrometer

Injector 1 Injector 2 Injector 3

from autosampler

Injector 4

to detector

Injector 5 Injector 6 Injector 7 Injector 8

Figure 21 High-throughput flow injection mass spectrometry system. The enlargement shows the flow diagram for the eight-position column selection valve. Reprinted from Ref. [68]. Copyright (2001), with permission from the American Chemical Society.

483


Bead Reservoir Holding coil 2

LOV geometry 1

4

HC1

λ HC2

3

2 5 6 1

Fiber Optics

Solenoid valve

Holding Coil 1 MS

3-way solenoid valve Carrier / wash solvent

Waste Fiber Optics LOV geometry 2

6

Bead Reservoir

5

4

1 2 3

PEEK rod

MS

3-way solenoid valve Waste

Ion exchange column

Solenoid valve

Figure 22 Drawing of LOV systems with geometry 1 and geometry 2 configurations of the bead holding cell used for UV and ESI-MS measurements of biotin-containing conjugates. Reprinted from Ref. [69]. Copyright (2002), with permission from the American Chemical Society.

miniaturization of analytical systems. The principle of QCM detection is based on the frequency change of the crystal that is proportional to the mass change on the crystal surface. The quantitative relationship between the frequency shift and the mass change is given by Sauerbray equation [75], which is applicable for gas phase sensor, or by the Kanazawa and Gordon equation [76] which also considers effects induced by the contact with liquid apart from the characteristics of the quartz disks and active layers. As a sensitive surface mass sensor, the QCM has been widely applied as a sensor for gases and acts as the basis for operation of electronic noses. QCMs usually operated in range of 5–20 MHz. Sorption of analyte gas onto the selective sorbent layer on the quartz surface leads to mass change, which will shift the oscillation frequency of the crystal. The sensitivity of the QCM is comparable to that of another mass sensitive detection device, the surface plasmon resonance (SPR) sensor, but is less complicated and of relatively lower cost [77]. Apart from the QCM, other piezoelectric devices for mass detection include micromachined resonating piezoelectric membranes [78] and a surface acoustic wave (SAW) sensor [79], the latter being able to operate at a higher frequency than the QCM, meaning that better mass resolution can be achieved. The sorbent materials used determine the selectivity of these sensors. Recently, molecular recognition based on bioaffinity and molecularly imprinted

484


Frequency counter Injection valve

Computer

Waste

Waste

Sample loop Pump

Oscilator

Water bath 10 MHz crysal O-ring joint

Buffer Oscillator Flow-through cell

(a) 6. 0.100 μg/μl 5. 0.250 μg/μl

4. 0.500 μg/μl

100 Hz

3. 1.00 μg/μl

(b)

2. 1.50 μg/μl 2 min

1. 2.00 μg/μl

Figure 23 (a) Set-up of the FIA-QCM system. (b) Individual binding curves for different concentrations of human IgG with immobilized histidine. Reprinted from Ref. [82]. Copyright (2004), with permission from Elsevier B.V.

polymers (MIPs) [80] has been introduced which is very selective, especially to biomarker biomolecules of importance in medical applications. Incorporation of these piezoelectric detection systems into FIA or SIA systems leads to improved selectivity and sensitivity, and speed enabling real-time analysis of biospecific interactions such as immunochemical reactions, hybridization of DNA, interaction of nucleic acids with proteins and drugs, and interactions of protein with amino acid and drugs [81–83]. Unlike conventional immunoassay, this detection mode does not require the use of enzyme or fluorescence labeled tracers. An example of QCM-FIA set-up is shown in Figure 23a [82] for real-time kinetic analysis of the interaction between human tumor necrosis factor-a and its monoclonal antibodies. The freshly coated quartz crystal is mounted in the


485

flow-through system, connected with an oscillator circuit and a frequency counter. Typical sensor output signal is shown in Figure 23b for the binding of different concentrations of human IgG with immobilized histidine [84].

10. X-RAY FLUORESCENCE A FIA system with a solid phase extraction (SPE) minicolumn has been coupled to an energy dispersive X-ray fluorescence (EDXRF) detector for continuous operation. The set-up (Figure 24a) [85] included a flow cell (18 mL, 10 mm path length) which was placed in the spectrometer in the X-ray irradiation zone, and EDXRF Espectrometer

SPE column

PP

FC W

sv

s sv

(a)

PC

CS

ES

Cd

Pb

(b) 0

5

10

Pb

15

20

25

30 keV

Figure 24 (a) Experimental set-up used for FIA-SPE-EDXRF measurements. FC, flow cell; W, waste; PP, peristaltic pump; SV, selection valve; S, sample; CS, conditioning solvent; ES, elution solvent. (b) An analytical EDXRF spectrum collected during the first 30 s of an elution step; 20 mL of sample containing 20 mg mL1 each of Pb and Cd was flushed through the SPE column. Reprinted from Ref. [85]. Copyright (2007), with permission Elsevier B.V.

Table 2

Application of various detection methods in FIA or other flow modes of analysis

Detection method

Analyte

Mode

Matrix type

Basis for detection

LOD (units as quoted)

Sample throughput (injections h1)

Comments

Visual

Acetic acid

FIA

Vinegar

–

60

Very simple setup. The set can [17] be put on an overhead projector. The setup can also be employed for an assay of ascorbic acid using redox reaction with permanganate solution.

Conductivity

Ascorbic acid

FIA

Vitamin C tablets

–

90

A simple setup.

Conductivity

Acetic acid

FIA

Vinegar

Conductivity

Acidity

FIA

Fruit juice

Conductivity

Sulfur dioxide

SIA

Air

Acid-base indicator change in color of Bromothymol blue. Calibration graph is a plot of the period of color change vs log concentration of acid (FIA titration). Neutralization of the acid with flowing ammonia: following change in conductivity. Acid-base neutralization, monitoring change in conductivity of ammonia stream. The acid sample/standard was injected into a stream of ammonia with gaseous diffusion into acetic acid flow: change in conductivity. Soap bubble — a liquid thin film can trap SO2. Conductivity measure of the film is monitored.

–

–

80

37 ppbv

12

Reference

[5]

Various setups with various [6] component configurations resulting in different degrees of automation. [7] Acidity (expressed as citric acid content) is determined using simple setup with simple flow-through detector. The gas diffusion with improving selectivity. The soap solution contained [16] Triton-X 100, nonionic surfactant .The soap bubble is as gas-sampling interface. High ratio of surface area to volume of liquid is useful.

56

Piezoelectric impedance sensor-FIA method is advantageous over other detection modes employed in gas-diffusion FIA.

[86]

–

68 FIA, 20 SIA

A conventional spectrofluorimeter was used for detection.

[33]

–

50

Determination of the particle mass concentration in colloidal suspensions.

[23]

–

–

[87] The detector was demonstrated to be effective for the flow injection nephelometric determination of sulfate by precipitation as barium sulfate. – [88]

Carbon dioxide

FIA

Wine, beer

Light scattering

Anionic surfactants

FIA SIA

.

Light scattering

Sulfate

FIA

Water

Light Bile acids scatteringevaporative

FIA

Bio-fluids

Turbidimetry

SIA

Ground, surface The determination is based – waste waters on the reaction of chloride with silver ions and the subsequent measurement of the turbidity caused by silver chloride precipitation.

Chloride

Based on the use of gaspermeable membrane to separate CO2 and a piezoelectric impedance sensor to follow the conductance change occurring in the acceptor solution. Formation of a solid phase by association of anionic surfactants and protonated o-tolidine. Dodecyl-benzene sulfonic acid (DBS) was selected as the reference anionic surfactant. Precipitation as barium sulfate and detection using a low-cost flowthrough detector employing a laser pointer. Screening for bile acids by using evaporative light scatting detection.

0.01 mM

Conductivity

55–57

Table 2 (Continued ) Detection method

Analyte

Mode

Matrix type

Basis for detection



Comments

Reference

Turbidimetry

Tannins

FIA

Tea

Based on the precipitation reaction with copper(II) in acetate medium.

6.5 mg L1

90

[89]

Turbidimetry

Proteins

FIA

Animal cells

The systems were developed to provide reliable, rapid monitoring of relevant proteins in animal cell cultivation processes.

–

–

Turbidimetry

Sulfate

SIA

Natural waters and industrial effluents

10 mg L1

26

Turbidimetry

Phytic acid

SIA

Food

0.03 mg L1

20

Flow cytometry

Proteins

FIA

Fermentation fluids

–

–

Flow cytometry

Ca2+

FIA

Jurkat T lymphocytes

Turbidimetric determination of sulfate using barium chloride as reagent and measuring the absorbance of the formed suspension at 540 nm. The diminution of the calcium oxalate crystallisation reaction rate in the presence of phytic acid. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Flow injection cytometry.

The intermittent flow of the washing HNO3 solution allowed the online cleaning of the system. A limiting factor for online bioprocess monitoring, especially of high molecular weight components, is the availability of an appropriate sampling device. An alkaline buffer-EDTA solution was used to periodically redissolve accumulated barium sulfate precipitated in the system which resulted in high sensitivity, accuracy, and precision. The method was applied to the determination of phytic acid in food samples (after purification by anion exchange chromatography). –

–

–

Improved time resolution of kinetic cellular events in flow cytometry by using a coaxial flow-mixing device integrated within a flow injection system.

[90]

[91]

[92]

[93]

[94]

– Color encoded microspheres derivatized to capture particular biomolecules are temporarily trapped in a renewable surface separation column to enable perfusion with sample and reagents prior to delivery to the detector. RNA was captured and 0.1 ng total RNA detected on PNA coated Lumavidin beads.

–

A new method of automated sample preparation for multiplexed biological analysis that uses flow cytometry fluorescence detection.

[95]

–

[96]

Environmental

Coupling SIA to microsphere array and flow cytometry.

–

–

New methods for automated, direct nucleic acid purification and detection are required for the next generation of unattended environmental monitoring devices. Autonomous pathogen detection system.

FIA

Solution

b and g rays were monitored by GM and scintillation counters.

–

–

Silver

FIA

Solution

–

–

Vanadium

FIA

Solution

g-counting, neutron activation analysis with neutron source. Radiorelease technique involving reaction of vanadium with silver powder (Ag-110m). Monitoring the activity by flow-through g scintillation counter.

–

–

Flow cytometry

Biomolecules

SI-BI

–

Flow cytometry

Intact RNA

SI-BI

–

Flow cytometry, immunoassayRadioactivity

Bacillus anthracis, Yersinia pestis

SIA

Phosphorus-32, iodine-131

Radioactivity

Radioactivity

[97]

Four types of radiometric cells [50] for GM and scintillation (NaI/Te) counters. Performances of the cells were investigated. Continuous neutron activation [52] analysis was investigated. Suggestion for further investigation with commercial flow-through radioactivity counter.

[51]


Analyte

Mode

Matrix type

Radioactivity

Americium, plutonium

FIA

–

Radioactivity


FIA

Radioactivity

Yttrium

FIA

Radioactivity

Strontium

MSFIA

Radioactivity

Radium, strontium

SIA

Basis for detection

Sorbent extraction column (Eichrom TRU-resin), liquid scintillation detection. Nuclear waste Selective separation on a TRU-resin sorbent column, liquid scintillation counter, g and a spectrometry. Water and Online separation using biological column containing samples HDEHP adsorbed on a C18 support, low background proportional counter. Water, milk, soil Separation of stable and radioactive Sr using a solid phase extraction with Sr-resin, low background proportional counter. Synthetic Simultaneous Ra-226 and sample Sr-90: preconcentration and separation, isotopes isolation by sequential coprecipitation, low background proportional counter.



Comments

Reference

–

–

Separation profile was studied.

[98]

–

8

10 mg L1

–

To reduce the carryover, the [99] column was additionally washed with bioxalate and water between each sample run. Procedure is free from [100] interferences with other isotopes or elements.

10 mg L1

–

–

0.15 Bq L1 Ra226 1.0 Bq L1 Sr-90

–

[101] Sr-90 two measurements of the beta activity are needed in a time period of one day. This latter activity is calculated from its daughter Y-90, assuming an initial secular equilibrium between the two isotopes.

[58]

Radioactivity

Strontium

SIA

Radioactivity

Technetium

SIA

Radioactivity

Strontium

SIA

Radioactivity


MSFIA

Water, milk, soil Selective isolation of strontium from the sample matrix on an open tubular reactor with a wetting film phase, low background proportional counter. Caustic aged Automated microwavenuclear waste assisted sample samples treatment, oxidation procedure using peroxydisulfate in acidic solution to convert reduced technetium species to pertechnetate. Liquid scintillation counter/ICP-MS. Aged nuclear The separation on a sorbent waste extraction minicolumn containing a selective resin binds Sr-90, mixed with liquid scintillation cocktail for liquid scintillation counter. Combination of the Soil and multisyringe flow vegetable injection analysis and ashes, multipumping flow synthetic system techniques with samples of the TRU-resin for biological separation, oxidization type Pu(III) to Pu(IV) using NaNO2. Multiplanchet low background proportional counter.

0.07 Bq

–

Avoid analyte carryover and reduction of the resin capacity factor by using wetting film phase.

[102]

–

–

Microwave digestion enables control of Tc-99 speciation in such samples.

[103]

2.62 Bq

2

Worker safety is improved because solution-handling operations are fully automated and contained.

[54]

0.004 Bq mL1

2

High Fe(III) concentrations can cause a remarkable decrease in the Am(III) retention.

[56]


Analyte

Mode

Matrix type

Basis for detection



Comments

Reference

Radioactivity

Strontium, americium, technetium

SIA

Aged nuclear waste

–

–

With a renewable column carryover on the column from one separation to the next is eliminated.

[55]

FIA

–

–

–

Compare flow [104] microcalorimeter with standard spectrophotometric method for kinetic study.

FIA Thermometric Enzymecatalyzed t-ornithine, L-methionine, and fructose synthesis Thermometric Hydrogen FIA peroxide

–

Separation on column using Sr-resin, TRUresin, and TEVA-resin sorbent extraction materials. Liquid scintillation detector. Flow microcalorimeter (FMC)-reaction rate is determined from the substrate consumption as calculated from the decrease in thermometric response. An enzyme thermistor.

–

–

The enzyme-catalyzed [105] reactions were performed in a simple batch reactor with immobilized biocatalysts.

–

Immobilized enzymic (catalase) reaction heatinduced optical beam deflection.

0.025 M

8

Thermometric Carbon dioxide

Carbonate sample

Dual-phase gas-permeation 1 mM system from a liquid donor to a gas acceptor stream with a thermistor flow-through detector.

Knife-edge and a photodiode [106] detection system are expected to be developed as a novel thermooptical biosensor. [107] Determination of carbon dioxide (in the form of carbonate)

Thermometric Immobilized enzyme kinetics

FIA

60

Thermometric Fluoride

FIA

Thermometric Glucose

FIA

Thermometric Monosaccharides, disaccharides

SIA

Dynamic surface tension detector (DSTD)

Protein

FIA

Dynamic surface tension detector

Ionic surfactants SIA


Urea, FIA GdmHCl, and GdmSCN

Fluoride tablets, Measurement of the mouth rinse enthalpy change upon adsorption of fluoride onto the ceramic hydroxyapatite using a thermistor-based flow injection calorimeter. Blood Glucose was determined by measuring the heat evolved when samples containing glucose passed through a small column with immobilized glucose oxidase and catalase. Mixture Use of miniaturized flowsolution through reaction calorimeters for enzymecatalyzed reactions. Protein sample Using FIA a pH gradient is blended in real time with a protein sample as the pH-dependent protein surface activity is measured by a dynamic surface tension detector (FIA-pH-DSTD). Solutions Study of the effects of the ion contents in solutions to the dynamic surface pressure of ionic surfactants Aqueous Measuring the changing solution pressure across the liquid–air interface of 4 mL drops repeatedly forming at the end of a capillary using FIA.

5 mM

–

–

[60]

0.5 mM

–

–

[108]

–

–

–

[109]

Continuous measurement, 2 s drop time

–

[110] An automated DSTD calibration procedure and data analysis method is applied, permitting realtime dynamic surface tension data to be obtained.

–

–

Simple fast screening, but also [64] a sensitive procedure for water quality determination.

–

–

Guanidinium hydrochloride (GdmHCl) and guanidinium thiocyanate (GdmSCN) changed surface tension of the solution.

[111]


Analyte

Mode

Matrix type

Basis for detection


Surfactants

FIA

Organic liquid


Milk proteins

FIA


Sodium dodecyl FIA sulfate (SDS) and polyoxyethylene-20cetyl ether Glutathione-SFIA transferase P1-specific inhibitors

– Measures the differential pressure as a function of time across the liquid– liquid interface of organic liquid drops (i.e., n-hexane) that repeatedly grow in water at the end of a capillary tip. – Separation by hydrophobic interaction chromatography, with 3 M guanidine hydrochloride (GdmHCl) as denaturing agent in the mobile phase. Measuring a differential – pressure across the liquid–air interface of growing drops.

Enzyme affinity detection (EAD)

Solutions

Complex mixtures


2 parallel EAD systems for – substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-HPLC.


Comments

Reference

–

–

[66]

–

–

[112]

–

–

[113]

–

–

[114]

Enzyme sensor

Fructosyl amine

FIA

Enzyme sensor

L-glutamate

FIA

Microscopy, fluorescence

Cell-based drug FIA discovery functional

Chinese hamster ovary cells


Cell surface antibody binding

FIA

Cells grown in monolayer

A polyclonal cell-specific – antiserum used to probe the cell surface was monitored by indirect immunofluorescence.

–


pH

BI

Cell

–

Microscopy

Cell calcium

FIA

Agonist

pH measurements in live – cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. Cellular response to – chemical agonists is essential in understanding the complex functions mediated by cell surface receptors.

Food

The sensor utilizes fructosyl – amine oxidase isolated from the marine yeast Pichia sp. N1-1 strain – The purified enzymatic extract was immobilised in glass beads, incorporated into an assembly with potentiometric detection. Flow injection-renewable – surface technique is the automated fluidic sampling, assay, and disposal of a suspended material

–

–

[115]

60

–

[116]

–

The use of microbeads as a disposable and renewable surface circumvents the problems of conventional mammalian cell functional assays that involve repetitive stimulations of the same group of cells. This work demonstrates the power of flow injection fluorescence microscopy for the study of immunochemical interactions on viable cells as well as its potential for screening cell surface antibodies. The cell biology and pharmacology are also stimulated with carbachol and the intracellular pHdependent fluorescence from the cells is recorded. —

[117]

–

[43]

[42]

[118]


Analyte

Mode

Matrix type

ESI-MS

Pyridinium chlorochromate (PCC)

FIA

Urine

ESI-MS

Polar components

FIA

ESI-MS

Hepatitis C viral FIA (HCV) RNA

ESI-MS

Anionic, cationic, nonionic surfactants

FIA

Waters

ESI-MS

Bacterial strains

FIA

Bacterial cells

Basis for detection


20 pg PCC 5 pg Formation of chromium Cr 6+ mL1 (Cr6+) in PCC with diethyldithiocarbamate and its extraction with iso-amyl alcohol. The quantitation was performed by selected ion monitoring at m/z 513. Commercial Diluted sample was injec- – fuel ted into an isocratic stream of 100% methanol at 0.2 mL min1. RNA target and Direct injection. – ligands

93 pg LAS 797 pg All surfactants were esterquat isolated by liquid–liquid extraction and quantified using labeled triethoxylated nonylphenol ([13C6]-NP3EO) and sodium dibutylnaphthalenesulfonate as internal standards. Axenically grown bacterial – cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream.


Comments

Reference

The present method for [119] analysis of Urine Lucks is recommended for use in forensic analysis because of its speed, high sensitivity, and high specificity.

–

–

–

–

Sample solutions were analy- [120] zed unfiltered to eliminate bias from selective sorption onto filter media. The mobile phase of 5 mM [121] ammonium acetate in 50% isopropanol maintained the noncovalent complexes. Simultaneous analysis of most [122] common surfactants by alternating both positive and negative ionization modes.

Acetonitrile contributes most significantly to the extraction process.

[123]

ESI-MS

Quinine

FIA

ESI-MS and ESI-MS/ MS

Simple sugars, oligosaccharides, and iso-alphaacids Free cholesterol (FC) and cholesteryl ester (CE)

FIA

Beer

FIA

EDTA plasma sample

FIA

blood

FIA

Citrus fruit

ESI-MS/MS

ESIMS/MS

ESI-MS/MS

Guanidinoacetate (GAA), creatine Thia-bendazole, Imazalil, O-phenylphenol

High throughput – analysis achieved via improvements to the design and operation of a Gilson 215 multiprobe liquid-handling system. Simple degassing, – dilution, pH adjustment and direct flow injection. 0.1 pmol An acetyl chloride derivatization method was used to convert FC to CE and analyzed by using a fragment of m/z 369 in a combination of selected reaction monitoring (SRM) and precursor ion scan for FC and CE, respectively. Extraction 0.30 mM GAA and formation of 0.34 mM butyl esters. creatinine Combination of stable isotopically labeled internal standards and a multiple reaction monitoring technique.

1 mg kg1 TBZ & IMA?? 5 mg kg1 OPP

–

–

45

60

4

Development of [68] applications to support very high throughput molecular weight identification, i.e., 5 min/96-well plate. Adding a second and optional [124] MS dimension for improved selectivity for beer characterization by fingerprinting. – [125]

Dried blood spots were extracted using methanol–water solution containing D3-Cr. Correction for the influence of directly injected sample extract without cleanup by using the internal standards labeled with a stable isotope.

[126]

[127]


Analyte

Mode

Matrix type

Basis for detection



Comments

Reference

ESI-TOF-MS

Histamine H2receptor binding ligands

Continuous flow and FIA

–

–

1 fmol

–

[128]

HPLC-ESI-MS Ovine caseins, whey proteins

FIA

Ovine milk proteins

–

MIMS

Ethanol, BTEX

FIA

Water

–

–

[130]

MS

Oxidation methionine residues

FIA

Monoclonal antibodies, recombinant proteins

– Complexity proteins associated with genetic polymorphism, post-translational changes (phosphorylation and glycosylation) and the presence of multiple forms of proteins. – BTEX in water in commercial samples of ethanolcontaining Brazilian gasoline was monitored online by FIA coupled with MIMS. – Lysyl endopeptidase digestion, LC/MS peptide mapping.

An automated SPE method directly coupled with mass spectrometry to detect basic and hydrophobic GPCR ligands using H2 receptor antagonists with high affinity. –

60

[131] MS detection allows for reliable peptide quantitation without baseline chromatographic separation of all the peptide peaks.

[129]

MS

Benzalkonium chloride

FI

Skin

MS

Neutral lipid

FIA

Microbial cells, biofilm communities

MS

Drugs and metabolites

FIA

Multiple drugs and metabolites

MS, ion trap

Digoxin and antidigoxigenin

FIA

Soluble orphan receptors

NMR

Beer

FIA

Food

Quantification was carried out using an external standard based on peak area summation of each benzalkonium ion. Separation of neutral lipid extract of microbial cells and biofilm communities by using high performance liquid chromatography/ electrospray/tandem mass spectrometry couple with FIA system. An online solid phase extraction liquid chromatography/ tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. A FIA bioassay to rapidly detect ligands for soluble orphan receptors in complex matrices was demonstrated. Measurements were performed using a 400 MHz NMR spectrometer using flow injection technology for automatic sample changing.

1.2 ng mL1

–

11.1 fmol mL1 ubiquinone

–

nM

–

[132] The method was applied to quantify DSQUAME tape from skin surface and extracted from the tape with methanol. Highest sensitivity is achieved [133] using flow injection analysis with multiple reactions monitoring wherein ammoniated molecular ions of specific isoprenologues. This assay was developed in [134] an effort to increase bioanalysis throughput and reduce the complexity of online SPE LC/MS/MS systems.

Z250 nM Digoxin –

—

–

1H NMR spectroscopy is [136] faster and requires simpler sample preparation.

–

[135]


Analyte

Mode

Matrix type

Basis for detection

Optical sensor Lead

FIA

–

Gallocynin immobilized in 0.075 mg L1 chitosan membrane has been studied as a sensor element of an optical sensor for lead.

Opto-electro chemical sensing

Iron(II), iron(III)

FIA

Tap water

Optosensing

Benzo[a] pyrene FIA (BaP)

Monitoring the absorbance of the sulfosalicylic acidFe(III) complex at 505 nm, Electrochemical flow cell for amperometric monitoring of the current due to the oxidation of Fe(II) to Fe(III) at potential of +1.0V vs SCE. The native strong room temperature phosphorescence (RTP) emission from the BaP recognized by the MIP The noncovalent MIP was synthesized using BaP as a molecular template.

Water



Comments

Reference

–

[137]

[138]

[139]

4.3 107 M Fe(III) 5.6 106 M Fe(II)

75

The response of the sensor was reproducible and can be regenerated by using acidified saturated KNO3 solution. –

10 ng L1

–

–

Optosensing

Labetalol

SIA

Pharmaceuticals and urine

Optosensor

Butylated hydroxytoluene

FIA

Cosmetics

Optosensor

Sulfide

FIA

Seawater, ground water, waste water

Optrode

Lead and cadmium ions Salmonella typhimurium

FIA

–

FIA

–

Piezoelectric biosensor

FIA

The analytical signal (native fluorescence) being monitored directly on sensing zone microbeads. The transient retention of this compound in a flow-through cell packed with C18 silica using ethanol:water mixture as a carrier. H2S from the donor channel of the GD-module passes into an alkaline receiver and the enriched plug merges with welldefined zones of the chromogenic reagents. A new H+ selective ketocyanine dye and a cadmium ionophore. The anti-Salmonella spp antibody was immobilized onto the gold electrode coated quartz crystal surface through a polyethylenimineglutaraldehyde (PEG) technique and dithiobis-succinimidyl propionate (DSP) coupling.

3.3 ng L1

–

2.0 mg L1

–

1.3 mg L1

–

Trace level of sulfide, compactness, versatility and lower sample/reagent consumption.

[46]

20 mg L1

55

[142]

–

–

Integrated waveguide absorbance optrode (IWAO). The PEG technique proved more successful for FIA applications than the DSP coupling.

5 mg mL1

–

The solid [140] support used was the nonionic silica gel C18, using 20% methanol–water (v:v) as a carrier. Solid phase UV [141] spectroscopic detection and its intrinsic absorbance monitored at 274 nm

[143]

[144]

Table 2 (Continued ) Detection method Piezoelectric shear wave

Analyte

Mode

Norepinephrine bitartrate

Matrix type

Basis for detection

Pharmaceutical preparations

Ion association complex with anionic surfactant.

Measurements were performed using a biotinylated antigen immobilized by streptavidin onto the gold surface of the quartz crystal and phages displaying recombinant antibodies or hPSTI mutants. Adsorption of dodecyl phenylsulfonate and interfacial ion-pair formation with epinephrine and L-dopa on silver electrode of quartz crystal microbalance.

Quartz crystal Human microbapancreatic lance secretory trypsin inhibitor (hPSTI)

FIA

Phage libraries

Quartz crystal Epinephrine microba(Ep) l-dopa lance

FIA

Pharmaceutical samples



–

–

1.22 mg mL1 Ep 1.05 mg m L1 L-dopa

120

Comments

Reference

The sampling frequency, accuracy and reproducibility are superior to those given for conventional analytical methods. The QCM was integrated into [145] a flow injection analysis system for the straightforward analysis of large sample numbers.

–

[146]

Quartz crystal Protein A microbalance

FIA

–

Poly (dimethylsiloxane) microfluidic analysis system based on mass sensitive detection.

–

–

Quartz crystal Human TNF-a microbalance

FIA

–

Antigen–antibody interactions.

–

–

Quartz crystal Protein microbalance

FIA

–

Changes in the resonant frequency.

–

–

[147] The sensor system was operated in a flow-through mode and the results were compared with measurements using an injection method, where a definite amount of solution is injected into a reservoir mounted on the QCM. QCM biosensor integrated in [82] FIA system was used for the real-time investigation of the interaction between human TNF-a (hTNFa) and its monoclonal antibodies. The generally good agreement found between biosensor data and the affinities known from conventional equilibrium enzyme-linked immunosorbent assay (ELISA) validates this analytical method. [83] QCM biosensor integrated into FIA system was used for the real-time investigation of molecular recognition between a protein and small molecular medicinal agents. Results indicated that the two drug ligands appeared quite different in this molecular recognition procedure although their structures were similar.


Analyte

Mode

Matrix type

Basis for detection


Quartz crystal Polycyclic FIA microbaaromatic lance hydrocarbons

–

Raman microscopy

Cyanogenic Glucosides

FIA

Plant tissue

Energy dispersive X-ray fluorescence

Lead, cadmium

FIA

Water

nM The antigen (benzo[a]pyrene-BSA conjugate, BaP-BSA) was immobilized through thioctic acid on gold coated quartz crystals, with a basic resonant frequency of 10 MHz for the detection of various polycyclic aromatic hydrocarbons. Flow injection surface– enhanced Raman scattering using a 532 nm laser. Using Dowex 50 cation1 mg Pb 1.8 mg Cd exchange resin as sorbent, and flushing the eluate through the flow cell for monitoring.


Comments

–

[148] Determination of BaP was performed in the flow injection system by using a competitive pattern, in which BaP reacted with the bound mAb10c10 causing frequency increases.

–

The SERS method was optimized by FI using a colloidal gold dispersion. Rapid method

–

Reference

[149]

[85]


505

was tested for determination of lead and cadmium after the SPE preconcentration step. Figure 24b illustrates the EDXRF spectrum obtained as the sample was eluted from the preconcentration column.

11. CONCLUSION This chapter summarizes some of the multiplicity of different, and in some cases, less commonly used detection methods that have been applied in flow injection and related areas of analysis. The examples cited highlight the advantages of the FIA approach in performing complex sample manipulation using relatively simple fluidics and detection systems of varying complexity. A summary table listing examples of many of these applications is appended (Table 2).

ABBREVIATIONS BI C4D

Bead injection Capacitively coupled contactless conductometric detection DIPD Dynamic interfacial pressure detector DSTD Dynamic surface tension detector EDXRF Energy dispersive X-ray fluorescence ELSD Evaporative light scattering detector ESI-MS Electrospray ionisation-mass spectrometry FFF Field flow fractionation GM Geiger–Mu¨ller counter HPLC High-performance liquid chromatography ICP-AES Inductively coupled plasma-atomic emission spectrometry ICP-MS Inductively coupled plasma-mass spectrometry LC Liquid chromatography MECA Molecular emission cavity analysis MIPs Molecularly imprinted polymers MS Mass spectrometry NMR Nuclear magnetic resonance NUNCt cell or chamber A specialized microscope slide for on-slide cell culture PVA Poly(vinyl) alcohol QCM Quartz crystal microbalances RI Refractive index SPE Solid phase extraction

506


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98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124


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PART IV Applications of Flow Injection Analysis


CHAPT ER

18 Food, Beverages and Agricultural Applications ´ V. To´th, Marcela A. Segundo and Ildiko Antońio O.S.S. Rangel

Contents

1. Introduction 2. Applications: Beverages 3. Applications: Plants and Vegetables 4. Applications: Milk and Dairy Products 5. Applications: Meat and Fish Products 6. Miscellaneous Food Products Abbreviations References

513 514 545 546 547 548 548 549

1. INTRODUCTION Food quality and safety are major issues nowadays. Owing to increased concern on public health issues, national and international legislation has imposed stricter regulations on food control, both regarding chemical and microbiological aspects [1–3]. This scenario has produced a major impact on both agriculture and food industry practices. Companies and governmental certifying and regulatory agencies in this sector are faced with an increasing number of parameters to be monitored and the need to detect ever decreasing concentrations. Meeting these requirements demands novel analytical methods that are sensitive, efficient, and which provide significant improvements in laboratory productivity. This situation calls for the development of fast and automatic analytical methodologies for the food and beverage sector. Foodstuffs can be considered a complex matrix for a number of reasons: they are seldom homogeneous, and a solubilization process is normally required before analysis. This makes the sample pretreatment process relatively complex, and usually labour intensive. Additionally, these pretreatments might alter the Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00618-1


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composition (namely the form of the analyte) of the foodstuff and impair the quality of the analysis itself. Moreover, food samples have a biological origin resulting in high temporal and spatial variability in analyte concentrations. Sample colour and turbidity may also make the analysis more difficult, due to the widespread use of spectrophotometric methods in this respect. In this context, flow analysis methods can be a powerful tool to overcome some of these difficulties and offer a relatively low-cost alternative. In fact, sample pretreatments such as sample digestion, using microwave, UV or ultrasound radiation-assisted processes can be efficiently carried out in flow systems, using an extremely limited amount of reagents and posing no danger to the operator. Mass separation processes (gas-diffusion, dialysis, ion-exchange) can also be performed in-manifold, allowing minimization of interferences and/ or analyte preconcentration. Regarding the instrumental measurement, flow methods make it possible to carry out all the necessary wet chemistry involved, including analyte derivatization and instrumental detection. Additionally, as kinetic time-based methods can be easily implemented, additional information can be obtained from the instrumental measurements. In this chapter, an overview of the flow methods described for the analysis of food, beverage and agricultural samples will be presented. The collection of publications was essentially obtained by using the search engine ISI Web of Knowledge. Owing to the large number of papers published so far on this subject (Figure 1), the decision was made to address only the advances reported since the year 2000. Information on previous works can be found in review papers published in the last decade focusing on different areas of food analysis [4–9], on particular flow techniques [10–13], on specific analytes [4,7,14–20] or on specific detection [21–24] and analyte-processing techniques [25–28]. As depicted in Figure 1a, the implementation of flow techniques in food analysis accompanies the trend observed for its application as analytical tool. Specific developments dealing with advances in sample pretreatment, such as digestion or mass separation methods, are not discussed in detail in this chapter, as these topics are the object of discussion in Chapters 6–9 of this book. Therefore, special emphasis will be given to the commodity involved. Considering the distribution of applications to specific classes of food (Figure 1b), the following categories were selected for review: beverages, milk and dairy products, meat and fish, fruits and vegetables and miscellaneous food products. The collection of publications from the year 2000 onwards is presented in Tables 1–6, where the main characteristics of the methodologies are summarized. The discussion that follows highlights some features of the flow systems, and some trends regarding the target analytes or groups of analyte.

2. APPLICATIONS: BEVERAGES A beverage is a drink specifically prepared for human consumption, other than water. Therefore, this designation includes alcoholic drinks (wine, beer, liquors, distilled spirits) and also coffee, fruit juices, tea and soft drinks, among others.

Food, Beverages and Agricultural Applications

515

Figure 1 (a) Evolution of flow-injection application to food analysis, and (b) distribution by commodity.

Owing to its liquid nature, this type of sample can be simply introduced into a flow system, without being weighed or solubilized. This aspect makes the automation of the whole analytical process easier, considering that any other pretreatment operation required can be included in the flow system before the determination of the target analyte. Furthermore, the possibility of direct sampling also allows direct, real-time monitoring of food processing, especially during must fermentation [29–34] or beer production [35].

516

Table 1 Some of the analytical features of flow methods for alcoholic beverages Matrix

Flow mode

Detection system

Working range

Reference

Alcohols Ethanol Ethanol Ethanol

Beer, liquors, wine Must Wine

FIA FIA FIA

Amperometry Amperometry Amperometry

[84] [31] [85]

Wine Beer, spirits, wine, Wine Non-alcoholic beer Wine, spirits Sake, wine Beer, distilled liquors, white wines Beer, distilled liquors, wine Wine Wine Beer fermentation broth Wine Wine Wine Must Wine Wine Distilled spirits Wine Beer, wine Wine

FIA FIA FIA FIA FIA FIA FIA

Density measurement FTIR UV-Vis UV-Vis UV-Vis UV-Vis UV-Vis

0.020–2.0 mM NA 0.01 103– 0.75 103 M 0–40% (v/v) 0.05–15% (v/v) 1–20% (v/v) 0–100 mM 10–30% (v/v) 0.04–100 mM 5 106–1 103 M

FIA

UV-Vis

0.5–30% (v/v)

[92]

FIA SIA SIA

UV-Vis Amperometry Amperometry

1.0–30.0% (v/v) 1–250 mM 0.15–30 mg L1

[93] [94] [35]

SIA SIA MCFA FIA FIA FIA FIA SIA SIA MCFA

UV-Vis UV-Vis Chemiluminescence Amperometry Amperometry Fluorimetry Potentiometry UV-Vis UV-Vis UV-Vis

0.008–0.024% (v/v) 0.03–0.30 mg L1 2.5–25% (v/v) NA 0.01–1 mM 2–8 g L1 20–500 mg L1 0.10–0.50% (v/v) 0.3–3.0 mM 2.0–10.0 g L1

[95] [61] [96] [31] [58] [59] [60] [61] [62] [63]

Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol Glycerol

[86] [87] [59] [88] [89] [90] [91]


Analyte

Antioxidant capacity ABTSd+ assay ABTSd+ assay ABTSd+ assay ABTSd+ assay Scavenging of H2O2 DPPHdAssay

UV-Vis UV-Vis UV-Vis UV-Vis Fluorimetry UV-Vis

Wine Beer, wine

FIA MSFIA

Chemiluminescence UV-Vis

Metals and metalloids Boron

Grape juice, wine

MCFA

Cadmium Cadmium Cadmium Cadmium Calcium Copper Copper Iron Iron Iron Iron Iron(III) Iron Iron Lead Lead Lead Lead

Wine Wine Wine Wine Wine Wine Wine Beer Beer Wine Wine Wine Beer Wine Wine Wine Wine Spirits

FIA FIA FIA FIA FIA FIA SIA FIA FIA SIA SIA SIA BI-FIA BI-FIA FIA FIA FIA FIA

Piezoelectric microbalance FAAS CV-AAS ET-AAS ICP-OES UV-Vis FAAS FAAS FAAS UV-Vis FAAS FAAS FAAS UV-Vis UV-Vis FAAS FAAS FAAS FAAS

Total phenolics Folin–Ciocalteu reducing assay

10–300 mM 4–250 mM NA 0.001–0.008 M 0.001–0.01 M 0.25 104– 6.00 104 M 1 109–5 105 M 5–80 mg L1

[41] [42] [43] [44] [44] [45]

NA

[97]

NA r7 mg L1 r300 ng L1 r1.0 mg L1 0–350 mg L1 NA 0.20–2.00 mg L1 NA NA 0.25–15.0 mg L1 0.10–6.00 mg L1 0.25–15.0 mg L1 NA 0.1–3.0 mg L1 1.0–500 mg L1 NA 0.5–15 mg L1 5–120 mg L1

[64] [65] [66] [67] [98] [64] [99] [100] [100] [99] [101] [101] [100] [102] [68] [64] [69] [70]

[46] [47]

517

FIA FIA SIA SIA SIA MSFIA


Beer Wine Beer Wine Wine Beer, wine

518

Table 1 (Continued ) Matrix

Flow mode

Detection system

Working range

Reference

Lead Lead Magnesium Manganese Mercury Potassium Zinc Zinc

Wine Wine Wine Wine Wine Wine Beer Wine

FIA FIA FIA SIA FIA SIA FIA SIA

HG-AAS ICP-AES UV-Vis FAAS CV-AAS Potentiometry UV-Vis FAAS

r10 mg L1 0.15–1,000 mg L1 0–350 mg L1 r3.00 mg L1 2–50 mg L1 NA NA r1.50 mg L1

[71] [72] [98] [99] [103] [104] [105] [99]

FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA SIA FIA MCFA FIA

Amperometry Amperometry Amperometry UV-Vis Fluorimetry Chemiluminescence Chemiluminescence Amperometry Amperometry Amperometry Fluorimetry UV-Vis Amperometry Fluorimetry UV-Vis UV-Vis UV-Vis UV-Vis Fluorimetry

0.2–8 mM 0.05–20 mM 3–50 mg L1 1–80 mg L1 1 106–1.6 104 M 0.1–10 mM 5–50 mM 5 106–1 103 M 0.02–1.0 mM 0.02–1.0 mM 0.05–1.5 g L1 0.1–1.0 g L1 1 105–4 104 M 0.02–1.5 g L1 0.05–1.0 g L1 0.01–0.15 g L1 0.5–4.0 g L1 0.5–10.0 g L1 0.01–1.20 mM

[106] [107] [75] [108] [109] [110] [111] [32] [112] [112] [113] [113] [32] [113] [113] [114] [37] [115] [116]

Organic acids and conjugate ions Acetate Wine Acetic acid Wine Ascorbic acid Wine Ascorbic acid Beer D-gluconate Noble rot wine Lactate Beer Lactate Beer L-lactic acid Wine, must D-lactic acid Beer, sake, wine L-lactic acid Beer, sake, wine L-(+)-lactic acid Wine L-(+)-lactic acid Wine L-malic acid Wine, must L-(–)-malic acid Wine L-(–)-malic acid Wine L-(–)-malic acid Wine Tartaric acid Wine Tartaric acid Wine L-tartrate Wine


Analyte

FIA

HPLC-UV-Vis

Anthocyan index Flavanoid fraction Flavonols (total)

Wine Beer Wine

FIA FIA FIA

Phenolic compounds Polyphenol index

Beer Wine

FIA FIA

UV-Vis Amperometry Adsorptive stripping voltammetry Amperometry Amperometry

Polyphenol index

Wine

FIA

UV-Vis

Polyphenol index Polyphenol index Polyphenolic (three fractions)

Wine Wine Wine

FIA SIA FIA

UV-Vis UV-Vis Evaporative light scattering

Beer Must Beer, wine White wine Must Wine Wine Beer Brandy, white wine Wine Beer fermentation broth

SIA FIA FIA FIA FIA FIA FIA FIA FIA FIA SIA

IR Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Chemiluminescence UV-Vis Amperometry

Sugars Carbohydrates Fructose Glucose Glucose Glucose Glucose Glucose Glucose (bonded) Glucose Glucose Glucose

0.5–16 mg L1 1.0–60 mg L1 20–500 mg L1 NA 0.03–1.0 mg L1

[117]

0.025–14 mM 0.04–2.0 mg L1 (gallic acid) 0.001–0.100 mg L1 (caffeic acid) 4–22 units 3–18 units 20–70 units 5–200 mg L1 5–300 mg L1

[121] [122]

0.86–7.13 g L1 NA 2–2,500 mg L1 20–500 mg L1 NA 1 106–1 103 M 0.02–50 g L1 0.011–13.9 mM 0.0003–0.05 mM 1 106–1 103 M 5–750 mg L1

[126] [31] [127] [128] [31] [129] [130] [131] [132] [133] [35]

[118] [119] [120]

[123] [118] [124] [125]

519

Wine


Polyphenols Anthocyanins

520


Flow mode

Detection system

Working range

Reference

Maltooligosaccharides Reducing sugars Reducing sugars

Beer

FIA

ESI-MS

5–100 mM

[134]

Wine Wine

FIA SIA

UV-Vis UV-Vis

40–400 mM 2–25 g L1 20–140 g L1

[135] [36]

Sulfur dioxide Sulfite Sulfite Sulfur dioxide Sulfur dioxide Sulfur dioxide Sulfite Sulfite Sulfur dioxide Sulfur dioxide

Wine, grape juice Wine Wine Wine Wine Wine White wines Wine Wine

FIA FIA FIA FIA FIA FIA FIA FIA SIA

Amperometry Amperometry Amperometry Amperometry Amperometry Conductimetry UV-Vis UV-Vis UV-Vis

[38] [73] [74] [75] [76] [77] [78] [79] [80]

Sulfur dioxide

Wine

MSFIA

UV-Vis

1.0–5.0 mM 20–100 mM NA 0.25–15 mg L1 5–100 mM 1.0–500 mg L1 1–20 mg L1 1–200 mg L1 2–40 mg L1 25–250 mg L1 2–75 mg L1 10–250 mg L1

Sake, wine Beer Wine, must Wine Must, grape juice Beer

FIA MCFA FIA FIA SIA FIA

0.2–100 mM NA 0–25 mg L1 15–60 mg L1 28–140 mg L1 NA

[90] [136] [29] [30] [34] [50]

Wine

FIA

UV-Vis Potentiometry UV-Vis UV-Vis UV-Vis Piezoelectric microbalance CE–ESI–MS

NA

[83]

Others Acetaldehyde Acidity Ammonia Assimilable nitrogen Assimilable nitrogen Astringency and bitterness Biogenic amines

[81]


Analyte

Beer

FIA

Body and smoothness

Beer

FIA

Carbon dioxide Chloride Diacetyl Diacetyl

Beer Wine Beer Wine

FIA MSFA FIA FIA

Diacetyl Diacetyl

Beer Beer

FIA MCFA

Dissolved solids ‘‘Fingerprinting’’ ‘‘Fingerprinting’’ Histamine Laccase activity Phosphorus (total) Proline Proteins Sulfate Tannin–protein interaction Urea Urea Urea

Wine Beer Beer Wine, cider Wine, must Beer Wine Rice wine Wine Wine Rice wine Wine, must Rice wine

Note: NA, not given/not available.

FIA FIA FIA FIA FIA FIA FIA FIA SIA FIA

Piezoelectric microbalance Piezoelectric microbalance UV-Vis Potentiometry Amperometry Cathodic stripping voltammetry UV-Vis Adsorptive stripping voltammetry UV-Vis NMR MS Fluorimetry UV-Vis UV-Vis Chemiluminescence UV-Vis Turbidimetry FTIR

FIA FIA FIA

Fluorimetry UV-Vis UV-Vis

NA

[52]

NA

[53]

0.5–5 g L1 NA NA 1 108–1 105 M

[137] [138] [55] [56]

NA 5–600 mg L1

[55] [57]

0.999–1.026 g mL1 NA NA r2.0 mg L1 0.6–24.0 U mL1 2–20 mg L1 1 108–5 105 M NA 300–1,500 mg L1 NA

[123] [139] [140] [141] [33] [142] [143] [144] [145] [51]

1.0–100 mM 0–25 mg L1 0.016–1.0 mM

[146] [29] [39]


Bitterness

521

522

Table 2 Some of the analytical features of flow methods for non-alcoholic beverages Analyte

Flow mode

Detection system

Working range

Reference

Fresh fruit extracts, herbal infusions, tea Coffee, fruit juices, soft drinks, tea Fruit juices, tea Fruit juices, soft drinks, tea Fruit juices, soft drinks, tea Tea

FIA

Potentiometry

1 106–1 102 M

[48]

FIA

UV-Vis

10–300 mM

[41]

SIA MSFIA

UV-Vis UV-Vis

[43] [45]

MSFIA

UV-Vis

NA 0.25 104– 6.00 104 M 5–80 mg L1

FIA

Clark-type oxygen electrode (polarography)

0.1–1.5 mM

[49]

Organic acids and conjugate ions Ascorbic acid Fruit juices Ascorbic acid Fruit juices L-ascorbic acid Fruit juices Ascorbic acid Fruit juices Ascorbic acid Fruit juices Ascorbic acid Fruit juices Ascorbic acid Fruit juices Ascorbic acid Fruit juices Ascorbic acid Soft drinks Ascorbic acid Fruit juices

FIA FIA FIA FIA FIA FIA FIA FIA FIA FIA

Amperometry Amperometry Amperometry Amperometry Chemiluminescence FAAS FAAS FAAS UV-Vis UV-Vis

[75] [147] [148] [149] [150] [151] [152] [153] [108] [154]

Ascorbic Ascorbic Ascorbic Ascorbic

FIA FIA SIA FIA-BI

UV-Vis Voltammetry Voltammetry UV-Vis

3–50 mg L1 0.025–1.0 mM 5–100 mM NA 10–1,000 mM 0.2–34.5 mg L1 0.1–50 mg L1 0.4–20 mg L1 1–80 mg L1 2.0 106– 1.0 104 M 0.3–0.8 g L1 3–35 mg L1 NA 5.1–68 mM

Antioxidant capacity Total redox capacity ABTSd+ assay ABTSd+ assay DPPHd assay Folin–Ciocalteu reducing capacity Xanthine oxidase inhibitory activity

acid acid acid acid

Fruit Fruit Fruit Fruit

juices juices juices juices

[47]

[137] [155] [156] [157]


Matrix

0.1–8.0 mg L1 0.6–6.0 mM

[102] [158]

MPFS FIA FIA FIA FIA FIA

Chemiluminescence UV-Vis Potentiometry Potentiometry Amperometry Fluorimetry

r11 mM NA 1 104–1 101 M 1 103–1 101 M r20 mM 1 106–1 104 M

[159] [160] [161] [162] [163] [164]

FIA

Fluorimetry

1 106–2 104 M

[164]

FIA

UV-Vis

NA

[160]

Sulfur dioxide Sulfur dioxide Sulfite Sulfite Sulfur dioxide

Fruit Fruit Fruit Fruit

juices juices juices juices

FIA FIA FIA FIA

Amperometry Amperometry Conductimetry UV-Vis

0.25–15 mg L1 20–100 mM 1.0–500 mg L1 1–200 mg L1

[75] [73] [77] [79]

Fruit juices Fruit juice Tomato juice Fruit juices, soft drinks Soft drink Fruit juices Fruit juices, soft drinks Fruit juices, soft drinks Fruit juices

FIA FIA FIA FIA

Amperometry Amperometry Amperometry Amperometry

3–25 mM NA r100 mM r12.0 103 M

[165] [166] [163] [167]

FIA SIA SIA

Chemiluminescence Voltammetry Chemiluminescence

0.0003–0.05 mM NA 1 105–1 103 M

[132] [156] [168]

MSFIA

Chemiluminescence

0.090–2.7 mg L1

[169]

MSFIA

Chemiluminescence

2.5 106–1 103 M

[170]

Ascorbic acid Benzoic acid Citrate Isocitrate Lactate D-malate L-malate

Sugars Fructose Glucose Glucose Glucose Glucose Glucose Glucose Glucose Glucose

523

UV-Vis UV-Vis


FIA-BI MCFA

Sorbic acid

Fruit juices Fruit juices, soft drinks Fruit juices Orange juice Fruit juices Fruit juices Tomato juice Fruit juices, soft drinks Fruit juices, soft drinks Orange juice

Ascorbic acid Ascorbic acid

524

Analyte

Matrix

Flow mode

Detection system

Working range

Reference

Sucrose Sucrose

Fruit juices Fruit juices

FIA FIA

Amperometry Amperometry

NA 1–12 mM

[166] [165]

Others Acidity

Fruit juices

SIA

UV-Vis

[171]

Fruit juices Fruit juices, soft drinks Fruit juices, soft drinks

SIA-LOV MCFA

UV-Vis Potentiometry

0.2–1.0% (w/v) 0.5–2.5% (w/v) 0.21.2% (w/v) NA

FIA

Amperometry

Atrazine Bitterness

Orange juice Coffee

FIA FIA

Boron

Grape juice

MCFA

Cadmium Cadmium

Orange juice Orange juice

FIA FIA

Chemiluminescence Piezoelectric microbalance Piezoelectric microbalance ET-AAS CV-AAS

Acidity Acidity Artificial sweeteners (acesulfame-K, cyclamate, saccharine)

[172] [136]

3–30 mM (cyclamic acid) 1–10 mM (acesulfame-K) 0.3–3.5 mM (saccharin) 0.014–1.120 mg L1 NA

[173]

NA

[97]

r300 ng L1 r7 mg L1

[66] [65]

[174] [52]



10–300 mg L1

[137]

1–16 mg L1 15.0–150 mg L1 NA NA

[175] [176] [177] [54]

0.1 104– 2.5 104 M 0.03–1.0 mg L1

[178]

2.0–20.0 mg L1 5.0–50 mg L1 0.001–0.01 M

[176] [176] [179]

UV-Vis Mass spectrometry

0.1–1.5 U mL1 NA

[180] [181]

UV-Vis Anodic differential pulse voltammetry UV-Vis

1–12 mg L1 3 107–1 105 M

[175] [182]

NA

[105]

Soft drinks

FIA

Caffeine Calcium Cations ‘‘Classification’’

Cocoa, soft drinks, tea Coconut water Fruit juices Orange juice, soft drinks Tea

FIA SIA FIA FIA

Evaporative light scattering detector UV-Vis UV-Vis IC-conductivity detector Potentiometry

FIA

Amperometry

Infusions, tea, tomato juice Coconut water Coconut water Apple juice

FIA SIA SIA FIA

Adsorptive stripping voltammetry UV-Vis UV-Vis UV-Vis

Fruit juices Grape juice

FIA FIA

Cocoa, soft drinks, tea Fruit juices

FIA FIA

Soft drinks

FIA

Flavonoids Flavonols (total) Iron Magnesium Organophosphate pesticides Pectinesterase activity Proanthocyanidins oligomers Theobromine Tin Zinc


[120]


Caffeine

525

526

Table 3 Some of the analytical features of flow methods for fruits and vegetables Matrix

Flow mode

Detection

Working range or LOD

Reference

Pesticides Bitertanol Carbamate Carbaryl Carbaryl

Fruit, banana Vegetables Vegetables Vegetables

MCFA FIA FIA FIA

Fluorimetry UV-Vis Chemiluminescence Chemiluminescence

[189] [202] [194] [196]

Carbaryl Carbaryl

Fruits Vegetables

FIA FIA

ESI(MS/MS) UV-Vis

Carbofuran Carbofuran

Fruits, vegetables Vegetables

MSFA FIA

Amperometry Chemiluminescence

Chlorpyrifos

Fruits

FIA

Chemiluminescence

Dimethylarsinic Dimethoate

Vegetables Vegetables

FIA SIA

Fluorimetry UV-Vis

Diphenylamine

Fruits

MCFA

Fluorimetry

Dichlorvos

Vegetables

FIA

Chemiluminescence

DDVP 2,4-D Imazalil Malathion Malathion Methamidophos

Fruits Fruits Citrus fruits Grains, vegetables Fruits Vegetables


ESI(MS/MS) ESI(MS/MS) ESI(MS/MS) Fluorimetry ESI(MS/MS) Fluorimetry

LOD: 0.014 mg kg1 LOD: 3.5–25mg L1 30–100 mg L1 5–100 ng mL1, LOD: 4.9 ng mL1 0.002–5.0 mg g1 LOD: 0.4 ng LOD: 25 ng 109–107 M 0.06–0.5 mg mL1 LOD: 0.02 mg mL1 0.48–484 ng mL1 LOD: 0.18 ng mL1 LOD: 0.014 mg mL1 0.03–0.5 mg g1 LOD: 0.01 mg g1 0.25–5 mg kg1 LOD: 0.06 mg kg1 0.02–3.1 mg mL1 LOD: 0.008 mg mL1 0.002–5.0 mg g1 0.002–5.0 mg g1 0.2–5 mg mL1 20–2,000 ng mL1 0.002–5.0 mg g1 14–1,400 ng mL1 LOD: 1.7 ng mL1

[192] [200] [203] [195] [198] [183] [201] [188] [197] [192] [192] [191] [185] [192] [184]


Analyte

Methylcarbamates N-methylcarbamate o-Phenylphenol Propoxur

Fruits, vegetables

FIA-LC

Fluorimetry

LOD: 3–12 ng g1

[186]

Fruits Citrus fruits Vegetables

FIA FIA FIA

ESI(MS/MS) ESI(MS/MS) UV-Vis

[193] [191] [199]

Propoxur

Vegetables

FIA

UV-Vis

Organophosphorus Organophosphorus

Vegetables, grains Vegetables

FIA-HPLC SIA

Fluorimetry UV-Vis

Organophosphorus Thiabendazole

Vegetables Fruits

FIA MCFA

UV-Vis Fluorimetry

Thiabendazole

Citrus fruits

FIA

ESI(MS/MS)

0.01–0.7 mg mL1 0.4–10 mg mL1 1–10 mg L1 LOD: 0.15 mg L1 LOD: 0.4 ng LOD: 25 ng LOD: 4–12 ng mL1 0.03–0.5 mg g1 LOD: 0.01 mg g1 LOD: 3.5–25 mg L1 0.3–10 mg kg1 LOD: 0.09 mg kg1 0.4–10 mg mL1

Toxins Aflatoxin B1 Aflatoxin B1 Fumonisin B1 Fumonisin B1 Ochratoxin A

Fruits Barley, wheat Corn products Corn Barley, wheat

SIA-immuno FIA FIA-immuno FIA-immuno FIA

UV-Vis UV-Vis UV-Vis UV-Vis UV-Vis

LOD: 0.2 ng mL1 0.5–10 ng mL1 NA 1–1,000 ng mL1 0.5–10 ng mL1

[215] [216] [217] [218] [216]

Inorganic anions Chloride Nitrite/nitrate

Coconut water Vegetables

FIA FIA

Potentiometry UV-Vis

[219] [220]

Nitrite/nitrate Nitrite/nitrate

Vegetables Vegetables

FIA FIA

UV-Vis FAAS

4–1,000 mg L1 0.30–3.00 mg L1 (NO 2) 1.00–10.00 mg L1 (NO 3) LOD: 2.96 mg r20 mg L1 (NO 2) LOD: 0.07 mg L1 r30 mg L1 (NO 3) 0.14 mg L1

[200] [190] [201] [202] [187]

[221] [222]


[191]

527

528


Flow mode

Detection


Reference

Nitrate Nitrite

Vegetables Flour, wheat

FIA FIA


[223] [224]

Nitrate Orthophosphate

Vegetables Cereals

SIA FIA

UV-Vis UV-Vis

1.00–10.00 mg L1 1.0 106– 1.0 101 M 1.35–50 mg L1 r196 106 (P) M

Carbohydrates Fructose

Fruits

FIA

Voltammetry

[227]

Glucose

Fruits

FIA

Voltammetry

Starch

Flour, bread

FIA

UV-Vis

r60 mM, LOD: 1.2 mM r60 mM, LOD: 1.2 mM 0.05–9 g L1

Organic acids and conjugate ions Ascorbic acid Fruits, vegetables Ascorbic acid Vegetables Ascorbic acid Vegetables Ascorbic acid Vegetables Ascorbic acid Vegetables, fruits Oxalic acid Vegetables


Turbidimetry Chemiluminescence FAAS FAAS Fluorimetry UV-Vis

Oxalic acid

Vegetables

FIA

UV-Vis

Oxalate

Vegetables

FIA

Chemiluminescence

Phytic acid Pyruvate

Plant Onion

MPFS FIA

UV-Vis UV-Vis

LOD: 1 mg mL1 LOD: 1 1013 M 0.1–50 mg L1 0.3–60 mg mL1 LOD: 0.012 mg mL1 0.1–8.0 mg mL1 LOD: 0.04 mg mL1 0.1–8.0 mg mL1 LOD: 0.08 mg mL1 2 106–9.5 105 M LOD: 0.05 mg mL1 LOD: 1 mg L1 NA

[225] [226]

[227] [228] [204] [207] [152] [205] [206] [229] [230] [231] [232] [233]


Analyte

Metals and metalloids Aluminium Arsenic Boron

Crystallized fruits Seaweed Plant

Cadmium Cadmium Cadmium Cadmium

LOD: 0.1–0.8 mg L1 NA LOD: 0.05 mg mL1

[234] [235] [236]

UV-Vis FAAS CV-AAS FAAS

[237] [238] [65] [239]

Cadmium Copper Germanium Gold Lead Lead Lead Mercury Molybdenum Nickel Nickel

Powdered corn Plant Mung bean, kelp Apple leaves Vegetables Powdered corn Corn Vegetables Mung bean, kelp Plants, flour Plants

FIA MCFA FIA FIA FIA FIA FIA FIA FIA FIA FIA

UV-Vis FAAS UV-Vis ICP-MS TS-FF-AAS UV-Vis ICP-MS CV-AAS UV-Vis FAAS UV-Vis

Selenium

Cereals, bakery products Apple leaves Apple leaves Mung bean, kelp Apple leaves Powdered corn Corn

FIA

HG-GFAAS

5–50 mg L1 LOD: 0.014 mg g1 LOD: 0.02–0.40 mg g1 LOD: 0.014– 0.011 mg g1 0.05–3.0 mg mL1 LOD: 1 ng mL1 NA LOD: 0.64 pg mL1 5.2–300.0 mg L1 0.05–6.0 mg mL1 NA LOD: 0.86 mg L1 NA 5–250 mg L1 0.05–0.50 mg L1 LOD: 17 mg L1 LOD: 0.06 mg L1


ICP-MS ICP-MS UV-Vis ICP-MS UV-Vis ICP-MS

LOD: 0.82 pg mL1 LOD: 2.24 pg mL1 NA LOD: 0.05 pg mL1 0.05–2.0 mg mL1 NA

[243] [243] [242] [243] [240] [245]

Silver Tellurium Tin Uranium Zinc Zinc

[240] [241] [242] [243] [244] [240] [245] [246] [242] [247] [248] [249]


UV-Vis HG-AAS UV-Vis

Vegetables Vegetables, fruits Vegetables Legumes, fruits

MCFA FIA Continuous flow MCFA FIA FIA FIA

529

530

Analyte

Matrix

Flow mode

Detection


Reference

Others Antioxidant capacity Antioxidant capacity Antioxidant capacity Antioxidant capacity Formalin Glucosinolate

Fruits, vegetables Vegetables Herbs Vegetables Fruits Vegetables


Amperometry Amperometry Amperometry Chemiluminescence Amperometry Amperometry

0.1–0.5 mM 1.0–10 mg L1 NA NA LOD: 0.0129 mM 0.005–1.0 mM LOD: 0.002 mM NA LOD: 75 mM

[49] [208] [209] [210] [250] [213]

r196 106 (P) M 0.09–45.0 mg mL1 LOD: 0.05 mg mL1 r196 106 (P) M 1.6 ng mL1

[226] [214]

b-Glucan myo-inositol phosphate Phosphorus (total) Phylloquinone

Oat Fruits, legumes

FIA FIA-CE

Fluorimetry UV-Vis

Cereals Vegetables, fruits

FIA FIA

UV-Vis Fluorimetry

Phytate Synephrine

Cereals Herbs, fruits

FIA FIA

UV-Vis Chemiluminescence


[251] [212]

[226] [211]



Table 4 Some of the analytical features of flow methods for milk and dairy samples Analyte

Matrix

Flow mode

Detection


Reference

Antibiotics Gentamicin Nafcillin Oxytetracycline

Milk Milk Milk

FIA-immuno FIA FIA

Amperometry Phosphorescence Voltammetry

[254] [256] [255]

Streptomycin Tetracycline

Milk Milk

FIA LOV

Chemiluminescence Chemiluminescence

LOD: 100 mg kg1 LOD: 3.6 107 M 100 ng mL1 200 ng g1 LOD: 5.16 109 M LOD: 2.0 mg L1

Inorganic anions Chloride Chloride Chloride Nitrate/nitrite Nitrite

Milk Milk Milk Dairy Milk

FIA SIA MSFA SIA FIA

Potentiometry Potentiometry Potentiometry UV-Vis Potentiometry

Nitrite

Milk, cheese

FIA

Potentiometry

Milk Milk Milk Milk Milk Dairy products Pasteurized milk, buttermilk, lowlactose milk

FIA FIA FIA FIA FIA FIA FIA

Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry Amperometry

4–1,000 mg L1 0.01–0.25 M NA LOD: 0.15 mg L1 1.0 106– 1.0 101 M 1.0 106– 1.0 101 M

[219] [281] [138] [272] [273]

LOD: 0.1 mM 0.1–20 mM LOD: 0.2 mM LOD: 0.1 mM 0.05–10 mM LOD: 0.06 mM 1–100 mM

[165] [277] [165] [165] [277] [280] [274]

[224]


Sugars Fructose Galactose Galactose Glucose Glucose Glucose Lactose

[257] [258]

531

Matrix

Flow mode

Detection


Reference

Lactose Lactose

Cheese whey Milk and instant dessert powder Milk Milk Different types of milk

FIA FIA


1–30 g L1 LOD: 0.5 mM

[275] [279]

FIA FIA FIA


[165] [277] [276]

Milk-based and sugar candidate artificial certified reference materials (CRMs) Milk-based and sugar candidate artificial CRMs

FIA

UV-Vis

LOD: 0.8 mM 0.2–20 mM 3.0 105– 1.0 103 M LOD: 9.6 106 M 0.01–0.80% (w/v)

FIA

UV-Vis

0.01–0.80%(w/v)

[278]

MCFA FIA

HG-AFS AAS

LOD: 1.67 ng g1 LOD: 0.014 mg g1

[260] [261]

FIA FIA

UV-Vis Electrochemiluminescence

[240]

FIA FIA

Potentiometry FAAS

0.05–3.0 mg mL1 8.0 106 to 1.0 104 M LOD: 2.0 106 M 104–102 M LOD: 2.5 mg L1

[262] [263] [264]

FIA

ICP-MS

LOD: 0.64 pg mL1

[243]

Lactose Lactose Lactulose

Monosaccharides

Oligosaccharides

Metals, metalloids Bismuth Cadmium Cadmium Calcium

Calcium Chromium (III) Gold

Milk shakes Solid and semisolid milk Milk powder Milk

Whole milk Non-fat milk powder Milk powder

[278]


Analyte

532


Iron Iron Lead Manganese(II)

Milk powder, infant formula Milk

FAAS

LOD: 0.60 mg g1

[265]

Closed-loop FIA FIA FIA

UV-Vis

NA

[266]

UV-Vis FAAS

0.05–6.0 mg mL1 LOD: 1.1 mg L1

[240] [264]

MCFA

CV-AFS

LOD: 0.011 ng g1

[267]

FIA MCFA MCFA FIA FIA FIA

ICP-MS HG-AFS HG-AFS ICP-MS ICP-MS FAAS

LOD: LOD: LOD: LOD: LOD: LOD:

FIA FIA

UV-Vis ICP

0.05–2.0 mg mL1 Various

[240] [271] [252] [253] [282]

Others Aflatoxin M1

Milk

FIA-immuno

Amperometry

Aflatoxin M1 Antioxidant activity Antioxidant activity Choline

Cheese Milk

FIA FIA


20–500 ppt LOD: 11 ppt Subnanomolar NA

Milk

FIA

Amperometry

NA

[283]

Milk

FIA

Potentiometry

[284]

Choline

Milk

FIA

Amperometry

5.0 104– 5.0 103 M r0.5 mM

Mercury Silver Tellurium Tellurium Tellurium Uranium Zinc

0.82 pg mL1 0.57 ng g1 0.20 ng L1 2.24 pg mL1 0.05 pg mL1 0.3 mg g1

[243] [269] [268] [243] [243] [270]

[285]

533

Zinc Various metals

Milk powder Non-fat milk powder Milk, non-fat milk powder Milk powder Milk Milk Milk powder Milk powder Milk powder, infant formula Milk powder Powdered milk


FIA

534

Analyte

Matrix

Flow mode

Detection


Reference

Choline

Milk, milk powder, soy lecithin Milk

FIA

Amperometry

NA

[286]

FIA

0.20–0.45% (w/v)

[287]

FIA FIA FIA FIA

Piezoelectric microbalance Chemiluminescence Amperometry Amperometry Amperometry

LOD: 0.35 mg mL1 r50 mM 10–180 mM LOD: 4 mM

[288] [289] [290] [291]

FIA FIA SIA SIA SIA

Amperometry UV-Vis UV-Vis Conductimetry UV-Vis

LOD: LOD: LOD: LOD: LOD:

[280] [292] [293] [294] [294]

Fat matter Isoniazid Lactate Lactate Lactate Lactate Phosphorus Phosphorus Urea Urea

Milk Milk and yoghurt Dairy products Fermentation monitor Dairy products Milk Milk Milk Milk


0.1 mM 2 mg L1 2 mg L1 2.6 104 M 2.8 105 M



Table 5

Some of the analytical features of flow methods for meat and fish products

Analyte

Matrix

Flow mode

Detection


Reference

FIA

UV-Vis

LOD: 0.05 mg L1

[297]

Nitrite/nitrate Nitrite/nitrate

Frankfurter and dry sausages Cured meat Meat

SIA FIA

UV-Vis UV-Vis

[298] [220]

Nitrite/nitrate

Fish

FIA

UV-Vis

Nitrite Nitrite

Meat Sausage

FIA FIA


Nitrite

Sausage

FIA

Potentiometry

Nitrite Nitrate

Meat Meat

Continuous flow FIA

UV-Vis UV-Vis

LOD: 9 mg L1 LOD: 13 and 20 mg kg1 LOD: 0.01 and 0.025 mg mL1 LOD: 7.5 mg mL1 1.0 106– 1.0 101 M 1.0 106– 1.0 101 M 0.1–50 mg L1 LOD: 2.97 mg

Metals, metalloids Arsenic Arsenic Arsenic Cadmium

Fish Fish Seafood Meat

FIA FIA FIA FIA

HGAAS HGAAS HG-ETAAS FAAS

Cobalt

Fish and eggs

FIA

Chemiluminescence

Cobalt

Bovine liver, fish, mussel Pork liver Meat Fish

MSFA FIA FIA FIA

Inorganic anions Nitrite

[295] [273] [224] [299] [221]

UV-Vis FAAS FAAS

NA LOD: 0.6 mg g1 LOD: 0.8 mg L1

[242] [332] [333]

[330] [331]

535

[326] [327] [328] [329]

UV-Vis

LOD: 045 mg g1 LOD 0.34 mg L1 LOD 72.1 ng L1 LOD: 0.014 mg 60 mg1 10 fg mL1 to 50 pg mL1 LOD: 1.66 ng L1


Germanium Iron Lead

[296]

536


Flow mode

Detection


Reference

Lead Mercury Mercury Mercury Mercury Mercury Mercury Mercury Mercury Mercury Mercury Molybdenum Selenium Selenium Tin Zinc Various metals

FIA MCFA FIA MSFIA FIA FIA-HPLC FIA-LC FIA FIA FIA SIA FIA FIA FIA FIA FIA FIA

FAAS CVAAS CVAAS CVAAS VGAAS UV ETAS CVAAS CVAAS CVAAS CVAAS UV-Vis Amperometry HGAAS UV-Vis FAAS ICP

LOD: 1.0 mg L1 LOD: 4.8 mg kg1 LOD: 4–26 ng g1 LOD: 5 ng L1 LOQ: 55 ng g1 LOD: 10–25 ng g1 LOD: 6.8 ng L1 LOD: 57 ng g1 LOQ: 0.86 mg L1 NA LOD: 0.46 mg L1 NA LOD: 6 mg L1 LOD: 10 mg L1 NA LOD: 0.6 mg g1 Various

[334] [309] [308] [307] [306] [305] [304] [303] [246] [335] [302] [242] [336] [337] [242] [338] [271]

Various metals

Seafood Fish Fish Fish Fish Seafood Fish Fish Fish, seafood Seafood Fish Pork liver Fish Dry fish Pork liver Meat Bovine liver, mussel tissue Fish liver

FIA

ETV-ICP-MS

Various

[339]

Quality indicators Agmatine Biogenic amines Histamine Histamine Histamine Histidine Putrescine Trimethylamine

Fish Fish, meat sausage Fish Fish Fish Fish Fish Fish

FIA FIA-CE FIA FIA FIA FIA FIA FIA

Amperometry Amperometry Amperometry Fluorimetry Amperometry Chemiluminescence Amperometry Amperometry

LOD: 0.005 mM LOD: 0.2–0.6 mg mL1 LOD: 100 pmol LOD: 0.8 mg kg1 LOD: 2.2 mM LOD: 0.01 mM LOD: 5 mM 1.0–50.0 mM

[314] [322] [310] [311] [312] [340] [313] [315]


Analyte

Escherichia coli O15 Others Nitrosamine Nitrosodimethylamine Oxytetracycline Tetracycline

Seafood Fish Fish, hake Fish sauce Fish Fish, hake


Potentiometry UV-Vis UV-Vis UV-Vis UV-Vis UV-Vis

LOD: 0.05 mg mL1 NA 0.3–7 mg N L1 50–200 mM (N) NA 1.4–14 mg N L1

[316] [341] [318] [317] [342] [318]

Fish sauce

FIA

UV-Vis

50–500 mM (N)

[317]

Fish

FIA

UV-Vis

NA

[342]

Fish Meat Meat

FIA FIA FIA


LOD: 2–3 107 M NA 2 106–2 103 M

[321] [320] [343]

Poultry

FIA

Amperometry

105 CFU mL1

[324]

Poultry

FIA

Piezoelectric microbalance

[323]

Poultry

FIA

Amperometry

107–109 CFU mL1 or 106–1,010 CFU mL1 LOD: 6 102 cell mL1

Cured meat Cured meat

FIA FIA


0.8–2,000 ng mL1 LOD: 0.29 ng mL1

[301] [300]

Eggs Fish

FIA FIA

Voltammetry Chemiluminescence

NA 4 109– 4 107 g mL1

[255] [344]

537


[325]


Trimethylamine Trimethylamine Trimethylamine Trimethylamine Trimethylamine Total volatile basic nitrogen Total volatile basic nitrogen Total volatile basic nitrogen ‘‘Freshness’’ ‘‘Freshness’’ ‘‘Freshness’’ (hypoxantine/ polyamines) Salmonella typhimurium S. typhimurium

538

Table 6 Some of the analytical features of flow methods for food analysis, miscellaneous food products Analyte

Flow mode

Detection

Application range or LOD

Reference

Sugars Monosaccharides/ oligosaccharides Fructose Glucose

Honey, syrups

FIA

UV-Vis

0.01–0.80% (w/v)

[278]

Syrup Honey

MPFS FIA


[378] [132]

Glucose Glucose Glucose

Oily food Syrup Honey

FIA MPFS SIA

Amperometry UV-Vis Chemiluminescence

Glucose

Honey

SIA/FIA

Chemiluminescence

0.50–2.00% (w/v) 3 104– 5 102 mM 0–1.0 mM 0.50–2.00% (w/v) 1 105– 1 103 M, LOD: 1 106 M 0.01–1 mM, LOD: 4 mM

FIA

HG-AAS

LOD: 0.068 mg kg1

[380]

FIA

HG-AAS

LOD: 0.15 mg kg1

[380]

Boron

Foods (daily food intake) Foods (daily food intake) Vinegar

MCFA

NA

[97]

Cadmium Cobalt Copper

Honey Honey Vegetable oil

FIA FIA FIA

Piezoelectric microbalance FAAS FAAS FAAS

LOD: 0.5 ng g1 LOD: 0.18 mg L1 NA

[356] [357] [348]

Metals and metalloids Arsenic Antimony

[379] [378] [168]

[355]


Matrix

Iron Lead Selenium

SIA FIA FIA

UV-Vis FAAS HG-AAS

LOD: 0.31 mg LOD: 350 ng g1 LOD: 0.060 mg kg1

[349] [369] [380]

Zinc Various metals

Edible oil Sweeteners Foods (daily food intake) Vegetable oil Oil

FIA FIA

FAAS ICP-MS, FAAS

NA Various

[348] [347]

Artificial sweeteners Acesulfame-K

Sweetener

FIA

UV-Vis

[374]

Acesulfame-K Aspartame

Sweetener tablets Sweeteners

FIA FIA

Amperometry UV-Vis

Aspartame

FIA

UV-Vis

FIA

UV-Vis

10–200 mg mL1

[372]

Aspartame

Low-calorie dietary products Low-calorie dietary products Sweetener tablets

40–100 mg mL1, LOD: 11.9 mg mL1 1–10 mM 10–80 mg mL1, LOD: 4 mg mL1 5–600 mg mL1

SIA

Chemiluminescence

[373]

Aspartame

Sweetener

FIA

UV-Vis

Cyclamate

Sweetener

FIA

Turbidimetry

Cyclamate

Sweetener

FIA

UV-Vis

Cyclamate

Sweetener tablets

FIA

Amperometry

r350 mg L1, LOD: 2.16 mg L1 10–100 mg mL1, LOD: 5.65 mg mL1 0.015–0.120%(w/v), LOD: 0.006% (w/v) r3.0 mM, LOD: 30 mM 3–30 mM

[371]

[374]

[375]

[376]


Aspartame

[173] [370]

[173]

539

540


Flow mode

Detection


Reference

Saccharine Saccharine

Sweetener tablets Low-calorie dietary products

FIA FIA

Amperometry UV-Vis

0.3–3.5 mM 10.0–200.0 mg mL1

[173] [372]

Vinegar Sweetener Honey

FIA FIA FIA

Conductimetry UV-Vis UV-Vis

[77] [377] [42]

Honey

FIA

Amperometry

0.010–0.100 M r103 M 4–250 mM, LOD: 1.3 mM NA

Honey, propolis, royal jelly Sweets Bread

FIA

Amperometry

NA

[359]

FIA-BI FIA

UV-Vis UV-Vis

[157] [345]

Chocolate Soup Pasta Lard, butter, pasta Sweetener Dehydrated broths


UV-Vis Chemiluminescence UV-Vis Amperometry UV-Vis UV-Vis

5.1–68 mM 2 106– 2.1 105 M, LOD: 8 107 M 1–16 mg L1 0.02–0.12 (OD600) 0–2 mg mL1 0.1–0.5 mM r103 M 0.342–1.368 mg 100 mL1, LOD: 0.185 mg 100 mL1

Others Acetic acid Aniline Antioxidant activity Antioxidant activity Antioxidant activity Ascorbic acid Bromate

Caffeine Catalase activity Cholesterol Cholesterol Cyclohexylamine Creatinine

[358]

[175] [365] [361] [346] [377] [366]


Analyte

Glucose

Honey, vinegars Soup

FIA FIA

Fluorimetry Amperometry

Hydroxyl radicals

Oil

FIA

Fluorimetry

Iodine value Lipid hydroperoxide Lysine

Olive oil Oil

FIA FIA


106–1.6 104 M 0.1–15.5 mM, LOD: 0.08 mM 2.6 107– 4 105 M, LOD: 7.91 108 M 9–125 IV NA

Hydrolysate food samples Tomato paste, baby food Soup-formulas Food seasonings

FIA

Amperometry

1 103–5 105 M

[381]

FIA

Amperometry

0–0.1 nM

[382]

FIA FIA

Potentiometry Amperometry

[383] [362]

Soup

FIA

UV-Vis

2.5–75 mM 10–160 mg L1, LOD: 1.7 mg L1 r140 mM, LOD: 1 mM

Monosodium glutamate Oligomeric proanthocyanidin

Soup

FIA

Amperometry

[368]

Health foods

FIA

UV-Vis

Parabens Propyl gallate

Soysauce Dehydrated broth bar, olive oil

FIA FIA

Chemiluminescence Amperometry

0.1–15.5 mM, LOD: 0.08 mM 0.010– 0.20 mg mL1, LOD: 5 mg mL1 Various 9 107– 1.1 106 M

D-gluconate

L-lactate

[350]

[351] [352]

[367]

[384]

[363] [353]


L-glutamate Monosodium glutamate Monosodium glutamate

[109] [368]

541

542

Analyte

Matrix

Flow mode

Detection


Reference

P4R and N2N (dyes)

Sweets

FIA

Solid-phase UV-Vis

[385]

Synthetic antioxidants Sudan I Tetracycline

Fat foods

UV-Vis

Hot chilli sauce Honey

Continuous flow FIA FIA-HPLC

0.30–20 mg L1 (P4R) 0.02–3.0 mg L1 (N2N) 10–300 mg mL1


Chocolate Oil

FIA FIA

Oily food

FIA

Theobromine Total lipid hydroperoxides Water


[386] [364] [360]

UV-Vis Fluorimetry

LOD: 3 pg mL1 LOD: 0.9– 5.0 ng mL1 1–12 mg L1 NA

Amperometry

NA (0–65%)

[387]

[175] [354]




543

The analysis of alcoholic beverages can be a cumbersome process because ethanol can be a serious interferent in almost all detection systems. Calibration using standards containing ethanol is a frequent solution to this problem [36,37], but this then requires that the application be devised for a specific matrix, considering that beer, wine or spirits have very different ethanol content. The ethanol interference may be circumvented by performing a ‘‘blank’’ measurement, as suggested by Corbo et al. [38] for the amperometric determination of sulfite. Since ethanol is electroactive and also permeates through the gasdiffusion membrane, the pH of the donor stream was changed to provide an analytical signal proportional to the ethanol present in the wine samples, which was then subtracted from the signal corresponding to sulfite plus ethanol. Another strategy suggested by Iida et al. [39] involved the application of a hollow-fibre membrane containing a non-porous layer at its outer surface for selective diffusion of carbon dioxide in the enzymatic determination of urea. The analytes presented in Tables 1 and 2 illustrate the dual role that flowbased methods have in food analysis. These include flow systems devised for routine analysis, based on well-established methods. However, flow systems were also applied to novel analytical tasks, such as the determination of analytes related to sensory properties or to characteristics that contribute to a value-added product. Antioxidants belong to this last class of compounds, and there has recently been an increased demand for methods to assess the ‘‘antioxidant properties’’ or the ‘‘antioxidant capacity’’ of food products [40]. These methods include the evaluation of either the generic ‘‘reduction’’ capacity, or the determination of a specific analyte (ascorbic acid, vitamin E) or class of analytes (phenolic compounds, carotenoids). The most common of these methods is based on the scavenging of a coloured radical, namely 2,2u-azinobis(3-ethylbenzothiazoline-6sulfonic acid) (ABTSd+) or 2,2-diphenyl-1-picrylhydrazyl (DPPHd). The automation of these assays is definitely advantageous, as reported by several authors [41–49], because strict control of reaction time and media composition are necessary to achieve repeatable and comparable results. Labrinea and Georgiu [42] reported the use of gradient calibration to perform automated dilution of concentrated samples and to obtain analytical measurements at different assay times. In this way, information concerning the kinetics of ABTSd+ bleaching was obtained after a single injection. For the same assay, studies concerning the influence of pH (5.4, 7.4 or unbuffered) were carried out in an SIA system [43]. A thorough mixture between food samples, buffer and ABTSd+ was attained in a mixing chamber placed in a lateral port of the selection valve. Comparing the two endpoint batch method protocols, the DPPHd assay takes considerably more time (up to 2 h) than the ABTSd+ assay (10–30 min). In the multisyringe flow-injection analysis (MSFIA) system proposed by Magalha˜es et al., a stopped flow approach was adopted, and the data collected within the first 3 min of reaction was used to calculate the total DPPHd consumption for samples containing slow reacting compounds [45]. The results were comparable to those attained using the endpoint batch method, with a considerable reduction of the analysis time. The multi-channel features of the selection valve used in SIA were exploited to

544


implement two complementary determinations. The reagent reservoirs (ABTSd+, H2O2 and homovanylic acid) and detectors (spectrophotometer and fluorimeter) were connected to different lateral ports of the selection valve. In accordance with the routine protocol, the scavenging activity was measured against either ABTSd+ or H2O2 [44]. The application of flow systems to the determination of parameters that can be correlated to astringency [50,51], bitterness [52], body and smoothness [53] has also been reported. These reports illustrate how useful flow injection-based techniques are for these types of studies. The functioning of an FIA manifold can mimic that which occurs in the mouth, where the sensory receptors (similar to the detector in a flow system) are constantly washed by saliva (carrier). The sensory stimulus is equivalent to the sample plug dispersed in the carrier in a flowinjection system, which has a transient effect on the receptor because it is continuously rinsed by fluids. For example, Kaneda and co-workers applied a lipid-coated quartz crystal microbalance connected to a flow-injection system to simulate and study the electrostatic and/or hydrophobic interactions of the beer taste components with the tongue and throat surfaces [50,52,53]. The results obtained showed a good correlation with those obtained from a sensory evaluation panel. The implementation of a potentiometric sensor array in an FIA system to distinguish simple tastes and to classify food samples has also been described [54]. Furthermore, systems for determination of specific analytes that contribute to the typical sensory characteristics of wine and beers have been proposed. Worthy of mention are manifolds for the determination of diacetyl [55–57], a strong smelling compound that evokes a buttery aroma, and glycerol [31,58–63] that is used to confer smoothness to wine. New flow-based methodologies for routine monitoring of food safety aspects should be highlighted. These include systems proposed for the determination of heavy metals, such as cadmium [64–67] and lead [64,68–72]. In almost all of these proposed manifolds, in-manifold complexation of the target metal, followed by in-manifold solid-phase extraction, elution and detection by atomic or emission spectrometry was adopted. Chuachuad and Tyson adopted another strategy by using immobilized tetrahydroborate to generate volatile species of cadmium [65] or lead [71], which were further determined by chemical vapour–atomic absorption spectrometry. Sulfur dioxide, added as a preservative to wine and fruit juices, may cause allergic response in susceptible subjects. Besides the mandatory indication of its presence in many countries, its levels are defined by legislation [3]. Several automatic systems have been proposed for determination of this analyte [38,73–81], with most incorporating some sort of gas diffusion device to allow the separation of SO2 from the food matrix before direct electrochemical detection [38,73,75,77] or before further derivatization and spectrophotometric detection [78–81]. The automatic assessment of ethyl carbamate precursors has also been described. High levels of urea and assimilable nitrogen at the initial stages of wine production are related to the content of potentially carcinogenic ethyl carbamate in the final product [82]. Therefore, Gonzalez-Rodriguez et al.


545

proposed an FI–pervaporation system for monitoring urea and ammonia [29]. The automatic determination of assimilable N using a similar strategy [30] or an SIA system [34] is also possible. The application of FIA as a sample-handling tool is highlighted in the determination of biogenic amines by capillary electrophoresis–electrospray mass spectrometry [83]. In this case an FIA system was used to perform in-manifold filtration and exact volume delivery to vials placed at an automatic sampler.

3. APPLICATIONS: PLANTS AND VEGETABLES Modern agricultural economies are highly dependent on the use of pesticides. A pesticide is defined as any substance or mixture of substances intended for preventing, destroying, repelling or mitigating any pest. Pests can be insects, mice and other animals, unwanted plants (weeds), fungi or other microorganisms. Substances or mixture of substances intended for use as a plant regulator, defoliant or desiccant can also be considered as pesticides. Therefore, a large and ever increasing variety of more than 1,000 of these compounds can be applied to agricultural crops during plant development, post-harvest processing and transport. These compounds might potentially remain in foodstuffs, resulting in an elevated risk, especially in freshly consumed fruits and vegetables. Regulatory bodies [1–3] have established various maximum residue limits for pesticides in foodstuffs. The determination of these residue levels in vegetables and fruits is a difficult task, not only because of the low target concentrations, but also due to complexity and variety of the samples. For these reasons analytical procedures with high selectivity and sensitivity are required. In the last few years various analytical methods based on flow techniques have been developed for these purposes (Table 3). Flow systems exploiting the selectivity and sensitivity of fluorimetric detection for the determination of pesticides have been developed [183–189], with one even incorporating an automated separation of organophosphorus pesticides by high-performance liquid chromatography (HPLC) followed by flow-injection post-column derivatization [190]. Tandem mass spectrometry (MS/MS) has been used in conjunction with flow injection methods to solve various analytical problems in biological samples. The hyphenation with flow injection enables both the quantitative and qualitative analysis of certain analytes and complex mixtures with little or no clean-up procedure. The first of the tandem MS detectors is used to detect separated compounds from all of the ionized compounds based on mass differences, while the second is used for detection of the target analyte, representing an advantageous alternative for analysis of pesticide residues [191–193]. Other flow methods can make use of chemiluminometric [194–198], spectrophotometric [199–202] or electrochemical [203] detection systems. The advantages associated with the consumption of vegetables and fruits are well-known. The nutritional quality of these products is of great interest to consumers and producers, and the assessment of nutritional parameters using flow methods has received increased attention since the first applications of these

546


methods were published. These parameters continue to be the centre of attention as demonstrated by the various papers published on the determination of ascorbic acid [152,204–207], total antioxidant capacity [49,208–210] and other components with specific nutritional or health benefits [211–214]. The flow methods developed for the determination of ascorbic acid were based on the reducing capacity of the analyte [152,205,206], with detection limits of as little as 1013 M being achieved by signal enhancement of the chemiluminescence reaction of cerium(IV) with Rhodamine B [207]. The antioxidant capacity assays, described earlier for the beverage samples, in many cases can be applied with little modification to the analysis of fruits and vegetable extracts. Besides the nutritional value of vegetable and fruit products, their inherent ability to accumulate potentially harmful substances such as nitrates and heavy metals has also received considerable attention and this is reflected in the number of papers dealing with these analytes.

4. APPLICATIONS: MILK AND DAIRY PRODUCTS The aflatoxins are a group of structurally related toxic compounds produced by certain strains of fungi. Under favourable conditions of temperature and humidity, these fungi grow on certain foods and feeds, resulting in the production of aflatoxins. These toxins can be found in combination in various foods and feeds in various proportions; aflatoxin B1, however, is usually predominant and is the most toxic [3]. Aflatoxin M1 (AFM1) is the major metabolic product of aflatoxin B1 in animals and is usually excreted into the milk of dairy cattle and other mammalian species that have consumed aflatoxin-contaminated food or feed. The food and drug administration (FDA) has set the action levels for aflatoxins at 20 mg kg1 in all food products designated for humans, other than milk; in milk this level is lowered to 0.5 mg kg1. However the current maximum level set by the European Union is 0.05 mg kg1 for AFM1 in milk [2,3]. Thus, concerns about sampling, sample preparation and analysis still remain in focus when determination of aflatoxins at the parts-per-billion level is to be reached. Immunochemical flow methods have been developed on the basis of the highly specific affinities of monoclonal or polyclonal antibodies for aflatoxins assays. Badea et al. [252] have developed a flow-injection immunoassay system for the determination of AFM1 in raw milk, establishing a dynamic concentration range between 20 and 500 ppt AFM1, with a detection limit of 11 ppt. Siontorou et al. [253] describe electrochemical flow-injection monitoring of AFM1 in cheese samples using filter-supported bilayer lipid membrane sensors with incorporated deoxyribonucleic acid (DNA). Subnanomolar detectable toxin concentrations were reached, with a sampling rate of four samples per minute. Another group of key analytes of interest in monitoring food safety is the antimicrobial agents. These agents can be routinely administered to foodproducing animals to promote growth and for therapeutic and prophylactic reasons. This practice can lead to the introduction of such agents into the human food chain resulting in significant health risks, such as the development of


547

resistant bacterial populations and allergic responses in sensitive individuals. In addition, the milk industry can be subjected to significant losses deriving from the inhibitory effects of drug residues on the culturing/fermentation processes. As a result, regulatory authorities have stipulated maximum residue/safe tolerance levels (MRLs/STLs) in foods of animal origin [2,3] in the range of 4–200 mg kg1 for the different type of antibiotics. Analytical methods for successful routine analysis must not only meet the required limits of detections but also provide low-cost and robust alternatives to current methods. Various flow methodologies were recently developed for different antibiotics in milk samples [254–258], providing adequate analytical figures for the targeted antimicrobial agents. Other analytes measured by flow techniques in the area of milk and dairy products are the metal ions [240,243,259–271], nitrate and nitrite [224,272,273] and different carbohydrates [165,274–280]. The major effort in this area of research during the last decade has been focused on performing all the necessary sample pretreatment steps within the flow method. Table 4 summarizes some of the analytical features of these methods.

5. APPLICATIONS: MEAT AND FISH PRODUCTS Of the flow methods developed in the last years for analysis of meat and fish products, the determination of nitrate and nitrite is still one of the most common [220,221,224,295–299], probably due to the health concerns related to the formation of nitrosamine and its carcinogenity. Furthermore in the last decade, flow methods have also been developed not only for the precursors (nitrite and nitrate), but also for the nitrosamine content of food samples [300,301]. As it is pointed out in the annual reviews of atomic spectrometry, mercury continues to be the most common analyte to be determined by chemical vapour generation, and this is certainly the case in the area of food-related flow analysis. Mercury is an analyte with great importance due to its toxicity and its bioaccumulation in animal tissues. This, and the fact that chemical vapour generation is most efficiently carried out using flow systems, explains the large numbers of the articles dealing with this analyte [246,302–309]. Similar to the area of environmental geochemistry, the speciation of the different forms of mercury is gaining importance in food analysis, sometimes in the form of hyphenated FIA–HPLC systems [305]. Evaluation of freshness and quality of meat and fish products is based on sensorial evaluation. However these assays and protocols are complex and time consuming, involving a trained group of tasters and consequent elevated costs. Therefore the emerging area of development and application of the so called electronic tongue and nose – biosensor devices for recognition (identification, classification and discrimination), quantitative analysis and assessment of taste and flavour components – has been receiving increased attention. Of these components, biogenic amines are considered as useful biomarkers of food freshness. Flow methodologies have been developed for the quantification of

548


histamine [310–312], putrescine [313], agmatine [314] trimethylamine [315–318] content or for the assessment of a so-called freshness factor that incorporates the degenerative compounds of adenosine triphosphate (ATP) [319–321]. The developed flow procedures (Table 5) are simple in configuration and can make use of electrochemical, fluorescence, chemiluminescence detection methods, with good sensitivity, selectivity and precision, even allowing the direct introduction of the solid samples [322]. Biosensors have also been implemented in flow-injection systems with the aim to detect food pathogens such as Salmonella typhimurium and Escherichia coli. These methods are based on the separation of the target microorganism from the sample, followed by further concentration based on its highly specific reaction with immobilized antibodies. Afterwards, the detection is carried out on a piezoelectric cell [323] or amperometrically [324,325].

6. MISCELLANEOUS FOOD PRODUCTS The application to food products that do not fall into the previous sections are presented in Table 6. These include the analysis of bread [345], butter [346], chocolate [175], edible oil [347–354], honey [42,109,132,168,278,355–360], lard [346], pasta [346,361], seasonings [362–364], soup [362,365–368], sweeteners [173, 369–377], syrup [278,378] and vinegar [77,97,109]. In general, these flow systems were devised for other matrices and are discussed in the previous sections.

ABBREVIATIONS AFS ATP CE CRM CV-AAS DNA ESI-MS ET-AAS ETV-ICP-MS FAAS FDA FIA FTIR HG-AAS HPLC IC ICP-MS ICP-OES

Atomic fluorescence spectrometry Adenosine triphosphate Capillary electrophoresis Certified reference material Cold vapour atomic absorption spectrometry Deoxyribonucleic acid Electrospray ionization mass spectrometry Electrothermal atomic absorption spectrometry Electrothermal vapourization inductively coupled plasma mass spectrometry Flame atomic absorption spectrometry Food and drug administration Flow-injection analysis Fourier transform infra-red spectrometry Hydride-generation atomic absorption spectrometry High-performance liquid chromatography Ion chromatography Inductively coupled plasma mass spectrometry Inductively coupled plasma optical emission spectrometry


IR MCFA MPFS MS MSFA MSFIA NMR SIA TS-FF-AAS UV-Vis

549

Infra-red spectrometry Multicommuted flow analysis Multipumping flow system Mass spectrometry Monosegmented flow analysis Multisyringe flow-injection analysis Nuclear magnetic resonance Sequential injection analysis Thermospray flame furnace atomic absorption spectrometry Molecular absorption spectrometry

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CHAPT ER

19 Life Sciences Applications Jianhua Wang and Xuwei Chen

Contents

1. Introduction 2. Deoxyribonucleic Acid (DNA) Assays 2.1 DNA separation and purification from biological matrices 2.2 DNA quantification 2.3 DNA separation and amplification 3. Assays of Proteins, Peptides and Amino Acids 3.1 Quantitative assay protocols 3.2 Activity measurements 3.3 Separation of biomolecules 3.4 Protein immobilization 4. Immunoassays 5. Enzymatic Assays 5.1 Heterogeneous enzymatic assays based on microreactors 5.2 Homogeneous enzymatic assays 6. Cellular Analysis 7. Perspectives Abbreviations References

559 560 560 562 565 566 566 569 569 575 575 581 581 583 585 586 587 588

1. INTRODUCTION Flow injection analysis (FIA), based on automatic injection of a series of samples/ reagents into a continuous carrier stream, has been developed as a powerful technique for fluidic manipulations prior to detection. Unlike conventional solution handling procedures based on equilibrium of the reaction systems, FIA obtains the analytical data from dynamic processes taking place in the flow manifolds, thus the analysis time is greatly reduced and at the same time, better precision, higher sample throughput and reduction in sample consumption are achieved. It has provided a versatile and inexpensive methodology for automation of analytical procedures and the advantages mentioned herein could readily explain the explosive growth of publications concerning FIA [1]. Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00619-3


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Jianhua Wang and Xuwei Chen

In 1990, the conception of sequential injection analysis (SIA) was conceived in order to reduce the inconvenience that hindered the utilization of FIA as a routine analytical tool. Relatively large consumption of reagents in the FIA mode is avoided since only the required amounts of reagents are aspirated and carrier is not pumped continuously in the SIA protocol. At the same time the SIA methodology offers great advantages since the systems can be easily adapted to various analytical protocols by merely adjusting and controlling the flow parameters through a computer without changing the physical configuration. The bead injection analysis (BIA) scheme has been recently developed as a more flexible technique, in which manipulation of beads takes place in flow-based manifolds. The BIA technique not only offers high precision for beads delivery but also avoids carryover in repetitive sample handling. The automated transportation of solid materials within the flow system facilitates well their renewal whenever necessary and provides a high degree of repeatability when metering, packing and perfusion of beads with samples and reagents. In addition, some beads, such as Sephadexs and Sepharoses can be detected directly in situ by UV–VIS and fluorescence spectrometry, which allows real-time monitoring of binding and elution of analytes. The flow-based techniques outlined above have proved to be powerful fluidic manipulating approaches, which have been discussed extensively in a number of articles [2,3]. They also offer an elegant and versatile interface for various detection techniques [4,5]. In the automatic mode, the FIA/SIA systems play very important roles by replacing labor-intensive manual procedures. In addition, they are especially suitable for reliable long-term operations. Features such as low sample and regents consumption, reduced analysis time, favorable reproducibility and repeatability and minimal sample contamination in a closed processing system, obviously make the FIA and SIA techniques among the most suitable approaches in life sciences analysis. In the last decade, the three generations of flow analysis techniques (i.e., FIA, SIA and lab-on-valve (LOV)), have been widely employed in life sciences analysis, including immunoassays and assays of various target macrobiomolecules (e.g., nucleic acids, protein species as well as amino acids).

2. DEOXYRIBONUCLEIC ACID (DNA) ASSAYS 2.1 DNA separation and purification from biological matrices The extraction and purification of DNAs from a variety of complex sample matrices (real-world biological samples) is a critical process, which should provide pure solutions of the DNAs of interest for further biological investigations. These solutions should be free of interferences arising from coexisting soluble constituents. These biological investigations may include processes such as polymerase chain reactions (PCR) and DNA hybridization. At this point, solidphase extraction (SPE) based on the affinity adsorption and separation of DNAs has been proven to be the most straightforward, robust, simple and efficient

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purification methodology, which can readily be performed in an appropriate FIA/SIA manifold. An FIA procedure including ion-exchange purification of nucleic acids in a miniaturized expanded-bed column has been developed by Nandakumar et al. [6] for on-line monitoring of the concentration of plasmid DNA during the cultivation of E. coli. In this case, the sorbent material was treated as a stationary component, which was used repetitively for sorption-dissolution of DNA and was renewed only after numerous analytical runs. The introduction of a renewable surface technique in FIA/SIA systems involving SPE with regeneration of the sorbent material after each analysis was reported [7,8]. This approach facilitated efficiently the avoidance of a problem frequently encountered in conventional SPE operations, which stemmed from the contamination or deactivation of the sorbent surface after processing a large number of samples. In some cases the processing of numerous samples can also lead to the loss of functional groups or active sites. Chandler and coworkers developed an SIA system with a renewable separation column (SIA–RSC) for automating the purification of total DNA from complex matrices [9–11]. A rotating rod, as depicted in Figure 1, was employed for the renewal of the solid phase, which was aimed at improving the reproducibility. According to the authors, this micro-column equipped with a rotating rod was capable to handle particulate materials without any clogging thus providing reproducible performance for a few months. This system was also adopted for studying

Figure 1 Schematic diagram of a rotating rod renewable filter (A). With the beveled rod in the trap position, beads with diameter larger than the leaky tolerance are collected in the microcolumn as the fluid flows continuously through the outlet port. To flush the beads to waste, the beveled rod is rotated at 1801 (B). Reprinted from [1]. Copyright (2003), with permission from Elsevier B.V.

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solid-phase nucleic acid binding chemistry [12]. Rapid evaluation of variables including solution compositions and temperatures of hybridization and elution was achieved. As the third generation of flow analysis techniques, LOV systems with their versatile channel design facilitate fluidic manipulation at the level of 0.1B100 mL, thus allowing surface renewal in a more flexible and precise manner. An LOV system, integrating a renewable micro-column, as illustrated in Figure 2, has offered significant advantages for DNA separation and purification in terms of improved sampling frequency, long-term stability and better precision over chipbased and capillary-based separation/purification systems utilizing the same principles [13]. In addition, the employment of BIA in an LOV system further facilitated real-time renewal of the micro-column thus leading to a significant improvement in the long-term reliability and robustness of operation. With a fluidic manipulation at the meso-fluidic level, the LOV-based DNA purification system also presents itself as an excellent sample pretreatment front end for microfluidic analysis systems such as micro-chip PCR and/or micro-chip electrophoresis. These systems provide a promising platform for the integration of DNA purification, PCR amplification and micro-chip electrophoresis into a compact system [13,14].

2.2 DNA quantification The accurate quantification of trace amounts of nucleic acid is a critical step in a wide variety of biological and diagnostic applications such as genetic diagnosis

Figure 2 Schematic diagram of the LOV meso-fluidic system with integrated demountable fluorescence flow cell, employed for DNA separation and purification (EtBr, ethidium bromide; LIF, laser-induced fluorescence; P1, P2, peristaltic pumps; SP, syringe pump; V1, three-way valve). Reprinted from [13]. Copyright (2003), with permission from Springer Science and Business Media.


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and forensic analysis. Considering the limited amount of source material for biological samples, rapid analysis protocols and the avoidance of cross contamination are crucially required. Obviously these requirements cannot be fulfilled by conventional manual procedures. FIA/SIA techniques show clear advantages for the quantification of DNA at trace levels because of the possibility for automatic fluidic manipulation and minimized cross contamination. A variety of detection techniques have been adopted for DNA quantification in flow-based analysis systems. These include spectrophotometry [15], spectrofluoromerty [16–21], chemiluminescence [22–26] and amperometry [27,28] as summarized in Table 1. Due to the controllable and highly reproducible dispersion in FIA systems, a higher sampling frequency is usually obtained and the reproducibility is improved, but the consumption of reagents is somewhat relatively high when a continuous flow of reagent and carrier takes place. This results in high analysis expenditures and the production of large amounts of toxic waste. In order to overcome these problems, some promising techniques have been developed such as the renewable drops technique [18]. In this technique the DNA and reagents solutions are delivered into a silica capillary tube by a peristaltic pump to form drops at the tip of the capillary tube where fluorescence measurements are conducted by employing optical fibers. This windowless detection system employing renewable drops provides a fresh reaction surface for each sample. This is of particular value for solving problems arising from irreversible reactions. At the same time lower reagents consumption is easily achieved. The versatility of in-valve spectrometric detection and precise fluidic manipulation make the LOV system a suitable alternative for reducing reagents and sample consumption. This has been well demonstrated in DNA quantification studies. A novel procedure with spectophotometric or fluorometric detection carried out in a meso-fluidic LOV system has been developed recently [15,21]. In the spectrophotometric detection mode, only 10 mL of reagent (crystal violet solution) and 5.0 mL of sample solution were required for each analysis; while in the fluorometric mode with laser-induced detection, the sampling volume was further downscaled to nano-liter levels, i.e., 600 nL, while at the same time much higher sensitivity (10-fold improvement) was achieved. Recently, an LOV procedure for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization was proposed by Edward and Baeumner [19]. UV and fluorescence detection has been exploited for monitoring the on-bead oligonucleotide hybridization as well as for the quantitative analysis of DNA strands with a linear dynamic range of 1–1,000 pmol. Song et al. [29] proposed an FIA manifold coupled to a ultra-sensitive surface plasma resonance (SPR) spectrometer for the detection of sequence-specific ultratrace levels of oligodeoxynucleotides and polydeoxynucleotides. A miniaturized flow cell (with a capacity of 4 mL) was constructed as an interface between the detector and the flow system. A detection limit at 54 fmol L1 was obtained, which implies a significant improvement in the detectable concentration levels by 2–3 orders of magnitude as compared to some of the other SPR methodologies.

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Table 1


Applications of flow-based techniques in quantitative assays of DNA

Detection method and comments

Calibration range

Detection limit

Sampling frequency

References

Absorbance, decoloration of DNA on crystal violet, 591 nm Fluorescence enhancement of berberin after reaction with DNA, lex/em ¼ 362/ 531 nm Fluorescence enhancement of Hoechst 33258 after reaction with DNA Fluorescence quenching of TMB-da after adding DNA, lex/em ¼ 278/ 403 nm Fluorescence enhancement of fluorescein after reaction with DNA, lex/em ¼ 480/ 520 nm Fluorescence enhancement of Ru(bpy)2PIP(II) after reaction with DNA, lex/em ¼ 460/ 590 nm Fluorescence enhancement of ethidium bromate after reaction with DNA, lex/ em ¼ 473/610 nm Chemiluminescence of the Rhodamine BCe(IV)-DNA system

0.2–6.0 mg mL1

0.07 mg mL1

30 h1

[15]

0–12 mg mL1

7.3 ng mL1

60 h1

[16]

–

0.01 mg

–

[17]

0.03–8.4 mg mL1

10 ng mL1

–

[18]

1–1,000 pmol

1 pmol

–

[19]

0–4 mg L1

3.7 mg L1

60 h1

[20]

0.03–3.0 mg mL1

0.009 mg mL1

60 h1

[21]

1.0 10–8– 0.1 mg mL1 2.1 10–6– 0.21 mg mL1 2.6 10–5– 0.26 mg mL1

8.3 10–9 mg mL1

–

[22]

3.5 10–7 mg mL1

[23]

6.5 106 mg mL1

[24]


565


a

Detection method and comments

Calibration range

Detection limit

Sampling frequency

References

Chemiluminescence of the Ce(IV)-Na2SO3Tb(III)-fluoquinolone antibiotic system Chemiluminescence of the 9,10anthraquinone-2,6disulphonic acidDNA system Amperometric detection of the oxidation of bound guanine moiety Electrochemical detection of the adsorption/ desorption of DNA on polypyrrole (PPy)coated electrodes

0.04–10 mg mL1

7.8 mg mL1

–

[25]

0.04–5.5 mg mL1

17 ng

45 h1

[26]

–

460 pg

–

[27]

1–10 mg mL1

6.1 1016 mol

–

[28]

TMB-d, 3,3u,5,5u-tetramethylbenzidine dihydrochloride.

2.3 DNA separation and amplification Capillary electrophoresis (CE) has been proven to be a powerful technique for fast and automatic DNA separation when hyphenated with FIA as the sample introduction front end [30]. This coupling scheme has been exploited extensively. Wang et al. proposed a compact microchip-based CE system for DNA separation using laser-induced fluorescence (LIF) detection and incorporating a liquid-core waveguide [31]. Automatic sample introduction was readily realized in an SIA system through a modified split-flow interface, which allowed the release of gas bubbles thus improving the stability of the system. In order to facilitate the integration and miniaturization of this chip-based SIA-CE analysis system, a light emitting diode (LED), rather than a laser beam, was further adopted as the excitation source with lock-in amplifier to enhance the signal-to-noise (S/N) ratio [32]. The feasibility of this compact manifold has been demonstrated by the successful separation of 11 components of an F174 HaeIII DNA digest sample. All the 11 components in the sample were effectively separated in 400 s with satisfactory resolution, with an S/N ratio comparable to that obtained by employing an SIA–CE system with LIF detection. The vast potential of FIA and SIA systems in fluidic manipulation also provides automatic sample preparation protocols for DNA amplifications. At this point, Belgrader et al. [33] developed a reusable flow-through PCR system for

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continuous monitoring of infectious biological agents. They employed an SIA system as the sample introduction front-end, which could be used repetitively to carry out the sequential analysis of samples. This provided great advantages over conventional PCR schemes with disposable reaction tubes for single use. In addition, much lower sample consumption was achieved, i.e., a reagent volume of less than 10 mL resulted in significant reduction of the running costs. A continuous flow PCR amplification system consisting of an SIA sampling processor, a microfluidic PCR chip and a 3-temperature-zone heating block was recently described by Liu et al. [34]. The system was capable of performing continuous PCR amplification with a low carryover between neighboring/ stacking sample zones. In order to reduce the dispersion and flow resistance of the reaction solution in the channels of the microchip, which could be considered to be the main source of cross contamination, the same research group designed a spiral-channel flow-through PCR microchip reactor. This system allowed the successful continuous amplification of seven samples in 1 h without cross contamination between samples [35].

3. ASSAYS OF PROTEINS, PEPTIDES AND AMINO ACIDS The analysis of proteins, peptides and amino acids can provide abundant information for diagnosis of various diseases, and thus their quantitative assays are of great importance in biological investigations as well as in clinical applications. Flow-based techniques have gained extensive attention and become much more popular in the fields mentioned herein attributed to the simple instrumentation required, ease of operation, reliability and low running costs.

3.1 Quantitative assay protocols It has been well documented that the weak resonance light scattering (RLS) intensity of some dyes could be significantly enhanced in the presence of trace amounts of proteins. These dyes include Bromothymol Blue [36], Amide Black-10B [37], Eriochrome Black T [38] and Biebrich Scarlet [39]. Various FIAbased automatic procedures for protein quantification have been developed recently. As compared to the conventional analytical procedures, these procedures provide much faster and inexpensive assays of total protein species in biological samples such as urine and serum. Chemiluminescence is a very sensitive and selective detection technique, which is most suitable for hyphenating with FIA. This detection technique has been recently utilized extensively for the direct determination of amino acids prior to their separation [40–43]. The most important key point to the success in achieving satisfactory selectivity in chemiluminescence detection is the choice of an appropriate reaction system and the manipulation of the chemical conditions in order to yield a response from the species of interest only. Costin et al. [40] proposed an FIA chemiluminescence methodology for selective detection of proline, histidine, tyrosine, arginine, phenylalanine and tryptophan in the


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presence of other amino acids. Selectivity was achieved by the application of a number of chemiluminescence reaction systems in addition to the manipulation of the reaction conditions where a certain amino acid gives rise to a response as a result of a particular reaction only. This approach offers significant advantages over conventional methods as derivatization, separation or extraction are not required, which dramatically reduces the analysis time. On the other hand, however, the employment of a series of chemiluminescence reactions obviously makes the overall analysis much more complicated. The chemiluminescent reactions between some amino acids and carbonyl functional groups of humic acids were used in an FIA system for the selective determination of glycine and arginine [41]. Considerable selectivity for these two amino acids in the presence of other amino compounds was achieved with detection limits of 0.20 and 0.25 mg L1 for glycine and arginine, respectively, along with a sampling frequency of 115 h1. A fluorescent derivative of albumin participates in a chemiluminescence reaction with peroxyoxalate with imidazole as the catalyst. By conducting this chemiluminescence reaction in an FIA system, an assay procedure for albumin was developed by employing a micellar medium as carrier [44]. A detection limit of about 0.1 fmol for albumin was achieved. Electroanalytical systems have been widely employed for the assay of macrobiomolecules in different flow analysis setups. Based on the electrocatalytic oxidation of cysteine at a pretreated platinum electrode, a selective FIA method for the biamperometric determination of this compound in amino acid mixtures and human urine samples was developed [45]. This method was characterized by a sampling frequency of 180 h1 and was applied to the determination of cysteine in real samples without any sample pretreatment. The assay of cysteine has also been performed by employing an FIA amperometric detection system based on the reaction of amino acids with chloramine-T [46]. A linear calibration graph of up to 10 mg cysteine mL1 was obtained, along with a sampling frequency of 220 h1. Nanjo et al. [47] have proposed recently an FIA system with an enzyme reactor and a hydrogen peroxide electrode for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. In their studies, fructosyl–amino acid oxidase and two fructosyl–peptide oxidases were covalently immobilized onto an inert support in the enzyme reactor. The proposed FIA system responded linearly to the concentration of fructosyl valine over the dynamic range 7.8 106–5.8 104 mol L1. The authors also proposed a similar FIA system, comprised of an electrochemical detector with fructosyl–peptide oxidase reactor and a flow-through spectrophotometer for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cell [48]. The hyphenation of FIA sample processing with high performance liquid chromatography (HPLC) provides promising potentials for both the elimination of interfering sample matrices and separation of the target species. An FIA system coupled to an HPLC system has been successively employed for detecting low molecular mass advanced glycation end-peptides (AGE-P) in samples from 126 diabetic patients, 54 normal controls and 20 diabetic mice [49]. The variance

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coefficients for intra-assay and inter-assay were 1.2% and 6.3%, respectively, which suggested that the AGE-P assayed by FIA provided a better precision and recovery compared to the protocols based on batch enzyme-linked immunosorbent assays (ELISA) and fluorescence spectrometry. As is well documented in the literature, FIA is suitable for coupling with almost any type of a detection system. The performance of FIA systems coupled to electrospray ionization mass spectrometry (ESI–MS), tandem mass spectrometry and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry (ESI–FAIMS) has been investigated for the suitability of these techniques for the determination of underivatized amino acids [50]. The experimental results have shown that ESI–FAIMS–MS offered improved sensitivity and significantly better S/N ratio when compared to ESI-MS. This was mainly due to the elimination of the background noise and the partial or complete resolution of all potential isobaric overlaps arising from amino acids. These results suggested that ESI–FAIMS–MS should be the preferred method for the quantitative analysis of proteinogenic amino acids in real samples. A sensitive procedure for the quantification of total protein in human serum involving SIA sampling and fluorometric detection, based on the rapid reaction between fluorescamine and primary amino acids, was proposed [51]. A few microliters of sample and fluorescamine solutions were mixed and the reaction of proteins with fluorescamine gave rise to a blue-green–fluorescent derivative, which was subsequently excited at 400 nm and the fluorescence was monitored at 470 nm. By loading 5.0 mL of sample and 4.0 mL of 0.075% (m/v) fluorescamine solution, a linear calibration graph was obtained within 0.3–12.5 mg mL–1 along with a substantially improved detection limit of 0.1 mg mL–1 as compared to 10.0 mg mL1 for the conventional manual procedure based on the same reaction system. As the third generation of flow analysis systems, an LOV analyser provides vast potential in bioassays attributed to its unique structural characteristics. In one of the recent studies, protein coated Sepharose beads were introduced into the flow cell of an LOV, where the beads were trapped by the tip of an optical fiber and the changes of spectral properties on the beads surface was monitored in situ. A label dilution protocol was thus developed to discriminate between the selective and non-selective bindings [52] which not only provided a protocol for monitoring bioligand interactions in real time, but also presented a sensitive method for the determination of low levels of analytes of interest in complex matrices in immunoassays. As a model analyte, the determination of immunoglobulin G (IgG) was performed with a detection limit of 470 ng. Based on a similar principle, i.e., selective capture and release of the analyte of interest on an appropriate stationary phase, micro-affinity chromatography (m-AC) and micro-bead injection analysis spectroscopy (m-BIAS) have been developed. The detection modes of the two techniques are quite different from each other. Both techniques have been recently applied to the determination of IgG as a model analyte in the same LOV system [53]. The beads were retained up-stream in the flow cell of the LOV system. The absorbance of the eluted analyte was monitored post-column in the m-AC procedure, while the spectral


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changes of the beads’ surface were detected in m-BIAS mode. When employing a longer light path in the absence of light scattering m-AC exhibited higher sensitivity compared to that of m-BIAS, i.e., the limit of detection of the m-AC technique for IgG was 5 ng mL1, and that of the m-BIAS technique was 50 ng mL1. The main analytical characteristics of flow-based techniques applied to quantitative assays of proteins, peptides and amino acids are summarized in Table 2.

3.2 Activity measurements The transient features of the signal recorded under thermodynamically nonequilibrium conditions in an FIA system are most suitable for measuring activitybased properties of certain biological species or compounds and reagents. A rapid FIA assay protocol for determining the activity of the purified catechol-Omethyltransferase (COMT) from porcine liver using electrochemical oxidation, fluorogenic derivatization, and fluorescence detection was described by Aoyama et al. [54]. It was demonstrated that the kinetic parameters obtained by using this FIA procedure were similar to those derived from an HPLC system but the FIA approach offered a much higher sample throughput. Recently, Staggemeier et al. [55] developed an FIA procedure based on coupling a linear pH gradient system and a dynamic surface tension detection unit (DSTD) for protein surface activity measurements (Figure 3). This system allowed high sample throughput screening of protein surface activity at the air/ liquid interface as a function of pH. This method not only provided an innovative approach for probing the pH-induced conformational changes of proteins by exploring surface tension measurements, but represented also a further advancement of conventional methodologies based on spectrometric measurements. This research group also developed an FIA manifold incorporating in parallel a multi-dimensional DSTD system and a UV-VIS diode array absorbance detector [56]. The system was used specifically for studying the effects of chemical denaturants, such as urea, guanidinium hydrochloride, and guanidinium thyocyanate, on the surface activity of globular proteins at the liquid-air interface.

3.3 Separation of biomolecules During the last decade, quite a few investigations have been directed towards the hyphenation of FIA with CE. Fang’s and Karlberg’s groups have made significant contributions towards the development of this field independently [30,57]. Their pioneering work on the hyphenation of an FIA sample pretreatment front end with a CE separation system greatly enhanced both sample injection and separation efficiency. In addition, the reduced consumption of samples and reagents as well as the possibility to separate and analyse small molecules in complex matrices opened promising avenues for applications in biochemistry.

570

Applications of flow-based techniques for quantitative assays of proteins, peptides and amino acids

Analyte

Detection mode

Linear range

Detection limit

Sampling frequency

References

Protein

Rayleigh light scattering Rayleigh light scattering

7.0–70.0 mg mL1

3.75 mg mL1

26 h1

[36]

0.50–32.00 mg mL1 for HSA 2.00–36.00 mg mL1 for BSA 7–36 mg mL1 for HSA 8–44 mg mL1 for BSA 0.005–18 mg mL1 for HSA 0.008–16 mg mL1 for BSA 1 108–1 105 mol L1

0.11 mg mL1 for HSA

–

–

0.85 mg mL1 for BSA

90 h1

[37]

0.882 mg mL1 for HSA 2.507 mg mL1 for BSA 5 ng mL1 for HSA

– 90 h1 –

– [38] –

7.8 ng mL1 for BSA

–

[39]

4 109 mol L1 for proline 1 108 mol L1 for tyrosine 4 107 mol L1 for histidine

–

[40]

Protein

Protein Protein

Amino acids

Rayleigh light scattering Rayleigh light scattering

Chemiluminescence


Table 2

Chemiluminescence

1–30 mg L1

Tryptophan

Chemiluminescence

L-cysteine albumin L-cysteine Cysteine Valine

Chemiluminescence Chemiluminescence Amperometry Amperometry Amperometry

Histidine

Amperometry

AGE-P Protein IgG IgG

Fluorometry Fluorometry Spectrophotometry Spectrophotometry

6.0 107– 3.0 105 mol L1 0.2–80 mg L1 1.02–12 mg L1 4 107–4 105 mol L1 0.2–10 mg mL1 7.8 106– 5.8 104 mol L1 7.0 106– 1.1 104 mol L1 0.01–10 mg mL1 0.3–12.5 mg mL1 0.1–1.0 mg mL1 0.1–0.4 mg mL1

115 h1

[41]

50 h1

[42]

0.1 mg L1 0.38 mg L1 1 107 mol L1 0.06 mg mL1 –

60 h1 180 h1 220 h1 –

[43] [44] [45] [46] [47]

1.4 106 mol L1

–

[48]

– 0.1 mg mL1 5 mg mL1 (mAC) 50 mg mL1 (mBIS)

– 40 h1 – –

[49] [51] [53] [53]


Amino acids

1 107 mol L1 for arginine 7 106 mol L1 for phenylalanine 2 106 mol L1 for trytophan 0.20 mg L1 for glycine 0.25 mg L1 for arginine 1.8 107 mol L1

571

572


Pump Sample

Computer Injection Valve

30 μL/min

60 μL/min

Waste

pH meter

Solenoid Valve

Mixing Coil 30 μL/min

Po Pressure Sensor

pH Gradient System

Capillary Sensing Tip and Drop

Air Supply Air Burst Capillary (Pneumatic Detachment)

Drop Collection Vessel

Figure 3 Schematic diagram of the FIA-pH-DSTD instrument configuration. The pressure sensor, connected to the tubing with a sidearm, measures the differential pressure across the liquid/air interface with respect to atmospheric pressure, Po, of the forming drops. Reprinted from [55]. Copyright (2005), with permission from the American Chemical Society.

When an SIA system is employed, the discrete zones of samples and/or reagents disperse into each other, thus resulting in a reproducible zone penetration. Consequently, the reaction product is formed in a well-defined area of concentration gradients and this provides reproducible analytical results, thus making SIA systems suitable candidates for on-line sample pretreatment such as pre-column derivatization. An FIA-based split-flow sample introduction system was developed and coupled to a CE system through a falling-drop interface [58]. A sampling throughput of up to 144 h1 was achieved along with a 2% carryover and an RSD of 3.2% by continuously introducing a series of 30 mL sample solutions containing a mixture of fluorescein isothiocyanate (FITC)-labeled amino acids. The same research group developed later an SIA micro-chip-based CE system for the separation of amino acids which incorporated a split-flow sampling unit, similar to the one mentioned above and integrated onto the micro-chip [59]. Sequential introduction of a series of 3.3 mL sample solutions containing a mixture of FITC-labeled amino acids gave rise to a carryover of 2.5% at a sample throughput of 48 h1. Baseline separation of FITC-labeled arginine, phenylalanine, glycine and FITC in sodium tetraborate buffer was achieved within 8–80 s. An on-column polymer-imbedded graphite inlet electrode for CE coupled on-line to an FIA system by using a poly(dimethylsiloxane) interface was proposed [60]. The electrode consisted of a conductive polyimide/graphite imbedded coating immobilized onto the CE column inlet. This integrated


573

electrode gave the same separation performance as a commonly used platinum electrode. The on-line FIA–CE system was used with electrospray ionization-time of flight-mass spectrometric detection. The authors validated this hyphenation technique by a successful separation of three peptides (methionine–enkephalin, neurotensin, and substance P) in an electrolyte consisting of 50% formic acid/ ammonia and 50% acetonitrile. The on-line coupling of SIA and CE via an in-line injection valve for automated derivatization of amino acids and peptides has recently been described [61]. Dichlorotriazinylaminofluorescein served as the derivatization agent, enabling sensitive laser-induced fluorescence detection of the derivatives. When using des-tyr(1)-[met]-enkephalinamide as the model analyte, on-line electrophoretic analysis was achieved. Glycine was selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For enkephalin, good reproducibility, linearity and a favorable limit of detection of 30 ng mL1 were achieved. Wu et al. [62] reported the successful hyphenation of mSIA–LOV with a CE system, where the LOV acted as the sampling ‘‘front end’’ for the CE setup, and this integrated mSIA–LOV–CE system was used for in situ protein derivatization [63], as illustrated in Figure 4. All the necessary micro-fluidic manipulations such as sampling, fluorogenic labeling, and CE capillary regeneration were automatically performed by the mSIA–LOV unit. On-line fluorogenic derivatization of Islet proteins (insulin, proinsulin and c-peptide) was carried out with fluorescamine which was followed by successful CE separation and fluorometric detection. The RSD values for peak area, electro-migration time and peak height using 3.45 mmol L1 insulin injections were 1.3%, 0.5% and 2.8%, respectively. A miniaturized sequential affinity chromatography within an LOV system was developed for the separation of mouse IgG, chicken IgG and bovine serum albumin. An automatically renewable micro-column was integrated into the LOV module used for separation and quantification of biomolecules on Sepharose Protein A beads by absorbance measurements at 280 nm. This setup reduced greatly the sample and reagent consumption and the total assay time. In addition, a favorable limit of detection of 6.0 ng mL1 for mouse IgG was obtained [64]. Ogata et al. [65] reported on two other types of LOV–BIA geometries for investigating the automated selective capture and release of biotin-containing conjugates on immobilized streptavidin. The capturing and releasing procedure were monitored on-line by UV/VIS spectrometry and the dissociation procedure was simultaneously monitored by ESI–MS. The LOV–ESI–MS instrument was also used for repetitive assays of lysosomal beta-galactosidase in human cell homogenates. Fast analysis in 4.5 min for a full cycle and robust operation in 60 repetitive analyses were demonstrated thus making the transfer of the LOV–ESI–MS technology into clinical practice very promising. A similar procedure was used for the simultaneous measurement of the affinities of multiple ligands to proteins [66]. In this automated LOV mode, 1 mg of protein was sufficient for 35 repetitive analyses and the equilibrium dissociation constants (Kd) could be determined rapidly in the range of 105–107 mol L1.

574

Anode

Waste Sample To Isolation Valve Flow-through Port

LOV PMT

Spectrometer

Waste

2

Objective Lens

CE Buffer

1

6 5

Optical Fiber

3

6-way Selector Valve

4

Capillary Cathode

Reagent Epiluminescence Microscope Syringe Pump

0.1M NaOH

UV Source

0.1M Phosphoric UV Source Acid

Holding Coil

CE Buffer Reservoir LOV

Figure 4 Schematic diagram of an LOV–CE derivatization system for peptides. Reprinted from [63]. Copyright (2003), with permission from the Royal Society of Chemistry.


Spectrometer


575

3.4 Protein immobilization Immobilization chemistry is of key importance for the successful production of selective supports. Using an LOV–BIA manifold, Ruzicka et al. [67] studied the immobilization reactions of six proteins (albumin, ovalbumin, lysozyme, human IgG, ribonuclease A and cytochrome C) on the surface of agarose beads by measuring the rate and yield of the coupling reactions. By exploiting the BIA technique, the currently recommended protocols for reductive amination were shortened from several hours to only a few minutes. The leakage of immobilized ligands was measured by in situ direct spectrometric monitoring of the captured beads. These results suggested that BIA spectrophotometry was a useful tool for quality control of agarose-based chromatographic supports. It could also be used for the optimization of a wide variety of immobilization chemistries, used for synthesis of chromatographic supports, immobilization of enzymes, and derivatization of biosensing surfaces. The real-time monitoring of protein immobilization by using this protocol resulted in the surprising finding that current immobilization protocols were far from optimal.

4. IMMUNOASSAYS Flow-based immunoassay protocols, especially FIA immunoassay (FIA–IA) and SIA immunoassay (SIA–IA) have proven to be very useful in eliminating the drawbacks of conventional immunoassay schemes, which are usually timeconsuming and labor-intensive. Due to their unique characteristics such as minimized sample consumption, capability of sample pretreatment, and the ease of automation when high sample throughput is pursued, FIA–IA and SIA–IA have been extensively employed in various fields, among which life sciences applications have attracted extensive attention. Both methodologies are very suitable for hyphenating with various detection techniques, such as electrochemical methods, fluorometry, chemiluminescence methods and spectrophotometry. The flow-based immunoassay with electrochemical detection is one of the most developed methodologies. So far, the majority of the amperometric flow immunoassay systems have involved an immunoreactor, in which the antibody-antigen incubation step and the enzyme reaction take place. Another separate amperometric detector is employed for the oxidation or reduction of the enzyme-generated electroactive product at the surface of an appropriate electrode. Therefore, the design and implementation of a unique sensing surface for facile ligand functionalizations and biospecific interactions are very critical. Screen-printed electrodes have been extensively explored in recent years for bimolecular immunoassay [68–72], due to their low price and satisfactory reproducibility. With the aim of simplifying the flow-based enzyme immunoassay systems with amperometric detection, a promising approach has been developed by combining the immunoreactor and the detection unit into a single device by

576


immobilizing the immunoreagent directly onto the electrode surface, which serves as an immunosensor. Valat et al. [73] have reported on a protein A-based FIA–IA immunosensor for enzyme immunoassay of rabbit IgG and mouse IgG. It has been demonstrated that this immunosensing system can be used repetitively for 30 assay cycles. It is also disposable whenever necessary. An immunosensor for rapid separation-free determination of carcinoembryonic antigen (CEA) in human serum was developed by co-immobilizing thionine and horseradish peroxidase (HRP)-labeled CEA antibody on a glassy carbon electrode (GCE) through covalently binding with glutaraldehyde (GA) linkage. This immunosensor showed good accuracy, acceptable storage stability and favorable precision when employed in an FIA system. After the system had been optimized, a detection limit of 0.1 ng mL1 for CEA was achieved [74]. Chemiluminescence has become a very attractive detection technique in FIA–IA applications because of its simple instrumentation, very low detection limit and wide linear dynamic range. HRP is commonly used in chemiluminesence detection by catalyzing the oxidation of luminol by hydrogen peroxide (H2O2). A chemiluminesence FIA system has recently been employed in the immunoassay of Estriol [75], a-fetoprotein [76,77], 17 b-estradiol [78] and CEA [79]. The performance of FIA–IA and SIA–IA with chemiluminesence detection in the determination of a-amino acids with an immunoreactor consisting of a flow cell packed with immobilized haptens was investigated by Silvaieh et al. [80]. The experimental results indicated that better repeatability and higher sampling frequency were obtained by SIA–IA. The corresponding detection limits were 1.01 ng mL1 for the FIA–IA system and 0.29 ng mL1 for the SIA–IA system, which were further improved to 0.22 ng mL1 and 0.036 ng mL1, respectively, by employing stopped-flow mode. Although bioluminescence is not employed as extensively as chemiluminescence in flow-based immunoassay systems, it is indeed a very useful tool for some specific purposes and certain analytes of interest. Ho and Huang [81] recently described an FIA–IA system based on bioluminescence with liposomal aequorin as the label, which utilized the binding-site-directed immobilization of anti-biotin antibodies in a microcapillary using protein A. This system allowed the detection of 50 pg of biotin, which was a 60-fold improvement in sensitivity as compared to a similar FIA–IA system with fluorescence detection. Among the various flow-through immunosensors, quartz crystal microbalance (QCM) immunosensors have started to play an important role, which they deserve. QCMs are developed by the immobilization of antigen or antibody onto the surface of a piezo-electric material. It is crucial to obtain satisfactory immobilization to ensure high sensitivity and stable response in practical QCM applications. Four types of approaches including physical immobilization, and three chemical approaches (i.e., thioamine thiolation and the use of periodateoxidized dextran-modified thioamine or a thiol-gold chemisorption-based self-assembled monolayer (SAM)) have been employed by Liu et al. [82] to immobilize human serum albumin (HSA) onto the surface of a QCM. The performance of these QCMs has been investigated in an FIA–IA system. It was observed that all four methods had lead to comparable detection limits of the


577

QCM–FIA immunoassay. However, the SAM-based approach generated the largest frequency shift and also yielded the largest linear detection range. This indicated that the use of thiolated long-chain fatty acid forming a SAM might potentially be of greater interest as a protein immobilization method in QCM– FIA applications. A conducting polymer entrapment (CPE) method for immobilizing immuno-proteins on QCM has been also proposed by the same research group [83]. A higher frequency shift compared to that obtained by physical immobilization has been achieved. Fluorescence has been a key immunoassay detection technique for many years, yielding a large variety of procedures in this field. A simple and specific FIA–IA procedure for the detection of CEA by timeresolved fluorescence has been developed by Yan et al. [84] and it has exhibited higher sensitivity than conventional immunoassays can offer. This procedure was based on a sandwich immunoassay format involving the immobilization of a monoclonal antibody into an immunoaffinity column acting as an immunoreactor in the corresponding FIA–IA system. The cleaved solution was detected by time-resolved fluorescence after the reaction between the immunocomplex in the immunoaffinity column and the enhancement solution that was used to cleave the Eu-labels from the immunocomplex. Serum samples containing CEA have been detected in a linear range 2.5–100 ng mL1 along with a limit of detection of 1.0 ng mL1. The analysis of a large number of human serum samples showed good agreement with the results obtained by an alternative electrochemiluminescence immunoassay approach. The proposed time-resolved fluorescence FIA–IA method could be further developed for fast clinical detection of serum containing different levels of CEA. Although the detection sensitivity of spectrophotometry is somewhat lower as compared to other spectrometric techniques, it is frequently employed in immunoassays and is indispensable for solving some specific problems. A spectrophotometric immunoassay protocol was developed for vitellogenin (Vg) [85]. This method utilized an SIA system equipped with a jet ring cell, in which the immunoassay was conducted by using primary antibody-immobilized on Sephadexs beads and an HRP-labeled secondary antibody. The major drawback of this system was that the time required for the immunoreaction, i.e., 3 h, was somewhat too long for a quantitative assay. This might be attributed to the slow colour-development reaction. In order to handle this issue, an SIA-based chemiluminescence procedure for the determination of Vg using magnetic microbeads coated with an agarose gel instead of Sephadexs beads was developed [86]. The assay time was significantly shortened, though 20 min were still required for completing the assay. Most of this time was needed for the incubation of the immunoreaction system because of the slow diffusion of Vg or the secondary antibody into the agarose gel coated on the magnetic microbeads. It was recently discovered that magnetic microbeads coated with polylactic acid facilitated the rate of the immunoreaction [87,88]. Thus, a rapid and sensitive sandwich SIA-based immunoassay for the determination of Vg was developed. The SIA system consisted of a syringe pump, a multi-position valve and a flowthrough immunoreaction cell equipped with a magnet and an amperometric

578


detector. Magnetic microbeads with an anti-Vg monoclonal antibody (primary antibody) immobilized on them were used as a solid support. After the primary antibody-immobilized magnetic beads were introduced and trapped in the immunoreaction cell, a Vg sample solution, an alkaline phosphatase (AP)-labeled anti-Vg polyclonal antibody (secondary antibody) solution and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. Vg was determined by the electrochemical detection of p-aminophenol (PAP), an enzymatic product of PAPP by the action of AP of the secondary antibody. A solution containing PAP, which was generated in the immunoreaction cell was transported to the amperometric detector where the oxidation current of PAP flowing through the working electrode was measured. The detection limit of the immunoassay was about 2–3 mg L1. The entire reaction time required for this system was reduced to less than 15 min. Two sampling approaches for flow-based chromatographic competitive binding immunoassay, i.e., the simultaneous and sequential injection methods, were investigated by Nelson et al. [89]. Both techniques used a column with a limited amount of antibody, subjected to a perfusion of sample and a labeled analyte analog. In the simultaneous injection mode, the sample and labeled analog were introduced at the same time into the column, while in the sequential injection mode the sample was injected first, followed by that of the analog. This resulted in different analytical characteristics of these two approaches. This study used chromatographic theory and data previously obtained by injecting HSA into an anti-HSA antibody column to compare the response, detection limit, linear range and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method provided a lower limit of detection. The simultaneous mode offered a broader linear range and a higher upper limit of detection. In flow-based heterogeneous immunoassays, a solid support is generally used to immobilize either the antibody or antigen, thus permitting the separation of free fractions from bound immunocomplexes. Usually, a relatively long time is needed for regeneration of the solid support, which results in a lower sampling frequency. At the same time, some irreversible changes of the surface characteristics of the immunoreactor caused by its repetitive use might deteriorate the reproducibility. Therefore, carrying out the immunoassay by appropriately handling of the solid support material is of great importance. Renewable surface techniques seem to be the most suitable approach. An SIA bead-based immunoassay system has been developed by Hartwell et al. [90] for the determination of hyaluronan (HA). The main purpose of this study was to automate the immunoassay by ensuring precise delivery of micro-volumes of reagents and precise timing of the incubation and washing steps. These operations were achieved by computer control of the flow system’s bi-directional syringe pump. The manifold was designed with the aims of (i) reducing back pressure from beads that acted as solid surfaces for immobilization of the target substance; (ii) reducing dispersion and dilution of the reagents during incubation and (iii) maximizing the signal while minimizing the incubation time. This was


579

achieved by introducing air segments to separate the reagent zones from the carrier stream and by using a suitable sensitive detector, i.e., an amperometric sensor. The amount of HA was determined using competitive ELISA-based technique where immobilized HA and HA in solution competed to bind with a fixed amount of biotinylated-HA-binding proteins (b-HABPs). Upon separation of the two phases, anti-biotin conjugated with enzyme and a suitable substrate were introduced to follow the binding reaction of the immobilized HA and b-HABPs, whose degree of binding was indirectly proportional to the amount of HA in solution. Varnum et al. [91] described an enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies. By employing the renewable surface technique, the analysis time was reduced to less than 10 min, while at the same time the sensitivity was found to be somewhat lower compared to the conventional batchwise ELISA. An SIA renewable surface heterogeneous fluorescence immunoassay system with chip-based micro-flow-through cell has also been developed by Zhu et al. [92] for the determination of human IgG in serum. Immobilized antibody was prepared by conjugation of sheep anti-human IgG antibody to protein A coated Sepharoses CL4B beads. FITC labeled anti-human IgG antibody was used as the second antibody. The immobilized antibody beads, serum and the second antibody were sequentially injected into the chip-based micro-flow-through cell where a sandwiched antibody–antigen conjugate with fluorescence probe was formed and the fluorescence intensity was measured in the cell using optical fibers. After the measurement, the beads were discharged and the cell was ready for the next operation cycle. A detection limit for IgG of 0.1 mg L1 was achieved at a sample throughput of 11 h1. RSDs of 1.7% and 5.2% were obtained for inter-day and intra-day determinations of serum samples containing 3.9 mg L1 IgG. As the third generation of flow analysis techniques, the unique configuration of LOV is an ideal platform for BIA and provides vast potential for facilitating renewable surface operations, which have been one of the most important issues in immunoassay development. Carroll et al. [93] have reported on a novel analytical method for the detection and study of GAD65 autoantibodies, which have been implicated in the onset of Type 1 diabetes. There is a clinical need for a rapid and automated assay of GAD65 autoantibodies. The work mentioned above has focused on exploiting the advantages of BIA for ELISA in an LOV system. The BIA ELISA scheme is a microscale technique that uses enzyme labeled secondary antibodies to detect the capture of target antibodies on immobilized antigen in the flow cell of the LOV manifold. A detection limit of 20 ng mL1 for GAD65 monoclonal antibody 144 compares favorably with the sensitivity and precision of a standard ELISA currently employed to detect GAD65 autoantibodies. Compared to the standard ELISA protocol, BIA ELISA offers a significantly reduced assay time and complete automation of solution handling and detection. Table 3 summarizes the characteristics of selected flow-based immunoassay applications.

580

Table 3 Applications of flow-based immunoassay systems Detection mode

Linear range

Detection limit

References

Alpha-fetoprotein

Amperometry

Alpha-fetoprotein Alpha-fetoprotein Interleukin-6 Carcinoembryonic Carcinoembryonic Carcinoembryonic Carcinoembryonic IgG IgG

Chemiluminescence Chemiluminescence Amperometry Amperometry Amperometry Chemiluminescence Time-resolved fluorometry Amperometry Amperometry

5–20 ng mL1 20–150 ng mL1 5.0–100 ng mL1 2.0–75 ng mL1 5–100 ng L1 0.50–25 ng mL1 0.5–3.0 ng mL1 3.0–167 ng mL1 1.0–25 ng mL1 2.5–100 ng mL1 30–700 ng mL1 –

Human IgG Estriol 17 beta-estradiol Biotin D-phenylalanine

Fluorometry Chemiluminescence Chemiluminescence Bioluminescence Chemiluminescence

0.3–7.0 mg L1 10.0–400 ng mL1 10.0–1,000 ng mL1 1 1011–1 103 mol L1 –

HSA Vitellogenin Vitellogenin Vitellogenin Hyaluronan Neurotoxin GAD65 autoantibodies

Quartz crystal microbalance Spectrophotometry Chemiluminescence Amperometry Amperometry Fluorometry Spectrophotometry

0.01–05 mg mL1 7.8–125 ng mL1 2–100 ng mL1 0–500 g mL1 1–5,000 ng mL1 – –

2 ng mL1 – 2.7 ng mL1 0.5 ng mL1 1.0 ng L1 0.22 ng mL1 0.1 ng mL1 0.5 ng mL1 1.0 ng mL1 3 ng mL1 0.02 mg mL1 (mouse IgG) 0.2 mg mL1 (rabbit IgG) 0.1 mg L1 5.0 ng mL1 3.0 ng mL1 50 pg 1.01 ng mL1 (FIA mode) 0.29 ng mL1 (SIA mode) 0.01 mg mL1 5 ng mL1 2 ng mL1 2–3 ng mL1 1 ng mL1 1.4 pg mL1 20 ng mL1

[68] – [76] [77] [69] [70] [74] [79] [84] [72] [73] – [92] [75] [78] [80] [81 – [82] [85] [86] [87,88] [90] [91] [93]

antigen antigen antigen antigen


Analyte


581

5. ENZYMATIC ASSAYS On-line enzymatic reactions taking place in flow analysis systems have been widely employed in order to facilitate routine biochemical analyses and applications in biocatalysis. FIA and SIA enzymatic assay approaches are excellent choices for bioassays because of the unique characteristics of the flow systems including low sample and reagent consumption and thus reduced analysis costs, ease of operation, as well, as fast analysis.

5.1 Heterogeneous enzymatic assays based on microreactors Most of the flow immunoassay systems use enzymes immobilized on microbeads or on the interior surface of microfluidic channels, whilst some employ dissolved enzymes in order to perform reactions in microfluidic systems. Flowbased analytical systems incorporating microreactors are generally characterized by outstanding repeatability and reproducibility, which can be attributed mainly to the elimination of the drawbacks of iterative batch mode operations. The characteristic features of FIA and SIA systems fit perfectly with the applications of enzymatic microreactors as described in a recent review article [94]. The most commonly used enzymatic microreactors prepared by the immobilization of an appropriate enzyme onto a suitable supporting material are used for the direct determination of biomolecules that act as substrates in enzymatic reactions. A large variety of detection techniques can be employed for monitoring the enzymatic reactions, e.g., amperometry, chemiluminescence and spectrophotometry. An FIA method for the determination of serine, using a mini-column containing immobilized serine dehydratase isolated and purified from rat liver, has been developed [95]. Ammonia produced from the enzymatic reaction was reacted with hypochlorite and phenol in alkaline medium yielding the blue indophenol anion, which was detected spectrophotometrically at 640 nm. A limit of detection of 0.01 mM along with a sample throughput of 25 h1 was achieved. The usefulness of enzyme microreactors for the determination of very low concentrations of amino acids has also been demonstrated recently with the determination of cysteine [96] and L-aspartate [97]. For the determination of cysteine, a rotating biosensor and stopped-flow technique were adopted in order to improve the detection sensitivity [96]. Nanjo et al. [48] described an enzymatic FIA method for rapid measurement of hemoglobin A(1c) (HbA1c). The FIA system was comprised of an electrochemical detector with a specific enzyme-reactor, i.e., a fructosyl-peptide oxidase (FPOX-CET) reactor, and a flow-through spectrophotometer for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cells. First, total hemoglobin was determined spectrophotometrically in digested samples and then the fructosyl valyl histidine (FVH) released from glycohemoglobin by the selective proteolysis was selectively determined using the electrochemical detector with the FPOX-CET reactor. This FIA

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system was automatically run at a sampling rate of 40 h1. The enzymatically determined HbA1c values deduced from the concentration ratio of FVH and total hemoglobin were closely correlated with the HbA1c values certified by the Japan Diabetic Society and the International Federation of Clinical Chemistry (IFCC). A miniaturized biosensor has been developed by Dutra et al. [98] for the determination of uric acid in biological fluids. The amperometric biosensor was prepared by using a carbon paste electrode modified with uricase from Arthrobacter globiforms and tetracyanoquinodimethane as the electron transfer mediator. When incorporated into an FIA system, it allowed 50 measurements per hour for uric acid in a range of 1–100 mmol L1 with a precision of 0.20% RSD. Among the various procedures for glucose determination, those based on enzymatic reactions have been widely employed. Generally, these procedures are based on the generation of hydrogen peroxide during glucose oxidation by glucose oxidase (GOD), which usually takes place in the immobilized microractorer incorporated into the flow manifold. The hydrogen peroxide thus generated can readily be detected by various techniques. An SIA renewable surface reflectance spectrophotometric system for the enzymatic determination of glucose in human serum samples has been developed by Wang et al. [99,100]. A built chip-based flow-through cell was used to trap the microbeads, which could readily be renewed for each analytical cycle. The analytical results agreed well with those obtained by the phenol-4aminoantipyrine method. An automatic flow procedure for the determination of glucose in animal blood serum using glucose oxidase with chemiluminescence detection and based on multicommutation was described by Pires et al. [101]. The flow manifold consisted of a set of three-way solenoid valves assembled to implement multicommutation. Glucose oxidase was immobilized on porous silica beads and packed in a minicolumn. The procedure was based on the enzymatic degradation of glucose, producing hydrogen peroxide, which oxidized luminol in the presence of hexacyanoferrate(III) and giving rise to chemiluminescence. The results were in agreement with those obtained by the conventional method (LABTEST Kit) at the 95% confidence level. Chen et al. [102] developed an amperometric FIA biosensor system for glucose assay where the biosensor consisted of a chitosan membrane from the carapace of the soldier crab where glucose oxidase was immobilized. The sensor signal was linearly related to glucose concentration with good sensitivity and reproducibility. A three-layer polydimethylsiloxane/glass microfluidic SIA system with stationary phase particles immobilized on one side of the channel wall was developed by Xu and Fang [103] for the chemiluminescence detection of glucose. A conventional SIA system was coupled directly to the microfluidic system. Hydrostatic delivery of the reagents was used to achieve efficient and reproducible sample introduction at 10 mL level. A detection limit of 10 mmol L1 glucose was obtained.


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5.2 Homogeneous enzymatic assays The capabilities of flow-based techniques for manipulating very small volumes of expensive enzymes also offers certain advantages in the use of soluble, rather than immobilized, enzymes in enzymatic assay systems. This approach facilitates the elimination or alleviation of typical drawbacks associated with the commonly used packed-bed or open tubular column enzyme reactors, such as fouling of the surface of the packed material, carryover effects, flow resistance, loss of binding sites or functional groups and the employment of harmful organic solvents during the immobilization process. A multisyringe flow injection analysis (MSFIA) manifold has been developed as a powerful tool for performing automated enzymatic assays in a renewable format using soluble enzymes [104]. The flow manifold is shown schematically in Figure 5. The MSFIA system involved four glass syringes connected in a block to Waste SV6

Off Autosampler On

HC

Off

SV5

On

KR On

RL W

SV1 SV2 SV3 SV4

PSM

Cobalt

Carrier

S1 S2

Luminol / NaOH

Enzyme

Off

S4

S3

Figure 5 Schematic diagram of the multisyringe FIA setup assembled for chemiluminescence determination of glucose at ultra-trace levels using soluble enzymes (S1–S4, syringe pumps; SV1–SV6, three-way solenoid valves; HC, holding coil; KR, knotted reactor; RL, reactor line; PSM, photosensor module; and W, waste). Reprinted from [104]. Copyright (2004), with permission from the American Chemical Society.

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the same step-by-step motor and coupled to three-way solenoid valves. This flow-based configuration was suitable for handling minute volumes of soluble enzymes. It also accommodated reactions with divergent kinetic and pH demands, which were used in the indirect chemiluminescence assay of glucose at ultra-trace levels. The procedure involved on-line glucose oxidase-catalyzed oxidation of beta-glucose in a homogeneous phase to beta-glucono-delta-lactone and hydrogen peroxide. Subsequently, the generated oxidant merged downstream with an alkaline zone of 3-aminopthalhydrazide and a metal-catalyst zone of Co(II) at a high flow rate aiming at warranting maximum light collection from the fast chemiluminescence reaction. For the enzymatic assay of glucose under optimal conditions, a sample throughput of 20 h1 and a detection limit of 72 mg L1 were obtained. The advent of LOV resulted in the construction of miniaturized SIA systems by integrating the sampling conduit and flow cell into a microfabricated compact structure mounted atop a multi-position selection valve. A higher sampling frequency is usually obtained in the LOV protocol since the sampling line present in conventional SIA systems has been eliminated through integration and miniaturization. This has been well demonstrated on enzymatic assays of glucose and ethanol [105]. Sampling frequency could be further increased by processing two sample injections simultaneously and by optimizing the assay protocol through flow acceleration in the LOV system [106], which is achieved by isolating the flow cell from the rest of the LOV system by turning the groove of the multiposition valve away from the flow cell port. Thus, the sample, reagent, and spacer of run #2 can be stacked into the holding coil, while the reacting mixture from run #1 is being monitored in the flow cell, as illustrated in Figure 6. The sampling frequency of this accelerated protocol is comparable to that of FIA and

Loading run #2 RUN #2

(a) HC

C S

RUN #1 Sp

R

(b)

RUN #2

HC

C

P

RUN #1 S

P R

Sp

P

Sending run #2 to flow cell

Figure 6 Accelerated mSI-LOV protocol (C, Carrier; S, sample; R, reagent; Sp, spacer; P, product; HC, holding coil; P, flow cell). (a) Stacking sample, reagent and spacer of run #2 into the holding coil during the stopped-flow measurement of run #1. (b) Sending the stacked zones of run #2 to the flow cell, to wash out the flow cell and start the measurement of run #2. Note that the extended volume of the spacer prevents intermixing of the sample/reagent zones of run #1 and run #2, and assists in washing out the flow cell. Reprinted from [106]. Copyright (2004), with permission from the Royal Society of Chemistry.


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the analysis time is reduced from 200 s required for traditional SIA format to about 30 s in the LOV protocol. By attaching a micro-reactor to one port of the LOV system to enhance mixing, enzyme kinetics and inhibition were investigated by the same research group using acetylcholinesterase (AChE) and antiotensin-converting enzyme (ACE) as model analytes [107]. The Michaelis constant (Km) for AChE and ACE obtained by this system agreed well with those reported in the literature. Ohgami et al. [108] proposed a microfluidic system for the analysis of the activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT). The system consisted of a glass chip with a microelectrochemical L-glutamate sensor and a polydimethylsiloxane sheet with a Y-shaped micro-flow channel. Sample solution and substrate solution for the enzymes were introduced from two injection ports at the end of the flow channel and then mixed immediately by diffusion in the mixing channel. The enzyme activities were measured rapidly without any other reagents. The relationship between the slope of the response curve and the enzyme activity was linear in the range 7–228 U L1 for GOT and 9–250 U L1 for GPT, respectively.

6. CELLULAR ANALYSIS Recently, Ruzicka and coworkers have focused their research on flow-based renewable surface techniques for cell-based assays by immobilizing the cells of interest onto the surface of an appropriate bead material [109–112]. The real-time renewal of the cellular materials is usually performed by employing the flow design of a jet ring cell. The rapid renewal of the beads allows the refreshment of the inspected cells and thus each assay can be conducted on a fresh set of cells. This makes in situ acquisition of information concerning the cellular activities feasible. In order to investigate the real-time cellular glucose consumption, which could be used to ascertain the effects of a hypoxic event in cells, Schulz and Ruzicka [113] developed a micro-SIA–LOV (mSIA–LOV) system with an integrated microbioreactor for real-time in situ determination of glucose consumption by live cells. The adherent cells were cultured onto microcarrier beads and packed into a renewable microcolumn within the mSIA–LOV system. Glucose sensing was performed through the use of a two-step nicotinamide adenine dinucleotide (NAD)-linked enzymatic process. The course of the assay was monitored in real-time by measuring the absorbance of NADH at 340 nm. This microsequential assay based on plug/nozzle design had a linear dynamic range for glucose of 0.1–5.6 mmol L1. The mSIA–LOV system allowed the assay to be carried out using only 40 mL of the enzyme reagent and 3 mL of sample. The technique was tested on a murine hepatocyte cell line (TABX2S) adhered to Cytopores beads. Rapid cellular glucose consumption was facilitated by a high cell density, which allowed a large number of cells (104–105) to be retained in a very small volume of 3 mL. In turn, this cell density resulted in the rapid depletion of glucose from the cell medium over a short time period of less than 2 min. Based on the same principle, cellular lactate extrusion rate was also

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determined by the same research group using a similar mSIA–LOV setup. A linear dynamic range 0.05–1.00 mmol L1 was achieved and the measurement could be conducted within 30 s [114]. Extracellular acidification rate (ECAR) is a key parameter for cell activity used for the evaluation of the factors that alter metabolic functions, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. By coupling the BIA technique, an exploratory study on ECAR measurement has been carried out by Erxleben et al. [115] in an LOV manifold. Two kinds of beads with different design were used in this investigation, Cytopores beads were used for cell culturing and trapped in the central channel of the LOV system, while Sephadexs beads were employed for covalently binding the indicator and they were retained in the flow cell. Hydrogen ions extruded from the cells were accumulated during a stopped-flow period and then detected by the change in absorbance of the pH indicator solution. The feasibility of this approach was demonstrated by measuring ECARs of the mouse hepatocyte cell line of TABX2S and the results agreed well with those obtained by using the Cytosensor system.

7. PERSPECTIVES The three generations of flow analysis techniques, i.e., FIA, SIA and LOV, have been well accepted as indispensable tools in the automation of analytical procedures, offering at the same time low sample and reagent consumption and high sampling frequency. Their potentials have been extensively exploited during the last decades, yet these techniques are far from being fully exploited so far. Although only very limited investigations have been directed towards flowbased cellular analysis, the integration of microbioreactors with live cells into flow systems has been demonstrated to be a very promising approach in this field. This not only provides an alternative for in situ investigation of cell activities, but also offers an attractive approach to adopting biological cells as the functional material in SPE for sample pretreatment. FIA and SIA have been proven to be powerful analytical tools for on-line sample pretreatment. The introduction of LOV has further enhanced the capabilities of flow-based techniques for clean-up of micro samples, which is of high importance in the analysis of biological samples. The development of labon-chip or micro-total analysis systems (mTAS) have attracted extensive attention. However, there is 3–6 orders of magnitude difference between the volumes used in mTAS and in conventional sample pretreatment systems and this has lead to the so-called ‘‘world-to-chip’’ interfacing problem, which has plagued the further development of the mTAS systems. The solution of this problem arising from the mismatch of the processed volume scales between the ‘‘world’’ and the chip has remained a challenge. At this point, LOV has provided a promising sample processing front end for mTAS systems. The assay of macrobiomolecules, such as DNA, peptides and proteins, in complex sample matrices has gained increasing interest. Thus, the potential of


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the on-line sample pretreatment capabilities of flow systems when coupled to BIA for matrix removal and analyte preconcentration or even for chromatographic separation by selecting appropriate sorbent materials and suitable eluents is large. This field obviously deserves further investigation.

ABBREVIATIONS m-AC ACE AchE AP b-HABPs m-BIAS CE CEA COMT CPE DSTD ECAR ELISA ESI-FAIMS FIA-IA FITC FPOX FVH GCE GOD GOT GPT HA HAS IFCC Km LIF PAP PAPP PCR RLS RSC S/N SAM SPE Vg

Micro-affinity chromatography Antiotensin-converting enzyme Acetylcholinesterase Alkaline phosphatase biotinylated-HA-binding proteins Micro-bead injection analysis spectroscopy Capillary electrophoresis Carcinoembryonic antigen Catechol-O-methyltransferase Conducting polymer entrapment Dynamic surface tension detection unit Extracellular acidification Enzyme-linked immunosorbent assays Electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry Flow injection analysis immunoassay Fluorescein isothiocyanate Fructosyl-peptide oxidase Fructosyl valyl histidine Glassy carbon electrode Glucose oxidase Glutamic-oxaloacetic transaminase Glutamic-pyruvic transaminase Hyaluronan Human serum albumin International Federation of Clinical Chemistry Michaelis constant Laser-induced fluorescence p-aminophenol p-aminophenyl phosphate Polymerase chain reactions Resonance light scattering. Renewable separation column signal-to-noise ratio Self-assembled monolayer Solid phase extraction Vitellogenin

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REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

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CHAPT ER

20 Pharmaceutical Applications Miroslav Pola´ˇsek

Contents

1. Introduction 2. Automated Analytical Flow Methods in Pharmaceutical Research 2.1 Pilot screening tests in the discovery of drugs of natural origin 2.2 Screening tests for drug–bioligand interactions 2.3 Study of drug–protein binding 3. Automated Analytical Flow Methods in Pharmaceutical Production and Drug Quality Control 3.1 Process monitoring during drug production 3.2 Assay of pharmaceutical formulations for the content of their active components 3.3 Automated drug dissolution, release and permeation tests Abbreviations References

591 594 594 595 596 599 599 600 610 613 613

1. INTRODUCTION Patients should have a high degree of confidence in the quality and effectiveness of their prescription medicines. However, every medicine carries possible benefits and risks. To ensure that the benefits and risks are balanced in favour of the patient, monitoring of drug effectiveness and safety must be comprehensive and incessant. Therefore, nowadays regulations concerning the quality of pharmaceuticals in a broader sense (covering all steps from drug discovery to the pharmacy shelf), as defined by authorized national institutions (such as the US Food and Drug Administration, FDA), are becoming stricter. As a result, the introduction of a new medicine to global markets is a rather costly and lengthy process (Figure 1). Practically no stages of the development of a new medicine

Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00620-X


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Miroslav Pola´sˇek

LARGE SCALE SCREENING EXPERIMENTS PRECLINICAL TRIALS

May take 1 year or more Laboratory experiments to determine if the compound selected is a prospective drug; takes 3-4 years; no patient involvement

CLINICAL TRIALS PHASE I PHASE II PHASE III

NEW DRUG APPLICATION TO NATIONAL REGULATORY AUTHORITY (NRA)

Takes 1 year; 20 – 100 human volunteers Takes 2 years; 100 – 500 human volunteers Takes 3 years; 1000 – 5000 human volunteers

Takes 1 year

NRA REVIEW & DECISION

Examines safety and efficacy data, may request additional studies Takes 2 – 3 years

MEDICINE AVAILABLE TO PATIENTS

If the drug is approved and registered

Figure 1 Simplified time line of a typical process involving the development of a new drug.

would do without selective, efficient, fast and reproducible analytical methods that help to make the drug development procedures more effective. Analytical methods play an important role in areas such as: (a) high-throughput screening and drug synthesis technologies; (b) bulk drug characterization (e.g., evaluation of acid–base properties, solubility and interaction with proteins); (c) acute and subchronic toxicity testing; (d) pharmaceutical formulation development; (e) dosage form stability testing; (f) pharmacokinetic and pharmacodynamic studies on drug absorption, distribution, metabolism and excretion which are based on the detection and determination of drugs in human body fluids; (g) identification of metabolites formed by chemical transformations of the parent drug molecules and (h) bioavailability/bioequivalence studies. Methods of pharmaceutical analysis are also necessary to control and ensure that all pharmaceutical formulations appearing on the market contain the correct amount of active compounds and that the content of specified impurities is within the acceptable limits (Figure 2). Nowadays pharmaceutical analysis relies generally on chromatographic separation methods but the assay of pharmaceuticals also requires the use of

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Pharmaceutical Applications

FIA, SIA, MCFIA

FIA, SIA, MCFIA

large scale drug production screening preclinical trials optimum drug candidate selected and isolated or small-scale synthesized

drug content assay content uniformity test dissolution rate study

FIA, SIA, MCFIA impurities

formulation development

drug T A B L E T

BULK DRUG

excipients

assay of the drug and impurities

SIC, FIA, SIA, MCFIA

impurities stability tests



clinical trials

SIC ?

Figure 2 Possible applications of analytical flow methods (FIA, SIA, SIC and MCFIA) in the development of a new drug and in quality/safety assurance (FIA: Flow Injection Analysis; SIA: Sequential Injection Analysis; SIC: Sequential Injection Chromatography; MCFIA: Multicommuted Flow Injection Analysis).

reagent-based techniques, rapid automated sample processing and solution handling. Automation and miniaturization of solution-based assays, as offered by flow injection analysis (FIA), sequential injection analysis (SIA), SIA-lab-onvalve (LOV) and other recently introduced analytical flow methods (e.g., sequential injection chromatography (SIC), multicommuted flow injection analysis (MCFIA) and multisyringe flow injection analysis (MSFIA)), have had a positive impact on performing many routine and research-related analyses in pharmaceutical laboratories. The first publications dealing with applications of FIA and SIA to the analysis of drugs and other biologically active substances emerged soon after the inception of these two flow technologies in 1975 and 1990, respectively. A relatively large number of mainly FIA procedures for automated assay of drugs have been successfully devised by adapting existing batch methods. Those procedures developed until 1996 were summarized in a specialized monograph on the use of FIA in pharmaceutical practice by Calatayud [1]. A short chapter devoted to pharmaceutical uses of FIA was included in a monograph by Trojanowicz [2]

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published in 2000. Several review articles that focused on FIA/SIA-based pharmaceutical analysis issues appeared in analytical journals between 2001 and 2007. Solich et al. presented an overview of FIA applications in pharmaceutical analysis published in two parts. Part 1 covered methods utilizing spectrophotometric and chemiluminescence (CL) detection [3] while Part 2 surveyed FIA methods based on other spectroscopic detection techniques and electrochemical detection [4]. Pharmaceutical implications of the introduction of miniaturized SIA-LOV and the new emerging technology of SIC were discussed in another review article by Solich et al. [5], published in 2003. Recently Pimenta et al. [6] have compiled an excellent and comprehensive review of pharmaceutical uses of SIA, including automated process analysis, drug dissolution and drug release testing. A recently published review on electrochemical detection in SIA [7] covers the detection of a number of common pharmaceuticals. Applications of FIA spectrophotometry to pharmaceutical analysis were summarized by Tzanavaras and Themelis [8]. These authors focused their review on homogeneous and heterogeneous FIA systems and FIA procedures involving automated sample pretreatment by liquid–liquid extraction, solid-phase extraction and on-line digestion or photolysis. CL applications of FIA and SIA in pharmaceutical analysis published between 2001 and 2006 were discussed in a review published in 2007 by Mervartova et al. [9]. Special emphasis was paid to the analytical figures of merit of the 211 methods reviewed and the corresponding sample matrix characteristics. This chapter intends to summarize various applications of automated analytical flow methods at different stages of the drug development process and in quality control of pharmaceutical products (dosage forms).

2. AUTOMATED ANALYTICAL FLOW METHODS IN PHARMACEUTICAL RESEARCH 2.1 Pilot screening tests in the discovery of drugs of natural origin The main goal of preliminary stage large-scale screening tests for discovery of drugs of natural origin is to find sources of compounds with desired biological activities (prospective drugs or lead structures). The candidate drugs can be found usually in wild or cultivated plants. For each plant species investigated extracts are prepared from various plant organs with solvents of different polarity and the individual extracts are analysed to find those possessing specific chemical activity or desired biological activity. For economical reasons only a limited number of promising extracts can be subjected to further detailed studies involving separation and identification of bioactive compounds by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, their isolation from the extract and subsequent activity screening at cellular or molecular biology level. The selection of suitable preliminary screening methods depends largely on the expected effects of the potential drug (e.g. anticancer, hypoglycaemic, hypotensive, antioxidation). The primary analytical challenge


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encountered with such studies is to select a method capable of analysing rapidly large series of discrete samples. In addition to high-throughput, the analytical method used must also be highly reliable. It should not fail to identify good drug candidates or produce false positive results. SIA methods based on proper modification of known batch methods can fulfill the requirements mentioned earlier. Thus, an automated SIA system equipped with a spectrophotometric diode-array detector was devised for rapid monitoring and evaluation of antioxidation and radical scavenging activity of plant materials [10]. This SIA method was based on the known reaction of the stable 2,2u-diphenyl-1picrylhydrazyl radical (DPPH) with antioxidants in organic or aqueous-organic media resulting in bleaching of DPPH. This reaction was suitable for performing routine screening tests for the presence of various antioxidants in a large series of lyophilized herbal or mushroom extracts. The sample throughput was 45 h1 and a substantial reduction of the consumption of organic solvents was achieved compared to the corresponding batch procedure. Later on Lima et al. [11] developed a similar spectrophotometric SIA system for measuring total antioxidant activity of beverages and foods. The reagent used was a coloured cationic radical generated by off-line oxidation of 2,2u-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) and a single analysis required about 5 min. Magalhaes et al. [12,13] utilized MSFIA with the DPPH reagent and spectrophotometric detection to automate the determination of total antioxidation capacity of selected food products with a sample throughput of 14 h1. Recently, Miyamoto et al. [14] have developed a combined FIA-SIA method with CL detection allowing to differentiate between antioxidation activity of analytes for the free radicals O 2 and NO, generated on-line. The quenching effect of the antioxidants on the O 2 and NO-induced CL of luminol was monitored and a single assay took 2 min. In principle, an FIA-SIA approach with appropriate chemistry and detection technique could be applied to the simultaneous examination of other chemical or biological effects.

2.2 Screening tests for drug–bioligand interactions In pharmacological screening the drug candidates can be identified and characterized at cellular or molecular biology level by biological tests called ‘‘functional assays’’. Functional assays permit to recognize if a potential drug inhibits or elicits certain biological response through interactions with living cell receptor sites. Normally such stimulus (dose)-response assays are conducted in manual batch arrangement which may cause poor repeatability of the experimental data collected (dose-response curves). This drawback was overcome by automation of the fluid/cell handling utilizing flow analysis techniques [15,16]. The conventional batch experiments are normally performed by repeated exposure of a single set of live cells or tissues to the drug examined. However, this approach may result in desensitization or even degradation of the biological material. Such a problem, inherent to conventional functional assays, was successfully solved by devising an automated flow analysis concept of renewable surfaces represented by live cells immobilized on the surface of

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micro-beads that could be reproducibly captured in the flow cell and subsequently removed from the system and replaced by a fresh portion after recording the dose-response curve [17–19]. Caroll et al. [20] devised a micro-scale label-dilution method for bioligand interaction studies using bead injection with SIA in lab-on-valve format (SIA-LOV) and spectrophotometric detection. The method discriminated between selective and non-selective binding and allowed real time monitoring of bioligand interactions, involving interactions between autoantibodies and their target molecules related to the research of diabetes. Later on Grate et al. [21] developed a flow technique for automated capture and release of colour-encoded micro-beads in a fluidic system. This approach used coupling of renewable surface methods with flow cytometry detectors for suspension array multiplexed analyses. This analysis involved a two-step sandwich immunoassay and a one-step DNA-binding assay. Automated SIALOV with bead injection and spectrophotometric detection was employed for monitoring extracellular acidification rates [22]. Such assays are used conventionally for the evaluation of factors that influence metabolic functions of cells caused by interactions with potential drugs or toxins. In this study micro-beads with captured live mouse hepatocyte cells and Sephadexs micro-beads with immobilized coloured pH indicators were transported and perfused directly in the SIA-LOV micro-fluidic system. The pH changes were detected by recording the changes of absorbance of the micro-beads with the immobilized pH indicators. Molecules taking part in intercellular communications play an important role as targets in new drug discoveries. Recently, an automated FIA system with on-line solid-phase extraction and simultaneous fluorescence and mass spectrometric detection was used to study the interactions of G proteincoupled receptors (e.g., Histamine H2-receptor) with inhibitors (e.g., fluorescentlabeled receptor ligands) as potential drugs [23]. The system allowed sensitivity down to 5 fmol of the ligands studied.

2.3 Study of drug–protein binding After administration, most drugs enter the blood stream where they are transported partly in a free (unbound) form and partly reversibly bounded to various blood components such as plasma proteins (albumin, globulins, lipoproteins and glycoproteins) and blood cells. Generally, the pharmacological effect depends on the intensity and kinetics of the drug–protein binding phenomena since just the unbound drug can reach the target organ or tissue and properly interact with the specific sites of the receptors targeted. Hence the plasma protein equilibrium binding constant is an important factor in characterizing the pharmacokinetic and pharmacodynamic properties of a drug. The actual concentrations of the unbound and bound drug molecules are also influenced by the drug metabolism (e.g., hepatic clearance, enzymatic hydrolysis, etc.) and biomembrane permeation rates. Hence the study of drug–protein interactions and evaluation of the corresponding binding parameters under equilibrium and non-equilibrium conditions, both in vitro and in vivo, is a crucial step in the preclinical and clinical trials within a new drug development scheme. A number of

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methods have been devised to study drug–protein interactions. Since the drug– protein binding is a reversible and relatively rapid process, the analytical procedure used for its evaluation should not disturb the established native binding equilibrium. Currently two analytical procedures, satisfying this requirement, are frequently used. One of them involves HPLC determination of the free drug after its physical separation from the protein-bound fraction by equilibrium dialysis, ultrafiltration, microdialysis, gel filtration, or solid-phase microextraction. The other procedure is based on the direct determination of the concentration of the free drug without a separation step by a detection method selective to the free drug (e.g., spectrophotometry, fluorometry, amperometry). The utility of combining FIA sample handling with on-line microdialysis for the study of drug–protein binding was demonstrated in several studies [24–27]. Shi et al. [24] used a microdialysis probe integrated in a FIA manifold to determine amperometrically the concentration of streptomycin not bound to bovine serum albumin (BSA) at pH 13. The data obtained were used for the determination of the streptomycin-BSA association constant and the number of binding sites. The same microdialysis-FIA setup, but with CL detection based on enhancement of the luminol-K3[Fe(CN)6] CL by the free drug, was utilized by Huang et al. to study the streptomycin-BSA binding equilibrium [25] and for determining the association constant of tetracycline with BSA [27]. Wang et al. employed a microdialysis-FIA system with CL detection of terbutaline, based on its CL reaction with permanganate and formaldehyde, to study the protein binding with BSA [26]. A typical microdialysis-FIA-CL setup is shown in Figure 3. The studies mentioned earlier have demonstrated that the microdialysis-FIA measurements do not disturb the drug binding equilibrium because just a negligible amount of the free drug is removed from the reaction

SP

Perfusate

15 cm P V

KH-1 R1

HV F PMT

BPCL

R2

MP CS501-3C

R3 1

2

Waste

Figure 3 Schematic diagram of a FIA setup for studying the drug/protein binding using CL detection with on-line microdialysis sampling (1: stirrer; 2: terbutaline sulfate/BSA mixture solution; SP: microdialysis syringe pump; MP: microdialysis probe; R1: water carrier; R2: KMnO4 solution (in H2SO4); R3: HCHO solution; P: peristaltic pump; V: injection valve; F: flow cell; KH1: Model KH-1 syringe micropump controller system; CS501-3C: CS501-3C super thermostat water bath controller; PMT: photomultiplier tube; HV: negative high-voltage supply; BPCL: computer controlled luminescence analyser. Reprinted from Ref. [26]. Copyright (2003), with permission from Elsevier.

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medium by sampling of the perfusate while the volume of the reaction mixture and hence the concentration of the protein remains unchanged. An FIA manifold coupled with an ultrafiltration unit was used recently for the study of the antiviral drug dehydroandrographolide succinate (DS)-BSA binding [28]. The concentration of the unbound DS in the ultrafiltrate liquid was determined by FIA-CL based on the oxidation of DS by [Fe(CN)6]3 in the presence of Rhodamine B as the CL enhancer. This allowed the calculation of the corresponding binding parameters. Recently Zacharis et al. published two papers reporting on drug–protein binding studies with the use of monolithic separation columns [29,30] integrated in an automated SIA setup equipped with UV and fluorescence detectors (Figure 4). A similar SIA system [28] was used for determining the binding rate constant of ciprofloxacin and BSA. These two compounds were incubated, unbound ciprofloxacin and the drug–protein complex were separated on the monolithic anion exchange column and determined by UV spectrophotometry and/or fluorometry. An SIA system incorporating a monolithic epoxy disk with immobilized BSA [30] was employed to study the naproxen-BSA binding. In this SIA system naproxen solution was aspirated towards the monolithic disc where the drug interacted with the immobilized BSA; the proteinbound fraction of the drug was retained on the monolithic disk while free naproxen was eluted and monitored by its intrinsic fluorescence. The binding constants and the number of binding sites, as well as other kinetic and thermodynamic parameters, including those characterizing the competition

CF

BSA

CIM RC

λmax=280nm

FL λoat=300nm λem=460nm Waste

HC Carrier

UV

SV NaCl AW

SP

Figure 4 Schematic diagram of the SIA-convective interaction media (CIM) instrumentation (Carrier: phosphate-buffered saline solution; SP: syringe pump (V ¼ 10 mL); HC: holding coil (130 cm/0.75 mm i.d.); SV: selection valve; CF: ciprofloxacin solution; BSA: bovine serum albumin solution; AW: auxiliary waste; RC: reaction coil (60 cm/0.75 mm i.d.), maintained at 371C; CIM: housing with two monolithic quaternary amine disks; UV: UV detector operating at 280 nm; FL: fluorescence detector (lexitation ¼ 300 nm/lemission ¼ 460 nm). Reprinted from Ref. [29]. Copyright (2006), with permission from Elsevier.


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between naproxen and alendronate in the BSA-naproxen-alendronate system, were evaluated by mathematical processing of the breakthrough curves (i.e., intensity of fluorescence vs. elution time). A different approach to the methodology of drug–protein binding experiments was proposed by Liu et al. [31]. An FIA system with integrated quartz crystal microbalance biosensor was used for real-time investigation of the molecular interaction between selected proteins and small drug molecules (i.e. sulfamethazine or sulfamethoxazole) immobilized on the gold electrodes of the piezoelectric crystals. The binding interactions of these two immobilized drugs with various proteins were monitored in solution by measuring changes in the resonance frequency of the corresponding modified crystal sensor of the microbalance (sensorgrams). The sulfamethazine-modified sensor showed specific interactions with Immunoglobulin G (IgG) only while the sulfamethoxazolemodified sensor exhibited considerable binding with trypsin and chymotrypsin but minute specific binding with IgG. Additional studies of the specific interactions between immobilized sulfamethazine and human IgG, goat IgG and mouse IgG were carried out and significant species-dependent differences were observed. The corresponding kinetic rate constants and equilibrium association constant for the sulfamethazine–IgG interactions were determined. A good example of expedient application of SIA in the study of biomembrane permeation rates was presented by Sklena´rˇova´ et al. [32]. The SIA technique was applied to automated pharmacokinetic study of the transporter-mediated passage of a model substrate (Rhodamine 123) through dually perfused rat placenta. The method was based on real-time fluorometric monitoring of Rhodamine 123 concentration in both the maternal and fetal compartments. The samples of perfusate were aspirated into the SIA system in 3-min intervals for 36 min and the effect of several inhibitors (e.g., azide, quinidine) of the P-glycoprotein transporter on the transport profiles of Rhodamine 123 was examined.

3. AUTOMATED ANALYTICAL FLOW METHODS IN PHARMACEUTICAL PRODUCTION AND DRUG QUALITY CONTROL 3.1 Process monitoring during drug production A comprehensive review on Process Analytical Technology (PAT) involving automation of biotechnological drug manufacturing, particularly fermentation in pharmaceutical industry, was published recently [33]. Actual PAT implementations involving feedback-controlled fermentation processes for biopharmaceutical production are used predominantly in the manufacturing of small molecule antibiotics rather than in large molecule (protein) fermentation processes. PAT can be conducted: (a) off-line — the analysis utilizing discrete samples is carried out in a laboratory away from the production process; (b) at-line — the analysis of discrete samples is carried out next to the production process; (c) on-line/in-line — continuous analysis takes place in a side stream

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and (d) in situ — continuous analysis takes place directly in the reaction vessel [33]. Analysis itself can be discrete, i.e., the analyte is measured periodically, or continuous, i.e., the analyte is measured continuously. FIA has become a successful example of an in-line PAT implementation owing to its versatility, possibility for miniaturization and ease of handling of a large number of discrete samples to be analysed in preset time intervals [34]. The potential of FIA in utilizing bioenzymatic analytical microbioreactors for glucose, lactate, ethanol, galactose and L-amino acid monitoring in cell culture media has been demonstrated recently [35]. Minireactors packed with appropriate immobilized enzymes were employed in at-line SIA-CL monitoring of glucose and lactic acid as the parent compounds and penicillin as the final product in the fermentation process of penicillin production [36]. The detection chemistry for glucose and lactic acid was based on the formation of hydrogen peroxide in the reactors with immobilized glucose oxidase and lactate oxidase, respectively and CL detection of hydrogen peroxide by reaction with luminol and K3[Fe(CN)6]. Penicillin was hydrolysed to penicilloic acid in an enzyme reactor packed with immobilized penicillinase and the penicilloic acid was detected through its quenching effect on the CL reaction between luminol and iodine. This method was adapted for the on-line SIA-CL monitoring of penicillin production [37]. Glucose was determined in the same way as in the batch method outlined earlier [36] while penicilloic acid was detected spectrophotometrically after reaction with iodine resulting in decrease of the absorbance of the blue iodine–starch complex. The SIA-CL system was used for on-line monitoring of the fermentation process for more than 2 weeks. The concentration of morphine was controlled by an off-line SIA-CL method in both aqueous and organic extracts of the poppy plant Papaver somniferum during the technological process of morphine isolation [38,39]. The detection chemistry was based on the CL reaction of morphine with potassium permanganate in aqueous sulfuric acid medium containing hexametaphosphate as CL enhancer.

3.2 Assay of pharmaceutical formulations for the content of their active components As mentioned earlier, the attempts to apply automated flow methods to the assay of drugs as bulk substances and/or active components of pharmaceutical formulations are as old as the history of FIA and SIA and the research papers devoted to this field are numerous. This topic has been repeatedly and excessively discussed since the introduction of FIA and SIA in a number of monographs and review articles [1–9]. Despite the proven merits of the flow methods in pharmaceutical analysis, they have not found yet wide acceptance by practicing pharmaceutical analysts because the assay of an active substance in a drug formulation is generally a matter of discrete analysis regulated by official protocols that normally do not require high degree of automation. The potential of flow methods in pharmaceutical analysis will be demonstrated in the subsequent paragraphs where selected recently published FIA, SIA, SIC, MCFIA and MSFIA applications in the analysis of real dosage forms will be discussed. For the sake of clarity, the applications will be arranged according to the flow


601

techniques employed and detection methods utilized, starting with FIA methods. A summary of the drugs assayed is presented in Table 1.

3.2.1 FIA with spectrophotometric detection Conventional UV detection at 292 nm without derivatization was used for the assay of lansoprazole in capsules with 10 mM NaOH as the carrier stream [40]. Similarly, cefuroxime axetil was determined in tablets at 281 nm with methanol-water (1:10, v/v) as the carrier stream [41]. Salbutamol was determined in tablets and oral solutions as a quinoneimine dye at 500 nm after derivatization with 4-aminophenazone and K3[Fe(CN)6] [42]. Al-Abachi et al. [43] determined amoxicillin in capsules and injections. Amoxicillin was derivatized by oxidative coupling with N,N-dimethyl-pphenylenediamine. On-line circulatory regeneration of Fe(III)–1,10-phenathroline complex as a reagent in a spectrophotometric FIA assay of ascorbic acid was employed to reduce the cost of analysis [44]. Peroxydisulfate was used to re-oxidize the Fe(II)–1,10-phenathroline produced. An inclusion complex between phenolphthalein and b-cyclodextrin, which is colourless in alkaline solution, was used as a spectrophotometric reagent for the FIA determination of fluoxetine in tablets [45]. The increase in the absorbance of the displaced phenolphthalein was measured at 554 nm. Spectrophotometric FIA procedure was proposed for the assay of methyldopa in tablets [46]. The method was based on absorbance measurements of the yellow molybdate–methyldopa complex at 410 nm. Penicillamine was assayed in tablets by FIA involving a solid-phase reactor for the in situ production of Co(II) that formed a coloured complex with penicillamine in alkaline medium with an absorption maximum at 360 nm [47]. The reactor was prepared by immobilization of cobalt carbonate on a polymer matrix and Co(II) was released by the passage of a sulfuric acid stream through the reactor. The concept of bead injection spectroscopy was implemented in a FIA system for the sensitive determination of promethazine or trifluoperazine in pharmaceuticals using Fe(III) and ferrozine as reagents [48]. A bead suspension of Sephadexs QAE A-25 resin was initially injected into a commercial flow-through optical cell to form a reusable flow through sensor. The Fe(II)–ferrozine complex was retained on the bead packing where Fe(II) was produced by the oxidation of promethazine or trifluoperazine by Fe(III). After measuring the absorbance of this complex at 567 nm, the beads were discarded and replaced by a fresh bead suspension. Mixtures of caffeine and theophylline were separated directly in a FIA manifold and determined by UV spectroscopy at 272 nm [49]. Initially, these analytes were aspirated into a separation minicolumn packed with C18 silica-gel beads that was linked to a flow-through cell packed with the same beads. Caffeine was retained on the minicolumn while theophylline was eluted by the carrier stream and its transient signal was recorded. Thereafter caffeine was eluted by aqueous 25% methanol and determined spectrophotometrically.

3.2.2 FIA with fluorescence detection Tricyclic antidepressants (e.g., imipramine, desipramine, amitriptyline, nortriptyline, clomipramine or doxepine) were determined in pharmaceutical formulations by an FIA-extraction fluorescence method [50]. The aqueous samples were

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Table 1 Survey of drugs determined by FIA, SIA or MCFIA in dosage forms Method/detection Calibration range


Dosage form

Reference

Acetaminophen Alendroic acid Amikacin Amoxicillin Aluminium Aluminium Azithromycin Amittriptyline Amoxicillin Ascorbic acid Caffeine Caffeine Cefuroxime axetil Chloramphenicol Creatine Dextrose Diphenhydramine L-dopa Dopamine Dopamine Dopamine Drotaverine Epinephrine S-enalapril Fluoxetine Fluvoxamine Gentamicin

FIA/AMP SIA/FL FIA/CL SIA/SP FIA/FL SIA/FL FIA/AMP FIA/FL FIA/SP FIA/SP FIA/SP FIA/MS FIA/SP FIA/AMP FIA/MS MCFIA/SP SIA/SP FIA/CL FIA/FL FIA/CL FIA/CL FIA/POT FIA/CL SIA/AMP FIA/SP FIA/SWADSV FIA/CL

50 30 n/a 25 120 72 n/a 60 120 60 12 30 70 n/a 30 90 5 100 24 135 100 n/a 100 75 80 120 n/a

Tablets Tablets Lyophilized injections Dry injections Tablets, suspensions Tablets, suspensions Tablets Tablets, capsules Capsules, injections Tablets, granules Capsules, tablets, syrup Tablets Tablets Capsules Tablets Haemodialysis solution Injections Injections Injections Injections Injections Tablets, injections Injections Tablets, raw substance Tablets Tablets Ointments

[64] [76] [53] [73] [52] [52] [65] [50] [43] [44] [49] [70] [41] [66] [70] [100] [75] [55] [51] [54] [55] [61] [55] [85] [45] [69] [58]

0.8–500 mM 0.13–10 mg L1 9.9–20 mg L1 10–60 mg L1 0.03–12 mg L1 0.1–4 mg L1 1–10 mg L1 0.25–3 mg L1 10–700 mg/mL 0.2–30 mg L1 1–16 mg L1 5–15 mg mL1 1–6 mM 0.1–20 mM 5–15 mg mL1 0.1–1 g L1 10–40 mg L1 0.6–10 mg L1 0.01–0.1 mM 400–3000 mg L1 0.6–9 mg L1 0.5mM–10 mM 0.5–5 mg L1 0.08–1.5 mM 0.05–10 mM 0.5–50 mM 3.9–30 mg mL1


Analyte

0.08–0.22 mM

70

Drops

[71]

FIA/POT FIA/FL SIA/FL SIA/CL SIA/FL FIA/SP SIA/SP FIA/SP FIA/CL FIA/AMP FIA/SWADSV FIA/SP SIA/POT SIA/AMP FIA/CL FIA/POT FIA/SP SIA/AMP FIA/CL FIA/SP FIA/CL SIA/SP FIA/SP FIA/SP SIA/CL SIA/POT FIA/RS

0.063–10 mM 0.25–3 mg L1 0.016–10 mM 0.1–10 mM 10–125 ng mL1 5.4–54 mM 3–42 mg mL1 50–200 mg L1 1.1–20 mg L1 0.35–100 mM 3–20 mM 5–60 mg L1 0.2–10 mM 0.2–6 mM 0.1–200 mM 0.7–1000 mM 0.5–8 mg mL1 0.12–0.6 mM 7–700 pM 0–74 mg L1 0.5–50 ng mL1 25–300 mg mL1 1–12 mg L1 0.5–10 mg mL1 0.5–100 mg mL1 1–50 mM 0.017–13 mg mL1

n/a 60 30 180 n/a 90 40 210 100 20 120 70 25 75 20 n/a 12 75 n/a 75 90 40 12 12 120 55 80

Tablets, drops Tablets, capsules Tablets, capsules Gels, ointments Tablets Formulations not specified Tablets Tablets Injections Tablets, drops Tablets Tablets Tablets Tablets, raw substance Tablets Tablets Tablets, syrup, cream Tablets, raw substance Tablets, capsules Tablets, oral solutions Tablets Dry injections Capsules, tablets, syrup Tablets, cream, syrup Tablets Tablets, oral solution Tablets

[63] [50] [77] [82] [78] [40] [74] [46] [55] [67] [68] [47] [83] [85] [59] [62] [48] [85] [57] [42] [56] [74] [49] [48] [81] [85] [72]

Notes: AMP, amperometry; CL, chemiluminescence; FL, fluorescence; MS, mass spectrometry; POT, potentiometry; RS, Rayleigh scattering; SP, spectrophotometry; SWADSV, square-wave adsorptive stripping voltammetry and TURB, turbidimetry.

603

FIA/TURB


Homatropine methylbromide Hyoscyamine Imipramine Indomethacin Indomethacin Labetalol Lansoprazole Metoclopramide Methyldopa Norepinephrine Paracetamol Paroxetine Penicillamine Penicillin G S-pentopril Pipedimic acid Piribedil Promethazine S-ramipril Rhein Salbutamol Terbutaline Tetracaine Theophylline Trifluoperazine Trimethoprim Valproate Verapamil

604


injected into a carrier stream containing 9,10-dimethoxyanthracene-2-sulfonate (DMAS) and HCl to form a protonated analyte-DMAS ion pair in the reaction coil. Thereafter, the carrier stream merged with a stream of dichloromethane and the mixture passed first through an extraction coil and then through a phase separator where the dichloromethane extract was directed towards the fluorescence detector. The fluorescence signal was measured at 448 nm (excitation at 265 nm). Seckin [51] devised an indirect FIA-fluorescence method for the determination of dopamine in injections utilizing the quenching effect of dopamine on the fluorescence signal of m-dansylaminophenyl boronic acid. The aluminium content was determined in tablets and pharmaceutical suspensions by an FIA-fluorescence technique based on the formation of Al3+–chromotropic acid complex, soluble in acidic aqueous medium [52]. The fluorescence signal of this complex was measured at 385 nm (excitation at 360 nm) and the sample throughput was 120 h1. An SIA approach employing the same derivatization reaction for Al was also reported by the same authors [52] but the method was less sensitive and the sample throughput was only 72 h1.

3.2.3 FIA with chemiluminescence detection A considerable fraction of the published FIA-CL methods have been used for drug assays [9]. Those methods are based on the inhibition or enhancement effect of the drugs analysed on the analytical CL reaction. For example, the FIA-CL determination of the aminoglycoside antibiotic amikacin in lyophilized injections was based on the fact that this drug inhibits strongly the CL emission of the oxidation of luminol by hydrogen peroxide in alkaline medium in the presence of Cu(II) as catalyst [53]. The inhibition is caused by the fact that amikacin interferes with the catalytic effect of Cu(II) by forming a fairly stable Cu(II)–amikacin complex. The net inhibition of the CL intensity of the luminol–H2O2–Cu(II) system was proportional to the amikacin concentration. Similarly, the inhibition effect of dopamine on the CL emission of the luminol-[Fe(CN)6]3 reaction [54] and the quenching effect of this neurotransmitter and other catecholamines (norepinephrine , epinephrine and L-dopa) on the luminol-iodine reaction [55], were used in the development of FIA-CL assays of these compounds in various dosage forms. However, the strong sensitizing effect of terbutaline on the CL emission of the luminol-permanganate reaction [56] and the intensity enhancement of the CL of the luminol-[Fe(CN)6]3 reaction by rhein [57] were utilized in the ultra-sensitive FIA-CL assay of these analytes. Fernańdez-Ramos et al. [58] proposed an FIA-CL method for the assay of gentamicin in ointments and liquid pharmaceutical formulations. The method was based on the off-line derivatization of gentamicin with o-phthalaldehyde with subsequent enhancement of the CL emission of the relatively complicated peroxyoxalate-hydrogen peroxideimidazole reaction, stabilized by sodium dodecylsulfate. The weak CL of the sulfur dioxide triplet observed when sulfite is oxidized electrochemically at a Pt electrode in sulfuric acid medium has been enhanced considerably in the presence of pipedimic acid. This finding was used in the development of an FIA-electrogenerated CL procedure for determining pipedimic acid in pharmaceutical dosage forms [59]. Recently, the prospects of the tandem UV


605

photodegradation-CL concept in the analysis of drugs that do not exhibit native CL were discussed [60]. Screening tests involving 97 pharmaceuticals were conducted to examine possibilities for their analysis using FIA or SIA with photo-induced CL [60].

3.2.4 FIA with electrochemical detection Almost all types of potentiometric and voltammetric detection techniques have been used in FIA-based drug analysis. Potentiometric FIA assay of drotaverinium chloride in tablets and infusions was performed with a carbon paste ion selective electrode (ISE) [61]. The electrode was based on a mixture of two ion exchangers, namely, drotaverinium-silicotungstate and drotaverinium-tetraphenylborate, dissolved in tricresyl phosphate as the pasting liquid. Piribedil was determined in tablets by FIA-potentiometry with a carbon paste ISE involving piribedil phosphomolybdate as the ion-exchanger dissolved in tricresyl phosphate as a solvent mediator for the paste [62]. Badawy et al. [63] employed an ISE with PVC membrane containing hyoscyamine tetraphenylborate (or phosphotungstate) as the anion-exchange electro-active components for the FIA-potentiometric assay of hyoscyamine in tablets and drops. A carbon film resistor electrode operated at +0.6 V vs. a Ag/AgCl reference electrode was utilized in the FIA-amperometric assay of acetaminophen in tablets [64]. The carbon film electrode provided good selectivity and excellent sensitivity for this analyte in relatively complex commercial drugs without requiring any surface modification and sample pretreatment. An FIA-amperometric method allowing determination of azithromycin in tablets was devised by Palomeque and Ortı´z [65]. The indicator glassy carbon electrode was operated at +0.9 V vs. a Ag/AgCl reference electrode and the transient oxidation current of azithromycin was measured in a carrier stream consisting of the Britton-Robinson buffer solution (pH 8.0). Rapid electrode fouling was overcome by introducing an online clean-up step, involving washing the electrode surface with 2-propanol, in the FIA system. The interference from dissolved oxygen in the FIA-amperometric assay of chloramphenicol was overcome by using preanodized screen-printed ring disk carbon electrode [66]. This allowed separation of the irreversible reduction step of the nitro group of chloramphenicol to the corresponding hydroxylamine from the subsequent reversible oxidation of hydroxylamine to its nitroso derivative. The preanodization treatment helped to lower the overpotential of the electrochemical reaction of chloramphenicol and favoured its selective detection under aerobic conditions. An FIA setup with integrated microfluidic sensor involving a micro-cell modified with horseradish peroxidase was employed for the determination of paracetamol in tablets and drops [67]. Paracetamol was initially oxidized in the enzyme cell by hydrogen peroxide to N-acetyl-p-benzoquinoneimine which was reduced downstream to hydroquinone at a glassy carbon electrode maintained at 0.1 V. Nouws et al. [68] devised an FIA-square-wave adsorptive stripping voltammetric method for the determination of paroxetine in pharmaceuticals. The method was based on the on-line pre-concentration and reduction of paroxetine accumulated at a mercury drop electrode operated at 1.55 V vs. a Ag/AgCl

606


reference electrode in a borate buffer at pH 8.8. Presence of dissolved oxygen did not interfere considerably with the analysis and the sample throughput was 120 h1. A similar method was also developed for the FIA assay of fluvoxamine [69].

3.2.5 FIA with miscellaneous detection methods The concept of FIA with mass spectrometric (MS) detection suitable for drug analysis was tested recently by Wade and Miller [70]. An FIA manifold linked to a MS detector was used for quantitative assay of caffeine and creatine as the active ingredients in tablets. Samples (20 mL) of the dissolved tablets, spiked with internal standards (i.e., 1-phenylalanine for caffeine and guanidineacetic acid for creatine), were transported by the carrier stream of H2O-acetonitrile-formic acid (50:50:0.1) with a flow rate of 0.6 mL min1 to the MS detector. Positive electrospray ionization was used and single ion (M+H)+ monitoring was conducted at m/z 195 for caffeine and m/z 166 for 1-phenylalanine or m/z 132 for creatine and m/z 118 for guanidineacetic acid. The samples were analysed over the analyte concentration range 5–15 mg mL1. Accurate quantitation was achieved by determining the ratio of the analyte response, measured as peak area, vs. the response of the corresponding internal standard. Owing to the outstanding selectivity and low detection limits inherent to MS detection, FIA-MS is very suitable for the analysis of formulations with low content of the active ingredients or of drugs that do not possess distinct chromophores or electrophores. An FIA-turbidimetric procedure exploiting merging zones was devised for determining homatropine methylbromide in liquid pharmaceutical preparations [71]. The determination was based on the precipitation of homatropine methylbromide with silicotungstic acid in aqueous acidic medium. The turbidity measured as the absorbance signal at 410 nm was proportional to the concentration of homatropine methylbromide. Xu et al. [72] employed recently an FIA manifold coupled to a spectrofluorometer, equipped with a fused silica flow cell and operated at the same excitation and emission wavelengths (293 nm), for measuring the transient signal of Rayleigh scattering from the verapamil-tungstophosphoric acid ion-association complex. This complex was formed upon injection of verapamil samples into a carrier stream of pH 1 (HCl) containing 0.1 mM 12-tungstophosphoric acid. The Rayleigh scattering intensity was proportional to the concentration of verapamil.

3.2.6 SIA with spectrophotometric detection The spectrophotometric SIA methods for drug analysis are very similar to the corresponding FIA applications. A certain advantage of SIA is the fact that colour reactions based on more complex derivatization chemistry or including sample pretreatment (such as pre-separation of the analyte or removal of interfering matrices by liquid–liquid or liquid–solid extraction) do not necessitate the construction of complicated multichannel FIA manifolds. Moreover, stoppedflow techniques and procedures based on in situ formation of unstable reagents or pH gradients are easier to implement with SIA rather than with FIA


607

techniques. Thus, an SIA method not requiring analyte derivatization or separation was devised for the assay of amoxicillin in pharmaceuticals in the presence of interferents [73]. The SIA setup with a diode-array spectrophotometric detector and an analysis program sequence, allowing generation of a pH gradient in the flow system, was utilized to obtain the spectra and concentration profiles of the components in the sample. Amoxicillin was resolved and quantitated by chemometric procedures (i.e., multivariate curve resolution with alternating least squares). An illustrative example of a drug assay by SIA based on stopped-flow measurement of an unstable coloured intermediate reaction product is the nonequilibrium determination of metoclopramide and tetracaine in tablets or injections [74]. Metoclopramide and tetracaine form unstable red intermediate compounds with absorption maxima at 495 and 572 nm, respectively, when being oxidized in sulfuric acid medium by potassium dichromate in the presence of sodium oxalate as catalyst. The change of the absorbance of the intermediate products was measured in a stopped-flow regime to quantitate these analytes at mg mL1 concentration levels. The concept of the SIA-LOV technique was exploited for the extraction-spectrophotometric assay of diphenhydramine in injections [75]. Three solutions (i.e., sample, bromocresol green in aqueous phthalate buffer at pH 3 and chloroform) were sequentially aspirated into a coil attached to the central port of a conventional multiposition selection valve where the extraction process was performed. Thereafter, the aqueous and organic phases were separated in a conical separation chamber unit attached to one port of the valve. This unit was in fact a modified disposable tip of an automated pipette. The absorbance of the chloroform extract containing the diphenhydramine-bromocresol green ion-association complex was monitored at 415 nm. The system demonstrated the possibility of costeffective on-line automated extraction on micro-scale. Numerous other pharmaceutical applications of SIA with spectrophotometric detection can be found in the recently published review [6].

3.2.7 SIA with fluorescence detection Owing to its inherent higher sensitivity and selectivity compared to most spectrophotometric methods, fluorescence detection is widely used in automated SIA assays of drugs in pharmaceutical formulations [6], as well as in drug dissolution studies. A stopped-flow SIA method with fluorescence detection for the determination of alendronic acid in tablets was based on the reaction of this drug with o-phthalaldehyde in the presence of 2-mercaptoethanol at pH 10.2 (borate buffer) [76]. The sample and reagent zones were aspirated into the holding coil and then propelled to a reaction coil where the overlapped zones were stopped for 60 s to gain maximum sensitivity. The intensity of fluorescence of the derivatized alendronic acid was measured at 455 nm with excitation at 340 nm. Pinto et al. [77] devised an SIA-fluorescence method using pulsegenerating solenoid micropumps to enhance mixing of the analyte and reagent zones. The usefulness of this technique was demonstrated by the SIA assay of indomethacin in tablets and capsules [77]. Indomethacin was hydrolyzed in 0.1 M NaOH solution containing 20 mM hexadecyltrimethylammonium bromide and the fluorescence of the hydrolytic products was measured at 358 nm with

608


excitation at 278 nm. A fluorescence optosensing element based on a flow cell packed with silica C18 micro-beads was used for the sensitive SIA-fluorescence determination of labetalol in tablets and urine [78]. After aspiration of the sample plug into the aqueous carrier solution containing 20% methanol and 0.1 M NaOH, the labetalol plug was propelled to the flow cell where the analyte was retained for a predetermined period of time and then eluted with the carrier. The transient fluorescence signal of the retained labetalol was monitored at 420 nm with excitation at 330 nm. The limit of detection was 3.3 ng mL1.

3.2.8 SIA with chemiluminescence detection Unlike the well-established FIA methods for drug analysis involving CL detection, SIA methods with this detection technique have not yet found wider acceptance in the analysis of the active ingredients in dosage forms. Several local anesthetics (e.g., procaine, benzocaine and tetracaine) were determined in injections by SIA with CL detection using acidic permanganate as the reagent and formic acid as the enhancer [79]. The permanganate-based CL approach was employed for the assay of sulfonamides in tablets with glutaraldehyde as the enhancer [80] and trimethoprim with hexametaphosphate as the enhancer [81]. Indomethacin was determined in ointments and gels in a recent study [82] by SIA with CL detection which involved the use of the Ru(III)–2,2u-bipyridine complex. This unstable reagent was prepared in situ in the SIA system from the more stable Ru(II)–2,2u-bipyridyl complex by its oxidation with Ce(IV). Generally, the SIA methods with CL detection are highly sensitive, selective and rapid. In most applications of this type the sample throughput exceeds 100 h1. For a wider concentration range the calibration curve can be parabolic which is one of the minor problems that can be encountered sometimes with these methods. In contrast to FIA where the continuous flow of the reagents allows the background CL emission to be offset electronically before the sample injection, in SIA it is necessary to carry out blank injections to calculate the net CL intensity.

3.2.9 SIA with electrochemical detection The potentiometric SIA assay of Penicillin G in tablets was based on the use of a tubular poly(vinylchloride) ISE modified with Mn(III)-5,10,15,20-tetraphenylporphyrinate, 2-nitrophenyloctylether and sodium tetraphenylborate as the Penicillin G sensor [83]. The linear calibration range was 0.2–10 mM Penicillin G and the sample throughput was 25 h1. The electrode’s pasting liquid consisted of the two ion exchangers drotaverinium-silicotungstate and drotaverinium-tetraphenylborate dissolved in tricresyl phosphate. Incorporation of the Mn(III)-5,10, 15,20-tetraphenylporphyrinate ionophore into ceramic sol-gel membranes based on methyltriethoxysilane allowed fabrication of potentiomertric ISE suitable for the SIA assay of valproate in tablets and syrups [84]. The method was characterized by a calibration range 1–50 mM and a sample throughput of 55 h1. An amperometric biosensor was prepared by immobilization of L-amino acid oxidase in carbon paste and applied to the enantioselective SIA assay of S-enalapril, S-ramipril or S-pentopril in raw bulk substances of these drugs [85]. The carbon paste electrode was operated at 0.65 V vs. a Ag/AgCl reference


609

electrode in a 0.1 M NaCl carrier stream flowing at 3.6 mL min1. The calibration curves were rectilinear for 0.08–1.5 mM S-enalapril, 0.12–0.6 mM S-ramipril and 0.2–6 mM S-pentopril. The sample throughput was 75 h1.

3.2.10 Sequential Injection Chromatography and related techniques SIC, which is a hybrid SIA-low pressure chromatographic technique, and was devised by coupling a conventional SIA setup to a monolithic separation column, is discussed in more detail in Chapter 10. Practically all publications concerning SIC that have appeared since 2003 are devoted to the analysis of one or more active ingredients and excipients (e.g., preservatives) in pharmaceutical formulations. For example, SIC with spectrophotometric UV detection was applied to the assay of methylparaben, propylparaben and triamcinolone acetonide in a cream [86], salicylate and methyl salicylate in an ointment [87], diclofenac, methylparaben and propylparaben in an emulgel [88], paracetamol, caffeine and acetylsalicylic acid in tablets [89], ambroxol hydrochloride and doxycycline in capsules and tablets [90], naphazoline nitrate and methylparaben in nasal drops [91], betamethasone and chloamphenicol in eye drops [92], ambroxol hydrochloride, methylparaben and benzoic acid in syrups and drops [93], and salicylic acid and triamcinolone acetonide in topical solution [94]. An FIA manifold coupled with a monolithic separation column and spectrophotometric diode-array detector was utilized for the separation and quantitation of sweeteners, preservatives and antioxidants in cosmetics and food samples [95]. An FIA setup incorporating a monolithic column and CL detection, based on tris(2,2u-bipyridyl)ruthenium(III) and acidic permanganate as the reagents, proved to be useful for the separation and determination of six opiate alkaloids (morphine, pseudomorphine, codeine, oripavine, ethylmorphine and thebaine) and four biogenic amines (vanilmandelic acid, serotonin, 5-hydroxyindole-3acetic acid and homovanillic acid) in human urine [96]. Gonza´les-San Miguel and co-authors [97,98] coupled a multisyringe burette system equipped with a diode array UV detector to a monolithic separation column to determine amoxicillin, ampicilin and cephalexin in pharmaceutical formulations.

3.2.11 Multicommuted Flow Injection Analysis An MCFIA manifold coupled to a spectrophotometric detector was employed for successive automated determinations of ascorbic acid, thiamine, riboflavine and pyridoxine in multivitamin formulations [99]. The flow manifold was designed with eight computer-controlled three-way solenoid valves for independent handling of samples and four different reagent solutions. A 25-fold lower reagent consumption relative to analogous FIA methods was achieved. Knochen et al. [100] devised an automated spectrophotometric MCFIA method for the assay of dextrose in parenteral and haemodialysis solutions. The MCFIA manifold involves three computer-controlled three-way solenoid valves, one carrier flow channel and one reagent channel. The reagent was a mixture of glucose oxidase, peroxidase, 4-hydroxybenzoate and 4-aminophenazone and the reaction product was a quinoneimine dye with absorption maximum at 505 nm. The linear calibration range was 0.1–1.0 g L1 and the sample throughput

610


was 90 h1. The MCFIA concept was combined with a flow-through multioptosensor comprising a miniature on-line precolumn and a spectrophotometric flow cell, both packed with C18 silica-gel beads. The system was used for the automated assay of salicylamide and caffeine in capsules and pellets. Upon sample injection into a carrier stream of pH 8.6 (ammonia buffer) caffeine was strongly retained on the C18 beads placed in the minicolumn while salicylamide passed through. It reached the sensing zone where it was temporarily retained on the beads placed in the flow cell where it developed its transient absorbance signal and was subsequently eluted by the carrier. Thereafter, caffeine was eluted from the minicolumn with aqueous 25% MeOH introduced into the flowing system for 200 s and detected similarly to salicylamide . The sensor responded linearly in the range 2–30 mg mL1 for salicylamide and 1–14 mg mL1 for caffeine. A single analysis took about 8 min. A similar spectrophotometric MCFIA system allowed the separation and quantitation of caffeine, salicylamide and propyphenazone in capsules, tablets and pellets [101].

3.3 Automated drug dissolution, release and permeation tests The in vitro dissolution tests of pharmaceutical formulations play an important role in the quality control process of solid or semisolid pharmaceuticals and therefore they are included in many international pharmacopoeias. These tests should provide basic information on the dynamic parameters of the dissolution process represented by the so-called dissolution profiles. Such dissolution profiles allow assessing of the availability of the active principles being liberated from the finalized formulation and hence checking the reproducibility of the manufacturing procedure. The congruity of dissolution profiles of a generic formulation and the original medicine is often required as one of the criteria considered in the registration process of generic products. Most of the pharmacopoeial dissolution tests are based on manual sampling of an aliquot of the given dissolution medium from the dissolution apparatus after predefined period of time elapsed from the beginning of the test. The analytical information acquired in this manner can meet requirements postulated by a pharmacopoeial article but obviously it will not be sufficient to define in detail the kinetics of the dissolution process, needed in the research and technological design of new dosage forms. This problem can be solved by continuous automated monitoring of the concentration of the liberated active ingredients in the dissolution media by exploiting well-established FIA or SIA techniques. The pioneering work on the application of SIA with spectrophotometric detection to dissolution tests of solid dosage forms was reported by Liu et al [102,103]. This approach allowed to obtain the detailed dissolution profiles of ibuprofen tablets, sustained-release capsules and controlled-release matrix tablets [102] and also to monitor simultaneously the liberation of acetylsalicylic acid, phenacetin and caffeine from compound aspirin tablets [103]. Later on Solich et al. [104] published a series of papers on the use of SIA with fluorescence detection in dissolution tests of solid pharmaceutical preparations based on measuring the native fluorescence of the corresponding active ingredient, i.e., bumetanide [104], ergotamine tertrate [105]


611

and prazosin [106]. This approach was extended to the simultaneous acquisition of dissolution profiles of two active ingredients from a single formulation. Mathematical processing of overlapping UV spectra was exploited in the automated FIA monitoring of the ‘‘global’’ and ‘‘individual’’ dissolution profiles of amoxicillin-bromhexine capsules and amoxicillin-clavulanic acid coated tablets [107]. Recently, an MCFIA technique with spectrophotometric detection using the same chemometric approach was devised for evaluating the dissolution profiles of sulfamethoxazole-trimethoprim tablets and capsules and captoprilhydrochlorothiazide tablets [108]. Simultaneous monitoring of the liberation of ascorbic acid and rutin from a composed tablet was achieved by a hybrid SIAsolid-phase extraction technique with UV detection using a minicolumn packed with C18 silica-gel sorbent. Unlike ascorbic acid, rutin was temporarily retained on this sorbent and so these two active ingredients were successfully separated [109]. Automated release testing of active ingredients from semisolid pharmaceutical formulations and their permeation through membranes imitating human skin was achieved by linking a conventional Franz diffusion cell with an SIA

Figure 5 Schematic depiction of an automated apparatus for simultaneous measurement of dissolution and permeation. Sampling ports are indicated with capital letters, D for dissolution, A for apical and B for basolateral. The multiposition valve and its port assignment were as follows: Port 1 was connected to the waste; Port 2 was connected to the autosampler for aspirating the standard solutions; Port 3 was connected to the Krebs-Ringer buffer supply for replenishing the volumes taken from the basolateral compartment; Port 8 was connected to the fluorescence detector PMT-FL and Ports 4, 5 and 6 were assigned to Sampling Ports B, A and D, respectively. Reprinted from Ref. [114]. Copyright (2007), with permission from Elsevier.

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permeated amount (μg)

100

permeated amount (μg)

concentration (mg ml-1)

concentration (mg ml-1)

setup. The permeation profiles were measured with fluorescence detection for gels or ointments containing indomethacin [110] or salicylic acid [111,112]. Klimundova´ et al. [112] reported on a real-time data collection and processing approach in tests involving the simultaneous use of three Franz cells [112]. The methods outlined above [110–112] were characterized by a high sample throughput of 120 h1 and a relatively long running time of 6 h. An SIC setup linked to a Franz cell was employed for the automated simultaneous measurement of the release of two active substances (i.e. lidocaine and prilocaine) from a cream [113]. The

0

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60 time [min]

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Figure 6 Comparison between manual sampling and analysis by HPLC (lower diagram) (n ¼ 5) and automated sampling and fluorescence detection using SIA (upper diagram) (n ¼ 7). Closed circles (K) represent the concentration at site D (see Figure 5), open circles (J) the concentration measured at site A (see Figure 5) (both primary ordinate) and triangles down ( ) represent the permeated amount of propranolol hydrochloride (secondary ordinate). Reprinted from Ref. [114]. Copyright (2007), with permission from Elsevier.

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acceptor medium was sampled in 10.5 min intervals for 4 h. Monitoring of dissolution of propranolol from a single tablet combined with simultaneous permeation of the released propranolol through a monolayer of Caco-2 cells was achieved by devising a modular setup involving a commercial SIA analyser and a dissolution and permeation unit (Figure 5) [114]. The dissolution and permeation data, collected and processed automatically, were in good agreement with those obtained by manual sampling and subsequent HPLC analysis as shown in Figure 6.

ABBREVIATIONS BSA CL DMAS FIA HPLC IgG ISE LOV MCFIA MS MSFIA PAT SIA SIC

Bovine serum albumin Chemiluminescence 9,10-Dimethoxyanthracene-2-sulfonate Flow injection analysis High performance liquid chromatography Immunoglobulin G Ion selective electrode Lab-on-valve Multicommuted flow injection analysis Mass spectrometry Multisyringe flow injection analysis Process analytical technology Sequential injection analysis Sequential injection chromatography

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CHAPT ER

21 Industrial and Process Analysis Applications Celio Pasquini and Ma´rcio V. Rebouc- as

Contents

1. Introduction 2. The Advantages and Weakness of Flow Analysis Applied to Industry 3. Process Analysers Based on Flow Systems 4. Selected Applications of Flow Analysis to Industrial and Process Analysis 4.1 Determination of moisture 4.2 Determination of bromine index and bromine number 4.3 Determination of acidity/alkalinity and pH 4.4 Metallic species 4.5 Other industrial applications 5. Conclusion Abbreviations References

617 619 627 629 630 631 632 633 633 635 636 637

1. INTRODUCTION Industrial activities place high demands on chemical analysis. An overview of the issues associated with such activities is shown in Figure 1. While raw material and final product quality control may still rely on classical off-line analytical methods, others such as process and effluent control frequently require robust, fast, low cost and automated or mechanised methods. Modern industry relies on instrumental analytical methods performed by analysers. These automated instruments should ideally have the following characteristics: operate in on-line or in-line mode, supply the chemical information strictly in real-time (fast response), Comprehensive Analytical Chemistry, Volume 54 ISSN: 0166-526X, DOI 10.1016/S0166-526X(08)00621-1


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Health/safety monitoring Product quality control

Raw materials quality control

INDUSTRIAL ACTIVITIES

Effluents treatment and control

Process control and/or monitoring

Figure 1 The issues generated by industrial activities which are highly demanding for chemical analysis.

be free of mechanical moving parts, be capable of long-term unattended operation, be able to perform automatic sample preparation, be automatically self-calibrating, contribute negligibly to the waste generation of the plant (ideally, to be in accordance with green analytical chemistry principles [1]), to provide an unambiguous indication of system failure (alarm), be robust to changes in the process streams.

Of course, the previous chapters have shown that any analytical method based on flow analysis concepts naturally presents many of the characteristics listed earlier, and has the potential to fulfil many others. Since the introduction of the flow analysis concept by Skeggs in the middle of the 1950s [2] there has been much effort aimed at matching flow methods to the analytical demands of the industry. Some of these efforts have been as straightforward as adapting the flow method to work adequately with a new matrix of interest, and in some cases this simply involves the use of the laboratory analysis in the plant, but in off-line mode. On the other hand, other attempts have entailed profound alterations to analytical processes involving the flow analysis concept, such as those introduced by the use of Sequential Injection Analysis (SIA) [3]. It is not an easy task to classify the large number of approaches to flow analysis found in the current literature, and Table 1 lists some but not all of the varieties of real applications and includes those that have strong potential for future use in industrial and process analysis. The objective of this chapter is therefore to evaluate the ability of various flow analysis strategies to meet the challenges of industrial and process analysis, and to indicate the direction of further and necessary development in this area. Due to its increasing interest, the use of flow techniques for process analytical technology (PAT) will drive most of the discussion. Furthermore, the chapter makes reference to a number of selected works in which it is possible to identify a

Industrial and Process Analysis Applications

619

Table 1 Flow modes already employed or with potential to be employed in industrial and process analysis

Multi-segmented continuous flow (CF) Flow injection analysis (FIA) Mono-segmented flow analysis (MSFA) Sequential injection analysis/lab-on-valve (SIA-LOV) Flow-batch (FB) analysis Multi-commutation flow injection analysis (MCFIA) Multisyringe flow injection analysis (MSFIA) Reverse flow injection analysis (r-FIA) Batch injection analysis (BIA)

real contribution toward the use of the flow analysis principles for industrial and process analysis.

2. THE ADVANTAGES AND WEAKNESS OF FLOW ANALYSIS APPLIED TO INDUSTRY The field of industrial analysis employs four types of process monitoring. Figure 2 shows a schematic diagram of these four approaches. The most simple is off-line monitoring which requires the sample to be collected and sent to the laboratory. In the at-line approach the analyser is brought close to the process in the plant. However, the sample is still collected and transferred to the analyser manually. The on-line monitoring consists of an automatic sampler capable of supplying fresh samples representative of the process status to the analyser, which is attached to the process stream. Finally, process monitoring can be performed by using the in-line approach where a probe directly accesses the sample, and produces a signal related to the analyte content or process parameter. The relevance to process control increases as the time delay between sampling and the production of the analytical result decreases. Therefore, it is easy to justify the present search for suitable for at-, on- and in-line analytical methods. In-line monitoring involves the most direct and fast interaction of the analyser with the process. However, the system must detect the analyte as it is in the process stream or batch reactor. If a sensor is not available for direct monitoring, then sample must be extracted from the process and treated by an external system before detection on-line with a flow-based analyser, in order to ensure that the concentration determined is representative of the process at that time. Figure 3 represents the various types of flow systems in their simplest configurations, interfaced in-, on- and at-line to an industrial process. One of the greatest advantages of the flow analysis employed for process monitoring is the nearly direct interface with liquid streams. Therefore, the use of flow analysis systems is more relevant for at-line and on-line monitoring, because they can

620

At-Line

Off-Line

sample sample Analyser 12.3

12.3

In-Line

Figure 2 Representation of the four types of industrial process monitoring.

X

12.3

On-Line


LABORATORY


CF

621

air

D

FI

D

MSFA D

air SIA/LOV D

FB

MCFIA MSFIA D

D

r-FI D BIA D

Figure 3 Schematic diagram showing how the diverse flow analysis systems can be coupled to an industrial process through an on-line configuration. The symbol X represents an on-line sampling port.

provide sample pre-treatment before detection. This characteristic, common to all flow concepts, constitute another of their great advantages. The coupling of a flow system to a process stream, although frequently straightforward, deserves attention because the stream cannot always be directly

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admitted to the flow system. This occurs, for example, when the stream contains solids in suspension or when its pressure and/or temperature are very different from ambient conditions. Under such conditions, the formation of small gas bubbles inside the manifolds is quite common, and these can be trapped inside the detector or cause changes in the dispersion in unsegmented systems, impairing the measurement of the analytical signal. Even though flow analysis systems were designed specifically for processing liquid samples, solid samples such as metallic alloys can also be handled by an at-line configuration. This is done, for example, after sample on-manifold electrodissolution of the metallic species present in a disk of the alloy, after applying a suitable electrical current [4–6]. The resulting analyte solution is commonly analysed by spectrophotometry, ICP-AES or AAS. Gaseous analytes have also been determined directly by flow analysis systems [7]. Some of the applications can be of great interest for the industrial activities, mainly those related with the operation of boilers and their exhausting gases. Table 2 shows a comparison of the principal modes of flow analysis, highlighting their relative strengths and weaknesses for use in industrial process analysis. A relative basis for comparison is necessary because the importance of each parameter for each flow system mode is not the same. For instance, the presence of mechanical moving parts is a non-desired characteristic shown by all the flow systems listed in Tables 1 and 2. However, the effect of these mechanical parts on the overall performance of the analyser is distinct among the systems. It is possible to observe that all systems described so far present most of the desired characteristics to suit industry requirements. At this point, it is important to distinguish between the characteristics required for off-line analysis from those for at-line and on-line analysis, the last two being more suitable for process control. The robustness of a flow analyser can be considered as an overall figure of merit of the system associated with its long-term operation and immunity to both intrinsic variables of the system (such as flow rates, reagents and standards stability and detector performance) and ambient or process variables (such as temperature, pressure, analyte concentration and presence of corrosive gases and dust). Off-line and at-line applications of flow systems are less demanding in terms of robustness; self-calibration and a failure alarm system are not mandatory. The laboratory instrument is always being monitored by the technician in charge. The principal characteristic of the flow systems which enables them for off-line determinations in industry is, perhaps, their ability to perform the treatment of the sample in order to convert it to a form suitable to the detection system. Also, for off-line industrial applications, the reduction of sample and reagent consumption, lowering laboratory waste management and fast sample throughput are also considered when a decision for automating a given analytical method is to be taken. The different types of flow analysis show relative advantages and disadvantages, which must be carefully weighed by the person responsible for the laboratory automation/mechanization, before the decision for the adoption of

Table 2 Comparison among various types of flow systems regarding their suitability in attend the requirements for industrial and process analysis. The (+) and () denote favorable or non-favorable characteristics, respectively Desirable characteristic for industrial and process analysis At or on-line operation

Fast response time

Presence of mechanical parts

Robustness

Sample preparation

Selfcalibration

Waste generation

Failure warning

Tolerance to changes in process stream

CF FIA MSFA SIA-LOV FB MCFIA MSFIA r-FIA BIA

+++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++

+++ +++++ ++++ +++ ++++ +++ +++ +++++ +++++

+++ +++ ++++ +++++ ++++ ++++ +++ ++ +++

+++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++

+++ +++++ +++++ ++++ +++++ ++++ ++++ ++++ ++++

+++++ +++ +++++ +++ +++++ +++ +++ +++ +++


Flow system type

623

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one or other flow system is taken. For example, the air-segmented flow systems (continuous flow (CF) and monosegmented flow analysis (MSFA)) are characterized by their higher sensitivities. If this is a crucial feature of the analysis, these systems need to be considered. Furthermore, a comparison between both segmented flow systems reveals that MSFA requires a simpler analyser and employs usual devices employed by flow injection analysers (FIAs). The person involved in the definition of the most favourable flow system to fulfil the necessities of the industrial laboratory should be free of preconception about the different flow systems. It is important to note that the initial criticism of a given type of flow analysis approach, usually on the occasion that a new approach is proposed, causes a reaction from the researchers working in the development of the so said depreciated system. The consequences are that shortly thereafter the ‘‘jeopardized’’ system will find a way to overcome its limitations. For instance, modern analysers based on multi-segmented CF, originally introduced by Skeggs in the 1950s, do not have the large sample consumption that characterized the early generation of instruments. Also, the removal of air bubbles is no longer necessary in modern equipment, which is nowadays all computer controlled. The detection can be carried out for each liquid segment and the analytical signal can be digitally constructed. The subject of on-line process analysis, employing flow techniques, is more complex. The requirements are more restrictive and the robustness is decisive. The pioneer CF system [2], as mentioned earlier, has the advantage of high sensitivity, resulting from the restriction of the longitudinal dispersion imposed by the air bubbles. Simple reactions are promptly realized and the typical sample throughput of 60 samples hour1 is enough for many applications. Sample and reagent consumption are in the range of millilitres or lower. The main problem with the CF systems is the lack of adaptability. New manifolds need to be considered to accommodate a new chemistry for the determination of a new analyte. Parallel configurations were evaluated in the past (mainly for clinical analysis) as a solution to this problem. However, the complexity of the analyser reflects directly in a lack of robustness. The system is prone to suffer from direct effect of changes in flow rates and recalibration must be carried out frequently. The FIA [8] is remarkably simple when compared with its predecessor, the CF analyser. A single line manifold is possible and no one could propose a simpler flow manifold, so far. The FIA technique can be adapted to determine a number of analytes of industrial interest providing a direct integration with the process. However, due to its unidirectional operation mode, the system also lacks in versatility and, as a rule, this flow mode also requires one manifold per analyte. Robustness is also jeopardised by the dependence on flow rates which can be enhanced by the fact that usually a FIA system operates in a kinetic regimen, distant from the chemical equilibrium of the determination reaction and, therefore, more dependent on reproducible timing. Small bubbles that form inside the system can represent a serious problem when they are caught inside the detection cell. After all, it is a nuisance and unproductive to have to disassemble part of the flow manifold simply to remove trapped air bubbles.


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The MSFA system [9] was proposed as a hybrid of CF and FIA. The dispersion is also restricted by air bubbles sandwiching only one sample segment (50–250 mL). The generation of the front and end air bubbles is provided by the sampling valve with no need of extra pumping tubes. Long residence times can be achieved and sensitivity is one of the strong characteristics of this type of system, mainly when the chemistry involved in the determination is kinetically restrictive. Because of the presence of the inserted air bubbles in the manifold, it becomes immune to small air bubbles formation during sample processing. The localisation of the sample in MSFA is easily done by detection of the liquid/air interfaces during monosegment transport inside the manifold, using noninvasive optical switches. The segment can be taken to the cell and return to any point of the analyser for further processing before the definitive detection is performed. This strategy has been explored to perform titrations using MSFA [10]. Because of the possibility of sample localisation in the manifold, a MSFA analyser can be constructed to determine many analytes while reducing significantly the reagent consumption once its addition to the sample can be carried out just inside the monosegment during its passage by a certain point of the manifold [11]. Since MSFA works under chemical and physical equilibrium conditions, it is less dependent on flow rate variation, similarly to CF. The main problem with MSFA when operating in process analysis is the monosegment stability during its transport through the manifold. The presence of certain surface-active compounds in the sample can cause bubble rupture, destabilising the monosegmented sample pattern and loosing the determination. Of course, a warning can be provided to account for that fact. Perhaps the most significant breakthrough toward the use of flow analysis for industrial process monitoring and control was achieved by the SIA concept [12]. A SIA-based analyser is simple and versatile. It interfaces directly with the process and allow for sequential operations necessary for sample treatment followed by sample analysis. The system can be adapted to determination of a number of analytes without any modification in the manifold. That is, it is highly versatile. Mechanical devices are present, and it is, as in the other cases, a limitation regarding process analysers. However, these devices (basically a piston or peristaltic pump and a multi-port valve) are robust and the SIA concept allows for the reduction of their effect on the precision of the analytical results. Although a SIA system is dependent on flow rates, the sample process protocol can wait until at least the chemical equilibrium is achieved before the sample is directed to the detection module of the analyser. A SIA system will perform slower than a FIA system and other flow systems when more than one chemical sequential pretreatment of the sample is necessary to convert the analyte into a detectable form. Recently, the SIA concept has gained attention as a reliable way to perform pretreatments of interest of process analytical protocols [13]. The evolution of the SIA is the Laboratory on a Valve (LOV) concept which incorporates the use of reagent immobilized on tiny beads which are transported in suspension into and out of the flow manifold [14]. The beads can be accumulated in a small column in the valve to perform a sample treatment or be directly the medium in which the detection of an adsorbed analyte is made. The concept of renewable solid reactors

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is powerful and facilitates the extension of the concept to bioprocesses monitoring. The Flow-Batch (FB) concept of mechanization/automation gave rise to a true hybrid analyser [15]. The batch part inheritance of the analyser is a low volume (ca 1.0–2.0 mL) chamber magnetically stirred to which a number of liquid streams tubing are directed. In its flow part, usually a peristaltic pump and three-way micro-solenoid valves (up to six) are used to control the access of the fluids to the cell. The cell works as a batch reactor and sample additional pre-treatment can be automatically made outside, before the final reaction is processed. Electrochemical detectors can be assembled directly inside the cell or the reaction mixture can be aspirated to a flow-cell for final detection. Because of its hybrid characteristic, FB is, like MSFA, largely immune to the formation of bubbles. On the other hand, because the sample and reagent volume proportions are attained by time control of the solenoid micro-valves, the results are affected by nonproportional changes in flow rates. The FB system can be used for analysis of viscous liquids and suspensions, which are difficult to mix and require mechanically assisted mixing. However, the use of many electromechanical devices and pumping tubes impart a lack of robustness and this is a source of concern considering the application of FB systems for process analysis. Recently, a FB system has been proposed where sampling is not based on the time/flow rate ratio. Instead, the sample is injected as in a FIA system and carried to the batch cell. The system allows for precise sampling which is independent of the carrier flow rate. This type of system is suitable for automation of titration-based determinations such as bromine number and bromine index of hydrocarbons produced by the petrochemicals plants [16]. The multi-commutation flow injection analysis (MCFIA) concept [17] was introduced with the aim of reducing reagent and sample consumption, while keeping the advantages of easy process interfacing and high sample throughput. The MCFIA manifolds are assembled by using a number of three-way solenoid micro-valves usually connected to a common point that is aspirated through a peristaltic pump. The adequate operation of the solenoid valves provides sampling, reagent addition and reaction processing before the mixed zone is sent to the detector. The system operates by cycling the valves and admitting small adjacent portions of solutions (1–5 mL) which are mixed during their transport through the system manifold. The flexibility of a MCFIA system can be high. The main limitation of MCFIA systems to process industrial analysis is related with the multiple mechanical devices (electromechanical micro-valves) employed. Also, opportunistic bubbles can cause serious problems and request external intervention with a periodicity far beyond the acceptable for a process analyser. Multi-syringe flow injection analysis (MSFIA) [18] was developed based on the use of a number of small volume syringes and a multi-way port to drive the fluids in a manifold that is versatile and can interface directly with the industrial process. The major weakness of this approach is the use of many mechanical devices (syringes and valves), which are not compatible with the robustness required in a process analyser.


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Beside those already mentioned, there are some other modes of flow still waiting to be further explored regarding their use in industrial analysis. Two examples are reverse-FIA (r-FIA) [19] and Batch Injection Analysis (BIA) [20]. r-FIA presents the possibility of directing the sample flow straight to the detector while injecting a small volume of reagent to promote a reaction for determination. In fact it reverses the usual sampling strategy in which a discrete volume of sample is collected and introduced in the flow analyser to be processed. The system is capable of achieving higher sensitivity due to the reversal effect of the dispersion of the reagent in the sample stream. The BIA concept employs a large reservoir of a suitable solution mechanically stirred where, usually, an electrochemical detector is immersed. Usually a micropipette is employed for sample introduction over the indicator electrode. However, Figure 3 shows the possibility of using a FIA manifold to impel the sample to the detector surface where it is detected before being diluted by the large volume of the solution surrounding the detector. Sample pre-treatment can be accomplished, after injection, on the way to the detector.

3. PROCESS ANALYSERS BASED ON FLOW SYSTEMS As has been mentioned earlier, on-line analysers have been widely used in industry as they can provide real-time process monitoring [21]. Despite the fast development in this area, there are some at-line and off-line analyses which present great potential for replacement by on-line analysers. The major concern for most users throughout the world is their long-term reliability, which is strongly dependent on the equipment design, sampling system performance and process nature and stability. Each component of the system is usually well designed and works perfectly under controlled circumstances but one should be aware that the whole analyser must be tested and tuned to the very right operation under real process conditions. This is not as straightforward as one might expect. Beyond that, care must be taken to ensure the quality of the results by statistical equipment validation, periodic calibration and statistical quality control. The most common process analysers are based on the determination of analytes where there is a significant change in some physical property (electrical, optical, magnetic, thermal, etc.), that can be measured by, for example, chromatographs, conductometers, viscometers, pH metres, etc. However, there are some applications where a series of chemical reactions and/or sample pretreatments are required for analysis. In such cases, flow-based equipment would be ideal for fast and automated determinations, replacing the classical bench methods. Despite this, the use of flow analysis concepts in process analyser design is quite rare [22]. Some equipment is designed in such a way as to resemble a flow analysis system; however, they lack most of its main advantages. Therefore, flow systems have been used to introduce the sample into a carrier containing the reagent, which passes through a coil where the reaction takes place in the path to

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the detector. Sample pre-treatment and additional reagents may be added to the flow. The operation of most process analysers relies on the classical principle of reaching the chemical equilibrium and the whole equipment is designed on the basis of such a premise. Ruzicka and Hansen [23] mentioned three approaches to applying FIA in the continuous monitoring of industrial processes, based on sample injection, standard injection and reagent injection modes. In the first mode, which is also the most common approach in bench systems, the process stream is sampled and injected into the analytical circuit by an injection valve. The analyte present in the sample aliquot is mixed with the reagent and the reaction product is detected generating a peak in the equipment display. The second approach precludes the use of an injection valve, since a continuous flow of the process stream is maintained through the system. The readout is therefore a continuous curve whose height varies with the analyte concentration in the stream. The reagent injection mode (r-FIA) is a hybrid design between the first two. The process stream continuously flows through the system but the reagent is injected only when a measurement is required. In this way reagent consumption is reduced compared to the previous mode. In all modes a calibration standard can be pumped in order to calibrate the analyser, and therefore the stability of the standard over time must be ensured, taking into account that the standard is usually placed in a shelter in the plant or field. The sample injection approach has the advantage of allowing periodic system washing, preserving the analyser integrity and minimising the risk of memory effects. The need for an injection valve, however, reduces the robustness of the flow system compared to the reagent injection mode. In the standard and reagent injection systems the continuous flow of the process stream may reduce the lifetime of components and/or cause analyser failure depending on the nature of the sample. Although such systems, particularly the standard injection mode, are supposed to perform better in terms of following fast changes in the process, one should be concerned about the amount of time required for the analyser to respond to low concentrations immediately after transmitting a highly concentration sample stream. A suitable scheme, therefore, should be designed taking into account all specific features of the process and the sample. Knowledge of the sample composition, the presence of interferents and the nature of the sample (aqueous, organic) are all necessary in order to initiate the design of a system. The process conditions, such as temperature, pressure, expected range of analyte concentration and variability, may also play an important role. Additionally, precision, accuracy and quantification limit requirements are expected to have a decisive influence on the system concept, and are surprisingly often neglected. The need for unattended continuous operation is a critical obstacle to be overcome. Flow-manifolds are usually composed of many components that may face problems when samples containing some unexpected constituents (particulates, dissolved gases, etc.) are delivered into the system. The aggressive nature of the samples or of the industrial environment may also demand specific design features in order to reach the required robustness of the process analyser.


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Depending on the characteristic of the process stream, care also must be taken to prevent high pressure to damage the system. Most of common sources of problem in on-line analysers can be handled with proper sampling system (including filters, temperature conditioning cabinets, debubblers, etc.) and/or appropriate flow technique, but they must be known in advance. Trojanowicz [24] reviewed some commercially available instrumentation for FIA. Two main suppliers were noticed for instruments dedicated to process analysers: Eppendorf [25] and Ionics (now GE Analytical Instruments) [26]. Other suppliers, such as Thermo [27], ABB [28], Hach [29], Applikon [30], Tytronics [31], Polymetron (now Hach) [29], FIAlab Instruments [32], also produce on-line analysers based on flow analysis principles. Eppendorf designed equipment, called EPAS, which can be used for different applications: peroxide content, acetic acid and sulfuric acid in organic streams, free fatty acid in edible oil streams and ammonia and copper in effluents. The silica analyser is one of the most common flow-like process analysers [27–29]. The sample is propelled with a peristaltic pump and the silica reacts with ammonium molybdate producing a yellow complex. Oxalic acid is also admitted to suppress the phosphate interference. Finally, ammonium ferrous sulfate is added to provide a final reduction to an intense blue complex whose absorbance is measured. A proper surfactant is mixed to the carrier to minimise air bubble formation. The system is usually designed with a series of successive coils to ensure all reactions are completed before reaching the detector. Other examples are applications in sodium and fluoride analyses [27]. These systems are based on ion-selective electrode detection but require previous steps for degassing, pH adjustment and/or decomplexing agent addition. In a commercially available sodium analyser, the pH adjustment is achieved by permeation of ammonium through a sample coil immersed into an ammonium hydroxide reservoir. In an oil-in-water process analyser [30], the aqueous sample passes through a solvent extraction system in which the hydrocarbon compounds are solubilized into a suitable reagent, and the oil content is thereafter measured by an infrared detector. The equipment usually incorporates an additional manifold for solvent recovery. Similar approaches have been applied to chloride, ammonium, phosphate and nitrate determinations. Automatic titrators, with a wider range of applications have also been placed in the market [31,33].

4. SELECTED APPLICATIONS OF FLOW ANALYSIS TO INDUSTRIAL AND PROCESS ANALYSIS It is possible to find a large number of works in the literature dealing directly or indirectly with the application flow analysis to solve industrial analytical problems. In this chapter, a representative and selected number of works have been collected in order to provide the reader with a series of illustrative examples of the potential benefits of flow analysis applied to industrial analysis.

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Specific applications to important industrial fields, such as food, beverage, agricultural, life science, bioprocess and pharmaceuticals, have been described in previous chapters of this book. In this section, the selected works have been arranged by the industrial applications they address and the various flow concepts employed are compared.

4.1 Determination of moisture The determination of moisture or water content constitutes one of the most common determinations made in a countless number of sample matrices. For instance, the Karl Fisher titration method and its variants are used for at least 500,000 determinations a day in the most diverse industries such as pharmaceutical, petrochemical and fuel. The determination of moisture is of fundamental importance to monitor the distillation process during the production of organic solvents. This high demand has impelled the development of many automatic systems based on flow analysis. The FIA concept has been applied to the determination of water using spectrophotometric detection of the iodine consumed by the injected sample [34–40]. The system is very simple but needs to be calibrated by introducing water standards. The proposed FIA systems can perform between 60 and 120 determinations h1 of moisture in samples of organic solvents and/or petrochemical products. Working ranges are between 0.01 and 0.2% (v/v) of water. The use of potentiometric detection for water determination in solvents has been described [41]. The FIA-potentiometric system shows superior performance when compared with the spectrophotometric detection. Small sample volumes, down to 50 mL, are necessary, and the use of calibration standards with similar composition to the sample matrix is required. Water in solvents has also been determined down to mg L1 levels by employing near infrared (NIR) spectrometry [42,43]. In these cases, the use of a FIA system to deliver the injected sample to the detection cell warrants a large signal-to-noise ratio while providing the automatic cleaning. Recently, the volumetric Karl Fischer titration has been processed in a MSFIA system and applied to the determination of water in anhydrous and hydrated ethanol (an important agro-industrial product and a green substitute for fuels derived from petroleum) [10]. The system performs a true titration of a small (40–300 mL) sample volume introduced into an air carrier stream maintained free of moisture after passage through a molecular sieve column. Detection of the titration endpoint is made by biamperometry. Therefore, turbid solutions and samples with a variety of viscosities and refractive indices can be promptly processed. The MSFIA-Karl Fischer titrator does not require the use of standards for calibration. Some other additional advantageous characteristics of the system are its independence of flow rate, and low sample and reagent consumption. However, as for any system based on true titration, the time necessary for sample analysis is relatively high (3–5 min). The system has been applied to moisture determination in commercial ethanol in the range 0.01–0.5% (v/v).


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4.2 Determination of bromine index and bromine number The determination of the bromine number (BN) (gram of bromine reacting with 100 g of sample) and bromine index (BI) (milligram of bromine reacting with 100 g of sample) is a frequent measurement carried out mainly by the petrochemical industry. The measurement is associated with the amount of olefins present in the sample, an important parameter for product postprocessing by the polymer and other industries. It also poses some challenges to flow analysis because the samples are of different types presenting different densities, viscosities and refractive indexes. Furthermore, the samples are not aqueous and a degree of attention should be paid to the durability of the pumping systems. A Flow Injection system has been developed aiming at the determination of BN [44] allowing for a high sample dilution, if necessary, and using coulometric generation of bromine. However, this system suffers from some of drawbacks already mentioned, being dependent on flow rate, requiring at least one calibration step and not being able to deal with the presence of a gas phase during the titration process. This is especially true for systems employing spectrophotometric detectors to follow the titration progress. Figure 4 shows a recently proposed analyser whose main characteristic is robustness [16]. The system operates under the FB concept resulting in no dependence on flow rates, since the sample volume is defined by the flow part of the analyser which employs a FIA-based sampling system. The sample volume (50–300 mL) is quantitatively transported by the carrier reagent solution, containing the bromine precursor (Br) to a batch cell (2.0 mL total volume) containing the electrodes employed for coulometric generation of Br2 and the electrode pair for biamperometric monitoring of the titration. The hydrogen

Carrier/ reagent solution

L

V2

C V1 S

Sample

I

Sample

Waste

Recycle/ or dilution

Figure 4 Robust flow-batch coulometric titrator for determination of bromine index and bromine number in petrochemical products. I, sampling device; L, sampling volume (sample loop); V1 and V2, three-way electromechanical valves; S, magnetic stirrer; C, titration cell. All wetted parts are made of Teflons. A peristaltic pump with Vitons tubes is employed to impel the fluids.

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generated inside the titration cell do not interfere with both the titration reaction and the detection system. True titration is performed and the IB or NB is determined from the stoichiometric bromine necessary to react with the olefins present in the sample. Dilution can be controlled by diverting the carrier flow through valve V1 and transporting only a fraction of the dispersed sample to the titration cell. However, the adoption of this last procedure will reduce the analyser robustness because the operation is dependent on flow rate and requires previous calibration of the system. The reagent consumption is about 1.5 mL per sample and a titration can be performed in about 3 min for BI determinations. The system described earlier is a good example of a result came out from the efforts directed to the development of a robust flow system that can operate unattended for long periods of time, without the necessity of calibration standards and independently of flow rate.

4.3 Determination of acidity/alkalinity and pH Acidity/alkalinity and pH are among the most common and relevant analytical measurements made in industrial processes and laboratories. The flow analysis concepts have been applied to facilitate this measurement directly through the use of potentiometric [45,46] and spectrophotometric detection [47,48]. Alternatively, the acidity can be determined by flow-based (FIA, SIA) pseudo-titration based on concentration gradients produced by dispersion [49–51] or by means of true flow titration [15,52,53]. The concept of true titration, in the present context, is associated with the use of only one standard (titrant) solution of known concentration and stoichiometry of the titration reaction to calculate the analyte concentration. The use of FIA and SIA systems for pH measurements using pH sensors allow for reproducible sampling operation and automatic sample dilution and ionic strength adjustment. The calibration made by using standard buffer solutions is also carried out in flow systems, resulting in a high sample throughput (W120 samples h1) and high precision (typical, 0.1 pH units). Because the sample is washed out by the carrier solution, the sensor is maintained under a stable condition contributing for its performance and durability. The pseudo-titration in FIA systems can be used to determine acidity and alkalinity. However, it possesses a clear drawback in terms of process analysis: the necessity of calibration standards and frequent recalibration to correct flow rate variations. On the other hand, the system is fast and can provide almost real-time results for process control. A new approach to flow titration based on flow rate ratio has been described in which the principle of compensating errors is applied to avoid the lag time between the compositional change and its detection [54]. The system, when applied to a process stream with slow change in concentration of acid or base can reach unbeatable speed (3 s per titration), reproducibility (0.2–0.6% RSD) and titrant volume consumption (12 mL per titration). However, the system is based on flow rate ratios between titrant and sample streams and this is a source of weakness in the system.


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The adaptation of a true titration to the MSFA concept has also been demonstrated recently [55]. The analyser exploited in full the FB hybrid characteristic of the MSFA and the titration can be conducted in accordance with the IUPAC definition for titration. The sample volume (40–100 mL), titrant concentration and reaction stoichiometry are the only parameters needed for determination of acidity/alkalinity. A possible drawback of this type of flow titrator is in the relative long time necessary to perform a titration (2–5 min). Specific applications of flow systems for off-line determination of acidity and acid content in samples of industrial interest include the use of a monosegmented system for total acidity in vinegar [56], acidity in metallurgical solutions using MSFIA [57], total acidity in silage extract using MCFIA [58], total acidity in soft drinks and fruit juices using pseudo-titration in a SIA system [49,59], and, recently, a FIA pseudo-titration performed in non-aqueous media for the determination of free fatty acids in samples of palm oil [50].

4.4 Metallic species The industrial interests in the determination of metallic species is mainly associated with the necessity of effluent monitoring for contamination and, for metallurgical industries, with the quality of the final product and monitoring of alloying processes (see Figure 1). There is a myriad of works dealing with the automation of the determination of metallic ions in solution in low concentration by flow analysis, which can be adapted for monitoring of industrial effluents. However, there is a special series of works dedicated to the direct determination of solid alloys employing electrolytic dissolution coupled to FIA systems [4–6,60–62]. These works represent a significant effort in the direction of process control through the interface of flow systems for at-line monitoring of solid samples. A FIA system was proposed for electrolytic dissolution of solid steel samples and spectrophotometric determination of soluble aluminium in steels (0.01–0.13% w/w) [4] and molybdenum (0.7–2.7% w/w) [60]. Typical precision is in the order of 1–2%. These are single element determination. However, the same technique has been also coupled to ICP OES [6], electrothermal atomic absorption spectrometry [61] and plasma mass spectrometry [62] for multielement determination of metallic species in metallurgical products. The use of electrometric techniques, such as stripping voltammetry [63] can extend the concept to a more suitable analyser for at-line multielement analysis of solid metallurgical products.

4.5 Other industrial applications Other applications of flow analysis to solve industrial analytical problems can be found dispersed in the literature. The subject of applying SIA as an alternative approach to process analytical chemistry was reviewed some time ago [12] where its ability to adapt, without any deep change in the manifold, to the determination of many different analytes of industrial interest was highlighted.

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Although it is an unquestionable advantage of SIA over others flow analysis concepts, apparently the process engineers do not consider the flexibility as something extremely necessary. The culture of the industry still remains ‘‘one analyser for one analyte’’. FIA has been recently employed for monitoring paper mill industrial waters using a voltammetric electronic tongue. Principal component analysis (PCA) was employed to classification of samples while neural networks were used for the characterisation and evaluation of chemical demand of oxygen, conductivity and pH [64]. The on-line monitoring of alkali, sulfide and dissolved lignin during wood pulping has been evaluated in a FIA system by using attenuated total reflection-ultraviolet spectroscopy (ATR-UV) [65]. The FIA approach was effective in protecting the ATR probe from fouling in alkaline pulping liquors. The FIA approach has also been employed for the determination of surfactants in industrial products and wastes by potentiometric detector developed to selectively sense dodecylbenzene sulfonate down to 5 107 mol L1 [66]. Monitoring of dyes and dyebaths in the tanning and textile industries has been performed by using flow analysis. When the dye concentration of a dyebath is monitored in real-time, it allows the control of the rate of exhaustion and of chemical addition. Corrective actions can be taken, also in real-time, to ensure that the dyeing process is performing well. It has been demonstrated that the FIA determination of indigo in dyebaths is more rapid, precise and accurate than conventional titrametric methods [67]. Real-time measurement of dyes in a dyebath have been performed by both FIA and SIA, the major difference being in the sampling technique used [68]. SIA has been applied with second-order treatment for the determination of dye exhaustion in tanning effluents [69]. Linear calibrations were obtained in the 5–30 mg L1 range, with a correlation coefficients of 0.999 for each dye, and detection limits of 2.6, 3.9 and 2.1 mg L1 for Acid Red, Acid Brown and Acid Orange, respectively. Flow analysis has been used for the evaluation of the exhaustion of industrial degreasing baths based on the determination of total grease and surfactant contents [70]. The proposed system is an advantageous alternative to manual procedures and achieves the determination of both parameters in ca. 15 min, allowing for prompt decision about replacement or otherwise of the degreasing agents. The same problem has been recently addressed by using an ATR-FTIR membrane-based sensor for the simultaneous determination of surfactant and oil total indices [71]. The system is based on a liquid–liquid extraction of the analytes through a polymeric membrane into an organic solvent layer that is in close contact with the internal reflection element and continuously monitored. Samples are automatically processed using an on-line SIA system that is coupled to the FTIR. Multivariate calibration based on Partial Least Square (PLS) regression was employed to determine both parameters. Although rare, some reports can be found in the literature that describe the application of flow analysis in the polymer industry. In two relevant works, a FIA system was employed for polymer analysis of ethylene propylene diene monomer (EPDM) elastomers [72,73]. Solutions of the polymer were introduced into a flowing mobile phase which was monitored by an array of three


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detectors: a right-angle laser light-scattering unit, a differential refractive index detector and a differential pressure viscometer for the solution characterisation of EPDM elastomers.

5. CONCLUSION Flow analysis is a powerful tool for industrial process control and is playing a growing and significant role in PAT. In comparison with other process analysers, flow systems present the advantage of pre-processing the sample which makes their scope of applications wider. Flow analysis can also serve as a sample pre-treatment interface for common analytical instruments and detector systems employed in process analysis. The role of the sampling interface is to provide pre-treatment of the sample before it can be delivered to the main analysis system. Further research is necessary in this aspect of the use of flow analysis, in order to develop very robust interfaces that can operate unattended for long periods. Because robustness is an essential feature for a flow analyser used at-line or on-line in process control, significant further applied research is required to overcome the main drawbacks to long-term operation of these analysers. These drawbacks are associated with the dependence of the analytical result on the flow rate, the formation of gas bubbles due to the system operation or gas generating reactions and the requirement for recalibration using stable standards that are matrix matched to the sample. Only through the minimisation of these weaknesses in flow analysis will the concept become more acceptable to those responsible for monitoring of industrial processes and for the optimisation of their performance. The dependence of flow rate can be minimised by employing more robust devices. Inert syringe and piston pumps are more reliable than peristaltic pumps in terms of resistance to organic solvents and highly concentrated acids and bases. Peristaltic pumps are mechanically robust, but they require the use of flexible tubes which must be replaced periodically, and tubing wear is the main cause of drift in flow rate during long-term analyser operation. Strategies can be incorporated to the flow system in order to reduce or eliminate the dependence of the analytical signal on flow rates. This is perfectly achieved using hybrid systems such as MSFA and FB where the liquids are processed by a flow system up to a stage where the flow rates do not affect the analytical signal. This has been recently demonstrated for a FB system employed for the determination of bromine number and bromine index in petrochemical products [16]. It is also important to emphasise that sampling a known and reproducible volume of sample through a sample loop (as initially proposed by the FIA concept), is fairly independent of flow rate, while a volume sampled by timing a device such as solenoid valve will be dependent on the flow rate. The first must be the preferred choice whenever possible to better fit of the robustness criterion.

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The problems associated with providing appropriate and stable calibration standards is a more complex one. Some efforts have focused on the preparation of standards by dilution from a single stock solution of the analyte. This, in fact, can help when the stock standard is stable. When possible, the use of true stoichiometric-based analytical methods can totally overcome the requirement for many standards. For instance, the use of true flow titrations can preclude the use of multiple standards and if a coulometric method can be adopted use of standards will be totally unnecessary. In addition, the use of coulometric flow titrations ensures that the system is immune to changes in flow rates. Matrix matching between standards and sample must also be considered, especially if there are significant changes in the sample matrix during the industrial process. A major part of the problems associated with matrix mismatching in FIA, SIA, LOV, MCFIA and MSFIA systems is due to the interfering signals produced by differences in the refractive indices of injected zones and carrier (the schlieren effect) that occur because of the concentration gradients generated by dispersion. The CF, MSFA and FB concepts are less dependent on matrix matching because they produce homogeneous mixtures between sample and reagents prior to detection. Application of flow analysis can improve the performance of direct spectroanalytical methods such as infrared and NIR spectrometry, and electrochemical techniques such as voltammetry and potentiometry. However, its most valuable contribution to industrial and process analysis occurs when its potential for sample pre-treatment is fully utilised to provide the analytical information necessary for the process control. Nevertheless, even if sample pre-treatment is not required, the use of flow systems can confer greater advantages than direct reading of a process stream. These advantages are associated with automatic sample dilution, automatic cleaning of the flow-cell and in some cases, the exploitation of the sample gradient generated by dispersion in the flow manifold. The advent of miniaturised flow analysis systems (mTAS) may, in the future, allow for the entire analyser to be immersed in a process stream or batch reactor. Finally, some researchers suspect that information on the successful application of flow analysis to industrial process analysis is largely unpublicized and unpublished, because of commercial-in-confidence restrictions. While to some extent this may be true, the limitations of current flow analysis systems and methods to real industrial problems are recognised [22]. There is therefore great scope for reflection, innovative thinking and positive action in overcoming these limitations to effective flow-based process analysis.

ABBREVIATIONS ATR-FTIR ATR-UV BI BIA BN

Attenuated total reflection-Fourier transform infrared spectroscopy Attenuated total reflection-ultraviolet spectroscopy Bromine index Batch injection analysis Bromine number


CF EPDM FB MCFIA MSFA MSFIA NIR PAT PCA r-FIA SIA-LOV

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Continuous flow analysis Ethylene propylene diene monomer Flow batch analysis Multi-commutation flow injection analysis Monosegmented flow analysis Multi-syringe flow injection analysis Near infrared spectrometry Process analytical technology Principal component analysis Reverse flow injection analysis Sequential injection analysis-Lab-on-Valve

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CHAPT ER

22 Environmental Applications: Atmospheric Trace Gas Analyses Kei Toda and Purnendu K. Dasgupta

Contents

1. Introduction 2. Collection of Trace Gases 2.1 Membrane-based diffusion scrubber 2.2 Chromatomembrane cell 2.3 Drops, films and bubbles for the collection of gases 2.4 Multichannel scrubber 2.5 Collection of aerosol particles 3. Integration of a Gas Collector into a Flow Analysis System 3.1 Flow injection analysis system 3.2 Sequential injection analysis (SIA) 3.3 Hybrid flow analyser 3.4 In situ monitoring by stopped-flow 4. Flow System Miniaturization for Atmospheric Analysis 4.1 Micro gas analysis system (mGAS) 4.2 Conductometric system for ammonia 5. Illustrative Examples 5.1 Formaldehyde 5.2 Atmospheric H2O2 5.3 Atmospheric NH3 5.4 Atmospheric H2S and SO2 5.5 Atmospheric NOx 6. Applications to Breath Analysis 7. Ancillary Systems for Field Monitoring 7.1 Calibration of gas analysis system 7.2 Liquid flow control 8. Conclusions Acknowledgments References


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Kei Toda and Purnendu K. Dasgupta

1. INTRODUCTION Measurement of environmental samples is one of major application areas of flow analysis. Potable water, natural water and wastewater, soil leachates and pore water, atmospheric trace gases/particle samples have all been analysed by flow analysis. Water-soluble gases can be easily measured by coupling a suitable gas collector to transfer the gas to the solution phase. In this chapter, we describe how to collect gaseous species, how to measure gases and the application of such techniques to air analysis. Whereas many discrete samples are of interest in water analysis, the primary thrust in the analysis of air samples is automated continuous analysis. Techniques developed for trace gas analysis are useful not only for atmospheric analysis but also in many other areas, e.g., for analysing breath gases in clinical applications.

2. COLLECTION OF TRACE GASES In yesteryears, impingers and (impregnated) filters were the principal collectors for atmospheric gases as the first step to measurement. Filters have been used both in the active (flow-through) and passive (diffusion-based collection element) modes. Sampling with either impingers or filters takes a long time and with filters, laborious manual extraction processes are typically needed before measurement. For convenient measurement of air constituents, effective and simple gas collection techniques should be coupled to flow analysis [1–3]. Here we primarily concentrate on membrane-based gas scrubbers. Continuous wetted denuders have been extensively discussed elsewhere [2].

2.1 Membrane-based diffusion scrubber A membrane-based diffusion scrubber (DS) was first reported by Dasgupta [4]. Sample air and absorbing solution are separated by a gas permeable membrane, such as porous hydrophobic or an ionic hydrophilic membrane. Analyte gas molecules diffuse to reach the membrane surface, and then either (a) diffuse through the pores to be captured by the receptor solution on the other side [5,6] or (b) is captured by the hydrophilic membrane surface itself upon diffusion to the wall and then permeates through the polymer membrane to the receptor solution on the other side [4,7]. Theoretical considerations on mass transfer to the walls of a cylindrical tube, where the tube wall acts as a sink, were first presented by Gormley and Kennedy. For a cylindrical DS, if the tube wall behaves as a perfect sink, the collection efficiency f for an analyte gas of diffusion coefficient D (m2 s1) being sampled at a volumetric flow rate of F (m3 s1) is expressed as Equation (1) for a denuder of length L (m): f ¼ 1 0:819 expð3:657p DL=FÞ

(1)

The Gormley–Kennedy equation applies well to systems where (the stationary or flowing film on) the inner surface of the tube is a good sink for

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the analyte gas. Although qualitatively the same considerations apply to the membrane-based DSs, the membrane surface (especially a porous hydrophobic membrane surface) is far from a perfect sink for gases of interest. Some investigators have advocated the incorporation of a sink efficiency (e) parameter to compensate for this [8]: f ¼ 1 0:819 expð3:657pDL=FÞ

(2)

The value of e was determined experimentally (based on the best fit to Equation (2)) to be 0.3 for H2S with 0.1 M NaOH, and 0.02 and 0.1 for CH3SH with 0.1 and 0.3 M NaOH, respectively. Small gas molecules have high D values and are relatively efficiently captured in a scrubber collection system unless e is too low. The collection of large gas molecules, which obligatorily have much lower diffusion coefficients, is typically less efficient. Particles, the diffusion coefficients of which are orders of magnitude lower than those of gases, are essentially not collected by diffusion-based collection systems. The collection of gases can be made selective by the choice of the sink. For example, with an acidic scrubber liquid the collection of acid gases is inhibited due to a low e whereas that for basic gases f is enhanced and vice versa. As Equation (2) suggests, the collection efficiency increases with increasing scrubber length and decreasing sampling rate. While the cylindrical geometry is the simplest, other geometries are often more efficient. A rectangular or parallel plate geometry has been much studied. For rectangular parallel sink surfaces each with width W and S being the separation between the plates (S{W), f is given by Equation (3): f ¼ 1 0:914 expð7:54 DLW=FSÞ

(3)

For an annular denuder [9] in which both the outer wall of the inner tube (di) and the inner wall of the outer tube (do) are effective sinks, Ali et al. [10] derived Equation (4) by analogy with Equation (3) meant for parallel plates. The equation for an annular denuder is thus [11] f ¼ 1 0:914 expð7:54yDL=FÞ

(4)

y ¼ pðdo þ di Þ=ðdo di Þ

(5)

where

For the annular DS, however, only the inner tube wall is the sink and the outer wall is inert. No explicit equation exists to readily compute collection efficiency for such a system. Numerical solutions for the analogous heat transfer case were developed by Lundberg et al. [12], and methods to apply this to annular DSs have been developed [13,14]. Diffusion-based collectors with a flowing film of liquid as the sorbent are typically referred to as wetted denuders. Both cylindrical [15] and parallel plate [16] denuders collect ionogenic atmospheric gases of interest with high efficiency. Typically the collected analytes in the liquid effluent are concentrated by a preconcentration column and then introduced into an ion chromatograph to measure anions/cations. The ion chromatography (IC) method provides simultaneous

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measurement of various ions. However, if sensitive specific chemistry is available, flow analysis without preconcentration or separation is simpler and allows measurement with very little lag time. In this chapter, we have chosen not to discuss methods that involve chromatographic/electrophoretic separation in detail. In sensitive flow analysis methods for gases, a small scrubber liquid volume is desirable and the scrubber itself can be used as the sample loop of a traditional FIA analyser. The annular geometry, with a membrane tube in a jacket tube, where the liquid and the sample flows are through these respective channels, is often used for flow analysis-based atmospheric gas determination. It is possible to attain respectable collection efficiencies with the simple annular scrubber design. Porous hydrophobic membrane tubes are commonly used. Regardless of precise design, collection efficiency is importantly governed by the length of the membrane tube. The collection efficiency is a function of the tube length. By using two serial scrubbers with very different lengths, large dynamic range measurements by the same analysis system are possible, even though the dynamic range of the analysis system itself may be far more limited. Such a system is equivalent to simultaneously operating an instrument at multiple gain settings. This approach has been demonstrated for the measurement of H2S via its reaction with fluorescein mercuric acetate (FMA) [17]. The -SH group reacts with fluorescent FMA and renders it essentially nonfluorescent [18,19]. Two serial membrane scrubbers, comprising of porous polytetrafluoroethylene (pPTFE) tubes of effective lengths 1 and 30 cm, are used in series as shown in Figure 1 left. At the operative flow rates, the collection efficiency of the 1-cm scrubber is 1/10th of that of the 30-cm scrubber. This arrangement achieves both high dynamic range and good limit of detection (LOD) even with this quenching chemistry. Normally, high blank negative signal methods like this cannot simultaneously attain a good limit of detection and a large dynamic range. This is because to measure high concentrations, higher reagent concentrations are needed which results in a higher blank signal, increases blank noise and compromises the LOD. But in the serial scrubber system H2S can be measured over a four orders of magnitude concentration range, from 0.03 to 250 ppbv with a single flow line. Some selectivity can be brought about by the choice of the membrane as well. Whereas all molecules diffuse across the porous membrane, a hydrophilic Nafion membrane exhibits much less transport for the less polar CH3SH compared to H2S. Simultaneous measurement of H2S and CH3SH can thus be performed using parallel pPTFE and Nafion scrubbers arranged as shown in Figure 1 right. Such a system was used for the monitoring of the headspace of a septic tank, and the data were compared with cryocollection followed by gas chromatographic analysis with sulfur flame photometric detector. The two methods produced comparable data. However, the flow method was not only much more convenient and needed less capital equipment; it provided continuous measurement with good time resolution.

2.2 Chromatomembrane cell The chromatomembrane cell (CMC), introduced by Moskvin and Simon in 1994 [20] has unique structural features [21]. Comprised of a biporous membrane, the

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Figure 1 Flow-based sulfur gas measurements by using diffusion scrubbers (DSs). Instrument system for a wide dynamic range determination of H2S and SO2 (top left), and for the measurement of H2S, CH3SH and SO2 (top right). F, inlet filter; 3SV, three-way solenoid valve; IC, iodinated activated charcoal/soda lime column; OC, oxalic acid column; WS, water saturator; FM, flow meter; B, flow buffer bottle; P, air pump; PC, pressure control circuit; PS, pressure sensor; RB1, FMA reagent bottle; RB2, H2SO4/H2O2 reagent bottle; V, stop valve; LFR, liquid flow restrictor; FD, fluorescence detector; CD, conductivity detector; WB, waste bottle; PS, SPS and NS, gas diffusion scrubbers with pPTFE, short pPTFE and Nafion tubes, respectively. Response curves (a) and calibration curves (b) obtained with the dual diffusion scrubber/ detector (bottom left). In panel (a), the response of the short DS (1 cm) is shown as the solid line and that of the longer DS (30 cm) is shown as the dashed line. In panel (b), calibration curves are obtained with longer DS (&) and the shorter DS with (J) and without ( ) a reaction coil (0.86 mm i.d., 100 mm long). 1 mM FMA was used as the reagent throughout. Demonstration of simultaneous determination of sulfur gases in the headspace of a large septic tank (c). Lines are data obtained by the instrument and 10-min averages are shown as flat level lines. The bold lines are data obtained by the serial Tedlar bag sampling, Tenax preconcentration, and gas chromatography with flame photometric detection. The FMA concentration used was 10 mM. Reprinted from Ref. [17]. Copyright (2004), with permission from the American Chemical Society.

3

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absorbing solution is held in macropores while gases are sampled through the micropores. In the process, the sampled gas is captured in the absorbing solution. The absorbing solution is then pushed out by a carrier stream and introduced into the flow analysis system. Subsequently, the CMC has been applied for the measurement of NO2 [22,23], SO2 [24] and HCHO [25] by the Motomizu group. The sample air must be introduced into the CMC gradually. Because the absorbing solution volume held in the CMC is very small, even a small air sample volume is sufficient to provide a very good LOD. The air sample (typically 20 mL) is introduced by a syringe at a rate of 7 mL min1. Figure 2 shows the flow system used for NO2 measurements and typical signal peaks for

Figure 2 Schematic of an FIA system combined with a three-hole CMC and a response chart. RS, reagent solution (sulfanilamide (20 g L1) +/N-(1-naphthyl)ethylenediamine (NED) (0.5 g L1)+concentrated HCl (25 mL)); AS, absorbing solution (2 g L1 triethanolamine (TEA) solution); P1, double-plunger pump (each flow rate: 0.05 mL min1); P2, peristaltic pump (0.5 mL min1); P3, syringe type pump (7 mL min1); V1 and V2, six-way valve; SL, standard sample (NaNO2) loop; M, mixing joint; DG, degassing unit; RC, reaction coil (0.25 mm i.d. 50 cm); W, waste. The response curves are for 20 mL of 9, 18, 27, 36 and 60 ppbv NO2. Reprinted from Ref. [23]. Copyright (2002), with permission from Elsevier B.V.


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9B60 ppbv NO2. Though the sample volume is only 20 mL, good signals are obtained. The measurement time for one sample is 5–6 min and LOD is 0.9 ppbv.

2.3 Drops, films and bubbles for the collection of gases Drops of liquid provide unusual gas collectors that can provide the basis of in situ analysis for many gases. Alternatively, the drop can be sent back to the analysis system, which must obligatorily operate on a microscale. Once the measurement is done, the drop can be renewed by a flow system. This can be as simple as a reagent bottle placed at a height with a solenoid valve in the line. That a single drop can be used to monitor gas streams is a topic of general interest [26]. The broad topic of gas absorption by a moving spheroidal drop is of interest because this is the process that describes the washout of soluble gases from the atmospheric column [27]. Liu and Dasgupta first introduced the concept of a liquid drop as a gas sampling interface [28]. They collected ammonia in an acidic drop and then withdrew the drop into a capillary scale, electroosmotically driven sequential injection system for colorimetric analysis based on the indophenol blue reaction. Cardoso and Dasgupta [29] extended the concept to the collection and in situ measurement of NO2 using a supported liquid film/droplet using the Griess reaction. Films/drops supported on a loop are important microscale gas collectors that have been advantageously used with capillary scale analysis systems [30–33]. In situ electrochemical detection of gaseous hydroperoxides where the electrodes support the collection film has been reported [34] and similar electrochemical sensors have been reviewed [35]. Replacement of the film is carried out by a flow system. Recently breath ammonia was determined by a thin film of sulfuric acid held at the tip of two concentric tubular electrodes as shown in Figure 3; the titration of the acid by breath ammonia was continuously monitored conductometrically by the two electrodes [36]. Although most work has been done with stationary drops/films, continuously forming and falling drops can be used equally well. In situ measurement of low ppb levels of gaseous chlorine was demonstrated [37] by such a technique. Low levels of H2S and HCHO have been sensed in a single drop, respectively, by fluorometry [38] and colorimetry [39]. Cardoso’s group in Brazil have developed several drop-based analysis methods for HCHO [40], total aldehydes [41], SO2 [42] and NH3 [43]. The boundaries between a collection film and a drop is made diffuse by the more recently introduced continuous filmrecirculating drop approach [44] — such systems can provide an extremely high degree of sample concentration. The gas–liquid collector/equilibrator and responses in 0B50 ppbv NH3 are shown in Figure 4. Drop-based analysis has been reviewed by Liu and Dasgupta [45,46]. A bubble is a hollow film with a very large surface to volume ratio. It should therefore be an excellent gas collector/concentrator — direct conductometric measurement of sub-ppm levels of SO2 gas has been demonstrated for a nonionic soap bubble doped with H2O2 as oxidant to convert SO2 into conducting H2SO4 [47]. The soap bubble device and its performance is shown in Figure 5.

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Sample gas B A

D A

C D details

Air and liquid aspiration B Conductivity circuit

C Absorbing solution

Conductance Detector Output, V

Blank 0.8 18

38

0.6

900 700

60 500 300 200 129 85

85 129 0.4 0

200

100

300

Time, s

Figure 3 A liquid-film-based conductivity detector for breath analysis. (A) Outer stainless steel tube; (B) PTFE insulator tube; (C) inner stainless steel tube; (D) film-forming area. Lower panel shows response of the liquid-film NH3 sensor. NH3 concentrations: blank, 18, 38, 60, 85, 129, 200, 300, 500, 700 and 900 ppbv. Reprinted from Ref. [36]. Copyright (2006), with permission from the American Chemical Society.


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LO HS N2

T

LI S NI Gas out

SS

J

Gax in

C 49.4 pptv

60 min

24.7 pptv

12.3 pptv

"Zero Air" System blank no air sampling

Figure 4 A gas–liquid collector/equilibrator: N1, N2, 1/16 in. nuts; N3, 1/8 in. nut; T, PEEK tee; LI/LO, liquid inlet/outlet lines; S, PEEK sleeve; HS, heat shrink tube; J, jacket; C, bottom cap. The inset shows details of the stainless steel SS tube tip. Lower panel shows calibration plot in the 0–50 pptv region. Note that the first peak in a series always tends to read higher due to flow disruption when concentration is changed. Reprinted from Ref. [44]. Copyright (2000), with permission from the American Chemical Society.

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Figure 5 Bubble film conductometry. Experimental setup for bubble (upper). Bubble head BH produces a bubble in the box with retractable stainless steel electrodes E touching the bubble; water W is maintained at the bottom to humidify the bubble chamber; it also collects waste that is periodically emptied through a bottom port, not shown. To produce bubble, soap solution SS is aspirated through Teflon filter TF and delivered by solenoid valve pump SVP through capillary SC. Compressed air is then metered through mass flow controller MFC, through solenoid valve SV, and coiled elastomeric tubing CT. Lower panel shows temporal conductance traces of bubbles exposed to different concentrations of SO2 for 10 min. Electrodes were not washed between runs. Reprinted from Ref. [47]. Copyright (2006), with permission from the American Chemical Society.


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There is little real difference between gas/vapor collection into a drop by active sampling of ambient air or suspending a drop over the headspace of a sample and collection of the analytes by passive diffusion [48]. These techniques have come to be called single drop headspace microextraction techniques, and are becoming increasingly popular [49,50]. There is also no conceptual difference between analyte molecules being extracted into a drop from a gaseous matrix or from another, immiscible liquid matrix. Extraction of analyte molecules from an aqueous liquid into a nonaqueous liquid drop was first introduced over a decade ago [51,52]. This has now come to be known as single drop microextraction (SDME); it is widely used, and may sometimes have advantages over solid phase microextraction (SPME) techniques [53].

2.4 Multichannel scrubber In a membrane-based scrubber, a thinner solution layer is preferred to obtain a greater air to liquid concentration ratio. To make sampling more effective, it is desirable to use a wider membrane area. The use of a large membrane area and thin and uniform solution layer thickness needs structural support. A multichannel arrangement, most conveniently created by microfabrication, is often used [54,55]. The membrane is sandwiched by two plates on both of which channels are formed. The solution channel has a shallow depth: 0.2B0.5 mm. The channel for air is usually 1B3 mm in depth. Normally the air inlet is set perpendicular for facile machining. With such an air entry design, particles may deposit on the membrane surface. This scrubber is therefore particularly applicable for gas sampling in a nearly particle-free environment (e.g., microelectronics fabrication facilities) or when there is no likely interference from particle deposition or it is permissible to use a particle filter in the sampling line that would not result in removal of any gas of interest.

2.5 Collection of aerosol particles Aerosol particles play an important role in the atmospheric environment; the effect of particles to human health is often of equal or greater concern compared to those of pollutant gases. The total surface area provided by small atmospheric particles is large and this acts as nucleation sites and as sites for heterogeneous chemical reactions. The diffusion coefficient of even a 0.1 mm particle is 3–4 orders of magnitude smaller than typical atmospheric gas molecules of interest; as a result, particles are essentially not collected by diffusion-based collectors. Particles pass through a DS without reaching the collection surface. In other words, the DS is a selective collector for gas molecules. In contrast, particles can be collected on filters by active sampling. However, if so collected, the filter must be extracted with water to analyse by flow analysis. Instruments that use alternating filters (one is sampling while the other is being extracted and then dried) have been described [56,57]. Particles can also be collected continuously by impaction after growing them with steam [58,59]. The use of a continuously wetted packed bed has been explored but it is not very efficient [60]. Particles can also be collected by

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electrostatic means [61,62] but continuous extraction is not of course feasible: high voltage and conducting liquids are not compatible. Except in cases where the desired analyte cannot or does not occur in the gas phase, gases are generally removed before particle collection. Water mist, that helps continuously extract a hydrophobic [63] or hydrophilic [64] filter, has been successfully used. For example, aerosol Ce(III) was collected by steam condensation approach and measured by fluorometric flow injection analysis (FIA). The Ce(III) in the particle collector effluent was enriched on a weak acid cation exchanger preconcentration column and then eluted with 0.5 M H3PO4. Cerium(III) is strongly fluorescent, the fluorescence of Ce(III) was measured at 350 nm with excitation at 256 nm [65] with sub-pmol m3 detection limits. Aerosol protein was similarly collected, concentrated on a silica-based preconcentrator and measured by FIA after its reaction with Coomassie Blue G with low nanogram detection limits [66]. The measurement of strong acidity in aerosols was similarly accomplished. The particle collector effluent was concentrated sequentially on a cation exchanger and an anion exchanger, which respectively constituted the injection loops of cation and anion analysis subsystems. In the first system, non-H+-cations (primarily NH+4 ) were conductometrically determined as the corresponding hydroxide by elution with a strong acid plug with conductivity suppression using a hydroxide-form anion exchanger (concentrations are low enough that NH4OH is essentially completely ionized). In the second system, total (strong acid) anions were conductometrically determined by elution with a carbonate/hydroxide-based eluent and continuous suppression by a cation exchanger fiber suppressor. Aerosol strong acidity was determined on the basis of charge balance: Hþ equivalents present ¼ Sanion equivalents Snon-Hþ cation equivalents with an 8–10-min cycle, the LOD was 7–38 nmol m3 [67]. Aerosol Cr(VI) was collected on alternating filters. After sampling, the filter was washed with 10 mM NaOH, the extract neutralized with a membrane-based continuous cation exchanger (an alkaline solution will not be efficiently concentrated on a weak-base exchanger) and preconcentrated on a weak-base anion exchange preconcentrator column. Preconcentrated Cr(VI) was eluted by alkaline 0.1 M NaClO4 solution, reacted with sym-diphenylcarbazide (DPC) and the color was measured by a light emitting diode (LED)-based detector at 555 nm. Two sets of filter collection/extraction units were arranged in this system. While one collected the air sample, the other one was washed with NaOH and dried with clean hot air for the next 6.5 min so that it was ready for sampling at the end of the 15 min cycle. The LOD was 5 ng m3 for Cr(VI) [57]. Particle collection and analysis has been discussed in more detail elsewhere [68].

3. INTEGRATION OF A GAS COLLECTOR INTO A FLOW ANALYSIS SYSTEM 3.1 Flow injection analysis system A typical FIA system is conveniently applicable to air analysis in manual mode. After sampling, the absorber/scrubber solution, whether the sampling is


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conducted by an impinger, a denuder or a scrubber [69,70], can be injected into an FIA system. However, a fully automated arrangement, without manual intervention, is clearly preferred. Silva et al. [71] measured CO2 by a monosegmented flow system and conductivity detection. Air bubbles (100 mL each) were introduced from two sample loops, and CO2 was extracted into a water segment sandwiched by the air segments. The increase in the conductance of the intervening water segment was directly measured without liquid/air separation. Satienperakul [72] et al. determined CO2 by a colorimetric method with pH indicator. The gas sample (300 mL) was injected into an acidic solution stream, and passed across a porous membrane whence CO2 is transferred into an acceptor stream bearing a colorimetric indicator. In both of the above methods, the gas sample is directly introduced into the liquid stream. Such methods are suitable for analyte gases such as CO2, which typically exist in samples of interest at high concentrations. Aldstadt et al. developed a method for determining the gaseous arsenic compound trans-dichloro(2-chlorovinyl)arsine, commonly called Lewisite. This compound is of interest in remediation of hazardous waste sites as well as disposal of chemical warfare agents [73]. Lewisite in ambient air is collected into a NaOH solution in a membrane-based scrubber and becomes inorganic As(III) by hydrolysis. The collected As(III) is measured by flow-based amperometry with a wall-jet gold electrode. Takayanagi et al. developed a unique flow method for real-time atmospheric O3 determination [74]. Aged alkaline chromotropic acid (CA) reacts with O3 at the air–liquid interface and emits chemiluminescence (CL). The CL actually occurs from some derivative of CA, as yet not fully characterized, and not from CA itself. The CA reagent (0.5 mM) is rendered alkaline by NaOH solution and flows through a coil while exposed to UV irradiation; this results in the production of the active intermediate that produces intense CL with ozone. The solution bearing this intermediate flows on a nearly transparent wettable screen placed directly atop a transparent window behind which a photomultiplier tube (PMT) is located. Air is sampled into the enclosure in a manner that it directly contacts the solution layer and ozone in the air reacts to produce CL that is detected by the PMT. Even though it is a wet method, the response is very fast (B0.1 s) with high sensitivity (LOD 0.04 ppbv). The advantage of a continuous flow format, notably FIA, is that signals from discrete samples appear as peaks on a baseline. Much as with aqueous samples, a separate blank measurement is generally not necessary.

3.2 Sequential injection analysis (SIA) Although capillary scale SIA coupled to a drop collection system was introduced early on [28], few reports on gas analysis using SIA have appeared thus far. The biggest advantage of SIA is automated periodic gas analysis. Toda et al. [54] used three membrane-based serially connected multichannel scrubbers. In the direction of flow, the scrubbers utilized HCl, triethanolamine (TEA) and TEA absorbing solutions, respectively. The first collected HONO gas selectively and quantitatively. The second collected NO2. Prior to the third scrubber, the gas stream was made to pass through a KMnO4 bubbler to oxidize NO to NO2.

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The third scrubber then collected the NO2, originally present as NO. All gases were collected as nitrite. The absorbing solution was then aspirated into a syringe and transferred to a reactor. The three gases were measured by a single fluorometric system using the same diazo coupling reaction with C-acid (3-amino-1,5-naphthalenedisulfonic acid).

3.3 Hybrid flow analyser In the above two sections, we have separately delineated the merits of both FIA and SIA. Automation with SIA is simpler. Reagent consumption is also minimized in SIA: reagent is consumed only when needed. However, FIA is suitable for trace level analysis; it can provide a near continuous indication of where the baseline lies. To interpret the signal in an SIA system one must take into account the blank signal which can often be high due to the reagent blank and which can be further complicated by incomplete mixing and the Schlieren effect. In the so-called hybrid flow analyser [75], the strong points of both of these techniques are combined. Two syringe pumps are used to treat sample and reagent as shown in Figure 6. The majority of the time the pumps operate in FIA mode, pushing carrier solution and reagent solution to obtain signal peaks like in FIA. Good response signals with a small blank are obtained as shown in Figure 6. Aqueous and atmospheric ammonia was determined by this system and o-phthalaldehyde (OPA) chemistry. The time for one cycle was 8 min and LOD was 0.135 ppbv for gaseous NH3.

3.4 In situ monitoring by stopped-flow In flow-based analysis, the biggest component is a pump to deliver solutions at constant rates. If the system is operated in the stopped-flow mode, accurate flow control is not needed and the replacement solutions can be delivered by simple ways, e.g., gravity, etc. This greatly helps miniaturization of the analysis system to make it field-portable and affordable. One key operation involved is real-time monitoring of the chemical signal at the gas collector while no liquid flow occurs. The majority of the drop-based analysis systems operate in this mode. In the following, some of the others in situ detection systems used for gas analysis are introduced.

3.4.1 Long path length absorbance monitoring with simultaneous gas collection In flow-through solution phase absorbance measurements, the path length is rarely greater than 1 cm. Larger path length cells increase the sensitivity but typically so much light is lost to the walls that increase in noise wipes out any benefits. The advent of the liquid core waveguides (LCWs) that essentially behave as liquid-filled optical fibers permits the fabrication of small volume long path cells with good light transmission characteristics [76]. Amorphous Teflon fluoropolymer (Teflon AF) has become commercially available. It has a refractive index smaller than that of water and constitutes the basis for most LCW cells.

Figure 6 A hybrid flow analyser for ammonia determination (W1, W2, water; FL, fluorescence detector; DS, cylindrical diffusion scrubber; SV, solenoid valve (NC, NO, normally closed and open ports); SC, sample holding coil; MB1, MB2, mixed-bed resin columns; MC, open-ended mixing coil; AP, airpump; bottles A, B, reagents A, B.) and a typical system output (gas-phase samples: 0.00, 1.78, 4.15, 5.95, 8.19 and 10.75 ppbv NH3, 8 min per peak). Reprinted from Ref. [75]. Copyright (2006), with permission from the American Chemical Society.

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Teflon AF is highly permeable to many gases. If sample air passes on the outside of a thin-walled AF tube containing a gas absorbing chromogenic reagent, the analyte gas permeates through the AF material and reacts with the reagent to produce color. For example, chlorine was measured with acidic tetramethylbenzidine solution with a 15-cm AF 2400 tube (15 cm L 0.279 mm i.d. 0.533 mm o.d.) [77]. With much thinner wall custom-fabricated tubes, sensitive sub-second response times were demonstrated. The same work also demonstrated measurement of NO2 by the Griess reaction at levels relevant to ambient air. Subsequently differential measurement of NO2 and HONO by the same chemistry was shown to be feasible by the same chemistry. This was based on their different permeabilities through Teflon AF with two different tubes of somewhat different dimensions being simultaneously used [78]. A Teflon AF based LCW tube is useful not only for absorbance measurements but also for luminescence measurements [79]. Gas phase hydrogen peroxide has been measured at the parts per trillion level by a chromogenic Ti(IV)-porphyrin reagent using discrete scrubbers and a 5 cm LCW absorbance cell [80]. If the dissolved solids content of an aqueous reagent is very high, the refractive index of the solution can be higher than that of a standard fluorocarbon, notably fluorinated ethylene propylene copolymer (FEP Teflon). Recently Teshima et al. took advantage of this to construct an LCW cell with an FEP Teflon tube [81]. Acetone in breath was sampled. The chromogenic reagent formulation contained a very high concentration of NaOH. The solution refractive index exceeded that of FEP Teflon and permitted the use of a 10 cm FEP Teflon tube as an LCW [81]. The LCW cell is also particularly amenable towards luminescence detection. CL generated within the bore of an LCW [82] (or fluorescence generated by photoexcitation transverse to the axis of the LCW tube [79]) is effectively collected by a photodetector, often coupled by an optical fiber, placed at the end of the tube. Despite its many virtues, Teflon AF is very expensive and the manufacturer puts on restrictive regulations governing its use. Porous Teflon (pPTFE) tube and porous polypropylene (pPP) tubes were examined for the purpose of simultaneous gas collection and absorbance measurements [83]. Light transmission through the porous tube is not as good as that of a Teflon AF tube. Still, light transmission is sufficient to use several centimeters of a porous polymer tube as the absorbance cell. Comparison of light transmission is shown in Figure 7a. Though a long cell is not practical with the porous tubes, response with a 5 cm porous tube cell is actually both much larger and much faster than that with commercially available Teflon AF tubes (Figure 7b). Using the porous membrane tubes, both continuous flow and stopped-flow modes were tested. Absorbance (A) obtained is proportional to the optical path length (L) in the stopped-flow mode (Equation (6)) and to the square of the length (L2) in the continuous flow mode (Equation (7)). A ¼ 4kLCg t=d

(6)

A ¼ kpdL2 Cg =F

(7)

Relative light intensity


1

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(a)

0.1 0.01 1E-3 1E-4 0

50

100 Tube length (mm)

150

200

0.8

Absorbance

(b) 0.6 170 mm AF tube 0.4 0.2

50 mm PP tube

40 min

0.0

Figure 7 Light transmittance characteristics through water-filled tubes. (a) Light attenuation as a function of tube length: E, Teflon AF 2400 (1.064 mm 1.270 mm); K, Poreflon (2 3 mm); ’, Poreflon (1 mm 2 mm); 7, ePTFE (1.016 mm 1.270 mm). (b) Despite poorer light throughput, porous membrane tubes provide better performance than Teflon AF as a result of their substantially superior gas transport properties. Dashed line indicates the start of exposure to 240 ppbv O3. Its concentration was 2.5 mM. Reprinted from Ref. [83]. Copyright (2003), with permission from the American Chemical Society.

The constant k is dependent both on the diffusion coefficient of the gas and the effective sink efficiency that governs the gas uptake by the membrane; e is the molar absorptivity, Cg the gas concentration, t the stop time, d the tube diameter (assumed to be much greater compared to the wall thickness) and F the solution flow rate. Faster and larger response was obtained by stopped-flow compared to continuous flow. Figure 8 shows responses in the two modes of stopped-flow operations. The particular chemistry resulted in decrease of absorbance as ozone reacted with an indigotrisulfonate dye. The data on the left were obtained when the liquid refill was programmed to occur soon as a preset low absorbance value (ca. 0.3 AU) was reached, using a simple voltage comparator based circuit. The refill frequency was higher when the gas concentration was high. Interestingly, this mode used less reagent. The total volume of the liquid waste generated was a direct measure of the average ozone concentration. The right panel data were obtained with constant period of refill (9.5 min sampling and 0.5 min refill). Here, the decrease in absorbance of the composite waste solution from that of the original reagent was a measure of the average ozone concentration.

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VT L1 RB

ST T F

DF PD

L2 SF

T

MT

SV

Control Electronics Display Data Acquisition

W 0.3

0.34 (a)

(b)

0.2

Absorbance

0.32

25 ppbv

25 ppbv

58 75 ppbv ppbv 40 min

20 min 0.28

63 ppbv

0.1

0.30

160 ppbv

0.0

Figure 8 Schematic diagram of the ozone collector detector system and its responses. The liquid was made to flow by gravity and flow on/off was controlled by a solenoid valve, SV. RB, reagent bottle; T, plastic tee; MT, 1.75 mm i.d. 2.40 mm o.d. Accurel PP tube; W, waste; L1, L2, red (600 nm) and IR (850 nm) LEDs; SF, large optical fiber (core 1.5 mm), source fiber; DF, detector fiber; PD, photodiode/op-amp; VT, charcoal vent trap; F, 25-mm laptop PC style suction fan; ST, 30-cm3 syringe body, all was housed in an opaque plastic enclosure. Response at three different concentrations with automated 30 s reagent refill period (a) as a set absorbance (B0.3 AU) is reached and (b) after a preset time of 9.5 min. All data are in stopped-flow mode. Air flow velocity B100 cm s1. Reprinted from Ref. [83]. Copyright (2003), with permission from the American Chemical Society.

4. FLOW SYSTEM MINIATURIZATION FOR ATMOSPHERIC ANALYSIS A miniature flow analysis system has many advantages: small reagent consumption, fast response and applicability to situations where only small


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sample volumes are available [84]. Even for gas analysis, there are practical merits as shown below. Ohira developed a microsystem for SO2 analysis in 1990s. Two glass plates, on each of which 25 sets of microchannels were fabricated, were used to sandwich a gas permeable flat membrane with the channels aligned with each other [1]. Two sets of platinum conductivity electrodes were fabricated on the glass plate in the liquid flow channel. Conductivities of upstream and downstream of the gas absorbing area were compared and converted into SO2 concentration. This system was difficult to fabricate at that time. The same authors subsequently developed an SO2 determination system with the channel formed with a Teflon gasket and operated in the stopped-flow mode without any liquid pump [85]. Platinum electrodes were microfabricated in the very small scrubber volume (only 800 nL). This system has been successfully applied to near-real time measurement of SO2 at the Aso volcano museum (Japan). The Korenaga group has developed microdevices for fluorometric measurements of NO2 with 2,3-dimethylnaphthalene (DAN) [55] and SO2 with N-(9acridinyl)maleimide (NAM) [86]. In both devices, a porous glass plate (1 mm thickness) [87] was used for gas collection.

4.1 Micro gas analysis system (mGAS) Toda’s group recently introduced a micro gas analysis system (mGAS) [88]. There has been much effort on the development of micro total analysis systems (mTAS). This is especially true for bioanalyses, because reagents can be very expensive and only small amounts of samples are generally available. Although dubbed a total analysis system, in most cases it is only the mTAS chip itself that is small in mTAS. The liquid handling device and detection systems are not necessarily small. A true miniature gas analysis system includes gas sampling, gas collection, liquid handling and detection subsystems, all in an integrated miniature package, hopefully with the particular merit that it is easily fieldable. For true fieldability, the entire system should be operable by a battery in the field. Any really useful field gas analysis system must be sufficiently sensitive to measure low ambient concentrations. The first mGAS contained a zigzag microchannel scrubber comprising of a 200 mm wide 50 mm deep 200 mm long channel in a polydimethylsiloxane (PDMS) block, covered with a thin PDMS film. A technique to prepare a very thin PDMS film on the microchannel was developed. The channels were covered with a custom-made 7 mm thick PDMS film. Gas flux into the receptor is inversely proportional to the membrane thickness and such an ultrathin membrane provides a very good gas transport. The gas molecules permeate through the membrane and accumulate in a solution held in the channel. In other words, a thinner solution layer is preferred for high sensitivity measurements. The collected analyte concentration in the solution Cs is inversely proportional to the solution layer thickness d and membrane thickness t according to the

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following equation. Cs ¼

k0 Cg T k Cg T ¼ d t d

(8)

Here, T is the collection time, k the permeation constant of the membrane material and ku is apparent permeation constant for the membrane. A superior mGAS has since been developed [89]. The microchannel was arranged as sets of hexagons like the top of a honeycomb as shown in Figure 9a. The solution flowed through the microchannels in a manner that it was spread over the whole area. This device had a wider collection area than the previous design and provided excellent performance with thin porous flat membranes that facilitated gas transport. Liquid flow into the scrubber was accomplished by micropumps that were driven by 200 mA pulses (20% duty cycle at 1 Hz). Two systems for H2S and SO2 measurement were arranged in a single 10 cm 9 cm plate. This included liquid micropumps, a fluorescence detector for H2S, a conductivity detector for SO2 as well as scrubbers and a zero-gas generation bed as shown in Figure 9b. The instrument alternated between zero and measurement modes under the control of a three-way solenoid valve attached to the bottom of the plate unit. Figure 9c presents the simultaneous response from the two detectors obtained for a mixture of 2 ppbv H2S and 10 ppbv SO2. Zero and sampling times were 3 and 2 min, respectively, and data were obtained every 5 min. This operation was suitable for low-level gas measurements, as zero drift was continuously checked. Truly continuous measurements could also be performed without sending the instrument to the zero mode. The LOD for H2S was 0.1 ppbv, and that for SO2 was 1 ppbv without large interferences [89,90].

4.2 Conductometric system for ammonia Continuous SO2 determination by conductometric measurements was discussed above. Ammonia is a basic gas that can also be determined by conductometry. Conductometric breath ammonia sensing by a thin acid film has already been discussed [36]. Timmer et al. developed a microdevice for ammonia measurement [91]. Ammonia was collected into a NaHSO4 solution through a membrane, and then transferred to a selector where NH3 was regenerated by alkalinization with NaOH and the liberated ammonia was collected into water and the conductivity from NH+4 and OH thus generated was measured [91]. The outline of the device is shown in Figure 10a–c. The signal trace for stepwise decrease in NH3 concentration from 9.8 to 0.3 ppmv NH3 is shown in Figure 10d. Selectivity to NH3 was achieved by collection of NH3 in an acidic solution and re-vaporization by basification. The same group developed another microdevice for conductometric NH3 determination [92]. Air and purified water were introduced into a microchannel together. Good mass transfer took place between the air and the liquid segments in a small channel and water-soluble species were transferred to the liquid phase.


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Figure 9 Micro gas analysis system (mGAS). (a) Honeycomb structure microchannel scrubber, (b) flow diagram of the whole mGAS for the determination of H2S and SO2 and (c) responses for a mixture of 2 ppbv H2S and 10 ppbv SO2. SCB, sodalime/charcoal bed; MP, micropumps; HS, honeycomb scrubbers; FD, fluorescence detector; CD, conductivity detector; 3SV, 3-way solenoid valve. The bottom data were obtained with a solenoid valve operated to have a 3 min zero signal and a 2 min sampling period. Reprinted from Ref. [89]. Copyright (2005), with permission from the Royal Society of Chemistry.

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Figure 10 Conductometric device for NH3 measurement. (a) Cross-sectional view of the device, (b) glass chip for the separator, (c), conductivity electrode and (d) response to 9.8B0.3 ppmv NH3. Reprinted from Ref. [91]. Copyright (2004), with permission from Elsevier B.V.


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This method is similar to the monosegmented CO2 measurement method described previously [71]. A flow restrictor in the liquid line ahead of the conductivity detector and a PTFE membrane were incorporated before the gas outlet to introduce the bubble-free solution selectively to the detector. Measurement of 0.6–9.4 ppmv NH3 in N2 was demonstrated.

5. ILLUSTRATIVE EXAMPLES Many of the above-described methods and instruments based on them have excellent performance and are applicable to real measurement problems and have been so used. Near-real time trace gas measurements have been performed by flow-based analyses and the application base continues to grow. Selected examples are discussed here.

5.1 Formaldehyde Formaldehyde plays a paramountly important role in atmospheric chemistry. Extensive field measurements of atmospheric HCHO have been performed by several groups. Fluorometric methods are preferred for high sensitive measurements. Dasgupta’s group started with 2,4-pentanedione [93,94] and several data intercomparisons with other direct spectroscopic methods (tunable diode laser spectroscopy, Fourier transform infrared spectroscopy, etc.) have been published [95–97]. They subsequently used 1,3-cyclohexanedione (CHD) [98] to get greater sensitivity and also used it in the field [99] with LCW-based fluorescence detectors. However, they returned to the pentanedione reagent [100] because of small but discernible interferences in the CHD method from H2O2 for samples with very high H2O2 to HCHO ratios. Others have also used pentanedione as the preferred fluorogen [101]. Reactions with 5,5-dimethylcyclohexane-1,3-dione [70] have also been attempted for high sensitivity measurements; interferences, if any, have not been characterized. Detailed trends in atmospheric HCHO levels have been successfully monitored by flow analysis [96,99]. Air was sampled by a Nafion membrane DS and the collected HCHO was converted to fluorescent diacetyldihydrolutidine by adding ammonium acetate and pentanedione and putting the mixed stream through a heated reactor en route to a fluorescence detector. The instrument is shown schematically and photographically in Figure 11. The HCHO level decreases in the nighttime and increases in the daytime: much of the HCHO is secondary photochemical product in the atmosphere formed from the reaction of terminal olefins and ozone. Formaldehyde in the background air largely originates from the oxidation of isoprene, the dominant hydrocarbon emitted by vegetation. A typical diurnal trend in Houston is shown in Figure 12 [97]. This work compares the summertime HCHO levels in five major U.S. metropolitan areas, Nashville, Atlanta, Houston, Philadelphia and Tampa and examines their diurnal pattern. Observed HCHO levels were the highest in Houston, reaching

662 Kei Toda and Purnendu K. Dasgupta

Figure 11 Liquid phase flow schematic of the HCHO analyser. DS, diffusion scrubber; V, two-way, four-port valve; I, liquid phase injector (200 mL volume); T1, T2, mixing tees; A, 10 mM H2SO4; B, ammonium acetate buffer; P, pentanedione reagent; DP, debubble port (normally closed, used for removing a trapped bubble in the detector by injecting methanol); D, fluorescence detector; CR, capillary restrictor to apply backpressure on detector. Reprinted from Ref. [100]. Copyright (2005), with permission from Elsevier B.V.


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Figure 12 HCHO mixing ratio measured at Houston regional monitoring site-3 by the Nafion DS instrument vs. HCHO mixing ratios measured in La Porte by a differential optical absorption spectrometer operated by the University of California at Los Angeles with two different beam paths. The tic marks correspond to midnight. Sundays were August 20, 27, etc. The ME beam path is 475 m at 2 m height; the WT beam path is 1.9 km at an average height of 23 m. Reprinted from Ref. [97]. Copyright (2005), with permission from the American Chemical Society.

a maximum level close to 50 ppbv while the overall median mixing ratio was 3.3 ppbv. In contrast, in a mid-size city in Japan, Kumamoto, which is near the coast, the daytime HCHO mixing ratio is several ppbv and decreases at night to reach below 1 ppbv in the morning [102]. Not only on the ground, but it is also possible to analyse vertical and horizontal distribution of HCHO by deploying flow-based HCHO measurement systems in an aircraft [96,100]. Formaldehyde levels generally decrease monotonically with altitude; photochemically active plumes from tall stacks constitute an important exception. Aside from atmospheric chemistry, HCHO is a carcinogen and is therefore of interest as a health hazard. Relative to ambient air, HCHO levels are generally elevated indoors; offgassing from compressed wood products, particle boards and other building materials are considered to be a particular problem in residential settings. Formaldehyde, at least in part, contributes to the so-called sick house syndrome. In Japan, the maximum permissible HCHO level is 80 ppbv in indoor air. But even in a concrete laboratory setting where there are few offending wood products, typical HCHO levels are of the order of 10 ppbv. In this work, methyl-2-benzothiazolone hydrazone (MBTH) was used for the

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Figure 13 Indoor monitoring of HCHO. HCHO was measured in the laboratory (w 7.2 m d 8.5 m h 3.3 m). A small gas stove was turned on at 12:02, and at 16:36 it was turned off and the windows were opened. The symbol indicates the HCHO values automatically taken every 5 min by the flow-based instrument. The horizontal lines were obtained every 30 min by a batch process based on the AHMT (4-amino-5- hydrazino-3-mercapto-1,2,4-triazole) method. Reprinted from Ref. [102]. Copyright (2005), with permission from Elsevier B.V.

colorimetric measurement of HCHO. Formaldehyde was collected with two DSs, comprised of pPP membrane tubes, connected in parallel. When one DS collected HCHO, the carrier solution went through the other for determination. Thus high sensitivity and high throughput were obtained even with colorimetry. When a small gas stove was turned on, the HCHO level started to increase and reached over 30 ppbv. When the stove was turned off and all the windows opened, the HCHO level decreased abruptly to return to the original level as shown in Figure 13 [102]. Concurrently, HCHO was measured by a conventional method; HCHO was collected by an impinger containing a solution of 4-amino-3-hydrazino-5-mercato-1,2,4-triazole. The results from the two methods agreed well but the flow-based method provided higher sensitivity and much better time resolution.

5.2 Atmospheric H2O2 Hydrogen peroxide is one of the more important atmospheric oxidants; it is particularly involved in the oxidation of dissolved SO2 to form H2SO4. Fluorometric flow-based methods have been used from early on for the measurement of gas phase H2O2 [93,103]. A typical reaction involves the oxidation (often oxidative dimerization) of a nonfluorescent substrate RH to fluorescent R-R by hydrogen peroxide, mediated by a catalyst. A peroxidase enzyme has been most commonly used but photocatalysis is also applicable [104]. Many inexpensive heme protein preparations can substitute peroxidase enzymes very effectively [105]. Like HCHO, H2O2 is collected with high


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efficiency by a hydrophilic Nafion membrane DS [106]. Generally these methods cannot differentiate between H2O2 and the H2O2-HCHO adduct, OHCH2HO2, hydroxymethylhydroperoxide (HMHP). However, with such a membrane, collection efficiency for methylhydroperoxide (MHP) is very low compared to that for H2O2 or HMHP and the latter two can be selectively determined [107]. The attainable LOD for H2O2 with a liquid core waveguide LED excited fluorescence detector is excellent — in the low double digit parts per trillion level with thiamine as the substrate that is oxidized to fluorescent thiochrome [106]. It is also possible to measure the collected H2O2 with excellent sensitivity by luminol CL using a LCW-based CL detector [107]. In a subsequent paper [108], these authors used a dual scrubber system: a Nafion scrubber that collects only H2O2/HMHP and a porous membrane scrubber that collects MHP as well. The Nafion DS effluent was processed with hematin catalyst chemistry that responds only to H2O2/HMHP while the porous membrane DS effluent was passed through a MnO2 catalyst that selectively destroyed H2O2/HMHP. The MHP in the latter effluent was then measured with a peroxidase catalyzed system. The system used a unique dual fluorescence detector based on two alternately pulsed LEDs where the emitted fluorescence from each cell was collected by optical fibers and brought to the same photodetector. The signals were then separated by software. The system schematic, raw and separated detector signals, typical response and field data are shown in Figures 14–16. Peroxide measurement instrumentation has been put on aircraft, even on airships (blimps) [109]. Huang et al. collected peroxide by a glass coil collector [110]. In this case, all water-soluble species are collected into scrubbing water. After collection, the scrubbing water is injected into an FIA system. Firstly, the injected sample goes through a reversed phase miniature column to separate the peroxides. The peroxides react with p-hydroxylphenylethanoic acid in the presence of horse radish peroxidase (HRP) to form a fluorescent dimer [102]. Over an extended measurement period it was confirmed that the H2O2 mixing ratio ranged from 0.18 to 6.96 ppbv, higher in summer and lower in winter. Organic peroxides were found only in summer (MHP 0.07–0.29 ppbv in June–September, HMHP 0.07–0.34 ppbv only in September). Atmospheric organic peroxides concentrations appeared to be related to temperature as well as H2O2 levels and increased above 251C. Unlike H2O2, the organic peroxides exhibited a positive correlation with relative humidity. The peroxides contained in rainwater were also examined. In summer, the H2O2 level was sometimes over 50 mM and only in summer were MHP and HMHP detectable (found at levels r2 mM) in rainwater.

5.3 Atmospheric NH3 Ammonia is by far the dominant gaseous atmospheric base and is highly water soluble. It can therefore be collected into water easily. Genfa and Dasgupta [111] developed an FIA system for aqueous NH+4 ion using the reaction with OPA. The OPA reagent reacts with NH3 or primary amino acids and sulfite as a reducing agent to produce fluorescent products. The chemistry was used with a porous membrane DS with optional in-loop preconcentration to measure

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Figure 14 Analytical system schematic. N, Nafion Tube; E, ePTFE Teflon membrane tube; V1, V2, six way injection valve; P, peristaltic pump; AP, air pump; AC, activated carbon column; SV, three-way solenoid valve; FM, flow meter; NL, 23 gauge hypodermic needle (supplementary flow); FC, Flow controller; F1, F2, flush/debubble ports (normally closed); TP, trap column packed with active carbon to protect reagents and water from contamination; T1–T4, mixing tees; M, MnO2 packed column; R1, R2, thermostated reactors; D1, D2, LCW fluorescence detectors. Reprinted from Ref. [108]. Copyright (2003), with permission from the American Chemical Society.

atmospheric NH3 with an LOD of 45 parts per trillion by volume (pptv) [112]. Others have adapted and refined this further and showed that sensitive measurements with good time resolution were possible [113,114]. With careful thermostatting, the latter authors reported an LOD of 10 pptv with a time resolution of 10 min and concluded that with some further work, it would be possible to lower the LOD further. Later these types of instruments, along with wetted annular denuders, have been used to measure concentrations of gaseous ammonia across the Tenerife islands to assess the effect of cloud processing on the marine budget of reduced nitrogen compounds [115]. The continuous filmrecirculable drop collector [44] is hard to surpass in terms of its abilities to preconcentrate, however; an LOD of 4.5 pptv was reported.


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Figure 15 Instrument response. (a) PMT response as seen by the PC, consisting of the composite signal from two channels. The sample contained 2.0 ppbv H2O2 and 3.4 ppbv MHP. The lower and upper envelopes of the composite trace constitute the individual signals for H2O2 and MHP, respectively. (b) Software-isolated signal for the two channels. Reprinted from Ref. [108]. Copyright (2003), with permission from the American Chemical Society.

This same ammonia determination chemistry was coupled with a wet effluent diffusion denuder to measure atmospheric NH3 together with HNO2 and HNO3, the latter two being measured by anion chromatography [116]. The measurements were made in a grassy area near agricultural fields where NH3 was in excess over HNO3. One week of data are shown in Figure 17. Daily maxima of NH3 were 10B50 ppbv, much higher than those of HNO3 (0.4B0.9 ppbv). Ammonia reacts with HNO3 to form aerosol NH4NO3.

5.4 Atmospheric H2S and SO2 Sulfur gases are important pollutant gases. The acidic sulfur gas, SO2, is emitted during combustion of fossil fuels and is also produced in the atmosphere by oxidation of reduced sulfur gases such as H2S, CH3SH and dimethyl sulfide, etc. These divalent sulfur gases are mostly produced by natural biogenic activities. Sulfur dioxide adversely affects respiratory health. Sulfur dioxide is also the major precursor to acid rain. Sulfuric acid nuclei thus formed serve as cloud condensation nuclei (CCN) and play an important role in global climate issues. Monitoring of SO2 and reduced sulfur gases is therefore important.

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Figure 16 (a) System output for 2 ppbv H2O2 and 0.23–3.44 ppbv MHP. 3-min sample, 7-min zero. (b) H2O2 and MHP data from Philadelphia, PA, June 27, 4:00 pm to July 31, 7:40 am. Reprinted from Ref. [108]. Copyright (2003), with permission from the American Chemical Society.


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Figure 17 Concentrations of NH3, HNO2 and HNO3 during the first campaign from August 31 to September 6, 1992. Measurements were made at two heights above the ground: 3.20 and 1.60 m, respectively. Reprinted from Ref. [116]. Copyright (1996), with permission from Elsevier B.V.

While anthropogenic emissions of sulfur gases are of greater global concern, volcanoes are a major localized source of sulfur gases in some countries, including Japan. Volcanic gases SO2 and H2S were monitored for two years at Aso Volcano Museum, 1 km west of the fumarole of Mt. Aso, Japan [117]. The relationship between the gas composition/concentration and volcanic activity was investigated. When the volcano was active and nearing eruption, the concentrations of both gases increased. In addition, the ratio of SO2 to H2S increased. This was probably because of increased temperature, the oxygen bound in rocks was set free to oxidize H2S. A gas distribution map around the fumarole, based on a field-portable analyser carried around the crater, is shown in Figure 18 [17]. There was strong wind from 10 to 15 m s1 on that day. High concentrations were observed near the fumaroles. Downstream of the fumaroles, both gases were observed at high levels at a particular point where air flow was blocked by a cliff wall and flowed down a small ravine. Interestingly, the SO2/ H2S ratio increased downstream of the fumarole due to the oxidation of H2S to SO2, even within the short transport time of only several minutes. Soil sediment might be an important natural source of sulfur gases, especially marine sediments. Sulfate, which is the second major anion in sea water, is supplied to the coastal tidal flats twice a day. Azad et al. [118] monitored the emission of H2S and SO2 from tidal sediment in Ariake Sea, Japan. Ariake Sea is

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Figure 18 Walk-around monitoring of H2S and SO2 carried out around Mt. Aso (Japan) on November 12, 2002. The dotted line indicates the route the instrument was carried, and the black and grey solid lines are SO2 and H2S concentrations along the route, respectively. S, start point; N, Nakadake peak; P, Sunasenri plains; E, end point; F, active fumarole. Reprinted from Ref. [17]. Copyright (2004), with permission from the American Chemical Society.

a unique closed sea and there is a huge tidal flat (207 km2, B13% of the sea area). Sea water cannot be seen from the seashore in the low tide period due to large tidal flat as shown in Figure 19. The researchers brought a portable flow-based gas analyser to the tidal flat and investigated seasonal and daily variations in H2S and SO2. Both gases are emitted in higher concentrations in the summer. Interestingly, the two gas emission patterns have different features. The emission of H2S is the highest in nighttime with silty mud being the dominant source. However, the SO2 emission is the highest in daytime with sandy sediments being the dominant source. This was the first study to show that SO2 is emitted from marine sediments, while most extant literature regard sediments as only a sink for SO2, not as a source.

5.5 Atmospheric NOx There are several oxides of nitrogen and related compounds like HONO and HNO3 that are present in the ambient atmosphere. There is frequent interconversion among these species; it is therefore important to monitor each component simultaneously for atmospheric chemistry studies. The Griess– Saltzman reaction, sometimes called the Griess reaction is simple and useful chemistry for nitrite. It has been much used for the measurement of gaseous NO2 [22,23,29,79].

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Figure 19 Sulfur gas flux measurements (mgS m2 h1) at tidal flat of the Ariake Sea, West Japan. (a) Silty muddy and muddy sandy tidal flats are shown in the map. (b) The campaign site in the tidal flat at Higashiyoka. Measurements were performed during the day at Higashiyoka from February to October 2004; and at Sumiyoshi from April, 2004 to January, 2005. The seasonal variations of sulfur fluxes as H2S and SO2 from (c) muddy and (d) sandy sites are shown below. Note that the bars for H2S emission are enlarged ten times. In each campaign, the measurements were performed 10 times. Reprinted from Ref. [118]. Copyright (2005), with permission from Elsevier B.V.


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As previously mentioned, HONO, NO2 and NO were measured by SIA [54]. In an intercomparison, data for NO2 and NO were concurrently obtained by a O3-CL based commercial instrument. For the most part, the SIA and the CL data agreed well (Figure 20). For the NO results, the SIA data sometimes lagged behind the CL instrument results, apparently because of memory effects. One potential solution is to use a dry sorbent for the selective removal of NO2 and a similar low-memory (presumably dry) converter for NO to NO2, the latter conversion can be accomplished by ozone introduction. The three gases were monitored for a week. The data showed that HONO level increased just after rain, presumably due to hydrolytic conversion of NO2 followed by release as the water evaporated. Zhou et al. used 2,4-dinitrophenylhydrazine (DNPH) for the measurement of HONO collected with a segmented flow coil collector [119]. The product absorbance was measured at 309 nm. DNPH itself has absorption at 309 nm and DNPH reacts with aldehydes as well. After the reaction coil, the products were preconcentrated on a C18-silica column placed in the sample loop and then separated on a miniature C18-silica column (4.6 mm 5 cm) with 35% acetonitrile as an eluent. Peaks appeared in the order of unreacted reagent, nitrite derivative and formaldehyde derivative. A later effort by the authors utilized the Griess–Saltzman reaction with N-(1naphthyl) ethylenediamine and sulfanilamide [120]. The system was similar to the DNPH-based system, but used two coils; one measured HONO directly and the effluent from the other was coupled with a Cd-reduction column to additionally measure HNO3. A similar approach has been advanced much earlier 60 40 20 0 19:39

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by other authors where a wet denuder effluent was preconcentrated, NO 2 and NO separated on a low-pressure ion exchange column and put through a 3 Cd-reduction column prior to the Griess reaction and colorimetric detection [121]. The more recent system is shown in Figure 21. The product was measured at 540 nm. Figure 21 right panel shows one week measurement of HONO and HNO3 by the flow-based system together with NOx measurements by a commercial CL-based analyser. The HONO concentration reached up to 1.7 ppbv in the early morning due to overnight accumulation and local formation during the rush hour. Nitrous acid is decomposed by photolysis and HONO reaches a minimum of 0.1 ppbv in the afternoon. On May 3, the afternoon was cloudy; the HONO minimum level (B0.4 ppbv) was higher than other days. The ratio of HONO/NO2 was between 0.02 and 0.03 and the authors suggested that the heterogeneous hydrolytic conversion below was the dominant process for the formation of HONO during the measurement campaign. 2NO2 þ H2 O þ surface ! HONO þ HNO3

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The observed HNO3 level was several tens of pptv to 2 ppbv. Increased insolation intensity and photochemistry resulted in the highest levels of HNO3 around noon. After this, HNO3 level decreased by dry deposition and dilution with air. In contrast to HONO, HNO3 levels in the afternoon of May 3 were lower than other days due to lower solar radiation influx.

6. APPLICATIONS TO BREATH ANALYSIS Except for its high moisture content and a need to deal with the same, analysis of breath components are subject to the same considerations as analysis of ambient air. The applications of breath analysis for clinical diagnostic purposes have been steadily increasing. Beyond immediate effects of dietary constituents, the composition of exhaled breath reveals much about the physiology of an individual. Monitoring of breath constituents has the promise of being the least invasive means of disease diagnostics. Acetone contained in breath was measured by flow analysis [81]. Acetone was collected by a porous membrane DS and reacted with alkaline salicylaldehyde to produce yellow color. Absorbance was measured by an LCW-based long path detector with an LED light source. The breath acetone concentrations for nose and mouth exhalations were 21777 and 23079 ppbv, respectively, not markedly different from each other. Ketogenesis or ketosis involves the production of ketone bodies; these are primarily composed of acetoacetate, hydroxybutyrate and acetone. One volunteer had a modest meal at 8:00 p.m. and was given only water to drink until measurements were made on him at 11:30 a.m. the next day for the next 2 h via a face mask based continuous sampler. The acetone level started at 100 ppbv and reached 183 ppbv by 1:30 p.m., representing an 83% increase. Breath NH3 is a useful marker for halitosis, Helicobacter pylori infection, kidney and liver dysfunction. Breath NH3 was successfully measured by a unique concentric conductometric electrode system in stopped-flow mode [36].

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Figure 21 Schematic diagram of a two-channel system for the measurements of ambient HONO and HNO3. Solutions: #1, 1 mM phosphate buffer solution at pH 7; #2, 180 mM NH4Cl buffer solution at pH 8.5; #3, reagent solution containing 4 mM SA (sulfanilamide), 0.4 mM NED (N-(1-naphthyl)ethylenediamine) and 50 mM HCl. Typical operation conditions: air sampling rate, 2 L min1; solution flow rates, 0.24 mL min1; derivatization time, 5 min; derivatization temperature, 551C. The data on the right are time series of HONO (D), HNO3 (J), NO (—) and NO2 (—) obtained in downtown Albany, NY, between April 30 and May 5, 1999. Reprinted from Ref. [120]. Copyright (2002), with permission from Elsevier B.V.


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Figure 22 Results of continuous measurement of breath NH3 using mask sampling before and after protein lunch (a) and without protein (b). ’ NH3 level obtained by the liquid film sensor, line was obtained by OPA (o-phthalaldehyde) system.

Breath ammonia, put through a cartridge of solid NaOH pellets that reduces the moisture content without uptake of NH3, neutralizes a dilute H2SO4 film formed on the surface of the electrodes and changes the conductivity. This device can collect NH3 without memory effects and detects NH3 without interference from CO2, etc. If a subject is given a protein meal and breath NH3 is monitored, the levels go up after a lag time and then slowly decrease again (Figure 22). Both the delay between intake and the ammonia peak maximum and its clearance time are believed to have information on metabolic processes, specifically liver and kidney functions.

7. ANCILLARY SYSTEMS FOR FIELD MONITORING 7.1 Calibration of gas analysis system Gas analysis instruments need calibration with standard gases. In the laboratory, a gas standard from a cylinder is diluted with purified air or an inert gas with the help of mass flow controllers. It is not practical to carry gas cylinders and/or gas dilution systems to the field. However, for long-term atmospheric measurements,

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periodic calibration is a must. Liquid phase calibration is acceptable only to a degree as this does not check the inlet system or any changes in collection efficiency. Permeation devices are sometimes used for the calibration. Liquefied standard gas is placed in a sealed tube with at least part of the fluid in contact with a polymeric permeable surface, most typically Teflon. As long as the tube is held at a constant temperature, the vapor pressure inside the tube is constant and the vapor permeates out through the permeable wall at a constant rate. The permeation rate, which typically varies in the range of ng min1 to mg min1, can be determined by periodic weighing. An inert sweep gas such as purified (or at least analyte-free) air flows at a constant rate outside the permeation tube, thus generating a constant concentration of the gas. In one field instrument for measuring H2S, the calibration source for H2S, a permeation wafer device, was placed in an aluminum block heated at 401C by a siliconized heater affixed to the block. The power requirement was small enough for the entire instrument to work off a car battery. The instrument was used for measuring the H2S concentrations in an oil field [18]. Standard gases can also be made from solution. The principles have been elaborated early on [122–124]. In situ gas generation is typically performed in the sampling line just before the gas collector [125]. Acid gases, such as SO2 and H2S can be formed by making the gas source solutions (sulfite and sulfide, respectively) acidic. Ammonia gas can be generated by the same way but by alkalinizing. The mixed solution is introduced into the honeycomb structure microchannel-based standard gas generator as shown in Figure 23. The generator is essentially the mGAS scrubber operating in reverse [93]. This device also exhibits excellent performance for trace gas generation. As the data shown in Figure 23, calibration gas can be formed either in the zero-gas mode or the measurement mode. The generated gas concentration can be set by changing the source solution concentration. Unlike Henry’s law-traceable sources [121–123], the gas concentration can be varied by the flow rate of source solution. The flow rate can of course be controlled by using any pumping system that conveniently allows flow variation.

7.2 Liquid flow control Miniature components are needed for field use. Detectors can be decreased in size by using all solid state components, e.g., LED sources and photodiode sensors, etc. In field measurements, it is vital to reduce reagent consumption. It is necessary to control liquid flow, especially at low flow rates. Pneumatic pumping of a reagent where reservoir pressure is maintained constant by the help of a miniature compressor and a pressure sensor by a feedback control circuit is convenient and has been often used [17,18,88]. Recently, controlled electroosmotic flow (EOF) has been advocated for flowbased air analysis [126]. In mTAS and mGAS, PDMS and glass chip are used to fabricate channels. Channel surfaces have silanol groups, and EOF can be generated by applying appropriate voltage between solution reservoirs. In electrophoresis-based analysis, high voltage is simply applied between two


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Figure 23 Liquid flow system for gas generation with microchannel gas desorber (top) and typical response signal of mGAS coupled with the micro gas generator (bottom). All pictures of the parts are at the same scale. R1, R2, source and desorbing solutions; MP, micropump; MC, mixing coil (0.97 mm i.d. 50 cm); GD, microchannel gas desorber; BP, back pressure tube (0.3 mm i.d. 100 cm). Bottom chart: A 50 ppbv H2S test gas was introduced into the system. In the zero mode, H2S contained in the sample was completely removed from the air to obtain a baseline. In both the zero and sample modes, micropumps were activated to generate 50 ppbv H2S. The responses to the generated gas were obtained at the zero baseline and the measurement signal, respectively. Reprinted from Ref. [125]. Copyright (2007), with permission from Elsevier B.V.

solution reservoirs. In gas analysis systems, the purpose is to achieve constant liquid flow rather than to establish an electric field. To monitor these very low flow rates, a thermal flow sensor has been developed for PDMS channels [126]. A thermally conductive thin (25 mm) Kaptons film covers the microchannel. A micro Peltier device is placed on the Kapton film to cool the fluid flowing through the channel. Two platinum resistor-based temperature sensors are placed in the fluid channel, one upstream and one downstream of the Peltier. Without the flow, the two Pt sensor temperatures are same. However, temperature difference between them increases in direct proportion to the flow magnitude. The temperature difference is monitored with the help of an

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instrumentation amplifier operating in the differential amplifier configuration. The sensor is sufficiently responsive to accurately monitor mL min1 liquid flow rates. The flow sensor is attached in the reagent flow line and the magnitude of the EOF is monitored. The EOF is then controlled by feedback of the flow sensor signal to control the applied high voltage, as shown in Figure 24. When the set signal was changed from zero to 15 mL min1, the system applied high voltage which varied itself until the flow reached the desired value and then the flow rate remained constant under active control. Miniature piezo valves have been developed for micro flow control [126]. The merit of a piezo-based valve is that it is applicable to any solution whereas highly conductive solutions cannot be used in conjunction with high voltage. The photograph in Figure 25a shows the piezo valve. The piezo valve unit is comprised of PDMS blocks and a silicone membrane. Therefore, this device is compatible with reagents/samples that can be used with PDMS or glass chip microdevices. The control performance is good; this is shown in Figure 25b. The response speed is less than 5 s and a stable liquid flow can be obtained in combination with the micro flow sensor. Of course, such flow control strategies can be used for any flow analysis applications — it is not limited to gas analysis.

8. CONCLUSIONS Flow-based analysis is alive and well in the arena of trace gas measurement. The key in such application is often the gas collection device that allows a high gas to liquid volume ratio and that permits facile automation for continuous analysis. New detectors have been developed that has made major performance improvements possible. Flow-based methods allow the measurement of many types of trace gases providing new information. Miniaturization of flow-based air analysis is increasingly happening permitting more extensive applications in real field analyses. Flow-based gas analysis methods can be applicable to breath analysis, leading to disease diagnostics. It may sometimes be advantageous to convert an analyte in the aqueous phase to the gas phase so as to provide matrix isolation and a much cleaner analysis. One recent example is the determination of As(III) and As(V) in natural water at sub-mg L1 levels after selective conversion to AsH3 [127–129].

ACKNOWLEDGMENTS The authors thank the Japan Society for Promotion of Science (JSPS) for support of the collaborative project between Japan and U.S. Some of the author’s work discussed herein were supported in part by this grant, by the US National Foundation through grants CHE-0731792 and CHE-0709994 and The United States Environmental Agency through RD-83107401. However, no endorsement of any of these agencies should be inferred.


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CHAPT ER

23 Environmental Applications: Waters, Sediments and Soils Paul J. Worsfold, Ian D. McKelvie and Grady Hanrahan

Contents

1. Challenges of Environmental Analysis 1.1 Legislative and Economic Drivers 1.2 Rationale for water, sediment and soil analysis 1.3 Water, sediment and soil matrices 1.4 Sample preservation and storage 1.5 The need to determine elemental speciation 1.6 Quality assurance and quality control 2. Instrumentation and Modes of Application 2.1 Laboratory instrumentation 2.2 On-line sample treatment: Filtration/dialysis, digestion, preconcentration/matrix removal 2.3 Field-portable and autonomous systems 3. Range of Sample Types 3.1 Waters 3.2 Sediments 3.3 Soils 4. Applications 4.1 Overview 4.2 Nutrients 4.3 Trace metals 4.4 General chemical quality parameters 4.5 Organic contaminants 5. Future Trends Abbreviations and Definitions References


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1. CHALLENGES OF ENVIRONMENTAL ANALYSIS 1.1 Legislative and Economic Drivers Environmental protection plays an increasing role in today’s society as evidenced by more stringent regulations and international agreements such as the OSPAR Convention, Kyoto Protocol and Montreal Protocol. National and trans-national regulations include the European Union (EU) Water Framework Directive, Australia’s National Water Initiative and the US-based Pollution Prevention Act (PPA) and the Clean Water Act (CWA). Their success requires proper implementation, e.g., reliable monitoring strategies, sound and integrated management and the development of innovative and cost-effective environmental technologies.

1.2 Rationale for water, sediment and soil analysis The cornerstones of effective environmental resource management are research, management and monitoring. Research is essential to improve the understanding of biogeochemical processes, especially with respect to the origins, speciation, transport, bioavailability, toxicity and ultimate fate of chemical species within aquatic and terrestrial ecosystems. Based on this knowledge, suitable resource management strategies for ecosystem protection and/or restoration can be defined and implemented. For example, short-term monitoring may be required to assess the status of an aquatic system with respect to particular environmental values, whereas longer term monitoring is desirable to assess changes in ecosystem function or the effectiveness of management strategies in maintaining or improving ecosystem health. High temporal resolution monitoring is required to assess the impact of short-term events such as storms and different spatial scales are needed to address local through to global processes. Implicit in this research-monitoring-management relationship is the need for reliable water and soil/sediment quality data. Such information can be obtained through the conventional process of manual or automated sample collection and subsequent laboratory analysis or, more desirably, by the use of on-site or in situ analytical instruments or sensors. Flow-injection and related-flow technologies offer a powerful analytical tool for both laboratory and field collection of such quality data.

1.3 Water, sediment and soil matrices The determination of chemical species in these differing environmental matrices presents unique analytical challenges, particularly the ultra-low concentrations and the varying composition and complexity of the sample matrices. Quality assurance of the data, particularly with regard to accuracy, is of primary importance but may be compromised by cost, time and practical considerations. The use of flow-injection analysis (FIA) hyphenated with other techniques offers many attractive features for addressing these challenges. Long et al., for example, used multi-syringe flow-injection lab-on-valve analysis with automatic

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on-line sample pre-treatment by renewable solid-phase extraction and detection by atomic fluorescence spectrometry (AFS) for the preconcentration and determination of arsenic in freshwaters [1]. Flow-through approaches for metals have also been developed for solid environmental samples. This is important because their mobility, bioavailability and environmental impact depend partly on their association with particulate matter and interactions at the solid–water interface. FIA assemblies incorporating packed micro-cartridges have been successful in determining metal partitioning and release rates for soils via column-leaching experiments [2,3]. Hyphenated FIA techniques can also be beneficial for determining organic species. For example, Mulchandani et al. measured organophosphate nerve agents in industrial wastewaters using FIA with an amperometric enzyme biosensor incorporating a novel immobilized enzyme reactor and a carbon paste working electrode [4]. The response was linear up to 140 mM with a detection limit of 20 nM for paraoxon and methyl parathion. Increasing awareness of the importance of the dissolved organic carbon (DOC) pool and the associated organic fractions of many environmentally important elements has led to the development of several FIA methods for the determination of DOC in aquatic systems using, e.g., UV photo-oxidation coupled with peroxydisulfate digestion and FIA with conductometric detection [5]. The method had a limit of detection of 0.8 mg L1 and was not affected by high concentrations of chloride ions.

1.4 Sample preservation and storage Traditional environmental monitoring approaches are based on discrete sampling methods followed by laboratory analysis. Ideally, samples should be analysed in situ or immediately after collection; however, this is not always possible due to the lack of field-based instrumentation or delay after sampling from remote locations. Under these circumstances, it is imperative that sample integrity is maintained between the time of sample collection and analysis. This is especially true when investigating the biogeochemical cycling of nutrients and trace metals. An appropriate sample-preservation protocol must therefore be adopted in order to minimize changes in sample composition due to physical, chemical and biological processes that can occur during sample collection and storage [6,7]. Aspects that need to be considered include:

Storage container (type and size) and cleaning procedure. Sample matrix. Filtration technique. Chemical addition and physical treatment. Storage temperature.

Tables 1 and 2 provide further details of sampling and storage protocols for nutrients and trace metals in natural waters, respectively [6–13]. It is difficult to select a universally applicable treatment protocol for all environmental species in the myriad of matrices encountered due to the variability in physico-chemical parameters. Gardolinski et al., for example,

688

Species (as reported)a

Matrix

Storage container

Filtration technique

Storage temperature

Chemical addition/ physical treatment

Cleaning procedure

Reference

DRP TON

Riverine, estuarine, coastal

HDPE

0.45 mm cellulose acetate membrane filters

41C, 201C, and 801C

0.1% (v/v) chloroform

[6]

Silicic acid

Riverine, coastal Stream

HDPE

Not reported

201C

Not reported

Nutrient-free detergent, 10% (v/v) HCl for 24 h followed by ultrapure water rinse Not reported

Polyethylene

1.2 mm GF/C for ammoniacal and oxidized N; 0.45 mm membrane filter for dissolved P Not reported

41C, 201C

Acidification (pHo2.0) with H2SO4 for nitrogen samples only

Nutrient-free detergent followed by hot tap water rinse

[9]

41C, 181C

With/without HgCl2

Not reported

[10]

Ammoniacal N Oxidized N Total Kjeldahl N Dissolved P Total P Phosphate Nitrate Silicate a

Open ocean

Polypropylene

DRP, Dissolved reactive phosphorus; TON, Total oxidized nitrogen.

[8]


Table 1 Selected examples of preservation methods for nutrient species in natural water samples

Table 2 Selected examples of preservation methods tested for trace metal species in natural water samples Matrix

Storage container Filtration technique

Storage temperatures

Al, Cd, Cu, Fe, Mn, Ni, Pb, V, Zn

Riverine

Polyethylene, PTFE

0.45 mm polyethersulfone capsule filters

Not reported

Al, Cd, Cu, Ni, Pb, Zn

Riverine, rainwater

HDPE, polypropylene

0.45 mm membrane filters

Tributyltin Triphenyltin

Coastal

Polycarbonate and Pyrex glass

Not filtered, both liquid–liquid and solid-phase extractions performed

Cd, Cu, Pb

Open Ocean

FEP, PTFE

0.45 mm cellulose nitrate membrane filters

Chemical addition/ physical treatment

Cleaning procedure

Reference

[7] Bottles soaked with 1:1 (v/v) HNO3 overnight followed by rinse with ultrapure water [11] Room Acidified with Bottles soaked with 1:1 (v/v) temperature HNO3 to pH 1.0 HNO3 for 1 week followed by rinse with ultrapure water Room Acidified with Bottles soaked in [12] temperature, 0.1% v/v HBr saturated Na2Cr2O7 41C before in H2SO4 for 24 h extraction followed by rinse with double distilled water [13] 201C Not reported Bottles soaked with 1:10 (v/v) HNO3 overnight followed by rinse with ultrapure water Acidified with HNO3 to pHo2.0


Species (as reported)

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showed that freezing samples for dissolved reactive phosphorus determination from chalk-based catchments can lead to the co-precipitation of inorganic phosphorus with calcite after thawing of the samples [6]. Trace metal behaviour is influenced by such factors as temperature, pH, salinity, flow, hardness, DOC, dissolved oxygen, redox potential and total suspended solids [14,15]. In addition, water samples containing target organic constituents need special consideration due to issues such as volatilization, sorption, transformation reactions and leaching of contaminants (e.g., phthalates) from plastic sample containers [16]. There are additional challenges when considering soil and sediment samples, such as whether to prepare and store samples dry or wet. Studies have shown that the drying process can affect the kinetics of the extraction of trace metals (limiting information on bioavailability) in such samples [17,18]. Additionally, drying can alter the extraction yield of common organic constituents [19] and toxicity results from bioassays [20,21]. Anoxic sediment samples require nontraditional preservation techniques including sub-sampling under oxygen-free environments [22].

1.5 The need to determine elemental speciation Speciation relates to the identification and quantification of specific physicochemical forms of an element and operationally defined protocols are used to identify forms such as ‘‘labile’’ or ‘‘bioavailable’’ [13,23–27]. The determination of individual trace metal species, e.g., different oxidation states or individual organometallic species such as methyl mercury and tributyltin [28] is often necessary because total concentrations are poor indicators of bioavailability, toxicity to organisms and mobility in natural systems [13,29]. In many cases only a relatively small fraction of the total dissolved metal concentration exists in the free hydrated state or complexed with inorganic ligands, while a large fraction is complexed with organic ligands [30,31]. Factors influencing the speciation of inorganic metals include pH, temperature, major ion composition (e.g., metal– carbonate complexes) and ionic strength [32]. The determination of the various physico-chemical forms of the major nutrients is necessary to study loading and dynamics in natural water systems due to their role in eutrophication and photosynthetic and decomposition processes [27]. Nitrogen speciation, for example, can be operationally defined as total nitrogen (TN), total particulate nitrogen (TPN), total dissolved nitrogen (TDN), dissolved organic nitrogen (DON) and dissolved inorganic nitrogen (DIN) [33]. Such species are part of the global nitrogen cycle and are involved in biochemical transformations including fixation, nitrification and ammonification. Phosphorus occurs in aquatic systems in both particulate and dissolved forms and can be operationally defined as total phosphorus (TP), total reactive phosphorus (TRP), dissolved reactive phosphorus (DRP) and total dissolved phosphorus (TDP) [34]. It is generally recognized that orthophosphate (equated with DRP) is the most readily available species [35], although some dissolved organic and condensed phosphorus and particulate phosphorus species may also be utilized by algae and bacteria [36]. The silicon cycle consists of relatively few


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forms and the element is found primarily as a constituent of various silicate minerals, often combined with iron, magnesium and calcium [34]. FIA techniques are widely used for investigating nutrient speciation due to their inherently attractive features such as rapid sample throughput, on-line sample-treatment capability, good precision, low-reagent consumption and flexibility [37]. Such FIA techniques are also suitable for field-based analyses allowing high temporal and spatial resolution determinations without the need for problematic storage and preservation procedures.

1.6 Quality assurance and quality control Quality assurance (QA) and quality control (QC) are gaining awareness due to legislative demands and the complexities of modern environmental analysis [38,39]. Environmental monitoring programs, especially those involving complex matrices, speciation requirements and determination at ultra-trace concentrations, are vulnerable to errors in the sampling, storage and analysis components. In an environmental context, QA ensures that the collected data are of sufficient scientific credibility to permit statistical interpretations that can lead to good management and policy decisions. QC takes a two-fold approach in that it requires both monitoring of the QA process being studied and elimination of the causes of sub-standard performance. Data not based on good QA/QC are prone to error and can lead to flawed environmental management decisions. To ensure credibility and reliability in the environmental monitoring process, investigators need to follow a careful and planned program involving the tasks outlined schematically in Figure 1. Planning and design: Investigators need to define the environmental species (and concentrations) to be determined, technique used, method, location (spatial variability), frequency and timing (temporal variability) of sampling and the analytical technique used. Careful attention must also be paid to proper cleaning and preservation procedures prior to sampling. Certification of methods and investigators should also be performed. Field sampling and analysis: Sampling or field-based analysis will follow the planning and design process and consider all factors discussed above. When sampling, investigators should take replicate samples to ascertain precision of the sampling method, perform field blanks, check standards and collect samples in areas that are homogeneous in nature. Such procedures are also necessary when analysing in the field. Spiking of samples in the field with representative analytes will allow losses during sampling, transportation and analysis to be properly identified. Care must also be taken to ensure proper sample pre-treatment (if needed), logging of related physico-chemical parameters (e.g., pH, temperature) and traceability through some form of sampling/analysis record keeping. Sample storage and preservation: Detailed discussion on sample storage and preservation is presented in Section 1.4.

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Figure 1 Key aspects of a good QA/QC program.

Data analysis & method validation: It is essential that quality statistical analysis (e.g., descriptive, trends, regression, multi-variate analysis) be performed on the large body of environmental data collected. Analytical inaccuracies can arise, thus independent analysis by at least two methods is advisable. Proficiency testing programs (e.g., inter-laboratory comparisons) and the use of certified reference materials during the analysis process are also vital in the overall method validation process. Reporting and decision making: Reporting is the last, crucial step in the environmental monitoring chain. Data must be archived in a systematic and easily accessible manner for timely decision making. Many commercial databases are currently available in which investigators can incorporate such information as the analyst, techniques used, validation of entered data, evaluation and interpretation of results and end users.

2. INSTRUMENTATION AND MODES OF APPLICATION 2.1 Laboratory instrumentation Flow analysis is an approach to mechanized analytical chemistry usually carried out inside narrow bore tubing. An aliquot of an aqueous sample is introduced into the flow system and pushed towards the detector by the carrier/wash stream. During transport through the analytical path, the sample undergoes dispersion and dilution, resulting in a well-defined sample zone that undergoes


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reproducible, on-line physical and chemical treatment, e.g., dilution, reagent addition and dialysis. Sample passage through the detector results in a transient signal that is recorded as a peak, the height of which is proportional to analyte concentration in the sample. FIA is one particular type of flow analysis that has been described in detail elsewhere in this book. In its most common form, FIA involves the injection of a liquid sample into a flowing carrier stream. En route, the injected sample zone can merge with one or more reagents, and/or be subjected to other physicochemical processes (e.g., dialysis, thermal digestion, photo-oxidation or enzymatic reaction) that generate species that are detected in one or more flow-through detection devices. Photometric detection in the visible region is the most common form of detection used for environmental applications, but other detection techniques such as UV spectrophotometry, fluorescence, chemiluminescence, atomic and mass spectrometry, potentiometry and voltammetry have also been used. The salient features of the technique for application to environmental samples (aquatic and terrestrial matrices) are: The sample volume is low. Typical sample volumes are 5–500 mL, although volumes in the nanolitre range can be handled in microfluidic systems. Reagent consumption is also low, typically 0.5–2.5 mL min1 per channel, thus minimizing waste and allowing ‘‘environmentally friendly’’ chemical analysis. This is important for remote and long-term deployments. Sample-conditioning techniques such as dialysis, gas diffusion and ion exchange are efficiently accomplished on-line. The analytical path is a closed, ‘‘clean room’’ environment and there is no physical contact between the sample and the external environment (and vice versa), thereby avoiding analyte losses and/or sample contamination. Sample management is reproducible. Conditions for sample handling are reproducible from one sample to the next, and this feature is particularly important for applications involving on-line sample conditioning such as sample digestion. Sample passage through the analytical path is fast (usually 10–180 s) and residence time in the manifold is short, allowing rapid and high throughput analysis. Flushing time, the interval between achievement of the maximum analytical signal and baseline restoration, is typically 10–120 s. The next sample can therefore be introduced without a long delay time and this permits a high sample throughput, typically 30–300 h1. This is particularly advantageous in relation to, e.g., high throughput laboratory analysis and high temporal resolution in situ monitoring. Flow-injection instrumentation involves simple components such as samplers, liquid drivers (peristaltic pumps, piston pumps, solenoid pumps), injection devices (rotary valves, injector-commutators), reactors and flow lines (usually narrow bore tubing), mixing chambers and flow-through detectors. As a rule, these devices are readily available in most laboratories devoted to chemical

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analysis. Regarding detection, almost all analytical techniques have been used in flow analysis, with a low flow cell volume and a short response time compatible with system dynamics being the most important detector requirements. A variant of FIA that can be advantageous for environmental applications is reagent injection (also called reverse FIA) in which sample is pumped continuously and reagent is injected into it. When more than one reagent is injected, this is termed multiple reagent injection or multi-commutation, and this can be achieved using multiple solenoid valves, solenoid pumps or syringe pumps. This approach minimizes reagent consumption to a few microlitre per analysis and is especially useful when the application involves longer term monitoring. In summary, flexibility of manifold design is one of the key features of FIA that makes it so well suited to environmental applications.

2.2 On-line sample treatment: Filtration/dialysis, digestion, preconcentration/matrix removal 2.2.1 Filtration/dialysis There are two main types of membrane separation devices used in conjunction with FIA. One separates components of the sample zone on the basis of physical size, i.e., conventional filtration type devices, and the other is used for analyte separation, e.g., dialysis, microdialysis, Donnan dialysis, gas diffusion, pervaporation, membrane extraction and gas permeation via diffusion scrubbers [40,41]. Membrane-based sample preparation and separation techniques are reviewed in detail elsewhere in this book (see Chapter 8). On-line filtration is particularly useful for reducing the time needed for sample treatment and minimizing the risk of contamination in trace analysis, e.g., for the determination of sub-nanomolar iron in open-ocean waters [42]. Filters with different pore sizes can be used to fractionate aquatic samples and typical pore sizes are 0.2 or 0.45 mm to obtain an operationally defined ‘‘dissolved’’ phase (sometimes loosely referred to as the bioavailable fraction) and 0.02 mm to split this into ‘‘colloidal’’ and ‘‘truly dissolved or soluble’’ phases. Whilst 0.2 mm is now preferred for the determination of dissolved nutrients because it is more effective at removing bacteria, from a practical point of view this considerably extends the time needed for sample treatment. It is also important to consider the effect of the filtration process on sample integrity. For example, 0.2 or 0.45 mm filtration can remove a significant proportion of the colloidal fraction [43] and can influence the observed trace metal speciation (particulate versus dissolved), particularly in turbid estuarine samples [44].

2.2.2 Digestion Digestion techniques are necessary in order to determine the total concentration of an element in environmental samples. The digestion technique must be able to release the element from biological material, e.g., algal cells and plant detritus, and from sediments [45]. Traditional methods of digestion for natural water samples include fusion, wet or dry ashing, autoclaving, UV photo-oxidation and microwave heating [45]. UV photo-oxidation can be used for marine and


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freshwaters but heating to 90–120 1C in the presence of acid may also be required. McKelvie et al. used an on-line UV photo-oxidation FIA technique for the determination of organic phosphorus species and found that results were comparable with a batch peroxydisulfate method [46]. Microwave heating is an increasingly popular approach to digestion and this can be coupled with FIA to provide a clean, efficient and rapid analysis [47]. For example, Silva et al. achieved quantitative recoveries of a range of metals in rocks using a combined FIA-microwave–ICP–MS approach [48]. Use of on-line digestion and extraction techniques is described in more detail in Chapter 6.

2.2.3 Preconcentration/matrix removal These two functions are often performed in parallel and utilize solid-phase resins packed in micro-columns and incorporated into the FIA manifold. A particularly good example of the analytical challenge that can be met with this approach is the determination of trace elements in open-ocean seawater, where the analytes are often present at sub-nanomolar concentrations in the presence of a vast excess of matrix cations. Chelating resins have entertained extensive interest in recent years for the preconcentration of trace metals from seawater [49]. The method of Landing et al. [50] has been most commonly applied for the synthesis of an 8-hydroxyquinoline (8-HQ) resin micro-column used for metal determinations. 8-HQ is selective towards transition and heavy metal cations relative to alkali and alkaline-earth cations. Earlier methods involved the immobilization of 8-HQ onto silica substrates, which offer the advantages of good mechanical strength, resistance to swelling and rapid overall exchange kinetics in column applications but they are unstable at high pH. The method of Landing et al. used the highly porous, mechanically and chemically stable, hydrophilic organic resin gel Toyopearl-TSK as the solid support. This consists of intertwined vinyl polymer agglomerates, which offer stability, high porosity and high hydrophilicity due to the presence of ether linkages and hydroxyl groups. The polymer itself exhibits no cation exchange capacity and does not concentrate dissolved organic species. A TSK-8HQ micro-column has been used in an FIA manifold with chemiluminescence (CL) detection for the determination of important micronutrient elements such as iron [42,51,52], manganese [53], cobalt [54] and copper [55]. A typical micro-column, containing a chelating agent, ion exchange resin or adsorbent, can easily be incorporated into an FIA manifold, and allow analyte preconcentration/matrix elimination to take place upstream of the detector. An example of the incorporation of a micro-column within a sophisticated FI–CL manifold for the determination of iron in seawater is shown in Figure 2. Careful buffering of the seawater as it passes across the column can also impart additional selectivity to discriminate between, e.g., different transition metals and different redox species of an element. Furthermore, partitioning between different physico-chemical forms of an element (e.g., between colloidal, acid leachable and organically bound fractions) is possible by careful manipulation of both the solid phase and the manifold conditions.

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Figure 2 Flow-injection manifold for the determination of iron in seawater. Reprinted from Ref. [42]. Copyright (2002), with permission from the American Chemical Society.

2.3 Field-portable and autonomous systems In addition to the usual analytical requirements of good accuracy, precision, selectivity and sensitivity, remote deployment places significant additional demands on any instrumentation. The following are desirable features: Rugged, portable and fully automated instrumentation. A contamination-free environment (including any reagents, containers and sampling apparatus). Ability to remove matrix ions, e.g., sea salts in the case of marine samples, or to perform matrix modification, e.g., digestion. Stability for long periods with respect to on-board reagents and standards, pumping devices, fluidics and detector response. On-board filtration and the prevention of biofouling due to algal or bacterial growth.


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Remote calibration, validation and maintenance. Minimization of power, reagent consumption and waste generation for longterm, autonomous deployments. The characteristics of FIA are ideally suited to meet these requirements [56]. There are however a number of important practical issues that must be considered when adapting laboratory-based FIA methods for field deployment. First, it is essential that systems have provision for on-line sample filtration, typically through 0.2 or 0.45 mm cellulose acetate or polycarbonate membranes in a tangential flow configuration [57]. This is important for speciation studies (dissolved/particulate) and for the prevention of blockage and fouling of the manifold lines. Second, the importance of reliable in situ calibration should be emphasized for high-quality analytical data capture. This is necessary because of instrumental drift and the inevitable deterioration of reagents over time, and is usually done by injection of a single standard at regular intervals through a solenoid-switching valve. In situations where the sample matrix is variable, e.g., a salinity gradient along an estuarine transect, the refractive index or schlieren effect can be problematic in FIA. The schlieren effect causes the appearance of a negative frontal peak followed by a positive peak when a sample blank with high ionic strength is injected into a carrier of lower ionic strength. This behavior occurs because of a lensing effect which is due to a combination of the parabolic geometry of the sample zone under laminar flow conditions, and refractive index differences between the sample and carrier or reagent streams. The schlieren signal can give rise to large errors, especially when low concentrations of analyte are determined [58]. Approaches to overcoming this effect include matrix matching, dual wavelength detection, large sample injection volumes and reflective [59] or multi-reflection flow cells [60]. In the last decade FIA has increasingly been used for in situ monitoring of environmental compartments, particularly natural waters. The drivers for this have been (1) the desire to better understand complex biogeochemical processes and hence the need obtain high-quality data with good temporal and spatial resolution and (2) the increasingly stringent legislation pertaining to the quality of surface and ground waters and the demand for high throughput analysis that this legislation requires. To this end, remotely deployed FIA instruments represent a low-cost option for obtaining near-continuous, quantitative data for a wide range of aquatic chemical parameters [37]. Key application areas for such technology include:

Elucidation of environmental processes and biogeochemical cycles. Study of chemical fluxes, pathways and fates. Testing and generating environmental hypotheses. Monitoring compliance with legislation, e.g., the European Union Water Framework Directive. Providing archival data and baseline surveys, e.g., for environmental impact assessment.

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Environmental protection, e.g., effluent discharges, supply intake protection, leachate monitoring. Budget studies, e.g., for catchment management. An example of the instrument design for a submersible FIA-based nutrient analyser and its application to mapping nitrate concentrations in the North Sea [61] is shown in Figure 3. Each of the potential application areas and deployment scenarios listed above places different requirements on the design considerations. Thus, for example, if fixed site monitoring is appropriate, such as for a point discharge into a river or within a water treatment works, it is likely that mains power would be available. In contrast, for budget studies at remote locations or monitoring of soil leachate a portable, battery-powered monitor would be essential. In the latter context, in situ monitoring is particularly important for those species (e.g., nitrate) which are highly mobile and pass relatively quickly between soil water, ground water and surface water, because a conventional (manual) monitoring scheme may not detect short-term changes due to, e.g., storm events. FIA technologies will also have a role to play in the emerging areas of autonomous underwater vehicles and sensor platforms that are part of global monitoring networks [62]. For example, Thouron et al. reported an autonomous nutrient analyser for oceanic, long-term in situ biogeochemical monitoring [63].

3. RANGE OF SAMPLE TYPES 3.1 Waters Waters are analysed for a variety of reasons, depending on their origins, beneficial uses and immediate and ultimate destinations. This requires methods for a host of different parameters over a wide range of concentrations (Table 3). The principal drivers for water analysis are: Protection of human health, both for drinking waters and primary contact. The assessment of the status of aquatic ecosystems, e.g., defining a reference condition, as part of the protection and improvement of natural and impacted aquatic environments. Provision of water of suitable quality for primary industries, such as stock watering, irrigation, or aquaculture, and for particular industrial processes. Monitoring and control of treatment processes designed to improve effluent quality and minimize the environmental impact of discharges.

3.1.1 Potable waters The parameters of concern in assessing the quality of potable waters fall into the following general categories [64], and focus on the provision of water that is safe for human consumption: Micro-organisms, including bacteria, protozoa, toxic algae and viruses. Physical characteristics including radionuclide activity, turbidity and electrical conductivity.

Standard inlet Sample inlet Power/data cable connector “O” ring 3 way switching valve

Pump 2: sample/standard

Injection valve Reduction column Reaction coil

'T' Piece

Distribution manifold

Pump 1: reagents Sample loop

Electronics

(a)

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Figure 3 (a) Photograph of the submersible flow-injection analyser with explanatory diagram showing location of manifold components. (b) Contour plot of surface nitrate distribution in the North Sea during the IMPACT cruise. Reprinted from Ref. [61]. Copyright (2002), with permission from Elsevier B.V. Also published in Ref. [37].


Flow cell / solidstate detector

Waste Carrier N1NED Sulphanilamide PVC end plate

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Figure 3 (Continued ).

Chemical parameters, including inorganic chemicals (e.g., nitrate and nitrite, heavy metals, cyanide, bromate and boron), organic compounds (e.g., hydrocarbons, pesticides and PAHs) and halogenated compounds derived from water disinfection (e.g., trichloro-methane and -ethane, trihalomethanes). Nutrients species such as nitrate and nitrite, along with microbiological parameters, are indicators of contamination from human or animal faecal pollution, and are monitored in nitrate-vulnerable areas. High nitrate concentrations may cause methaemoglobinaemia in infants and this is reflected in, e.g., the statutory requirement of the Water Supply (Water Quality) Regulations for 1 1 England and Wales that [mg NO 3 L /50]+[mg NO2 L /3]p1 [65]. Lead may be problematic in waters from aged-reticulation systems, and in some jurisdictions there are regulated, permissible concentrations for this metal along with other toxic metals such as Hg, Ni, Sb, As, Cd, Cr, Se, Sb, Cr and Cu [65]. For waters containing higher amounts of natural DOC, there is the potential for production of carcinogenic compounds such as trihalomethanes during chemical disinfection. Similar risks of carcinogenicity are associated with a range of pesticides, including aldrin, dieldrin and heptachlor. High-quality potable waters may also be used in industrial applications, e.g., in food and beverage production, as boiler cooling waters, textile and paper manufacture and in the microelectronics industry. In this instance, parameters such as total hardness (Ca+Mg), silicate and metals such as Fe and Mn are of interest because of their potential for scaling and staining.


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Table 3 Typical composition and concentration ranges for (a) major cations and anions and (b) other dissolved constituents in different waters (a) Class

Constituent

World average freshwater [209] mg L1 (mM)

World average seawater [210] mg L1 (mM)

Major cations

Na K Ca Mg Cl SO2 4 HCO 3

8.0 (0.35) 3.0 (0.08) 30 (0.75) 5.0 (0.21) 8 (0.23) 18 (0.19) 105 (1.72)

10,759 (468.0) 399 (10.2) 409 (10.2) 1,294 (53.3) 19,320 (544.9) 2,709 (28.20) 145 (2.38)

Major anions

(b) Class

Typical constituents

Typical concentration range

Dissolved gases Trace metals

O2, N2, CO2 Fe, Mn Cr, Cu, Hg, Pb, Zn, etc Humic and fulvic acids, proteins, lipids Carbohydrates, porphyrins, plant pigments Contaminants: PCBs, PAHs, pesticides

mg L1 mg L1–mg L1 mg L1–pg L1 mg L1

Organic materials

mg L1–mg L1 mg L1–pg L1

3.1.2 Fresh and marine waters The emphasis here is on the maintenance and improvement of natural ecosystems and understanding environmental processes. The principal classes of water-quality parameters measured are: Nutrients: Nitrogen and phosphorus are essential macronutrients for photosynthesis in aquatic systems. In freshwaters, phosphorus may be the limiting nutrient for primary production, while in marine waters nitrogen is more commonly limiting. Silicate may also be limiting for the growth of diatoms in some marine systems. Of interest in this context are the most bioavailable species: phosphate (as dissolved reactive phosphate), nitrate and ammonia. However, total P and N are often measured as indicators of potentially bioavailable nutrients. When concentrations of P or N become elevated, e.g., due to point source inputs of nutrients such as discharges from sewage treatment works, or diffuse inputs from agricultural land, eutrophication may result. This often results in excessive growth of nuisance algae blooms of cyanobacteria in freshwaters that are toxic to humans and animals, cause odor and taste problems in water supplies and a general loss of recreational and

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environmental amenity. In marine waters, eutrophication may be manifested by blooms of dinoflagellates. Dissolved organic matter, dissolved oxygen and biological oxygen demand: Natural freshwaters usually contain low concentrations of dissolved organic matter (DOM). Discharges containing higher concentrations of organic matter will cause oxygen depletion, and this can be deleterious to fish and invertebrate populations. For this reason the dissolved oxygen concentration is often used as a primary indicator of stream condition, along with parameters such as DOC concentration, biochemical oxygen demand (BOD) and chemical oxygen demand (COD). The concentration of DOC in marine waters is usually low because of salinity-induced aggregation and settling. Trace metals: Measurement of trace metals in aquatic systems is motivated by their potential toxicity to aquatic organisms (e.g., Hg, Pb, Cu, Zn, As, Cd, Cr, Se, Sb, Cu), or because of the limiting role of some micronutrients metals, such as Fe [42] and Co [66] in open-oceanic waters. This latter process is of topical interest because of the role of phytoplankton as a sink for atmospheric carbon dioxide. Organic contaminants: These are extremely important because of their potential toxicity and carcinogenicity, and include pesticides from agricultural sources, polychlorinated compounds of industrial origin, polynuclear aromatic hydrocarbons and phenols from combustion and petrochemical production, halogenated compounds derived from water treatment, and organotin compounds historically used in marine anti-fouling applications. An emerging area of concern is the presence and effect of endocrine disrupting substances in water, which includes some of those listed above, natural hormones, synthetic steroids and alkyl phenolic surfactants in domestic wastewaters, phytoestrogens in effluent from papermaking and phthalates from plastic industries [67]. Water from rivers and lakes may also be used for agricultural irrigation and stock watering, and the parameters of interest include salinity, sodicity (proportion of Na with respect to Ca and Mg), N and P concentrations because of potential ground water pollution and eutrophication issues, and metals, metalloids, pesticides and herbicides because of either toxic effects on plants or accumulation in grazing animals [64]. In the area of aquaculture, e.g., shellfish, crustacean and fish farming, there is interest in both the quality of the water required for these enterprises with respect to parameters such as nutrients and heavy metals, and the polluting effects of the wastes that they produce [68].

3.1.3 Wastewaters Domestic wastewaters contain high concentrations of oxygen consuming organic matter, suspended solids and nutrients as well as trace concentrations of metals and pesticides (see Table 4). The objective of sewage treatment is to reduce the BOD of the effluent, and to decrease the nitrogen and phosphorus concentrations to a level at which the discharge of treated effluents into receiving waters will not have significant environmental impact. Effluents may be disinfected by chlorination, UV irradiation or ozonation prior to discharge into receiving waters.

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Table 4 Typical chemical characteristics of strong, medium and weak domestic wastewaters [211] Concentration (mg L1)

Constituent

Total solids Suspended solids Nitrogen (as N) Phosphorus (as P) Chloride Alkalinity (as CaCO3) Grease BOD5

Strong

Medium

Weak

850 350 85 20 100 200 150 300

500 200 40 10 50 100 100 200

250 150 20 6 30 50 50 100

Tertiary treatment, e.g., chemical precipitation, may also be necessary to improve the removal of phosphorus or heavy metals. Chemical and microbiological monitoring is performed for compliance monitoring, for parameters such as Escherichia coli, total coliforms, pH, conductivity, suspended solids, BOD, dissolved oxygen, ammonia, total P and total N. The purpose of this monitoring is to ensure that effluent discharge does not exceed consented concentrations that are considered to be harmful to the receiving waters. However, it is usually performed on a limited basis (daily, weekly or even monthly in some cases), and it is arguable that the use of on-line measurement, even at hourly intervals, would provide a much better level of environmental protection. On-line process monitoring is increasingly used in wastewater treatment for biological nutrient removal (BNR) wastewater treatment to enable better process control. Optimization models for denitrification are assisted by the availability of real-time data for ammonia and nitrate concentrations, while similar data for phosphate concentrations aids in the optimization of P-removal by phosphateaccumulating bacteria. [69,70]. Measurement of BOD can also be performed on-line [71,72] to monitor the efficiency of organic matter oxidation. Given that activated sludge contains up to 25 g L1 of active bacteria, the successful sample pre-treatment and long-term stability and reliability of on-line monitoring systems employed in these environments is a critical factor for their more widespread adoption.

3.2 Sediments Sediments are a major sink for metals, organic pollutants and nutrients and they provide a time-integrated record of the various pollutants in an aquatic system. Contaminants and nutrients may also be released from sediments back into overlying waters in significant amounts due to diffusion from porewaters, in response to changes in pH, redox potential and salinity conditions, or as a result of bioturbation or physical re-suspension. However, the value of sediments as a means of assessing the ecological health of aquatic ecosystems has only recently been recognized, and sediment quality guidelines are now specified in some parts of the world for metals, metalloids and organics, but as yet not for nutrients [64].

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In situ measurements in sediments to date have been restricted to simple probes, e.g., Eh and dissolved oxygen, because for parameters such as nutrients, metals and organics, sample preparation involving extraction or digestion is necessary. Consequently, most sediment analyses are performed in the laboratory after manual sampling and sample treatment. However, with the recent development of membrane dialysis probes [41] and automated solid sample extraction systems [73] (see also Chapter 14), sediment porewaters and extracts could potentially be measured in situ using portable flow-analysis systems. Use of in situ or at-site analysis would be of particular advantage in the study of, e.g., phosphorus speciation in anaerobic sediments, where all sample manipulations and analyses must be maintained under anoxic conditions in order to avoid loss of sample integrity.

3.3 Soils Soil analysis is particularly driven by the need to assess and manage soil fertility, i.e., the ability of a soil to provide an adequate supply of nutrients and trace elements necessary for plant nutrition. Where fertilizers are applied or other soil amendments are made there is also the necessity to monitor their transport from agricultural land because of the potential for pollution of rivers and coastal waters. Nutrients such as nitrate, ammonia and phosphate, and the essential trace elements S, Cl, K, Ca, Mg, Cu, Fe, Mn, B and Zn are routinely measured [74]. Like sediments, soils must be digested or extracted prior to analysis, and there are numerous extraction methods proposed to assess different forms of these elements in different soil types. For example, fertilizer requirements are based on empirical soil tests designed to assess the amount of plant available elements present. These schemes typically involve extraction with: Neutral salt solutions, e.g., 0.01 M CaCl2 for K and P, 0.01 M Ca(H2PO4)2 for S, or 2 M KCl for inorganic N. Anion or cation exchange resins for P, S, K, Mg and Ca. Acids and bases, e.g., 0.005 M sulfuric acid or 0.5 M NaHCO3 at pH 8.5 (Olsen extraction) for P. Complexing agents such as EDTA or DTPA for Cu, Zn, Mn and Fe [75]. Soils are also tested routinely for metals, pesticides and other organic contaminants in order to avoid contamination in crops and livestock. Flowanalysis techniques are widely used in agricultural laboratories for the analysis of soil extracts due to their high sample throughput and their ability to cope with wide concentration ranges by on-line dilution. However, the need for sample extraction and digestion limits their direct application in the field. Unlike sediments, which may be anaerobic, and must be manipulated under oxygen-free conditions prior to analysis, soils are usually supplied for assay as air-dried samples, which simplifies their handling and analysis. Therefore the use of flow-injection or sequential-injection automated extraction and digestion systems for soils [73], offers even greater promise in this area. These aspects are discussed in more detail in Chapters 9 and 14. respectively. Similar


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applications for metals and organic contaminants may be complicated by the need for atomic or mass spectrometric detection systems that are field-portable. Another major area of interest in soil analysis is the need to measure metals, organic contaminants and radionuclides as part of the assessment and remediation of contaminated land. Such assessment involves a measure of the potentially mobile contaminants present in liquid, solid or multi-phase waste, and in many countries this takes the form of the toxicity characteristic leaching procedure (TCLP) [76]. This is a general, two extractant (pH 4.93 and pH 2.88) procedure for both volatile and non-volatile constituents that is followed by spectroscopic or chromatographic determination of individual analytes or classes of analytes. There is considerable scope for the automation of this and similar procedures by FIA and related techniques.

4. APPLICATIONS 4.1 Overview This section describes selected references since 1990 from readily available literature sources. It is not a comprehensive review of the literature but rather an overview that illustrates some of the attractive features of FIA for the quantitative determination of important analytes in waters, sediments and soils. For convenience the sample matrix, methodological details and key analytical figures of merit are presented in tabular form for the following generic classes of analytes: Nutrients (Table 5). Trace metals (Table 6). General chemical quality parameters (Table 7). Organic contaminants (Table 8). There are a number of major challenges facing the application of FIA to waters, sediments and soils, including:

Improving selectivity and speciation capability. Improving sensitivity and limits of detection. Overcoming matrix problems. Developing improved on-line sample-conditioning approaches, as part of the move to fully autonomous analysis. Improving the robustness of methods that are used in autonomous or portable analysis systems. Implementing ‘‘green’’ methods that are environmentally benign. The text below highlights some of the important generic features of FIA for the analysis of waters, sediments and soils from the specific applications cited in Tables 5–8.

4.2 Nutrients The most commonly used detection chemistry for phosphorus species involves the reaction of phosphate with acidic molybdate to form phosphomolybdic acid

Table 5

Selected examples of flow-analysis methods for the determination of nutrient species in waters, sediments and soils

Analyte Mode

Phosphorus species DRP FIA

Matrix type

Basis for detection

Detection


Sample throughput (inj h1)

Comments

Reference

Pristine fresh waters

Formation of phosphomolybdenum blue, tin(II) chloride reduction

Photometry-LED photometer

30 6

In-valve sample preconcentration

[100]

Formation of phosphomolybdenum blue, ascorbic acid reduction, ion paired with CTAB, on-line preconcentration on C18 Formation of phosphomolybdenum blue, tin(II) chloride reduction

Photometry-700 nm

0.6 mg P L1 0.1 mg P L1 3.2 mL sample preconcentration 1.57 nM

C18 column wash of ethanol to minimized schlieren effect

[101]

Photometry-710 nm, 1 m liquid core waveguide capillary cell Photometry-710 nm, 5 mm pathlength,

10 nM

18

Background correction at 447 nm

[103]

1 mg P L1

Photometry- 586 nm

4 mg P L1

54 continuous mode 60 kinetic mode 55

Photometry-882 nm

2g P L1

—

DRP

FIA

Seawaters

DRP

FIA

Fresh waters

DRP

SI–LOV

Lake, potable waters

Formation of phosphomolybdenum blue, ascorbic acid reduction

DRP

FIA

Potable waters

DRP

FIA

Estuarine, seawaters

Formation of phosphomolybdate-Rhodamine B ion pair Formation of phosphomolybdenum blue, ascorbic acid reduction

[212]

Strong interference from Al(III) Reagent injection manifold used to compensate for schlieren effect in waters of varying salinity

[96]

[106]

DRP

FIA

River waters

Oxidation of luminol by H2O2 produced by reac tion of phosphate with immobilized pyruvate oxidase G and peroxidase Oxidation of luminol by phosphovanadomolybdate Oxidation of luminol by phosphomolybdate

Chemiluminescence

96 nM

DRP

MSFIA

DRP

FIA

Potable, river, condensate waters Fresh waters

Solid-phase chemiluminescence Chemiluminescence

2 mg P L1

DRP

FIA, multicommutation



DRP

SIA

Seawaters

DRP

FIA

River waters

H2PO 4

FIA

Wastewaters

DRP, NOx, NH+4

FIA

Wastewatertreatment process waters

Interference from humic substances

[213]

11

High tolerance for silicate

[98]

0.03 mg P L1

180

[99]

Photometry-LED photometer (lmax ¼ 650 nm)

0.15 mM

225–380

Fluorescence quenching of Rhodamine B by phosphomolybdate

Fluorometrylex ¼ 470 nm, lem ¼ 550 nm

0.05 mM

270

Phosphomolybdenum blue, tin(II) chloride reduction Solenoid pump reagent delivery Cobalt wire ion-selective electrode Formation of phosphomolybdenum blue, tin(II) chloride reduction. Cd reduction followed by the Greiss reaction Gas diffusion, absorbance change of acid–base indicator

Photometry-710 nm, background correction at 447 nm Potentiometry

0.67 mM

2

Interfering metals removed by in-line chelating resin column On-line, continuous spatial measurements at sea On-line, conti nuous spatial measurements at sea Extended on-line temporal river monitoring

1 mM

—

—

ca. 9

Photometry-610 nm (DRP), 540 nm (NOx), 590 nm (NH+4 )

[122]

[97]

[121]

Interference from [214] chloride [70] On-line filtration and monitoring of process waters containing up to 4,000 mg L1 solids for plant optimisation

Table 5 (Continued ) Analyte Mode

Matrix type

Basis for detection

Detection



Comments

DRP

FIA

Wastewaters



0.05 mg P L1

6

TDP

FIA

Wastewaters

Microwave digestion, amperometric detection of phosphomolybdate

Amperometry

0.1 mg P L1

20

DRP, TDP

FIA

Potable, river, tap waters


3 mg P L1

—

TP

FIA

Wastewaters

UV photo-oxidation of organic P, and acid hydrolysis of condensed P. Formation of phosphate formed by oxidation of thiamine to thiochrome by phosphomolybdate Microwave digestion, formation of phosphomolybdenum blue, tin(II) chloride reduction

On-line filtration [120] and monitoring of final effluent. 7-day unattended operation [115] 0.08 M perchloric acid and 0.02 M peroxydisulphate required for adequate digestion of organic and condensed P species Si/P ¼ 70 and As/ [79] P ¼ 50 tolerated


0.09 mg P L1

7 4 replicates

TP

FIA

Wastewaters

UV-thermal digestion, formation of phosphomolybdenum blue, tin(II) chloride reduction


0.15 mg P L1

8 4 replicates

Optimum digestion achieved with 10 g L1 ammonium peroxydisulfate at pH 0.6 Optimum digestion achieved using digestion conditions of

Reference

[47]

[114]

DCP

FIA

APHP

FIA

Phospha- FIA tidyl choline

DRP

FIA

DRP, TDP FIA

Wastewaters

Ion-exchange separation, on-line hydrolysis of condensed P to orthophosphate, formation of phosphomolybdenum blue, tin(II) chloride reduction Natural Hydrolysis by immobilized waters, alkaline phosphatase, sediment formation of extracts phosphomolybdenum blue, tin(II) chloride reduction Natural Hydrogen peroxide from waters, hydrolysis of phosphatidyl sediment choline by co-immobilized pore waters, phospholipase C, alkaline sediment phosphatase and choline extracts oxidase detected with luminol Anoxic Formation of sediment phosphomolybdenum pore waters blue, tin(II) chloride reduction

Runoff waters, UV photo-oxidation of soil waters organic P, and acid hydrolysis of condensed P. Detection as phosphomolybdenum blue, tin(II) chloride reduction

5

Photometry-690 nm

10 mg P L1 HPO2 4 20 mg P L1 P2O3 7 20 mg P L1 P3O5 10 2.8 mg P L1

Chemiluminescence

0.14 mM

30

Photometry-690 nm

38 mg P L1

—

Photometry-690 nm

7 mg P L1

40

Photometry-690 nm

—

6 g L1 ammonium peroxydisulfate and 2.1 M perchloric acid 95–102% recovery [81] of spiked samples over 20–2,000 mg P L1 range Immobilized alka- [111] line phosphatase not inhibited by high concentrations of organic P [113]

[110] On-line oxidation of interfering sulfide by permanganate. No significant oxidation of organic P species Fe, Al, Ca interfere- [107] nce minimized by acidic photooxidation and cation exchange sample cleanup


Matrix type

Basis for detection

Detection



Comments

Reference

DRP

MSFIA

Soil extracts

Photometry-880 nm

0.3 mg P L1

15

Ammonium lactate–acetic acid extracts

[215]

H2PO 4

FIA

Soil extracts

Potentiometry

1 mM

—

DRP

FIA

Soil extracts

Formation of phosphomolybdenum blue, ascorbic acid reduction Cobalt wire ion-selective electrode Sequential injection dynamic extraction, detection as phosphomolybdenum blue, tin(II) chloride reduction

Photometry-690 nm

0.02 mg P L1

Nitrogen species TDN FIA

Wastewaters

UV photo-oxidation of organic N to nitrate, direct UV detection UV photo-oxidation of organic N to nitrate, detection by Cd reduction and Griess reaction

Photometry-226 nm

54 mM

—

85–100% oxidation efficiency

Photometry-540 nm

0.03 mg N L1

25

UV photo-oxidation of organic N to nitrate, detection by hydrazine reduction and Griess reaction

Photometry-540 nm, background correction at 320 nm

2 mM NO 2 8 mM NO 3

[117] Residual peroxydisulfate caused gradual degradation of Cd reductor [84] Cr(II) & Cr(III) interfere in reduction. Organic C interference in photo-oxidation. Inhibition of photo-oxidation at C/NZ20

TDN

FIA

Natural, wastewaters

TDN, NOx

FIA

Wastewaters

[216] [217] Solid samples sequentially extracted in NH4Cl (pH 7), NaOH and HCl for determination of the exchangeable Fe + Albound, and Cabound fractions [116]

TDN

SIA

Wastewaters

UV photo-oxidation of organic N to nitrate, detection by UV photometry

Photometry-226 nm, 0.6 mg N L1 background correction at 320 nm

30–40

NOx

FIA

Synthetic samples

Cd reduction and Griess reaction

Photometry-540 nm

—

—

NOx

FIA

River waters, sewage effluents

NOx and NO 2 reduced with Ti(III) and iodide to gaseous NO. Separated NO reacted with O3 producing gas-phase chemiluminescence NOx and NO 2 reduced with Ti(III) and iodide to gaseous NO. NH+4 reacted with ClO and volatile dichloramine thermally degraded to NO. Separated NO reacted with O3 producing gas-phase chemiluminescence Nitrate adsorbed on anion exchange resin, eluted with perchloric acid, detection by UV photometry

Gas-phase chemiluminescence

0.70 mg N L1 NO 3 0.35 mg N L1 NO 2

Gas-phase chemiluminescence

0.01 mM NOx, NH+4

20

Interference from chloride at 3.5% (w/w) NaCl

[85]

Photometry-201 nm

0.1 mg L1

17

[87]

Chemiluminescence

4 nM

10

No interference from 1 mg L1 1 NO 2 , 75 mg L Cl , 200 mg L1 1 PO3 4 , 50 mg L 1 SO2 ,15 mg L 4 Fe3+ Strong interferences from NO 2 and ClO

NOx, NH+4 FIA

NO 3

FIA

River, lake, mineral waters

NO 3

FIA

Potable, river waters

UV photo-reduction of NO 3 to NO 2 , reaction with H2O2 decreases luminol chemiluminescence

Higher concen[118] trations of oxidant required for high COD wastewaters [83] 35–100% nitrate reduction efficiency, depending on reductor used and geometry of column [218]

[86]


Matrix type

Basis for detection

Detection



Comments

Reference

NOx

FIA

Seawaters



0.45 mM

40

[109]

NOx

FIA



Photometry-LED photometer

1.4 mM

4

NOx, DRP, Si

FIA multicommutation

Seawaters

NH+4

FIA

Submersible field system, operated to 300 m depth, 2–351C, over salinity range 0–35 Submersible field system; used for tidal cycle monitoring Submersible system using solenoid pumps reagent delivery. Thirty nitrate profiles between 200 and 1,100 m acquired over a 15-day period Organic C interference removed with C18 resin

NH+4

NH+4

Cd reduction and Griess reaction. Formation of phosphomolybdenum blue, tin(II) chloride reduction Formation of silicomolybdenum blue, ascorbic acid reduction in presence of oxalate Natural waters Nessler’s reagent, cation exchange preconcentration

o0.1 mM NOx Photometric-LED photometer (lmax ¼ 525 nm, filt, 540 nm NOx, lmax ¼ 850 nm, filt, o0.1 mM DRP 850 nm DRP, filt. o0.5 M Si 810 nm Si)

Photometry-410 nm

3 mg L1

FIA

Fish farm waters

Photometry-660 nm

—

FIA

Fresh, saline waters

Fluorometrylex ¼ 370 nm, lem ¼ 418–700 nm

0.03 mM

Indophenol blue reaction, using salicylate instead of phenol OPA-sulfite

ca. 4

45

[128]

[63]

[89]

[219]

9

Negligible interference from primary

[92]

NH+4

FIA


Gas diffusion, OPA-sulfite acceptor stream


7 nM

30

NH+4

Reagent injection FIA

Waste, estuarine, seawaters

Gas diffusion, absorbance change of acid-base indicator


9 mg N L1 continuous mode 3 mg N L1 stopped flow mode 3–5 nM methylamines 20–40 nM NH3

135 60

NH+4 , FIA–IC methylamines

Natural waters Gas diffusion separation and preconcentration of volatile N species, ion chromatography with suppressor

Conductometrysuppressed

NH+4

Industrial effluents

Pervaporation, absorbance change of acid-base indicator

Photometry-590 nm

0.1 mg L1

11

Soil extracts

Indophenol blue reaction, using salicylate instead of phenol Gas diffusion, ammonia ion-selective electrode

Photometry

0.05 mg N L1

60

Potentiometry

—

80

Reaction with diacetyl monoxime thiosemicarbazide

Photometry

0.01 mg N L1

60

FIA

NH+4 /FIA

TN

FIA

Soil digests (Kjeldahl)

Urea-N

FIA

Soil extracts

4

amines, Hg2+. Slight interference from S2 No interference from volatile amines or salinity Interference from salinity not observed NH+4 poorly resolved from Na+ and K+ at higher concentrations Unfiltered samples, method tolerant of surfactants and particulates Potential interference from amino acids PVC electrode with sensor of nonactin in tris(2-ethylhexyl)phosphate

[93]

[94]

[220]

[221]

[222]

[223]

[224]


Matrix type

Basis for detection

Detection



Comments

Reference

Silicon species Silicate, SIA DRP

Wastewaters

Formation of silicomolybdate and vanadophosphomolybdate


0.9 mg Si L1 0.2 mg P L1

23

Sample segmentation with oxalic acid to avoid interference between silicate and phosphate Oxalic acid added to acid molybdate to avoid interference of silicate in P measurement

[225]

Silicate, DRP

SIA

Wastewaters

Formation of silicomolybdenum blue and phosphomolybdenum blue, ascorbic acid reduction

Photometry-660 nm

1 1 mg SiO2 3 L 3 1 0.1 mg PO4 L

74

Silicate

SIA

Ultrapure waters


0.06 mg L1

—

[227]

Silicate

FIA

Fresh waters

Fluorescence quenching of Rhodamine B due to ion pairing with silicomolybdate Oxidation of luminol by silicomolybdate

Chemiluminescence

0.35 mg Si L1

80

[228] Metal ion and phosphate interferences removed with cation and anion exchange columns, respectively

[226]

Table 6

Selected examples of flow-analysis methods for the determination of trace metal species in waters, sediments and soils

Analyte Mode

Matrix type

Basis for detection

Detection



Comments

Al

FIA

Potable, treated waters

Reaction with Pyrocatechol violet

Photometry-580 nm

o45 mg L1

2

Al

FIA

Lake waters

Formation of fluorescent Al-lumogallion complex


3.7 nM

—

Al

SIA

Fluorometrylex ¼ 354 nm, lem ¼ 481 nm Fluorometrylex ¼ 484 nm, lem ¼ 552 nm

60

FIA

Formation of fluorescent Al-8-HQ-5-sulfonic acid complex Preconcentration of Al on immobilized 8-HQ. Eluted Al forms fluorescent chelate with lumogallion

2.2 mg L1

Al

Drinking, treated waters Seawaters

[229] Field portable system. Interference by Fe(III) masked by reduction to Fe(II) and complexation with 1,10 phenanthroline No interference [230] from citric or fulvic acids, or Fe(III) [231]

ca. 0.15 nM

20

Al(III)

FIA

Waters

Al3+ differentiated from AlF+2 by complexation with 8-HQ

Atomic spectrometry-ICP-AES

2 mg L1

—

FIA

River water RM

Complexation of As(III) with PDC and preconcentration

Atomic spectrometry-ICP-MS

0.021 mg L1 As(III)

18

Shipboard application. Use of detergent to enhance fluorescence On-line isolation and 18-fold preconcentration of Al3+-8-HQ complex on XAD-2 resin mini-column No interference from organo-

Reference

[232]

[233]

[234]


Matrix type

As total, As(III), As(V)

Basis for detection

Detection

As(III)

FIA

As(III)

FIA

As, Sb, Se FIA

River, seawater Complexation of As(III) with RMs PDC and preconcentration in a knotted PTFE reactor. Total inorganic As measured after reduction of As(V) with l-cysteine. Spiked river Chemical (NaBH4) and electrochemical hydride generation waters

Watertreatment chemicals, riverine sediment RM

Generation of AsH3 which diffuses through PTFE membrane into an Ar gas acceptor. Chemiluminescence initiated by reaction with Generation of As, Sb and Se hydrides with NaBH4


0.029 mg L1 Total inorganic As

in a knotted PTFE reactor. Total inorganic As measured after reduction of As(V) with lcysteine

As total, FIA As(III), As(V)


Atomic spectrometry- 0.023 mg L1 As(III) AFS

32

Atomic spectrometry- 0.05 mg L1 ICP-AES

60

Chemiluminescence

0.6 mg L1

Atomic spectrometry- 0.037 mg L1 As Flame AAS 0.121 mg L1 Sb 0.131 mg L1 Se

300–450

—

Comments

Reference

arsenic species. Enrichment factor of 22 compared with conventional ICP-MS Enrichment factor of [235] 11

[236] Interference from Co(II), Ni(II), Cu(II), Zn(II) and Pb(II) when chemical hydride generation used Sb(III) interference [139] tolerated up to 40-fold excess

Thiourea and [237] L-cysteine used to reduce As(V) and Sb(V). KI-ascorbic acid used to mask Fe(III) interference

Cd

FIA

Wastewaters

Homogeneous crystalline double membrane electrode

Potentiometry

o 0.056 mg L1

15–20

Cd

FIA

Drinking waters


18 ng L1

—

Co

FIA

Seawaters

On-line preconcentration of Cd-8-HQ complex on activated carbon Co catalysis of the oxidation of DPD by H2O2 in presence of Tiron

Photometry-554 nm

1 ng L1

50

Co

FIA

Artificial seawaters, CRMs

Chemiluminescence

0.62 ng L1

7

Co

FIA

Spiked natural waters

On-line preconcentration of Co(II) on 8-HQ immobilized on silica gel. Eluted Co(II) detected with gallic acid-H2O2 chemiluminescence Sorption of Co(II) in a knotted PTFE reactor coated with PMBP

Atomic spectrometryElectrothermal AAS

8.1 ng L1

—

Co

FIA

Drinking waters

On-line preconcentration of Co on activated carbon


20 ng L1

—

Co, Fe

FIA

Seawaters

Co(II) catalyzed oxidation of pyrogallol by H2O2 in presence of methanol and surfactant Oxidation of luminol by dissolved oxygen catalysed by Fe(II)+Fe(III)

Chemiluminescence

5 pM Co(II)

—

40 pM Fe(III) +Fe(II)

On-line preconcentration of chlorocomplexed Cd by anion exchange (AG1 X-8) Enrichment factor of 80 for 50 mL sample Method suitable for shipboard deployment without sample pretreatment NASS and CASS CRMs analysed

[238]

Masking of Al(III) with fluoride, and Cu, Fe(II) and Fe(III) with thiourea Enrichment factor of 95 for 50 mL sample Shipboard application

[241]

[239]

[240]

[138]

[242]

[66]

Table 6 (Continued ) LOD (units as quoted)


Comments

Reference

2.0 mg L1 Cr(VI)

17

Enrichment factor of 25

[243]

o0.1 mg L1

30

[244]

Atomic spectrometry-Flame AAS

0.2 mg L1 Cr(III) 0.2 mg L1 Total Cr

—


20 ng L1

—

Cr(III) oxidized on-line with NaIO4 followed by complexation with PTQA Total Cr determined by reduction of Cr(VI) with ascorbic acid Cr(III) masked with CDTA. Enrichment factor of 250 Use of a porous core electroosmotic pump for carrier propulsion

Analyte Mode

Matrix type

Basis for detection

Cr(III), SIA Cr(VI)

Waters

Cr(III), FIA Cr(VI)

Cr(VI)–DPC ion paired with Photometry-546 nm ClO 4 and extracted into a solvent film on the inner wall of a PTFE tube, followed by elution with acetonitrile Oxidation of PTQA by Cr(VI) Fluorometryto form fluorescent product lex ¼ 360 nm, lem ¼ 500 nm

Mineral, tap, distilled waters, sediment RM Tap, mineral, On-line separation and river waters preconcentration of Cr(III) using PAPhA chelating resin

Cr(III), FIA Cr(VI)

Cr species preconcentrated on Amberlite XAD-16 resin as Cr-TAR

Detection

Cr(III), FIA Cr(VI)

Waters

Cr(VI)

FIAsandwich zone injection

Spiked waste- Reaction of Cr(VI) with DPC waters

Photometry-540 nm

—

—

Cu

FIA

Waters


0.6–1.5 mg L1

15–30

Preconcentration of Cu(II) on Dowex 50 W-X8

[245]

[246]

[247]

[248]

Cu

FIA

Seawaters

Cu, Ni, Mn

FIA

Seawaters

Fe total

FIA

Seawaters

Fe total + Mn

Reverse FIA

Treated waters

Fe total, Fe(II)

FIA

Seawaters

Fe(II)

FIA

Seawaters

Fe(II), Fe(III)

FIA

Tap, river, ground waters

Fe(II), Fe(III)

FIA

Natural waters

In-field sample loading on Amberlite XAD-4 impregnated with PAN. Minicolumns connected to laboratory FIA and eluted into AAS Formation of Cu and Ni PDC and Mn-8-HQ. Preconcentration in a knotted PTFE reactor Preconcentration of acidified sample onto NTA chelating resin. Fe-catalysed oxidation of DPD by H2O2 Fe and Mn catalyzed oxidation of luminol by KIO4


0.06 mg L1

—

Atomic spectrometryElectrothermal AAS

6.0 mg L1 Cu 7.6 mg L1 Ni 29 mg L1 Mn

26

Photometry-514 nm

0.024 nM

—

Chemiluminescence

3 ng L1

Preconcentration with cation exchange column. Fe(II) catalysed oxidation of brilliant sulfoflavin by H2O2 Fe(II) catalyzed oxidation of luminol

Chemiluminescence

0.45 nM

12

Chemiluminescence

8–12 pM

—

Formation of Fe(III)-PDC complex and preconcentration in a knotted PTFE reactor under pH controlled conditions Sorption and absorbance measurement of Fe(SCN)3 6 in flow cell packed with exchange resin


0.08 mg L1

21

Photometry-480 nm

80 mg L1

—

Field sampling and [249] preconcentration technique. Enrichment factor of 30 for 25 mL of sample Enrichment [250] factors of 44 (Cu), 21 (Ni) and 8 (Mn) for 30 s sample loading [136]

Used for in situ monitoring of Fe + Mn during water treatment

[251]

[51]

Shipboard deployment. Co interference masked with dimethylglyoxime Enrichment factor of 12 for 2.5 mL sample

[42]

In-valve oxidation of Fe(II)

[253]

[252]

Table 6 (Continued ) Comments

Reference

Analyte Mode

Matrix type

Basis for detection

Detection



Hg

FIA

River, lake, rain waters

Atomic spectrometry-Cold vapour AAS

10 ng L1

24

Hg

FIA

Tap waters

Atomic spectrometry-Cold vapour ICP-AES

4 ng L1

—

Enrichment factor of 200 from a 50 mL sample

[254]

Hg

FIA

Drinking waters

7

Inhibition also due to Cu2+ and Ag+

[206]

Hg

FIA

Waters, sediment/ soil digests

On-line microwave digestion of inorganic and organic Hg compounds. Amalgamation preconcentration Cloud point extraction of Hg(II)-5-Br-PADAP complex in non-ionic surfactant, PONPE 5. Reduction of Hg(II) with SnCl2 Inhibition of hydrolysis of urea with immobilized urease. Detected by a change in the acceptor stream pH of a gas diffusion FIA system due to decreased NH3 production Gas diffusion of Hg cold vapour into a KNO3 acceptor stream

Amperometry, gold electrode at +0.6 V

900 ng L1

12

[255]

Hg total

FIA

Natural waters

Hg vapour amalgamated on gold-coated piezoelectric crystal

Piezoelectric crystal

o0.3 mg L1

—

Atomic spectrometry-Cold vapour AAS

—

20 at 100 W and 20 kHz

Gas diffusion cell heated to 851C to enhance diffusion Regeneration of piezoelectric crystal by peroxydisulfate Eliminates the need for oxidants. Tolerant of hydroxyl scavengers at concentrations Z1,000 mg L1

Hg FIA total, Hg inorganic

Spiked Ultrasonic irradiation natural (sonolysis) used to convert waters, organic Hg into Hg(II). simulated Reduction with SnCl2 or wastewaters NaBH4

Potentiometry-pH

[131]

[256]

[134]

Hg, Hg FIA organic

Seawaters

On-line thermal oxidation of organic Hg to Hg(II) with Br/BrO 3 Mn2+ oxidized in PbO2 reactor to form MnO 4 Mn2+ catalyzed oxidation of 4,4-bis(dimethylamino)diphenylmethane with NaIO4 Mn2+ catalyzed oxidation of TCNQ

Mn

SIA

Mn

FIA

Tap waters, effluents Natural waters

Mn

FIA

Seawaters

Ni

FIA

Natural waters

Pb

FIA

Tap waters

Pb

FIA

Pb

FIA

Tap, pond, Preconcentration of Pb on river waters immobilized crown ether with a cavity size selective for Pb2+ (Pb-Specs) Seawaters In-field sample loading on Amberlite XAD-4 impregnated with PAN. Minicolumns connected to laboratory FIA and eluted into AAS

On-line preconcentration of Ni on activated carbon at pH 5.0. Eluted with HNO3 Complexation with 5,10,15, 20-tetra(4-N-sulfoethylpyridinium)-porphyrin

Atomic spectrometry-AFS

25 ng L1 Hg(II) 23 ng L1 CH3HgCl

—

Photometry-526 nm

0.62 mg L1

50

Photometry-602 nm

0.073 mg L1

60

Stopped flow kinetic method

[258]

Chemiluminescence

0.1 nM

10

[53]


82 ng L1

—

Shipboard application. Preconcentration using immobilized 8-HQ Enrichment factor of 80 from a 50 mL sample

Photometry-480 nm

10 mg L1


1 mg L1

—


5 ng L1

—

Oxidation efficiency ca. 100%

[135]

[257]

[259]

[260] Interference from Al, Cd, Cu, Mn and Zn masked with NH3/NH4Cl buffer. Fe inteference masked with acetylacetone Column eluted [261] with ammonium oxalate Field sampling and [262] preconcentration technique. Sample size up to 1,000 mL


Matrix type

Basis for detection

Detection



Comments

Pb

FIA

River, seawaters

Formation of Pb-PDC complex and preconcentration in a knotted PTFE reactor

Atomic spectrometry-Electrothermal AAS

2 ng L1

—

Pb

FIA

Tap waters

Formation of Pb-diethyldithiocarbamate complex and preconcentration in a knotted PTFE reactor On-line preconcentration of Pb isotopes for TOF-MS


0.2 mg L1

—

Enrichment factors [263] of 142 for a sample of 6.8 mL. Standard addition necessary for seawaters Enrichment factor [264] of 140 from a 10 mL sample

Atomic spectrometry-ICP-TOFMS Chemiluminescence

6 ng L1

—

Enrichment factor of 20

[265]

0.057 mg L1

—

Interference from DOM removed using C18

[266]

Atomic spectrometry-hydride generation-AAS

0.55 mg L1 (4 M HCl, 10% Lcysteine) 0.54 mg L1 (4 M HCl, 10% thiourea)

60

Interferences from [267] transition metals and hydride forming elements eliminated by addition of l-cysteine or thiourea

Pb, Pb FIA isotope ratios Pt(IV) FIA

Natural waters

Sb

Spiked well, seawaters

FIA

Spiked river waters

On-line separation of Pt(IV) on biosorption column. Pt(IV) catalyzed oxidation of luminol SbH3 generated by reaction between Sb and tetraborohydrate immobilized on strong anion exchange resin.

Reference

Se

MSFIA

Drinking waters

Generation of Se hydride by reaction with NaBH4

Se total, Se(IV)

FIA

Seawaters

Se(IV)

FIA

Surface waters

Se, As

FIA

Zn

FIA

Natural waters, plant digests River, estuarine, seawaters

On-line by microwave irradiation (210 W for 60 s) with NaBr to reduce Se(VI) Se(IV) co-precipitated with La(OH)3 and collected on a PTFE bead column. Precipitate dissolved with HCl and hydride produced with NaBH4 Sample alternately mixed with HCl or thiourea to reduce Se(VI) and As(V) prior to hydride production On-line separation of Zn from seawater matrix and preconcentration

Note: Metals are arranged alphabetically according to chemical symbol.

Atomic spectrometry-hydride generation-AFS Atomic spectrometry-hydride generation-AFS Atomic spectrometry-hydride generation-AFS

0.01 mg L1

84

[268]

5 ng L1 Se(IV) 4 ng L1 Total inorganic Se 5 ng L1 (3.4 mL sample) 3 ng L1 (6.8 mL sample)

—

[130]

Atomic spectrometry-hydride generationICP-MS Atomic spectrometry-isotope dilution ICP-MS

0.03 mg L1 Se 0.02 mg L1 As

20

[129]

0.014 mg L1

—

[132]

38

Enrichment factors of 11 and 20 for 3.4 and 6.8 mL of sample, respectively

[269]

Table 7

Selected examples of flow-analysis methods for the determination of general chemical quality parameters in waters, sediments and soils Matrix type

Basis for detection

Detection



Comments

Tap, river waters

Precipitation of K-18-crown6 complex - tetraphenylborate ion pair

Indirect photometry-250 or 274 nm

—

30

Natural Reaction with cresolphwaters, KCl thalein complexone soil extracts

Photometry-575 nm

—

160

Ca, Mg, SIA alkalinity

Waters

Photometry-570 nm (Ca, Mg), 611 nm (alkalinity)

0.32 mg Ca L1 0.03 mg Mg L1 1 5.1 mg HCO 3 L —

40 40 65

Ca, Mg

SIA

Mineral waters

Reaction with cresolphthalein complexone. Alkalinity determined by colour change in Bromocresol green due to HCO 3/ CO2 3 reaction with acetic acid Automated sample dilution and addition of La-releasing agent

[141] On-line filtration to remove precipitate. Sample treatment required to remove interference from Fe(II) [142] Masking with 8-HQ and EGTA for Ca and Mg determinations, respectively Masking with [143] 8-HQ and EGTA for Ca and Mg determinations, respectively

Atomic spectrometry-flame AAS

—

—

Ca, Mg

FIA

Natural waters

Decrease in free phosphate concentration in carrier due to formation of Ca3(PO4)2 or Mg3(PO4)2 detected by Co wire electrode

Indirect potentiometry

10 mM

—

Analyte Mode

Major cations and anions K FIA

Ca, Mg

FIA

Relative error between conventional method and SIA o3.6% Masking with 8-HQ and EGTA for Ca and Mg determinations, respectively

Reference

[144]

[145]

Ca

FIA

Ca, Cl

FIA

Cl

SIA

Cl

FIA

Natural waters

Cl

SIA

SO2 4

FIA

Ground, surface, wastewaters Natural, seawaters

SO2 4

FIA

Soil solutions

TIC, FIA HCO 3


Total CO2 3

River, sea, rain, distilled waters

FIA

Natural, borehole waters Natural waters Mineral, natural, ground waters

Use of ion-selective membrane electrodes for Ca and F Use of two ion-selective electrodes in series Chloride reacts with Hg(SCN)2. Released SCN reacted with Fe(III) forming Fe thiocyanate complex Chloride reacts with immobilized Hg(SCN)2 in a bead reactor. Released SCN reacted with Fe(III) forming Fe thiocyanate complex Formation of AgCl precipitate

Potentiometry

1.94 mM

60

[146]

Potentiometry

—

40

[147]

Photometry-480 nm

3 mg L1

37

[148]

Photometry-480 nm

14 mM

100

[149]

Turbidimetry-410 nm

2 mg L1

55

Formation of PbSO4 precipitate in an ethanol–water medium Sulfate reacts with Ba DMSA in dimethylsulfoxide giving a decrease in absorbance Gas diffusion of CO2 into acceptor stream

Turbidimetry-410 nm

1 0.3 mg SO2 4 L

35

[151]

Indirect photometry-668 nm

1 0.3 mg SO2 4 L

60

[152]

Acoustic wave impedance

10 mM

45

Gas diffusion of CO2 into acceptor stream of DNN5S acid–base indicator


1 mM

—

Interference from iodide and sulfide

Used to detect TOC after offline wet chemical oxidation

[150]

[153]

[154]


Matrix type

Basis for detection

Detection



Comments

pH PH

Seawaters

Injection of phenol red indicator

Photometry-433 and 558 nm

—

25

Shipboard opera[155] tion. Precision better than 70.005 pH units

River, pond, seawaters

I2 liberated in the Winkler method extracted on-line with 1,2-dichloroethane containing 2-thionaphthol, causing a decrease in fluorescence On-line Winkler reaction. Triiodide produced

Indirect fluorometry lex ¼ 288 nm, lem ¼ 355 nm

0.49 mM

18

Batchwise preparation of samples by Winkler method required

[156]

Photometry-440 nm

0.25 mg O2 L1

48

Method applied to on-line measurement of DO in sediment core reactors

[157]

Reverse FIA

Dissolved oxygen (DO) DO FIA

DO

FIA

Natural waters

DO

FIA

River, pond, tap waters

I2 produced by on-line Winkler reaction extracted with 1,2-dichloroethane containing 2-thionaphthol, causing a decrease in fluorescence

Indirect fluorometry-lex ¼ 288 nm, lem ¼ 355 nm

—

12

Seawaters

Oxidation of luminol by H2O2 with Co2+ catalyst in alkaline solution Reaction of periodate with H2O2 in alkaline solution

Chemiluminescence

10 nM (Ultrapure water) 5 nM (seawater) 5 nM

120

Hydrogen peroxide FIA H2O2

H2O2

FIA

Snow melt waters

Chemiluminescence

100

Reference

[158]

Portable system deployed shipboard Little interference from transition metals

[159]

[160]

H2O2

FIA

Seawaters

Oxidation of luminol by H2O2 with Co2+ catalyst in alkaline solution Oxidation of luminol by H2O2 with Co2+ catalyst in alkaline solution Reaction of H2O2 with an acridinium ester

H2O2

FIA

Rain, tap waters

H2O2

FIA

Rain, fresh, seawaters

Sulfide S2

FIA


Nitroprusside method, or MB method

S2 reacts with DPD in presence of acidic Fe(III) to produce MB that is adsorbed to C18 in photometric flow cell S2 reacts with DPD in presence of acidic Fe(III) to produce MB

Solid-phase photometry666 nm

2 nM

—

Chemiluminescence

0.5 nM

100

Chemiluminescence

58.1 nM (rain) 1.61 nM (surface water) 0.352 nM (seawater)

—

Little sensitivity to [163] organic peroxides

Photometry-538 or 745 nm

20 mg L1 (direct injection) o2 mg L1 (12 mL sample) 1.7 mg L1

30

In-valve gas diffusion for preconcentration

[164]

12

Elution with methanol and HCl

[165]

Photometry-LED photometer (lspectral band ¼ 600–700 nm)

0.15 mg L1

80

5

S2

FIA

Waters

S2

MSFIA

Mineral, tap, fresh, sea, wastewaters

Cyanide CN

FIA

Wastewaters

Gas diffusion preconcentration of sample. Reaction of diffused HCN with INA-PZ

Photometry-544 nm

3 mg L1

20

CN

FIA

Wastewaters

Pervaporation gas diffusion of HCN. Precipitation of S2 with Pb2+ prior to pervaporation

Amperometry, 0.05 V

1 mg L1

12–15

Little interference from ions other than Fe2+ Stopped flow mode

[161]

Chemiluminescence

[162]

[166]

Elimination of interferents such as CNS by silicone membrane No significant interference from 50 mg L1 S2 or 1000 mg L1SCN

[167]

[168]

Table 7 (Continued ) Comments

Reference

Analyte Mode

Matrix type

Basis for detection

Detection



CN

Wastewaters

Suppression of fluorescence by reaction of cyanide with Cu-calcein complex bound to Amberlite XAD-2


0.5 mM

10

[169]

On-line UV photo-oxidation of DOC. Gas diffusion of CO2 into phenolphthalein indicator On-line UV photo-oxidation of DOC. Gas diffusion of CO2 into cresol red indicator Near-UV TiO2 mediated photooxidation

Photometry-552 nm

0.1 mg C L1

45

[119]

Photometry-570 nm

0.05 mg C L1

8

[170]

Conductometry

1 nM

—

Photometry445 nm

1.5 mg COD L1

—

[172]

Photometry

0.5 mg COD L1

30

[173]

Chemiluminescence

—

—

[174]

Atomic spectrometry-ICP-AES, l ¼ 333.75 nm (LaII line)

30 mg L1

36

[175]

FIA-flowthrough opto-sensor

Dissolved organic carbon (DOC) DOC FIA Stream waters

DOC, DIC SIA

DOC

Fresh waters

FIA

Synthetic samples, algae Chemical oxygen demand (COD) COD FIA Well, river, wastewaters COD FIA Fresh waters

COD

FIA

Waters

Fluoride F

FIA

Waters

On-line microwave digestion with potassium dichromatesulfuric acid UV photo-catalytic oxidation with acidic potassium permanganate UV photo-oxidation with O3

Lanthanum/alizarin complexone/fluoride complex extracted into hexanol/ N,N-diethylaniline

Calibration using gaseous CO2

[171]

F

FIA

Waters

F

FIA

F

FIA

Natural, borehole waters Natural, borehole waters

Other halogen species FIA ClO 3, ClO 2

Drinking waters

ClO 3

FIA

Soil extracts

ClO2

Reverse FIA

Waters

ClO2

Reverse FIA

Waters

Br, BrO 3

FIA

Drinking waters

Immobilized periodate and luminol released by injection of sample containing F Use of ion-selective membrane electrodes for Ca2+ and F

Chemiluminescence

20 ng L1

120

[176]

Potentiometry

4.83 mM

60

[146]

Conversion of F to TMFS. Gas diffusion preconcentration; release of F and complexation with Zr-Alizarin

Photometry-520 nm

0.055 mg L1

17

Iodometric detection. Multivariate calibration required for quantification of ClO 3 Reaction of chlorate with excess KI to form I2/I 3. Amperometric detection of I 3 Reaction of chlorine dioxide with LB

Photometry-360 nm

Reaction of chlorine dioxide with Chlorophenol red On-line anion exchange separation of bromide and bromate prior to detection with ICP-MS

High tolerance for Al3+

[177]

Interference from chloramine and other oxidants Stopped flow mode used to enhance sensitivity Tolerant of ClO 3. Cl2 and ClO masked with oxalic acid

[178]

Indirect amperometry, +0.2 V

1.2 mM

25

[179]

Photometry

0.02 mg L1

-

Photometry

0.024 mg L1

60

[181]


0.13 mg L1

6

[182]

[180]

Table 8 Selected examples of flow-analysis methods for the determination of organic contaminants in waters, sediments and soils Analyte Mode

Surfactants Anionic FIA

Matrix type

Basis for detection

Detection



Comments

Reference

River, wastewaters

On-line preconcentration, detection with tubular electrode

Potentiometry

0.1 mM (0.03 mg L1)

10

No interference from chloride, nitrate, non-ionic surfactants

[184]

Polyoxylene sorbitan monoleate (Tween-80s) determined by fluorescence enhancement of Eosin B FIA-ESI-MS operated by alternating between positive and negative modes


1.7 mg L1

—

Nonionic

SIA

Natural waters

Anionic, catio nic, nonionic

FIA

Wastewaters

Mass spectro— metry-electrospray ionization

[185]

—

Samples isolated by liquid–liquid extraction Labelled triethoxylated nonylphenol and Na

[186]

6.5 min incubation

Screening application. Affinity column could be regenerated and

[187]

dibutylnaphthalenesulfonate used as internal standards Pesticides and herbicides AtrazFIA ine, diuron

Waters

Sequential competitive enzyme immunoanalysis. Atrazine competes with peroxidase-labelled atrazine for membrane-bound antiatrazine antibodies. Detected


ca. 0.01 mg L1

Sulfonurea herbicides

FIA

River waters

Atrazine FIA

Waters, soil extracts

s-TriFIA azines

Natural, tap waters

Carbaryl FIA

Natural waters Waters, soil extracts, grain extracts Waters, soil extracts

Carbaryl FIA

Paraquat FIA

by decrease in fluorescence of fluorogenic peroxidase substrate Photochemically induced fluorescence

Atrazine-selective membrane sensor, consisting of atrazine-phosphomolybdate ion-pair complex dispersed in PVC matrix Cyclic volatmmetry and amperometry using Cu and glassy carbon electrodes Oxidation of carbaryl by potassium permanganate Photoconversion of carbaryl to methylamine, detection with photogenerated Ru(bpy)2+ 3 Formation of a fluorescent paraquat-benzaldehyde charge complex

used W1,000 times Fluorometrychlorsulfuron lex ¼ 314 nm, lem ¼ 380 nm Metsulfuron-Me lex ¼ 322 nm, lem ¼ 378 nm 3-rimsulfuron lex ¼ 317 nm, lem ¼ 365 nm Sulfometuron-Me lex ¼ 290 nm, lem ¼ 341 nm Potentiometry

0.2 mg L1

Voltammetry

0.2 mg L1 aziprotryne


14.8 mg L1

—

[191]

12 mg L1

200

[192]

0.5 mg L1

—

[193]


56–80

Spiked recoveries of 90–106%

[188]

60

Near-Nernstian [189] response over range 102–105 M atrazinium ion

0.1 mg L1

0.2 mg L1

1.0 mg L1

0.3 mg L1

[190]


Matrix type

Basis for detection

Detection



Comments

Paraoxon FIA

Waters, soil extracts

Inhibition of immobilized acetylcholinesterase monitored by measurement of thiocholine produced by enzymolysis of acetylthiocholine iodide

Photometry-412 nm

—

6

[194] Enzyme reactor showed irreversible inhibition and required on-line reactivation with pyridine2-aldoxime-methiodide after each sample injection

Soil extracts

Biosensor utilizing immobilized bilirubin oxidase in the presence of excess NADH PCP oxidized 1,4-TCBQ which is oxidized by immobilized glucose oxidase. Process monitored by glassy carbon electrode Oxidative detection of phenols at an upstream coulometric electrode at +0.35 V


25 nM

—

[195]

Amperometry, glassy carbon electrode at +0.40 V

10 nM

ca. 12

[196]

Amperometry, +0.78 V

o0.1 mg L1

60

Acidified phenols diffuse across headspace and hydrophobic pervaporation membrane into alkaline acceptor stream


0.9 mg L1

5

Phenols PentaFIA chlorophenol PentaFIA chlorophenol

Contaminated soil extracts

Total FIA phenols

Industrial wastewaters

Phenols

Spiked lake waters

FIA

Removal of interferences by oxidative electrochemical reaction with an upstream electrode Method able to cope with particulate load

Reference

[197]

[198]

On-line derivatization of phenol to phenyl acetate prior to pervaporation through hydrophobic membrane, conversion to phenolate, and amperometric detection Reaction between phenolic substances and alkaline 4-aminoantipyrine On-line derivatization of phenols to phenyl acetates prior to diffusion through a silicone membrane into the mass spectrometer


25 mg L1

4

[199]

Photometry-510 nm

o0.05 mg L1

24

[200]

Mass spectrometryselected ion monitoring

0.5–20 mg L1

6

Tap, river waters

PAHs preconcentrated on C18 mini-column by FIA

Fluorometry: Laser inducedlex ¼ 355 nm, CCD detection, lem ¼ 370–530 nm

0.09–0.6 mg L1

Waters

NPA coupled with diazotised sulphanilamide (Griess reaction) to form azo dye; preconcentrated on C18 disk for reflectance detection Flow injection-atmospheric pressure chemical ionisation mass spectrometry

Reflectance photometry540 nm

1.1 mg L1

Phenols

FIA

Spiked river, effluent waters

Phenols

SIA

Wastewaters

Phenols

FIA–MIMS

Waters

Others FIA Polycyclic aromatic hydrocarbons (PAHs) 1-naph- MSFIA thylamine (NPA) Biomarkers

FIA-MS

Sediment extracts

Mass spectrometry

Determination of phenol, 2methylphenol, 4-chlorophenol, 4-chloro-3methylphenol, 2,4-dichlorophenol, 2,4,6trichlorophenol

[201]

[202]

14

[203]

60

Preparative HPLC [204] used to isolate biomarkers from sediment extracts

734


that is reduced with ascorbic acid or tin(II) chloride/hydrazine sulfate under controlled pH conditions to produce phosphomolybdenum blue, Table 5). However the acidic reaction conditions that favour the formation of phosphomolybdate and which are necessary to avoid interference from silicate (tartrate is also used to inhibit the formation of silicomolybdate) are known to cause hydrolysis of some organic and condensed phosphorus moieties, and for this reason the term reactive phosphorus is used in preference to ‘‘phosphate’’ [27]. There is an emerging interest in the role of organic phosphorus that stems from its relative abundance in aquatic and terrestrial systems, and the potential for its release and ultimate conversion to bioavailable forms [77]. The use of FIA systems capable of discriminating between organic and other forms of phosphorus provides a valuable tool for refining understanding of the aquatic phosphorus cycle. One convenient approach involves the photo-oxidation of DOP with alkaline peroxydisulfate in a FIA or SIA system with a PTFE capillary photo-reactor and low wattage UV source [46]. This approach selectively and rapidly (o60 s) oxidizes DOP to DRP that is then determined by photometric [78] or fluorometric [79] detection. The differentiation of dissolved condensed phosphates (DCPs) from DOP and DRP requires either selective weak acid hydrolysis before detection as DRP [80], or chromatographic separation of condensed phosphate species (e.g., tri- and di-phosphate) from orthophosphate, e.g., by use of anion exchange, prior to on-line acid hydrolysis and detection as phosphomolybdenum blue [81]. High and low molecular mass organic P species have also been separated on-line using gel filtration followed by detection as DRP after photo-oxidation [82] and applied to the characterization P in sediment pore waters and sediment extracts [77]. The almost universal flow-injection method for nitrate detection involves the use of a highly toxic Cd reductor column, followed by diazotization of nitrite, and coupling to form an azo dye. Despite widespread use, this method has a number of recognized problems which include incomplete reduction, over reduction and reductant poisoning by constituents of the sample matrix. These can result in variable nitrate reduction over the course of an analytical run, giving rise to poor selectivity for nitrate and nitrite [83]. The method also produces wastes containing Cd, and it is highly desirable that this method is replaced by a greener, more reliable method. Various authors have reported the use of hydrazine reduction as an alternative reductant [84], but this method is restricted to freshwaters and wastewaters. Other approaches include reduction of nitrate and nitrite using Ti(III) and iodide to NO with detection by gas-phase chemiluminescence [85], photoreduction of nitrate to nitrite with detection by chemiluminescence quenching of the H2O2–luminol reaction [86], and the anion exchange isolation of nitrate prior to direct UV detection [87]. However, none of these methods is suitable for nitrate detection in marine waters, and as yet there is no viable alternative detection chemistry that is as broadly applicable as the Cd reduction chemistry combined with the Griess reaction for the detection of nitrate. One promising approach which is being investigated by the United States Geological Survey, and which overcomes some of the problems, involves the use of off-line nitrate reduction with nitrate reductase followed by detection with the Griess reaction [88].


735

Photometric FIA involving the use of Nessler’s reagent [89] or the formation of indophenol blue (Berthelot’s method) [90] are used widely for the determination of ammonia in water and wastewater samples. Both methods use toxic reagents, and it is desirable from both environmental and occupational safety perspectives that these are replaced with more benign reagents. The indophenol blue method also suffers from interference by Mg2+ in marine waters, and an empirical correction based on Mg2+ concentration, pH or salinity must be applied to compensate for this effect [91]. These interferences can be avoided by the use of the OPA-fluorometric method, either directly [92] or after gas diffusion [93], or by gas diffusion with photometric detection [94]. The use of gas diffusion and/or fluorescence detection also avoids schlieren effects that are commonly encountered when FIA with photometric detection is used for estuarine samples. Various strategies have been reported for the improvement of sensitivity. These include the use of modified or alternative detection chemistries, on-line preconcentration, or improved detector optics. Phosphorus-limited waters can contain DRP concentrations ofo1 mg P L1, and approaches to improve sensitivity include ion-pairing of phosphomolybdate with reagents such as Malachite green [95] or Rhodamine B [96] to produce chromophores with higher absorptivity than phosphomolybdenum blue. Alternately fluorescence quenching of Rhodamine B by phosphomolybdate [97], or the oxidation of luminol by phosphovanadomolybdate [98] or phosphomolybdate [99] that produces chemiluminescence, also give enhanced sensitivity. In-valve preconcentration using anion exchange of orthophosphate was also shown to enhance sensitivity in freshwaters [100]. More recently Liang et al. [101] used a hybrid batch-flow injection system to perform solid-phase extraction of CTAB ion-paired phosphomolybdenum blue onto C18 which, after elution, gave nM detection of DRP in marine water samples. The selective and sensitive method for ammonia determination in marine samples described by Watson et al. [93] epitomizes some of the advantages of the FIA approach, viz.; the ability to condition samples/reagents on-line, perform phase separations to improve selectivity, and operate within a closed system. This involves the generation of ammonia at higher pH, followed by gas diffusion and detection by the OPA fluorescence method (Figure 4). This system utilized on-line reagent scrubbing to remove ammonia contamination from the alkaline reagent, and the FIA manifold was contained within a N2-purged chamber to avoid atmospheric contamination by diffusion through the PTFE manifold tubing. The use of long pathlength capillary cells to achieve enhanced sensitivity for species such as DRP has regained favour with the introduction of capillaries made of or coated with Teflon AF2400s and related products. These polymer capillaries have refractive indices less than water, and act as a liquid core waveguide (LCW) when filled with aqueous liquids. Unlike earlier systems that relied simply on the use of an extended optical pathlength [102] and suffered from scattering losses, there is little light attenuation even when long capillaries are used. Gimbert et al. have reported a LOD of 10 nM when a 1 m long LCW capillary cell was used in an FIA system for detection of DRP as phosphomolybdenum blue (Figure 5) [103]. However, because of the optical geometry, LCW detection systems are prone to pronounced schlieren effects. Several different

736


Sample

1.35 mL min

-1

Temperature controller 70 °C

Carrier

0.136 mL min

Waste

-1

NaOH

10% H2SO4 0.81 mL min-1 OPA

Gas-diffusion unit with teflon membrane Fluorescence detector

Peristaltic pump

Figure 4 FIA manifold for low-level detection of ammonia in marine waters. Note the on-line ammonia scrubber used to reduce the blank signal. Reprinted from Ref. [93]. Copyright (2003), with permission from the Royal Society for Chemistry.

approaches have been applied to overcoming the schlieren effect in FIA, including the use of large volume injections, dual wavelength measurement and correction [104,105], on-line matrix matching combined with reagent injection [58,106] and the use of reflective detection cells with an optical path that is less affected by refractive index differences [59,60] (Figure 6). In general, sample matrix problems commonly occur in water, soil and sediment analysis due to variations in sample ionic strength, the presence of coloured DOM such as humic and fulvic acids, or colloidal material that passes through membrane filters. In marine and estuarine samples the presence of ions such as Mg2+ and Ca2+ is often problematic and they have to be removed by cation exchange [107] or by masking with a chelating agent such as EDTA [80]. DOM can be removed on-line by solid-phase extraction [89,108], or multi-wavelength measurements can be used to perform background absorbance correction [109]. Matrix interferents such as sulfide or Fe(III) can be overcome by on-line oxidation [110] or reduction, respectively. Another possible approach where sample colour or colloidal content may vary from sample to sample is to use reverse FIA or multicommutation. In this mode, reagents are injected in an appropriate sequence into a continuously flowing stream of sample. This has the advantage that the analytical signal is imposed on a continuously measured sample background. However, a possible disadvantage is that reagent blank signals can be large because of impurities in the injected reagents, and LODs may suffer accordingly. The use of enzymatic reactions for selective hydrolysis by FIA techniques is an approach to nutrient speciation that has been only superficially explored to date. Shan et al. [111] described the use of immobilized alkaline phosphatase for the on-line hydrolysis of phosphomonoester species in waters (Figure 7), and similar approaches have been employed for the determination of phytase

737


Flow rate Sample loop = 500 mL (mL min-1) Reaction coil Carrier 1.2 30 cm (ultra-pure water) 0.3 Ammonium Molybdate 0.3 Tin(II) (a)

LWCC

Reaction coil 120 cm Detector

Chloride Waste

(b)

Figure 5 (a) Flow-injection manifold and (b) calibration data for high-sensitivity DRP analysis using a 1 m liquid core waveguide flow cell. Reprinted from Ref. [103]. Copyright (2007), with permission from Elsevier B.V.

hydrolysable phosphorus (PHP) [112], a fraction which includes myo-inositolhexakisphosphate which constitutes a significant proportion of naturally occurring organic phosphorus in sediments and sediment porewaters [77]. The phospholipid component of sediment and water organic phosphorus has also been detected using a flow-injection system with immobilized phospholipase C [113]. Similar FIA approaches using, e.g., nitrogenase and peptidase would be beneficial in the study of organic nitrogen speciation. Flow-injection photo-oxidation systems can also be used for the determination of total dissolved or total P, N and C species. Modification of the DOP system described above [46], involving the use of acidic oxidant [107] or an additional acid hydrolysis digestion stage [114] has been reported (Figure 8) and is now an approved standard method [80]. Similar TP digestion efficiency may be achieved using microwave digestion [47,115]. The same photo-oxidation approach has also been applied to the determination of TDN. The nitrate produced by

738


Figure 6 (A) Representation of the multi-reflection flow cell showing side entry and exit of the incident and emergent rays. (B) Comparison of schlieren responses obtained using the multi-reflection (MRC) and z-flow cells for either injections of saline water of varying salinity (S) into deionized water (normal FI), or injection of deionized water into saline water (reagent injection FI) for: (a) MRC, normal FI, S ¼ 100, (b) z-cell, normal FI, S ¼ 100, (c) MRC, reagent injection FI, S ¼ 100%, (d) z-cell, reagent injection FI, S ¼ 100, (e) MRC, reagent injection FI, S ¼ 25, (f) z-cell, reagent injection FI, S ¼ 25. The absorbance scales for FI traces (a), (b) and (e), (f) are offset by +0.006 and 0.006, respectively. Reprinted from Ref. [60]. Copyright (2003), with permission from Elsevier B.V.

photo-oxidation was detected by either direct UV absorbance measurement [116] or by the Cd reduction/Griess reaction method [117,118]. In both cases the high concentration of residual oxidizing reagent had an adverse effect by either producing a high background UV absorbance, or by accelerating the degradation of the Cd reduction column. This efficient FIA photo-oxidation process has also been described for the determination of DOC [119]. There are relatively few reports of successful field measurements of nutrients by FIA in portable or autonomous, unattended operational modes. Early field FIA systems tended to consist of slightly repackaged laboratory systems that

739


Figure 7 Flow-injection manifold for determination of APHP. IAPR, immobilized alkaline phosphatase-packed bed reactor; I, injection valve. Reprinted from Ref. [111]. Copyright (1994), with permission from the American Society for Limnology and Oceanography.

ml min-1 SA

0.69

D1

0.69

50 cm

6m

UV digestor (8 W)

6m

Thermal digestor (90 °C) 10 cm Accurel tubing Disposable Syringe Filters 60 μl

C1

1.24

C2

0.58

R1

0.86

R2

0.58

30 cm

30 cm

60 cm D

Waste

Red LED

Figure 8 FIA manifold for the determination of TP with UV/thermal digestion. SA, sample; D1, digestion reagent; R1, acid molybdate reagent; R2, tin(II) chloride/hydrazine sulphate reagent; C1 and C2, ultrapure water carrier streams. Reprinted from Ref. [114]. Copyright (1996), with permission from Elsevier B.V.

were not necessarily suitable for continuous use for extended periods, and consequently it was difficult to replicate the high precision and reliability achieved in the laboratory. In re-engineering flow-analysis systems for field application, the trend has been towards compact, low-power, low-reagent

740


demand systems, with minimal mechanical components that are packaged in robust housings [37]. In the wastewater area, Pedersen described the application of an autonomous flow-analysis system for simultaneous monitoring of DRP, NOx and NH+4 in activated sludge for process optimization during biological nutrient removal [70]. The system utilized tangential flow filtration and sample degassers to improve reliability during unattended deployments of 21 days. Benson et al. [120] similarly described an autonomous FIA system for monitoring secondary-treated sewage treatment effluents. This system used tangential flow filtration, sample degassing, hydrodynamic sample injection and LED photometers to achieve reliable operation during 7-day measurement cycles. The use of peristaltic pumps in FIA system for longer term, unattended field measurements is undesirable because flow rates vary as peristaltic pump tubes progressively wear. Hanrahan et al. [121] used solenoid pumps and miniature solenoid valves for the determination of DRP in the field. Phosphomolybdenum blue absorbances measured at 710 nm were corrected using the absorbance at a reference wavelength (447 nm) to remove signals due to pulsations from the solenoid pumps. A portable flow-analysis system described by Lyddy-Meaney et al. [122] embodied many of the desirable features for portable and autonomous analysers. Tangentially filtered sample was pumped using a single peristaltic pump through the manifold where microsolenoid valves were used to perform multiple microlitre reagent injections. Reagents were propelled by compressed helium for DRP analysis. The system was capable of ca. 240 measurements of DRP per hour, and when combined with data from a global-positioning system produced high-resolution chemical maps (Figure 9). Among the earliest applications of FIA to seawater analysis were the reverse FIA methods developed by Johnson and co-workers for the determination of DRP [123], NOx [124], ammonia [125] and silica [126]. The reverse FIA, or multicommutation approach, is particularly appropriate when long-term operation is required because sample is usually in ample supply and only minimal amounts of reagent are required and wastes generated. These early FIA analysers quickly evolved from off-line shipboard systems to on-line, submersible versions [127]. For example, submersible FIA systems for NOx have been described by Daniel et al. [109] for deployments at depths to 300 m, and by David et al. [128] and Gardolinski et al. [6] for tidal cycle monitoring (Figure 3). An autonomous nutrient analyser in situ (ANAIS) described recently by Thouron et al. [63] utilized solenoid pumps and micro-conduit manifolds for the determination of NOx, DRP and silica, and was used for profiling at depths of 200–1,100 m for periods of up to 15 days (Figure 10). The adoption of SIA systems for field-based use has been relatively slow, perhaps because these have been engineered mostly for laboratory use, and have seldom been optimized for trace analysis. However, an SIA system for the underway determination of DRP with a fast throughput of 270 h1 has recently been described [97], and it is likely that the versatility of this approach vis-a`-vis FIA will see its increasing use for nutrient measurements in field environments.


741

Figure 9 (a) Schematic diagram of the compact FIA for analysis of DRP. S, sample inlet; PP, peristaltic pump; TFF, 0.2 mm tangential flow filter; FS, differential flow splitter; PG, propellant gas and regulator; MC, mixing coil; FC, flow cell; V0, 2-way valve; V1, V2 and V3, miniature solenoid valves; R1, ammonium molybdate reagent; R2, tin (II) chloride reagent; Std, standard; W, waste; ——0.5 mm i.d. PTFE tubing for liquid flow; - - - 0.5 mm i.d. PTFE tubing for gas delivery; components under computer control. Reprinted from Ref. [122]. Copyright (2002), with permission from Elsevier B.V. (b) High-resolution DRP surface concentration map of Lake Victoria, Gippsland, SE Australia, 25 May 2005, produced from 987 measurements collected over 9.66 h. d d d. Cruise path. Unpublished data. Reproduced with permission of EPA Victoria.

742


Figure 10 (A) Scheme of the nutrient-flow manifolds for (a) NOx manifold (b) silicate and (c) DRP. P1-P7 are solenoid pumps, used to deliver sample, deionized rinse water and reagents R1, R2, etc., required for each of the detection chemistries. (B) Typical examples of raw data (A/D counts) for NOx, silicate and DRP samples (concentration of 6, 150 and 4 mM, respectively), from top to bottom. Reprinted from Ref. [63]. Copyright (2003), with permission from the American Chemical Society.

4.3 Trace metals FIA has been coupled with most atomic spectrometric detection techniques for laboratory-based determinations of metals. These include flame AAS, electrothermal AAS, ICP–AES and ICP–MS (see Chapter 14). Use of FIA as the means of sample manipulation has made hydride-generation and cold-vapour techniques into more practical methods with much higher reproducibility than was previously the case with manual systems. For example, Menegario et al. [129] achieved detection limits of 0.02 mg L1 for As and 0.03 mg L1 for Se using HCl or thiourea to reduce Se(VI) and As(V) prior to hydride production and ICP–MS detection. Enhanced sensitivity has been achieved for Se using atomic fluorescence detection. He et al. reported a detection limit of 5 ng L1 for Se(IV) in seawater and 4 ng L1 total inorganic Se following microwave irradiation with NaBr to reduce Se(VI) [130]. For mercury detection by the cold-vapour technique,


743

sensitivity can be enhanced (LOD of 10 ng L1) by incorporating cold-trapping or amalgamation steps [131], and such instrumentation is now commercially available (Table 6). Using FIA as a front-end sample-treatment facility also allows simple on-line manipulations such as the automated addition of releasing agent, preconcentration onto chelating resin micro-columns, matrix removal using suitable solid phases, sample dilution and transport in low salt carrier to minimize burner clogging. It is interesting to note that simple FIA manifolds are used to enhance the selectivity and sensitivity of all atomic spectrometric detectors, even sophisticated and expensive techniques such as ICP–MS. For example, Hwang et al. [132] used an FIA manifold incorporating a solid-phase micro-column to separate and preconcentrate zinc from a seawater matrix, and coupled it with isotope dilution ICP–MS detection to achieve a detection limit of 0.014 mg L1. Another important application of FIA for the determination of metals is the capacity to perform on-line sample digestion to break down particulate matter and colloidal material. This can be achieved using microwave [130,131] or ultraviolet [133] irradiation. These systems liberate bound metals in order to achieve total metal determinations. Ultrasonic irradiation [134] and thermal energy [135] have been used in a similar way to convert organic Hg into Hg(II) prior to detection. FIA has also been coupled with various molecular spectroscopic detection techniques, particularly spectrophotometry, fluorescence and chemiluminescence. To achieve low detection limits with spectrophotometric detection, catalytic methods are often used. Lohan et al. [136] combined preconcentration of acidified seawater samples onto an NTA chelating resin with iron-catalysed oxidation of DPD by H2O2 to achieve a detection limit of 0.024 nM for total inorganic iron. Molecular detection can also be interfaced with SIA systems. For example, Brach-Papa et al. [137] used the fluorescence of an Al-8-HQ-5–sulfonic acid complex to achieve a detection limit of 0.2 mg L1 for Al in drinking water. Chemiluminescence is becoming increasingly popular as a detection system for FIA due to an inherently low background signal (due to the absence of any source noise), wide dynamic range and simple instrumentation. As an example Hirata et al. [138] achieved a detection limit of 0.62 ng L1 for cobalt in seawater by combining preconcentration with the Co(II) catalysed gallic acid–H2O2 chemiluminescence reaction. Gas-phase chemiluminescence reactions are less common, but equally useful for detection of those analytes that form volatile hydrides. Lomonte et al. [139] reported a simple flow manifold (Figure 11) based on a combination of gas diffusion of AsH3 and ozone-generated gas-phase chemiluminescence for the determination of As at sub-microgram per litre concentrations. As well as providing analytical figures of merit that are competitive with atomic spectrometric detection, FIA with molecular detection is well suited to field deployment. Bowie et al. [140] compared a catalytic DPD spectrophotometric method with a luminol chemiluminescence method for the determination of low (sub nM) concentrations of dissolved Fe on-board ship in several oceanographic regions. The two manifolds are shown in Figure 12 and both included an immobilized 8-HQ column that was used to preconcentrate the analyte and remove the potentially interfering sea salt matrix.

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Light sealed box

Acceptor (argon)

Carrier (HCI) Reagent (NaBH4)

Peristaltic pump

Injection valve

Power supply

CL cell PMT

Mixing coil 2 Acceptor chamber Membrane Donor chamber

Waste

Mixing coil 1 Waste

Ozone generator Oxygen supply

Figure 11 Schematic diagram of the experimental GD–FI system for the determination of As(III) with gas-phase chemiluminescence (CL) detection. Reprinted from Ref. [139]. Copyright (2007), with permission from Elsevier B.V.

4.4 General chemical quality parameters Parameters in this category include the major cations, Ca and Mg (hardness), and the anions chloride, sulfate and bicarbonate/alkalinity in waters and soil waters (Table 7). Photometric methods for these have been described in both FIA and SIA modes, and the limits of detection achieved appear adequate for the analysis of most waters [119,141–182]. While a number of FIA potentiometric methods have been described for the determination of chloride [147,183], a commonly used and effective photometric method involves the use of Hg(SCN)2 [148]. The use of bead-immobilized Hg2+ as a means of avoiding the continuous use and release of toxic Hg2+ for chloride determination has recently been described [149] (Figure 13). A turbidimetric SIA method based on the formation of AgCl has also been described [150]. Perhaps one of the more challenging of these parameters to determine is sulfate. Commonly used ion chromatographic methods for sulfate involve longer elution times and are generally insensitive. The development of a more sensitive flow-based method for the determination of sulfate would be advantageous, e.g., in the study of acid rain, but the detection limits of current indirect photometric/turbidimetric methods are barely sub-milligram per litre [151,152]. FIA and SIA techniques involving gas diffusion or pervaporation through hydrophobic PTFE or silicone membranes have been described for the determina2 tion of analytes that form gaseous species when acidified (HCO [153] 3 /CO3 cyanide [167,168] and sulfide [164]) or derivatized (fluoride converted to TMFS [177]), and many cases offers an effective means of minimizing matrix interferences. An SIA manifold for the determination of dissolved inorganic and organic carbon that uses a low-power (8 W) UV source for the efficient photo-oxidative conversion of DOC to DIC is shown in Figure 14 [119]. Carbon dioxide produced by this process was detected by gas diffusion with photometric detection [170].


745

Peristaltic pumps

DIW water

1.6

Acid wash Sample

8-HQ column

1.6

Sample buffer

0.2 8-HQ

HCI eluent

Mixing loop

Injection valve Flow cell

1.6

Luminol

Photomultiplier

1.6

Data acquisition

mL min-1

(A)

Waste Peristaltic pump Sample Sample buffer HCI eluent

2.50 0.03

Short knitted mixing coil

8-HQ column 8-HQ

0.32 Injection valve

Reaction buffer DPD Peroxide (B)

0.32 0.06 0.10 mL min-1

2m knitted reaction coil

8-HQ

Data acquisition Spectrophotometer

20°°C water bath Waste

Figure 12 The two flow-injection manifolds used for the determination of Fe in seawater: (A) with chemiluminescence detection and (B) with spectrophotometric detection. Reprinted from Ref. [140]. Copyright (2004), with permission from American Society for Limnology and Oceanography.

COD is a commonly measured parameters that is used to assess the organic load of a water. The batch method typically involves a 2 h digestion time, but by the use of microwave or UV photo-catalytic digestion, this can be reduced to ca. 2 min [173]. Dissolved oxygen is also used to assess the impact of organic matter on respiration and photosynthesis in aquatic systems. It is commonly measured by either the Winkler titration or by the use of the DO electrode; both methods are time consuming and the latter method has a poor detection limit. Automation via FIA based on the Winkler reaction or on the oxidation of Leucomethylene blue, using either photometric or fluorometric detection has enabled detection limits as low as 0.02 mg L1 O2 to be achieved, at sample throughputs of up to 60 h1.

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D B

RS

R SPR W

C

L S

W

Figure 13 Schematic diagram of the flow-injection system used for chloride determination. C, carrier (water); W, waste; S, sample or reference solution; RS, Hg(SCN)2 regenerating solution; SPR, solid-phase reactor; RS, nitrate ferric solution; B, tubular coiled reactor; D, spectrophotometer (l ¼ 480 nm). Reprinted from Ref. [149] Copyright (2005), with permission from Elsevier B.V.

To HC Sample Cresol Red reagent

H2SO4

Std. / Sample AcidPeroxydisulfate reactant

Sulfuric reagent

Waste

570 nm

P

RC 2 6

HC

5

4

MV

7 IN

Carrier

OUT

3 8

1

2

Waste

GD RC1

SY Waste

Waste UV reactor

To UV reactor Sample

Acid-peroxydisulfate

Figure 14 SIA manifold for the sequential determination of DIC and (DIC+DOC). SY, syringe pump (2,500 mL); MV, multi-position valve; GD, gas diffusion unit; P, peristaltic pump; UV reactor-PTFE tube (350 cm–0.75 mm i.d.) coiled around UV lamp (15 W), HC, holding coil PTFE tube (500 cm–0.75 mm i.d.); RC1 and RC2, reaction coils consisting of coiled PTFE tube, RC1 (22 cm–0.75 mm i.d.) and RC2 (11 cm–0.75 mm i.d.); carrier, ultrapure water. Reprinted from Ref. [170]. Copyright (2005), with permission from Elsevier B.V.


747

Potentiometric electrodes operate reproducibly and rapidly in FIA or SIA modes and despite their relatively poor sensitivity compared with photometric detection methods, they can be effectively used, e.g., for the simultaneous determination of fluoride and calcium [146]. This chapter illustrates the advantages of FIA for maximizing the information obtained from transient ionselective electrode signals. These advantages also apply to voltammetric techniques. For example, Tue-Ngeun et al. [179] used indirect amperometry to determine chlorate in soil extracts by reaction with potassium iodide and detection of the triiodide produced (Figure 15). One parameter that has received considerable attention is hydrogen peroxide, because of its role as a natural oxidizing agent in the euphotic zone of the oceans. The predominant methods used for determination of H2O2 are chemiluminescence-based, usually involving the Co2+ catalysed oxidation of luminol [159,161]. One recently published method utilized reaction of H2O2 with an acridinium ester to produce chemiluminescence with maximum intensity at 470 nm. The manifold, incorporating a 10-port valve with automatic washing facility, is shown in Figure 16.

(a) Lab-Made Amperometer

Computer

Waste P2 WE

RE Waste AE

KI

MC

FC

I (b) HCl Water bath

P1 Standard/ Sample

MF-2052 Ag/AgCl electrode (BAS) Glass tubing (outer wall)

3 M KCl Frit

Figure 15 (a) The amperometric FIA manifold used for determination of chlorate: P1 and P2, peristaltic pump 1 and 2; I, injection valve; MC, mixing coil; AE, auxiliary electrode; WE, working electrode and RE, reference electrode. (b) A double-junction design reference electrode. Reprinted from Ref. [179]. Copyright (2005), with permission from Elsevier B.V.

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[1]

[5] 10

Carrier H2O

1.7 [2]

Acid wash

1.7

CL reagent

1.7

1 [4]

2

9

Waste 3

8

4

[3] 7

6

5

[6]

Waste Carbonate buffer

1.7

Figure 16 Flow-injection analysis manifold for chemiluminescence analysis. (1) Sample syringe (500 mL), (2) peristaltic pump (flow rates in mL min1), (3) acid wash loop (500 mL), (4) 10-port, 2-position injection valve, (5) sample loop (500 mL) and (6) flow cell. Reprinted from Ref. [163]. Copyright (2007), with permission from the American Chemical Society.

Application of FIA to this group of analytes has generally been limited to laboratory analysis with the exception of methods for H2O2 and cyanide. Given the importance of these chemical parameters as key indicators of environmental processes, development of field methods is a desirable objective.

4.5 Organic contaminants The area of organic contaminant analysis is dominated by separative techniques such as HPLC, GC and GC–MS that offer high selectivity and sensitivity [184–204]. For flow-injection methods to gain acceptance in this area, the advantages compared with extant separation techniques must be clearly demonstrated. Nevertheless, the one area where flow injection can be advantageous is in the speed of analysis. For example, an FIA potentiometric method for atrazine has been reported which is capable of 60 injections an hour at concentrations typically found in soil extracts. This is approximately 10 times faster than the equivalent HPLC method, and depending on the lifetime of the PVC membranes used in the electrode construction, such an approach may offer a viable approach for atrazine detection in the field [189]. Similarly, a sensitive method has been reported for the detection of carbaryl which is applicable to water, soils and grain extracts [192]. In this case, on-line photoxidation was used to convert carbaryl into methylamine which reacted with photochemically generated tris(2,2-bipyridyl)ruthenium(III) to produce chemiluminescence (Figure 17). With a sample detection limit of 12 mg L1 and an injection rate of 200 h1, FIA methods such as this should prove highly competitive with


PP

749

Ru(bpy)32+ UV

R1 R2 R3 R4

1.2

(IV)1

1.2

L1

1.1 D

1.1

L2

PC

(IV)2 UV Carbary1

W

Figure 17 Flow-injection manifold for the determination of carbaryl. PP, peristaltic pump (with flow rates given in ml min–1); R1, water; R2 ¼ 1.4 103 M potassium peroxydisulphate and 0.05 M phosphate buffer of pH 5.8; R3, 0.15 M phosphate buffer of pH 6.5; R4, water; (IV)1, (IV)2, injection valves; L1, L2, photo-reactors; D, luminometer; W, waste. Reprinted from Ref. [192]. Copyright (2003), with permission from Elsevier B.V.

chromatographic techniques, in terms of analytical performance, sample throughput and cost (Table 8). One approach that will increase in popularity is the use of flow-injection techniques capable of screening for the presence of a wide range of toxicants or other pollutants in surface and ground waters. This approach allows rapid identification of those waters that require detailed contaminant analysis, and enables a more strategic allocation of resources for chromatographic analysis. Examples of such approaches are the use of FIA for monitoring the respirometric response of E. coli cultures subjected to a range of toxicants [205], and the inhibition of enzymes such as acetylcholinesterase and urease that occurs when they are exposed to contaminants such as pesticides [194] or heavy metals [206]. Amador-Hernandez et al. [202] have also described a flow-injection screening technique for polycyclic aromatic hydrocarbons which involved preconcentration onto a C18 mini-column prior to elution and detection by laser-induced fluorescence (Figure 18). Identification and semi-quantitation of PAHs were achieved by the application of multi-variate statistics. Chromatographic analysis of organic contaminants usually requires extensive sample pre-treatment, involving filtration, extraction, distillation and/or derivatization, and any approach that can either avoid or automate these unit operations is highly desirable. For example, phenol determination has been performed by pervaporation flow injection, in which phenols were acidified and the resulting volatile species allowed to migrate across an air gap through a microporous hydrophobic membrane into an acceptor stream prior to detection by amperometry [198]. In a related method, less volatile phenols were converted on-line to volatile phenyl acetates that were then diffused across a pervaporation membrane prior to electrochemical detection [199] (Figure 19). A similar

750


W

S

L

RC C

Nd-YAG LASER FC

E

IV2 P PC

FOA I/O

DG SP

ICCD

Figure 18 FIA system for the screening of PAHs. Laser arrangement: L, lens; FC, flow cell; FOA, fibre optic assembly; SP, spectrograph; ICCD, intensified charge coupled device; I/O, multi-IO box; DG, delay generator; PC, personal computer. Dynamic manifold (within the dashed lines): S, sample solution; C, carrier solution; E, eluting solution; P, peristaltic pump; IV, injection valve; RC, retention column; W, waste. Reprinted from Ref. [202]. Copyright (2001), with permission from Elsevier B.V.

Valve B

Detector

R3

W Membrane

R2

Glass beads

35 °C

Valve A

R1

Pervaporation unit Pump 1

Mixing chamber

W Pump 2

Figure 19 Experimental set up for the rPFI on-line derivatization analysis of phenol: R1, phenol solution; R2, NaOH/NaCl solution; R3, NaOH/KNO3 electrolyte; valve A, 20 mL sample loop; Detector, amperometric with glassy carbon working electrode; +0.62 V (vs. Ag/AgCl). Reprinted from Ref. [199]. Copyright (2003), with permission from Elsevier B.V.

flow-injection approach has also been employed for the preparation of phenyl acetates derivatives for membrane introduction mass spectrometry (MIMS) [201]. The use of on-line derivatization followed by membrane separation confers even greater selectivity to the highly selective mass spectrometric analysis and enable quantification of designated phenols without interference from congeners. An alternative approach to isolation and separation is the use of chromatomembrane separations, in which a PTFE membrane containing both micro and macropores


751

is used to discriminate between polar and non-polar species. This approach has been used for extraction of PAHs and organic halogens from wastewater samples, prior to gas chromatographic separation and detection [207]. This approach has also been used for the determination of anionic surfactants in natural waters in the presence of humic acids [208]. Another means of enhancing selectivity is the use of enzymatic reactions. Labra-Espina et al. described the use of immobilized bilirubin oxidase (BOX) in the presence of NADH for the indirect fluorescence detection of pentachlorophenols [195]. The limited number of applications described in this section reflects the challenges of applying FIA technologies to the quantification of individual organic contaminants. The legislative requirements to fully characterize such species and the expense of the sophisticated laboratory techniques involved, creates a need for rapid field-based screening techniques. FIA has the potential to meet this need due to the attractive features described in Section 2.1.

5. FUTURE TRENDS FIA techniques have been described for the determination of numerous parameters in water and wastewater monitoring, as demonstrated by the examples listed in Tables 5–8. However, the routine rather than research application of these techniques, using commercial instrumentation, has been limited to a restricted number of parameters. The area where FIA has achieved the most widespread application is nutrient monitoring in natural waters and wastewaters. Another popular commercial application of FIA, due to its ability to handle small sample volumes in a closed system, is the determination of cyanide species in waste and environmental waters. Measurement of these parameters most commonly utilizes photometric detection and well-established derivatization chemistries (often adapted directly from segmented continuous flow analyser methods) that meet the accreditation requirements of statutory authorities such as the USEPA. While the importance of method accreditation is recognized from a regulatory perspective, it is arguable that this lengthy and involved process can impede the timely introduction of improved analytical methods. There are relatively few examples of robust field applications of FIA, and in part this is due to the difficulties associated with reagent and instrument stability, calibration stability, sample conditioning (e.g., filtration), biofouling and the intrinsic unreliability of some FIA components when translated from a benign laboratory environment. There is therefore a pressing need for sampleconditioning devices that are compatible with FIA systems and which can be deployed for extended periods in challenging sample environments. Whilst miniaturized flow-analysis systems, e.g., lab-on-a-chip or mTAS, offer obvious advantages with respect to reduced sample, reagent and waste volumes, the use of these microfluidic systems will place much higher demands on the performance and integrity of sample-conditioning devices. It is important to

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state that all instrument components and methods used for field deployment are thoroughly tested prior to deployment to ensure that they are fit for purpose. Finally, it should be stressed that the primary objective of the application of FIA to the analysis of waters, sediments and soils, either in the laboratory or the field, is to obtain high-quality chemical data for the purpose of improved understanding and protection of environmental systems.

ABBREVIATIONS AND DEFINITIONS 1,4- TCBQ 5-Br-PADAP 8-HQ mTAS AAS AFS APHP BNR BOD BOX CASS CCD CDTA CL COD CRM CTAB DCP DIC DMSA DNN5S DO DOC DOM DOP DPC DPD DRP DTPA EDTA EGTA

Tetrachloro-1,4-benzoquinone 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol 8-hydroxyquinoline or 8-quinolinol m-Total analytical system Atomic absorption spectrometry Atomic fluorescence spectrometry Alkaline phosphatase hydrolysable phosphorus. Phospho monoesters are hydrolysed to molybdate reactive P Biological nutrient removal Biological oxygen demand Billirubin oxidase Coastal Atlantic seawater standard Charge-coupled device 1,2 cyclohexane-diaminetetracetic acid Chemiluminescence Chemical oxygen demand Certified reference material Cetyltrimethylammonium bromide Dissolved condensed phosphorus. Requires acid hydrolysis for conversion to molybdate reactive P Dissolved inorganic carbon Dimethylsulfonazo(III) 4-(2,4 dinitrophenylazo)-1-naphthol-5-sulfonic acid Dissolved oxygen Dissolved organic carbon Dissolved organic matter Dissolved organic phosphorus. Requires oxidation to phosphate for detection as molybdate reactive P 1,5-diphenylcarbazide N,N-dimethyl-p-phenylenediamine Dissolved (molybdate) reactive phosphorus. Measures free orthophosphate plus labile condensed and organic P species Diethylenetriaminepentaacetic acid Ethylenediaminetetraacetic acid Ethylene glycol tetraacetic acid


ESI FIA GC GC-MS GD HPLC IC ICP-AES ICP-MS INA-PZ LB LCW LOD LOQ MB MIMS MRC MS MSFIA NADH NASS NOx NPA NTA OPA PAH PAN PaPhA PCP PDC PHP PMBP PONPE 5 PTQA PTFE PVC PVP RM rPFI SI-LOV SIA SPE TAR TCNQ

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Electrospray ionization Flow injection analysis Gas chromatography Gas chromatography-mass spectrometry Gas diffusion High-performance liquid chromatography Ion chromatography Inductively coupled plasma–atomic emission spectrometry Inductively coupled plasma–mass spectrometry Na isonicotinate-3-methyl-1-phenyl-2-pyrazolin-5-one Leucomethylene blue Liquid core waveguide Limit of detection Limit of quantification Methylene blue Membrane introduction mass spectrometry Multi-reflection cell Mass spectrometry Multi-syringe flow-injection analysis with multi-commutation Nicotinamide adenine dinucleotide (reduced) North Atlantic Seawater Standard Sum of nitrate+nitrite usually determined after reduction of nitrate to nitrite 1-naphthylamine Nitrilotriacetic acid o-phthaldialdehyde Polycyclic aromatic hydrocarbons 1-(2-pyridylazo)-2-naphthol Poly(aminophosphonic) acid Pentachlorophenol Pyrrolidine dithiocarbamate Phytase hydrolysable phosphorus 1-phenyl-3-methyl-4-benzoylpyrazol-5-one Polyethyleneglycolmono-p-nonylphenylether 2-(-pyridyl) thioquinaldinamide Polytetrafluoroethylene Polyvinyl chloride Poly(vinylpyrrolidine) Reference material reverse pervaporation flow injection Sequential injection-analysis-lab-on-valve system Sequential injection analysis Solid-phase extraction 4-(2-thiazolylazo)-resorcinol 7,7,8,8-tetracyanoquinodimethane

754


TDN TDP

TETA TIC TMFS TOC TOF-MS TP USEPA

Total dissolved nitrogen Total dissolved phosphorus. Requires a combination of oxidation and hydrolysis to convert organic and condensed P species to molybdate reactive P for detection Triethylenetetramine. Total inorganic carbon Trimethylfluorosilane Total organic carbon Time-of-flight mass spectrometry Total phosphorus. Unfiltered (or coarse filtered) sample digested, and detected as molybdate reactive P United States Environmental Protection Agency

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SUBJECT INDEX

ABTSd+ assay, 517, 522, 543–544 accuracy, 98, 132, 138, 160, 169, 171, 185, 240, 288, 335, 377, 399, 411, 423, 427, 464, 488, 502, 628, 686, 696 acetaldehyde, 348, 520 acetaminophen, 115, 414, 421, 602, 605 acetic acid, 144, 185, 252, 274, 352, 362, 369, 426, 436, 463–465, 467, 481, 486, 518, 540, 606, 609, 629, 710, 724, 752–753 acetylsalicylic acid, 134, 160, 278–279, 609–610 acidity, 32, 230, 388, 463, 486, 520, 524, 617, 632–633, 650 acousto-optic tunable filter, 427, 435 activated carbon, 180–181, 666, 717, 721 activity measurements, 559 adsorptive stripping, 269, 454, 519, 521, 525, 603, 605 aerosol particles, 639, 649–650 affinity chromatography, 23, 28, 30, 32–33, 37, 39–40, 42, 280 aflatoxin, 527, 533, 546 agarose beads, 30, 455 agmatine, 536, 548 agonist, 178, 471, 495 agricultural samples, 117, 514 air bubbles, 24–25, 37, 41, 319, 321, 324, 450–451, 457, 624–625, 651 air segmented continuous-flow analyzer, 24 Al, see aluminium aldehyde dehydrogenase, 348, 351 alendroic acid, 602 alendronate, 281, 599 alkaline phosphatase, 115, 147, 153, 709, 736, 739, 752 alkalinity, 41, 617, 632–633, 703, 724, 744 All Injection Analysis, 94–95, 105 alternating helical reactor, 70 aluminium, 136, 186, 188, 206, 346, 350, 427–428, 479, 529, 602, 604, 633, 689, 706, 709, 715, 717, 721, 729, 743 Am-241, 475, 477 amalgamation, 141, 720, 743

4-aminoantipyrine, 172, 198, 733 4-aminophenazone, 601, 609 5-amino-2,3-dihydro-14-phthalazinedione, 354 Amberlite XAD-, 172–173, 718–719, 721 Amberlitet IR 120(H), 184 ambroxol, 268, 274–275, 277–278, 609 americium, 490–492 amikacin, 602, 604 amittriptyline, 602 ammonia/ammonium, 3–5, 34, 101–102, 113, 144, 152, 162, 173, 182, 198, 211, 223–224, 269, 296–297, 347, 350–351, 382, 389, 391, 402, 446, 463, 476, 486, 496, 520, 545, 610, 629, 639, 645, 652–653, 658, 661–662, 665–667, 675–676, 701, 703–704, 708–710, 713, 721, 735–737, 740–741 amorphous fluoropolymers, 316 amoxicillin, 268, 270, 281–282, 360, 601–602, 607, 609, 611 amperometric, 71–73, 148, 221, 289, 364–365, 441, 444, 448, 450–452, 454–455, 457, 500, 543, 605, 608, 687, 708, 729, 733, 747, 750 ampicilin, 609 analyte preconcentration, 102, 171, 261, 376, 397, 399, 514, 695 analytical figures of merit, 98, 594, 705, 743 analytical-experimental models, 48, 65, 67, 71 anionic surfactants, 161, 164–165, 172–174, 470, 487, 751 annular diffusion scrubber, 641, 642 antibiotics, 281, 368, 531, 547, 599 anticancer drugs, 594 antidigoxigenin, 499 antioxidants, 280, 357, 542–543, 595, 609 antioxidation capacity, 595 antioxidation drugs, 594, 595 arsenic, 112–114, 124, 142, 181–182, 224, 395–397, 425, 463, 529, 535, 538, 651, 687, 716 artificial neural network, 133 artificial sweeteners, 524, 539

761

762

Subject Index

As, 112–114, 124, 142, 181–182, 224, 395–397, 425, 463, 529, 535, 538 As total, 9, 393, 690, 700, 716 As(III), 114, 142, 176–177, 187, 396, 651, 680, 715–716, 744 As(V), 114, 142, 177, 187, 396, 680, 716, 723, 742 ascorbic acid, 7, 88, 113, 174, 182, 268, 273, 276, 389, 463, 486, 518, 522–523, 528, 540, 543, 546, 601–602, 609, 611, 706, 710, 712, 714, 716, 718, 734 aspect ratio, 55, 57, 75 astringency, 520, 544 asymptotic solutions, 54 at-line, 599–600, 619–620, 622, 627, 633, 635 atmospheric analysis, 639–640, 656 atomic absorption spectrometry, 11, 30–34, 63, 73, 101, 119, 124, 136, 154, 171, 181, 198, 214, 270, 284, 312, 376, 395, 402–403, 544, 548–549, 633, 752 atomic emission spectrometry, 125, 136, 154, 181, 198, 376, 402, 462, 505, 753 atomic fluorescence, 30, 121, 138–140, 143, 154, 301, 344, 376, 402, 548, 687, 742, 752 atomic spectroscopy, 19, 28, 32, 34, 84, 105, 375, 378, 401 atomization cell, 396, 403 atrazine, 115, 524, 730–731, 748 attenuated total reflectance, 409, 420–421, 435 automatic sample dilution, 632, 636 autonomous system, 685, 696, 698 auxiliary electrode, 450–451, 747 axial dispersion, 28, 35, 53–55, 57, 65, 69, 73–74, 255 axial dispersion coefficient, 54–55, 57, 65, 74 axially dispersed plug flow model, 48, 54, 59, 74 azinobis(2u-ethylbenzothiazoline-3-sulfonic acid) (ABTSd+), 543 azithromycin, 602, 605 Ba, see Barium baby formula, 117 back extraction, 375, 379–382 background correction, 89, 99–100, 316, 319, 323, 330, 333–334, 364, 706–707, 710–712, 714 barium, 138, 188, 192–194, 468, 487–488, 725 barium sulfate, 468, 487–488 baseline noise, 335 baseline-to-baseline time, 63 Batch Injection Analysis, 105, 619, 627, 636 Beryllium, Be, 182

bead injection, 23–24, 27, 30–34, 39–40, 171, 270, 284, 378, 383–384, 402, 408, 441, 455, 462, 495, 505, 560, 596, 601 Bead Injection Spectroscopy, 30, 39–40, 601 beads, 27, 30–31, 39–41, 72, 86, 139, 146, 150–151, 183, 228, 252, 352, 360, 366, 383–390, 392, 399–400, 455–456, 480, 489, 495, 560–561, 596, 601, 608, 610, 625, 750 beer, 117, 139, 218, 312, 423, 487, 497, 499, 514–521, 543–544 benzo[a] pyrene, 500 benzoic acid, 166–167, 268, 274, 277–279, 431, 434, 523, 609 benzophenone, 277 berberine, 170 Bessel functions, 55 beverages, 101, 105, 117, 139, 224, 338, 350, 411, 425, 513–517, 519, 521–523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 595 biamperometric, 631 biamperometric monitoring, 631 bibliometrics, 111, 113, 115, 117, 119, 121, 123 bicarbonate, 744 bilayer lipid membrane sensors, 546 bilirubin oxidase, 732, 751 bioavailability, 134, 216, 377, 397–398, 592, 686–687, 690 biochemical oxygen demand, 702 bioequivalence, 592 biofouling, 696, 751 biogenic amines, 230, 280, 520, 536, 545, 547, 609 bioligand interaction, 34, 591, 595–596 biological materials, 135 bioluminescence, 97, 344, 353, 356–357, 362 biomarkers, 547, 733 biomembrane permeation, 596, 599 biomolecular assays, 28, 30 biomolecules, 102, 354, 357, 484, 489, 559 bioprocess control, 258, 476 biotin, 479, 483 ‘black-box’ models, 48, 58, 63–64 bitterness, 520–521, 524, 544 blood, 116–117, 141–143, 214–215, 219, 223–224, 252, 258, 269, 423, 425, 493, 497, 596 BOD, see also biochemical oxygen demand boron, 181, 364, 450, 517, 524, 529, 538, 700 boron doped diamond, 364 bovine serum albumin, 215, 231, 280, 284, 597–598, 613 Br, 720, 752 brackish water, 336

Subject Index

BrO 3 , 721, 729 bromine number, 617, 626, 631, 635–636 bromothymol blue, 152, 168, 338, 465, 486 BTEX, 432, 434–435, 498 bubble, 137, 322, 330, 464, 486, 625, 629, 645, 648, 661–662 bulk detector, 71 C18, see octadecylsilyl silica, 160 Ca, 24, 69, 182, 303, 450, 747 cadmium, 15, 30–32, 34, 113, 138, 177, 182, 185–190, 192–194, 231, 261, 269, 369, 392–394, 501, 504–505, 517, 524, 529, 532, 535, 538, 544, 643, 659, 672–674, 689, 700, 702, 707, 710–712, 717, 721, 734, 738 caffeine, 133, 160, 278–279, 412–414, 525, 540, 601–602, 606, 609–610 calcein, 348, 369, 728 calibration, 6, 8, 62, 82, 96, 102, 132–133, 139, 164, 173–177, 180, 182, 195, 203, 210, 219, 222, 250, 294–295, 315–316, 328, 332, 352, 408, 416–417, 419–420, 422–423, 427, 432, 435, 443, 449, 457, 463–465, 467, 486, 493, 543, 564, 602, 608–609, 622, 627–628, 630–632, 634, 636, 639, 643, 647, 675–676, 697, 728–729, 737, 751 capacitively coupled contactless conductometric detection, 463, 505 capillary batch injection, 87 capillary electrophoresis, 28, 33–34, 106, 150, 173, 203, 214, 230–231, 240–241, 269, 287–289, 291, 293, 295, 297, 299, 301, 303, 305, 315, 317, 322–324, 352, 369, 446–447, 463, 545, 548 Capillary-based flow-through cell, 323 carbaryl, 731, 748, 749 carbohydrates, 413, 457, 519, 528, 547, 701 carbon, 104–105, 138, 151–152, 154, 180–181, 221, 223, 316, 327, 364, 368–369, 417, 425, 447, 450–451, 455, 457, 470, 476, 487, 492, 521, 543, 605, 608, 666, 687, 702, 717, 721, 728, 731–733, 744, 750, 752, 754 carbon dioxide, 104, 223, 417, 476, 487, 492, 521, 543, 702, 744 carbon paste ISE, 605 catalase activity, 540 catalytic methods, 743 cathodic-stripping technique, 454, 455 Cd, see cadmium, 15, 30, 138, 143, 177 Cd reduction, 707, 710–712, 734, 738 CD-ROM, 15

763

Ce(IV), 180, 608 cefuroxime axetil, 601–602 cellular analysis, 559 cephalexin, 268, 281, 609 certified values, 131, 193 cetyltrimethylammonium bromide, 198, 348, 352, 362, 369, 752 Chalk’s Flow Analysis Database, 314 charge coupled device near infrared, 429, 435 charge-coupled devices, 318 Chebyshov polynomials, 61 cheese, 117, 531–533, 546 chelating disk, 187, 189 Chelex, 187 chemical kinetic phenomena, 47–48, 66–67 chemical oxygen demand, 144, 154, 702, 728, 752 chemiluminescence, 9, 15, 17, 19, 32, 96–97, 120, 146, 160, 198, 211, 224, 252, 267–270, 301, 343–344, 349, 352–354, 356–364, 366–367, 462, 472, 516–519, 521–524, 526, 528, 530–531, 534–542, 546, 548, 563, 594, 603–604, 608, 613, 651, 672, 693, 695, 707, 709, 711, 714, 716–717, 719, 721–722, 726–729, 731, 734–735, 743–745, 747–748, 752 Chi-squared distribution functions, 63 chloramphenicol, 274, 277, 602, 605 chloride, 3–4, 6, 9, 16, 32, 89, 91, 113, 138–139, 153, 197, 213–214, 217–218, 293–294, 352, 362, 369, 448, 466, 468, 470, 487–488, 497, 499, 521, 527, 531, 605, 629, 687, 703, 706–712, 725, 730, 734, 737, 739, 741, 744, 746, 753 chlorogenic acid, 368 8-chlorotheophilline, 166 choline, 354, 361, 421, 533–534, 709 chromatomembrane, 101, 159, 170, 192, 198, 639, 642, 644, 750 chromium(III), 31, 169, 182, 184, 187, 196–197, 251, 268–270, 354, 388–390, 400, 710, 718 chromium(VI), 31, 151, 169, 187, 197, 252, 268–270, 382, 387–390, 399–401, 650, 718 chromium, 31, 70, 104, 138, 151, 169, 182, 184, 186–190, 192–194, 196–197, 251–252, 268–270, 354, 382, 387–390, 393, 399–401, 427, 496–497, 650, 662, 700–702, 710, 718 chromogenic reagent, 148, 332–334, 337, 501, 654 chromotropic acid, 176, 181, 224, 350, 604, 651 chymotrypsin, 115, 599 cimetidine, 173

764

Subject Index

ciprofloxacin, 598 citrate lyase, 349 Cl, 177, 180, 218, 673 clinical analysis, 114, 116, 132, 624 ClO, 224, 711 cloud point extraction, 382 CN, 727, 728 Co, see Cobalt coal, 119, 137 cobalt, 30, 58, 64, 69, 73, 113, 148, 176–178, 182, 184, 186, 189–190, 192–194, 213, 258–259, 261, 269–271, 273, 281, 325, 347, 349–351, 354, 358–359, 365–366, 369, 388, 392–393, 427, 535, 538, 544, 601, 609, 690, 702, 709, 716–717, 719, 723–724, 740, 743 cocoa powder, 137 COD, see also chemical oxygen demand cold vapour, 138, 143, 154, 377, 393, 402, 548, 720 cold vapour atomic absorption spectrometry, 548 cold vapour atomic fluorescence, 138 cold-trapping, 743 collector, 192, 217, 251–252, 255, 261, 477, 639–640, 645, 647, 649–650, 652, 656, 665–666, 672, 676 colloidal sols, 431–432 components of spectrophotometric instruments, 311, 317 concentration gradients, 26, 51, 632, 636 conception of FIA, 3, 6 condensed phosphate, 147, 734 conductance, 443, 445, 463, 487, 646, 648, 651 conducting polymer entrapment, 577, 587 conductivity, 96, 101, 290–291, 294, 301, 443–446, 448–449, 463–464, 486–487, 525, 634, 643, 646, 650–651, 657–661, 675, 698, 703 conical mini-column, 181 constant-head reservoirs, 87 continuous film-recirculating drop, 645, 665 continuous filtration, 204–205, 235–236, 254–255, 257–258 continuous filtration using knotted reactors, 235, 255 continuous filtration with filters, 235, 255 continuous filtration with ultrasound assistance, 235, 257 continuous flow, 8, 10, 16, 25–26, 28, 43, 69, 89, 94, 98, 101, 114, 124, 130–131, 136, 154, 162, 191, 208, 212, 216, 247, 252, 254, 280, 313, 401, 431, 448, 498, 529,

535, 542, 563, 608, 619, 624, 628, 637, 651, 654–655, 751 continuous flow analysis, 10, 114, 124, 130, 154, 313, 637 continuous flow titration, 69 continuous liquid–liquid extraction, 204, 205, 235–237, 241, 261 continuum source atomic absorption spectrometry, 376, 402 controlled dispersion, 7, 25–26, 55, 84, 95 convection, 28, 47, 49–51, 53–55, 57–58, 82, 84, 90, 444 Convective Interaction Media, 271, 284, 598 convective-diffusion equation, 48, 52–53, 55, 58–59, 67–68, 71 conventional continuous liquid–liquid extraction, 204, 231 copper, 30–31, 35, 72–73, 113, 137, 143–144, 181, 217, 252, 347, 350, 473, 488, 517, 529, 538, 629, 695 coulometric generation of bromine, 631 coulometric, 71, 97, 441–443, 455–457, 462, 631, 636, 732 coulometry, 97, 442–443, 462 Cr, see Chromium Cr(III), see Chromium(III) Cr(VI), see Chromium(VI) creatine, 115, 497, 602, 606 Cresol Red, 104, 151–152, 728, 746 critical micelle concentration, 70 crops, 117–118, 545, 704 Cu, 138–139, 143, 162, 175–177, 186–190, 192–194, 218, 259, 268–270, 354, 364, 377, 382, 392–393, 604, 689, 700–702, 704, 716–719, 721, 728, 731 cyanide, 91, 700, 727–728, 744, 748, 751 b-cyclodextrin, 267, 601 cylindrical denuder, 641 cytometry, 461, 470–471, 488–489, 596 10,10’-dimethyl-9,9’-bisacridinium nitrate, 355 dairy, 117–118, 513–514, 531, 534, 546–547 Dean number, 55–56, 74 debubblers, 136, 629 definition of FIA, 8 degassing, 88, 151, 195–196, 497, 629, 644, 740 degreasing baths, 422, 634 delta-function, 59, 63–64, 70, 73 denuder, 188, 198, 640–641, 651, 667, 673 deoxyribonucleic acid, 546, 559–560 determination of moisture, 617, 630

Subject Index

dextrose, 602, 609 D-gluconate, 351, 518, 541 diacetyl, 521, 544, 713 dialysis, 9, 16, 42–43, 57, 101–103, 203–205, 207, 209, 211–223, 225–227, 229, 231, 236, 247–248, 297, 333, 396–397, 514, 597, 685, 693–694, 704 dialysis membranes, 213 diaryloxalates, 353, 356 dichlorotriazinylaminofluorescein, 299, 305, 347 diclofenac, 268, 275, 278, 609 diffuse reflectance, 427, 435 diffuse reflectance NIR spectrometry, 427 diffusion coefficient, 51–52, 59, 61, 63, 67, 74, 86, 640–641, 649, 655 diffusion mass flux, 51 diffusion scrubber, 159, 188, 191, 198, 639–640, 643, 653, 662, 694 digestion, 84, 104–105, 118, 129–131, 133, 135–153, 216, 335, 378, 397, 491, 498, 514, 594, 685, 687, 693–696, 704, 708, 720, 728, 737, 739, 743, 745 digestion efficiency, 737 digoxin, 115, 499 dimensionless form, 50, 52–54, 59 diode array detector, 154, 272, 281, 284, 316 diphenhydramine, 166, 602, 607 diphenyl-picrylhydrazyl (DPPHd), 543, 595 direct-injection high-efficiency nebulizer ,378, 381, 402 dispersion, 7, 9, 15, 23, 25–26, 28, 35, 48–61, 63, 65–66, 69–71, 73–74, 81–82, 84–87, 90, 95–96, 98, 105–106, 137, 228, 255–257, 294, 314, 319–322, 324, 332, 380, 451, 462–464, 470, 504, 563, 622, 624–625, 627, 632, 636, 692 dispersion coefficient, 53–55, 57, 63, 65, 74, 84 dispersion models, 48, 59 dispersion volume, 63 dispersive IR spectrometer, 411 displacement micropump, 89, 91 dissolution, 105, 117, 129–139, 141, 143, 145, 147, 149, 151, 153, 212, 249, 255, 259–261, 294, 388, 395, 397, 561, 591, 593–594, 607, 610–611, 613, 633 dissolution methods, 131 dissolution profiles, 132–133, 610–611 dissolved condensed phosphates, 734 dissolved inorganic carbon, 151, 154, 752 dissolved inorganic nitrogen, 690 dissolved organic carbon, 151, 154, 687, 728, 752 dissolved organic nitrogen, 690 dissolved organic phosphorus, 147–148, 752

765

dissolved oxygen, 346, 350, 363, 454, 605–606, 690, 702–704, 717, 726, 745, 752 dissolved reactive phosphorus, 688, 690 dithiocarbamates, 240, 426 diuron, 412, 730 DNA, 31, 150–151, 290, 347, 351, 364, 484, 546, 559–564, 596 DNA-binding assay, 596 dodecyl sulfate, 172, 428, 494 dodecylamine, 174, 361 Donnan dialysis, 205, 213–214, 216–217, 694 donor solution, 204, 209, 248 dopamine, 115, 359, 602, 604 dose-response curves, 595 double-humped peaks, 66, 68–70 Dowex 1X, 177, 182 Dowex 50W X, 178 drinking water, 115–116, 174, 177, 187, 268, 350, 698, 717, 720, 723, 729, 743 drotaverine, 602 drug discovery, 470, 495, 591 drug dissolution, 132–134, 591, 594, 607, 610 drug metabolism, 219, 596 drug release testing, 594 drugs, 114–115, 117, 129, 132–133, 219, 240, 252, 267, 276, 347, 434, 470, 484, 499, 591–594, 596, 599–602, 604–608 dual opposite end injection, 287, 296 dual wavelength measurement, 736 dual-conical microcolumn, 398 dyes and dyebaths, 634 dynamic micropump, 89–90 dynamic surface tension, 97, 461, 477, 480, 482, 493–494, 505 early years of FIA, 3, 11 electrical capacitance, 443 electroanalytical techniques, 211, 442 electrochemical dissolution, 105 electrochemical hydride generation, 393, 402, 716 electrochemical roughening procedure, 431 electrochemiluminescence, 97, 343, 358, 363, 365–369, 532 electrodialysis, 204–205, 212–214, 217, 298 electrohydrodynamic, 90, 106 electrokinetic injection, 287, 291–292, 294–296, 300–301 electrokinetic pumping, 288, 300, 302 electrolytic dissolution, 633 electromagnetic, 91, 139–141, 143, 311–312, 314, 441 electromagnetic induction heating, 139 electromechanical valves, 631

766

Subject Index

electronic tongue, 547, 634 electroosmotic flow, 89–90, 106, 289, 305, 676, 678 electroosmotic pump, 91, 304, 718 electrophoretically mediated microanalysis, 287–288, 302, 305 electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry, 568 electrospray ionization mass spectrometry, 33–34, 479, 548 electrostatic actuation, 91 electrothermal atomic absorption spectrometry, 30–34, 171, 376, 402, 548, 633 ELISA, see also enzyme-linked immunosorbent assays energy dispersive x-ray fluorescence, 485, 504–505 enrichment factors, 167, 185, 189, 214, 240, 247, 249, 256, 392, 394, 719, 722–723 enthalpimetric, 461, 476 enzymatic assays, 9, 16, 33, 38, 303, 559 enzymatic reactions, 147, 211, 351, 354, 365, 367, 369, 736, 751 enzyme affinity detection, 494 enzyme hydrolysis, 446 enzyme sensor, 495 enzyme-linked immunosorbent assays, see also ELISA epinephrine, 502, 602, 604 Escherichia coli, 537, 548, 703 b-estradiol, 174, 178, 576 ethanol, 113, 135, 154, 176, 181, 218, 298, 365, 368–369, 389, 391, 412, 414, 420–423, 425, 427, 429, 432, 434, 463, 498, 501, 516, 543, 600, 630, 706, 725 ethylene propylene diene monomer (EPDM) elastomers, 634 ethylmercury, 114, 181 ethylparaben, 277–278 excipients, 133, 593, 609 Exponentially Modified Gaussian (EMG) function, 65, 66, 74 Exponentially Modified Square Function, 66 Extracellular acidification, 33, 596 extraction, 15, 19, 30–33, 62, 100–101, 103, 105, 159–161, 163–171, 173, 175, 177–181, 183, 185, 187–189, 191–193, 195, 197–198, 204–205, 207, 211–212, 235–254, 258–259, 261–262, 265–268, 273–278, 283–284, 297, 323, 332–333, 340, 350–351, 358, 361, 368, 375, 378–384, 388, 394, 398–401, 417–418, 422, 430, 474–475, 485, 490–492,

496–497, 499, 505, 544, 560, 594, 596, 601, 604, 606–607, 611, 629, 634, 640, 649–650, 687, 689–690, 694–695, 704, 710, 720, 730, 735–736, 749, 751, 753 extraction coil, 100, 165–169, 178, 237, 239, 243, 261, 380–381, 418, 604 F–, 74, 84, 88, 90, 104, 149–150, 186–188, 252, 266, 298, 327, 329, 346, 361, 363, 369, 376, 382, 386–387, 402, 409, 418, 475, 479, 498, 503, 529, 549, 595, 599–600, 604, 617–620, 622, 627, 633, 722, 734, 740, 747, 754 failure warning, 623 Faradaic current, 450–451 Faraday’s law, 443, 456 fatty acids, 412, 633 Fe, see Iron Fe total, see iron, total, 719 Fe(II), see iron(II) Fe(III), see iron(III) ferrozine, 132, 601 FIA-CE, 297, 304, 530, 536 FIA-MS, 19, 479, 606, 733 fibrous alumina, 181 Fick’s first law, 51 field measurements, 661, 676, 738, 740 field-portable, 87, 652, 669, 685, 696, 705 filterless filtration, 236, 258, 261 filters, 133, 139, 170, 213, 235, 254–256, 261, 317–318, 328, 347, 435, 629, 640, 649–650, 688–689, 694, 736, 739 filtration, 101–103, 106, 138, 147, 204–205, 212, 235–236, 252, 254–261, 361, 391, 545, 597, 685, 687–689, 694, 696–697, 707–708, 724, 734, 740, 749, 751 FI-Microchip CE, 287, 299 finite-difference methods, 53 finite-element methods, 53 fireflies (Photinus pyralis), 357 first-order reaction, 67, 69–70 fish, 113, 139, 143, 174, 230, 426, 513–514, 535–537, 547, 702, 712 flame atomic absorption spectrometry, 119, 136, 154, 181, 198, 376, 402, 548 flame atomic emission spectrometry, 376, 402 flame furnace atomic absorption spectrometry, 376, 402, 549 flavonoids, 525 flow-batch coulometric titrator, 631 flow injection liquid–liquid extraction flow injection microscopy, 470–471 flow injection/membrane introduction mass spectrometry, 73

Subject Index

flow programming, 24, 26, 36, 283, 334, 380, 389 flow reversal, 26–27, 29, 36, 42, 44, 94, 96, 98, 165 flow system type, 623 flow titration, 69, 632, 636 flow-based extraction protocols, 398 flow-batch, 619, 626, 631 Flow-Batch (FB) concept, 626 flow-batch coulometric titrator, 631 flow-through measurement, 37, 317, 319, 329 flow-through radiometric cell, 475 flow-through tunnel cell, 422 fluorescamine, 347, 351 fluorescence, 30–32, 37–38, 90, 96–98, 101, 120–121, 134, 138–140, 143, 154, 161–162, 178, 211, 215–216, 224, 267–268, 270, 281, 283, 290, 299–301, 304–305, 343–352, 354, 356, 367, 376, 402, 427–430, 461, 472, 484–485, 489, 495, 501, 504–505, 548, 560, 562–563, 596, 598–599, 601, 603–604, 607–608, 610–612, 643, 650, 653–654, 658–659, 661–662, 665–666, 677, 687, 693, 707, 714–715, 726, 728, 730–731, 735–736, 742–743, 749, 751–752 fluoride, 213, 476, 493, 629, 717, 728, 744, 747 fluoxetine, 601–602 fluvoxamine, 602, 606 Folin–Ciocalteu reagent, 173, 178, 247 foods, 117, 139, 230, 350–351, 538–539, 541–542, 546–547, 595 formaldehyde, 196, 224–225, 351, 357, 362, 597, 639, 661, 663–664, 672 fosfomycin, 153 Fourier number, 52–54, 57–58, 75 Fourier sine series, 61 Fourier Transform Infrared, 258, 267, 284, 410, 435, 636, 661 fractionation studies, 398 Franz diffusion cell, 134–135, 278, 611 free radicals, 595 freon, 240 freshness, 537, 547–548 fruit juices, 32, 514, 522–525, 544, 633 FTIR, 97, 267, 284, 410–411, 416–420, 422–424, 426, 435, 516, 521, 548, 634, 636 furosemide, 268, 277 b-galactosidase, 480 galactose, 455, 531, 600 gallic acid, 362, 519, 717, 743 Gamma-distribution function, 66

767

gas analysis, 316, 423, 639–640, 651–652, 657, 659, 675, 677, 680 gas denuder, 159, 188, 191 gas diffusion, 16, 101, 104, 152, 191, 198, 203, 205, 207, 209–211, 213–215, 217, 219, 221, 223–227, 229, 231, 247–248, 297, 333, 463, 486, 544, 643, 693–694, 707, 713, 720, 725, 727–729, 735, 743–744, 746, 753 gas permeation, 694 gas-liquid collector, 645, 647 gas-liquid separation, 375, 378, 379, 392 gas-pressurized reservoirs, 87 Gaussian RTD, 65–66, 74 Gaussian shape, 54 Geiger-Mu¨ller (GM) counters, 475, 505 gel filtration, 147, 597, 734 gentamicin, 352, 361, 531, 602, 604 glass coil collector, 665 glassy carbon, 364, 369, 447, 450–451, 455, 605, 731–733, 750 glassy carbon electrode, 369, 451, 605, 731–733 glucose, 34, 69, 113–115, 218, 221, 267, 354, 359, 361, 365, 368–369, 412–413, 419–422, 428, 430, 446, 455, 476, 493, 519, 523, 528, 531, 538, 541, 600, 609, 732 glucosides, 434, 504 glycerol, 34, 467–468, 476, 516, 544 gradient techniques, 8 Green’s function, 67 Griess reaction, 144, 146, 645, 654, 670, 673, 710–712, 733–734, 738 ground water, 116, 501, 697–698, 702, 719, 725, 749 guanidinoacetate, 497 H2S, 101, 131, 211, 501, 639, 641–643, 645, 658–659, 667, 669–671, 676–677 haemoglobin, 354, 434 halogen species, 729 Hansen’s FIA-bibliography, 314 HCO 3 , 223, 426, 701, 724–725, 744 helically coiled open tubes, 47, 55 hemodialysis solutions hepatocyte, 596 herbicides, 702, 730–731 heroin, 365, 368 hexacyanoferrate, 17, 178, 357, 359, 455 Hg, see mercury High Performance Liquid Chromatography, 155, 265, 267, 278, 284, 430, 435, 499, 594, 613 high-affinity recombinant antibodies, 579

768

Subject Index

histamine, 498, 521, 536, 548, 596 HO-luminol reaction, 734 homatropine methylbromide, 603, 606 homogeneous membranes, 213 honey, 117, 359, 368, 538, 540–542, 548 horse kidney, 137 horseradish peroxidase, 115, 221, 351, 362, 370, 605 human immunogloblin G, 368 human pancreatic secretory trypsin inhibitor, 502 humic and fulvic acids, 701, 736 hybrid flow analyser, 639, 652–653 hydraulic models, 54 hydride generation, 11, 30, 112, 120–121, 136, 154, 198, 212, 240, 270, 284, 375, 392–393, 395, 402, 426, 446, 716, 722–723 hydrodynamic injection, 16, 52, 83, 92, 287, 290, 295 hydrogen peroxide, 113, 144, 347, 351, 354, 356, 359, 361, 367, 453, 455, 492, 600, 604–605, 654, 664, 709, 726, 747 hydrogen sulfide, 154 hydroxyquinoline, 346, 348, 350, 362, 369, 695, 752 hyoscyamine, 603, 605 hypobromite, 361, 363–364 hypochlorite, 354, 357, 363 hypoglycaemic drugs, 594 hypotensive drugs, 594 ICP-AES, 97, 105, 119, 136, 143, 154, 160, 177, 181–182, 185–190, 192–194, 197–198, 269, 376, 378, 382, 386, 397, 399, 402, 462, 505, 518, 622, 715–717, 720–722, 728, 753 ICP-MS, 33, 97, 120, 139, 143, 155, 160, 171, 175, 177, 181–188, 198, 269–270, 284, 376, 378, 381–382, 384–386, 391–392, 394, 397, 402, 462, 491, 505, 529, 532–533, 536, 539, 548, 715–716, 719, 723, 729, 753 ideally mixed tank model, 61–63 imazalil, 497, 526 imipramine, 601, 603 immobilized enzymes, 16, 455, 600 immunoaffinity chromatography, 40 immunoassay, 32, 120, 150, 252, 344, 352, 360, 362, 366, 368, 484, 546, 596 immunoglobulin G, 568, 599, 613, 260 immunoreactor, 575, 576, 577, 578 impingers, 640 in situ electrochemiluminescence, 343, 363 in situ measurements, 704

in situ monitoring, 319, 479, 639, 652, 693, 697–698, 719 in-atomiser sequestration, 394 indomethacin, 603, 607–608, 612 indophenol blue, 154, 645, 712–713, 735 inductively coupled plasma atomic emission spectrometry, see also ICP-AES inductively coupled plasma mass spectrometry, see also ICP-MS inertial forces, 50 infancy of FIA, 3, 7 injection volume, 81, 85–87, 92, 94, 98, 106, 250, 299–300, 304, 336, 468, 479, 697 in-line, 102–103, 147, 181, 224, 228, 468, 599–600, 617, 619–620, 707 input-output relationships, 48 integration window, 336 interference bands, 411 internal reflection element, 422, 435, 634 in-valve spectrometric detection, 563 Invisible College, 13–14 iodine-131, 489 ion associate, 161–162, 164, 169–170, 176, 181–182, 348, 350 ion chromatographic methods, 744 ion exchange, 34, 103, 140, 142, 159, 172, 177, 182–184, 277, 298, 350, 352, 360, 384, 396, 463, 473, 483, 488, 598, 605, 608, 650, 673, 693, 695, 704, 709, 711–712, 714, 717, 719, 722, 729, 734–736 ion-exchange membranes, 213 ion-selective electrode, 3, 72, 84, 98, 172, 269, 447, 449–450, 472, 605, 613, 629, 707, 710, 713, 725 iridium, 151, 184, 188, 394, 407, 409–411, 419, 423, 426–427, 462, 481, 519, 548, 656 iron, 31, 34, 89, 91, 113, 131–132, 136–139, 141, 143, 154, 173, 176, 180, 186, 188, 192–194, 216, 224, 240, 246, 269–270, 303, 354, 359, 433–435, 447, 452, 473, 491, 500, 517, 525, 533, 535, 539, 597–598, 600–601, 604, 689, 691, 694–696, 700–702, 704, 709–710, 715–717, 719, 721, 724–725, 727, 736, 743, 745 iron(II), 246, 269, 354, 359, 433, 435, 452, 500, 601, 715, 717, 719, 724 iron(III), 89, 154, 224, 269, 359, 452, 473, 491, 500, 601, 715–717, 719, 725, 727, 736 iron, total, 719 isobutylmethylketone, 169, 198, 402, 427 isoniazid, 115, 270, 360, 364, 534

Subject Index

Journal of Flow Injection Analysis Bibliography, 314 K, 701, 704, 724 Karl-Fisher titration, 630 ketoprofen, 278, 413 kinetic measurements, 81, 99–100, 314, 321, 337 kinetics, 18, 31, 47, 68, 72, 99–100, 105, 112, 210, 228–229, 244, 332, 349, 354, 376–377, 383, 386, 398, 402, 470, 492, 543, 596, 610, 690, 695 knitted coils, 96 knotted reactors, 235–236, 254–255, 261, 375, 380, 391 Kubelka-Munk theory, 72 L – glutamate, 585, 543 Lab on Valve, see also lab-on-valve Lab-at-valve, 31, 165, 198, 244 labetalol, 350, 501, 603, 608 lab-on-chip, 16, 28, 35, 91, 302, 304, 322, 324 lab-on-valve, 15, 23, 28, 30–34, 36, 84, 89, 95, 104, 171, 198, 210, 231, 247, 262, 270, 283–284, 299, 313, 322, 332, 347, 370, 375, 383, 403, 408, 449–450, 560, 596, 613, 619, 637, 686, 753 laccase activity, 521 lactate, 34, 115, 221, 349, 351, 354, 368–369, 455, 518, 523, 534, 541, 600, 710 lactic acid, 215, 269, 518, 600 Lambert-Beers law, 315, 328, 329 laminar flow, 29, 35–36, 49–50, 56–59, 67, 71, 75, 82, 84, 86, 95, 124, 257, 335, 444–445, 466, 697 lansoprazole, 601, 603 Laplace domain function, 61 Laplace transformation, 61 Laplace transforms, 53, 70 large injection volume, 336 laser induced fluorescence, 299, 301, 305, 351, 562, 565, 573, 587, 749 L-dopa, 502, 602, 604 lead, 10, 33, 113, 131, 136, 138–139, 181–182, 188, 216, 238, 252, 272, 282, 288, 290, 324, 337, 352–353, 425, 446, 475, 481, 500–501, 504–505, 517–518, 529, 533, 535–536, 539, 544, 546, 561, 594, 690–691, 700 LED detector cell, 330 LED photometers, 311, 740 LED, see also light emitting diode lensing effects, 337 LiChrolut EN, 172

769

LiChrolut RP-18e, 172 ligand pretreatment light emitting diode, 99, 106, 316, 340, 436, 650 light scattering, 258, 461, 467–470, 487, 505, 519, 525 limit of detection, see also detection limit limiting steady-state current, 71, 74 linear dynamic range, 98, 329, 563 linear flow velocity, 50, 53 lipid hydroperoxide, 541–542 lipid-coated quartz crystal microbalance, 544 liquid core waveguide, 97, 652, 665, 706, 735, 737, 753 liquid core waveguide, 97, 652, 665, 706, 735, 737, 753 liquid membrane-based extraction techniques, 235, 247 liquid type GM counter, 475 liquid–liquid extraction, 323, 340, 350, 379, 380, 422, 430, 594, 634, 62, 100, 101, 159, 160, 164, 165, 169, 170, 204, 205, 207, 211, 212, 236–238, 241, 244, 247, 253, 261 lisuride, 133 LOD, see Limit of Detection long path length absorbance monitoring, 652 long pathlength liquid-core waveguide cell, 327 Lorentzian function, 66 luminescence, 97, 343–347, 349, 351, 353–354, 356–357, 359, 361, 363–365, 367, 369, 597, 654 luminol, 17, 176, 181, 353–354, 359–360, 363, 367–369, 595, 597, 600, 604, 665, 707, 709, 711, 714, 717, 719, 722, 726–727, 729, 734–735, 743, 745, 747 macrobiomolecules, 560 magnesium, 6, 113, 131, 137–138, 145, 184, 188, 224, 297, 350, 358, 377, 394, 400, 412, 425, 504, 518, 525, 527, 535, 602, 612, 630, 676, 691, 700, 704, 714, 716, 724, 727, 744 magnetic particles, 150 magnetohydrodynamic, 90, 106 major cations and anions, 701, 724 Malachite green, 170, 735 manganese, 113, 138, 143, 177, 185–186, 188–190, 192–194, 269, 357, 363–364, 518, 533, 608, 689, 695, 700–701, 704, 719, 721 manifold variables, 81, 85

770

Subject Index

marine, 138, 154, 175, 357, 359, 495, 666, 669–670, 694, 696, 701–702, 734–736 marine bacteria, 357 mass spectrometry, 28, 33–34, 73, 97, 120, 125, 143, 149–150, 155, 171, 198, 231, 253, 269–270, 283–284, 376, 402, 461–462, 479, 482, 498–499, 505, 525, 545, 548, 594, 603, 613, 633, 693, 730, 733, 750, 753–754 mass transfer phenomena, 48–49, 58, 60 mathematical model, 47–50, 52, 54, 58, 60–61, 133 matrix matching, 94, 99, 336–337, 636, 697, 736 matrix removal, 34, 423, 685, 694–695, 743 Matthew Effect, 13–14 mean residence time, 52–54, 68, 74 membrane based scrubber, 649, 651 membrane extraction, 205, 247–248, 251–254, 262, 333, 694 membrane separator, 100 membrane-based separations, 375, 396 membraneless gas diffusion, 463 mercury, 31, 112–114, 121, 124, 136, 138–139, 141–143, 147, 154, 175–176, 181, 187–188, 198, 212, 240, 268, 270, 284, 350, 359, 377–378, 391–397, 402, 450, 454–455, 518, 529, 532–533, 535-536, 538–539, 547–548, 605, 690, 700–702, 720–721, 725, 742–744, 746 mercury speciation, 113 mercury, organic, 721 mercury, total, 720 mercury-CV system, 141 merging-zone, 68, 142–143, 152, 240 metabolism of toxic species, 116 metal or metalloid species, 379 metallic species, 393, 617, 622, 633, 690 metallurgical solutions, 633 metastable/transient constituents, 19 methylamine, 731, 748 methyldopa, 601, 603 Methylene blue, 131, 162, 198, 224, 430, 745, 753 methylmercury, 113, 181 methylparaben, 268, 274–275, 277–278, 609 methylsalicylate, 275, 278 metoclopramide, 603, 607 Mg, see magnesium micelles, 351, 382 Michaelis-Menten kinetics, 72 micro gas analysis system, 639, 657, 659 micro Sequential Injection Analysis, 33 micro Total Analysis Systems, 16, 150, 657 micro-affinity chromatography, 568

micro-bead injection analysis spectroscopy, 568, 587 micro-beads, 596, 608 micro-circle flow-through cell, 420 microcolumn, 27, 30–31, 34, 39–40, 43, 229, 273, 276–277, 339, 352, 382, 384, 389–390, 396, 398–401, 475, 561 microconduits, 15–16, 95, 322, 387, 400, 450 microdialysis, 203–205, 211–212, 216, 218–222, 231, 297, 304, 396–397, 597, 694 microfluidic, 23, 35–36, 43, 82, 89–91, 150, 241, 257, 303, 503, 562, 605, 693, 751 microporous membrane liquid–liquid extraction, 205, 247, 253, 262 microporous membranes, 93, 213, 223, 236, 749 microscopy, 461, 470–472, 495, 504 microTAS techniques, 28 microwave digestion, 105, 118, 136, 141, 143, 491, 708, 720, 728, 737 microwave irradiation, 143, 694–695, 723, 742 microwave sample preparation, 135 mid infrared, 408, 412, 421, 425, 436 milk, 117, 148, 215, 217, 269, 368–369, 420, 423, 490–491, 494, 498, 513–514, 531–534, 546–547 milk powder, 117, 420, 532–534 milk samples, 547 milliGATt pump, 89 miniaturization, 23, 28, 44, 89, 116, 124, 150, 228, 266, 282, 288, 299, 302, 407, 416, 435, 462, 481, 483, 593, 600, 639, 652, 656, 680 mixing device, 49, 70, 95–96, 124, 335, 488, 693 Mn, see manganese, 138, 143, 177, 185–186, 188–190, 192–194, 269, 608, 689, 700–701, 704, 719, 721 Mo(V), 169 Mo(VI), 169 modes of flow analysis, 354, 622 molecularly imprinted polymer, 172, 180, 198, 505 molybdenum, 6–7, 34, 147–148, 153, 169, 182, 529, 536, 633 molybdophosphate, 147, 170, 181, 198, 348, 350 monitoring of food processing, 515 monolithic chromatographic columns, 104, 282 monolithic column, 134–135, 265, 270–272, 279–281, 283, 499, 609 monolithic disk, 598 mono-segmented flow analysis, 619 monosodium glutamate, 541

Subject Index

morphine, 115, 361, 600, 609 multi syringe flow injection analysis, 375, 403, 626, 637, 753 multi-channel flow cell, 332 multicommutation, 87, 93–96, 98–99, 354, 361, 408, 410–411, 414–415, 420, 422, 427–428, 430, 435, 707, 712, 736, 740 multi-commutation flow injection analysis, 619, 626, 637 multiple reagent-injection, 93 MultiPump Flow Injection Analysis, 94, 270, 284 multi-reflection cell, 325, 327, 337–338, 753 multi-segmented continuous flow, 619 multi-syringe flow injection analysis, 375, 403, 626, 637 multisyringe liquid chromatography, 281, 284 multivariate curve resolution, 267, 270, 283–284, 607 multi-vitamin tablets, 139 myo-inositol phosphate, 530 N, N-dimethyl-p-phenylenediamine, 131, 752 Nafion, 72, 90, 213, 642–643, 661, 663, 665–666 Nafion membrane, 72, 90, 642, 661, 665 NaI/TI scintillation counters, 475 naphthylamine, 733, 753 naproxen, 267, 281, 598–599 naptalam, 173 National Research Council Canada certified reference materials, 138 Navier-Stokes equations, 55 near infrared, 311, 408, 410, 429, 435–436, 630, 637 Near-IR (NIR) spectrometry, 426 nebulizer, 138, 143, 251, 378, 381, 401–402 Nernst diffusion-layer, 72 Nessler’s reagent, 712, 735 neurotransmitter, 604 NH4+, see ammonia/ammonium nickel, 30, 113, 138, 143, 176–177, 181, 186, 189–190, 192–194, 218, 261, 269–270, 354, 388–389, 392–393, 427, 529, 647, 689, 700, 716, 719, 721 nicotinamide adenine dinucleotide, 215, 231, 348, 352, 369–370, 753 NIR, 97, 408–409, 426–430, 435–436, 630, 636–637 nitrate, 6, 113–114, 144–147, 240, 268–269, 293–294, 296–297, 304, 347, 420–421, 429, 433, 527–528, 531, 535, 547, 609, 629, 688–689, 698–701, 703–704, 710–712, 730, 734, 737, 746, 753 nitrate+nitrite, 753

771

nitrite, 91, 113, 117, 144, 146, 172–173, 179, 240, 269, 304, 347, 360, 362, 425, 473, 527–528, 531, 535, 547, 652, 670, 672, 700, 734, 753 nitrogen, 34, 105, 142, 144–146, 196, 251, 331, 362, 423, 425–426, 520, 537, 544, 666, 670, 688, 690, 701–703, 710, 737, 754 nitrogen dioxide, 196 nitrosamine, 537, 547 non-ionic, 147, 350, 382, 720, 730 non-polarized electrode, 443 norepinephrine, 502, 603–604 NOx, 137, 639, 670, 673, 707, 710–712, 740, 742, 753 nuclear magnetic resonance, 461, 480, 505, 549 numerical simulation, 48, 53, 60–61, 64, 68, 72 nutrients, 105, 114, 296, 685, 687, 690, 694, 700–705, 738 off-line monitoring, 619 oil-in-water, 629 oligosaccharides, 497, 532, 538 on-bead oligonucleotide hybridization, 563 o-nitroaniline, 166–167 o-nitrobenzoic acid, 166–167 on-line analysers, 627, 629 on-line electrochemical generation, 363 on-line filtration, 102, 258, 694, 707–708, 724 on-line interfaces, 222 on-line monitoring, 432–433, 561, 600, 619, 634, 703 open-closed FIA system, 69 o-phthalaldehyde, 604, 607, 652, 675 opiate alkaloids, 609 optode, see also optrode optosensing, 33, 172, 179, 268, 332–333, 500–501, 608 optrode, 72, 461, 472–473, 501 organic contaminants, 685, 702, 704–705, 730, 748–749, 751 organic extractant, 240, 244, 380 organic matrices, 135 organic nitrogen, 144, 146, 690, 737 organic phosphorus, 105, 146–148, 154–155, 690, 695, 734, 737, 752 orthogonal collocations, 53 orthogonal polynomials, 66 ortho-phthaldialdehyde, 347 Os, 151, 188, 243 oscillating first-order kinetic constant, 68 overpotential, 450, 605 oxaloacetate decarboxylase, 349 ozone, 146, 651, 655–656, 661, 672, 743–744

772

Subject Index

1,10-phenanthroline, 365, 715 PAH, 753 palladium, 181 paper mill industrial waters, 634 paracetamol, 154, 244, 275, 278–279, 413, 603, 605, 609 parallel plate denuder, 641 parallel-plate laminar flow, 57–58 parallel-tanks model, 73 paraoxon, 687, 732 paraquat, 731 paroxetine, 603, 605 partial least squares, 270, 284, 419, 436 Pb, 138–139, 141, 143, 175–177, 185–190, 192–194, 259, 261, 269, 382, 392, 426, 485, 504, 689, 701–702, 716, 721–722 Pb isotope ratios, 722 Pd–Mg matrix modifier, 137 peak height, 63, 66, 280, 293, 333, 644 Pećlet number, 52–53, 58–61 pectinesterase activity, 525 Peltier device, 677–678 penicillamine, 115, 601, 603 penicillin, 267, 600, 603, 608 penicilloic acid, 600 pentachlorophenol, 732, 753 periodate, 154, 354, 357, 360, 726, 729 permanganate, 354, 357, 361, 396, 486, 597, 600, 604, 608–609, 709, 728, 731 permselectivity, 215 peroxidases, 354 peroxydisulfate, 104, 141–142, 144–148, 151–153, 491, 601, 687, 695, 708–710, 720, 734, 746 peroxydisulphate, see peroxydisulfate peroxyoxalate, 174, 356–357, 361, 604 peroxyoxalate chemiluminescence, 356, 361 pervaporation, 73, 101, 203, 205, 207, 209–211, 213, 215, 217, 219, 221, 223, 225–231, 297, 359, 396–397, 545, 694, 713, 727, 732–733, 744, 749–750, 753 pesticide, 258, 425, 430, 473, 545 pH, 3, 16, 40, 96, 142, 152–154, 166, 172, 175–176, 178, 180–183, 185, 188, 196–197, 211, 216–218, 246, 248–250, 259, 269, 274–275, 282, 289, 304, 346, 350, 352, 366–367, 386, 388–389, 396, 399, 401, 417, 421, 431, 450, 457, 475, 493, 495, 497, 543, 596–597, 605–607, 610, 617, 627, 629, 632, 634, 651, 674, 689–691, 695, 703–705, 708, 710, 719–721, 726, 734–735, 749 pH modulation, 417

pharmaceuticals, 91, 117, 129–130, 132–134, 139, 154, 174, 268–270, 272–274, 350, 357, 359–361, 368, 421, 425, 501, 591–592, 594, 601, 605, 607, 610, 630 phase segmentor, 100, 169, 237–238 phase separator, 100, 159–160, 165–166, 169–170, 178, 237–238, 240–241, 244, 380–381, 417–418, 604 phenolics, 517 phenylbenzimidazole sulphonic acid, 277 phenylmercury, 114, 181 phosphate, 6–7, 9, 33–34, 88, 112–114, 131, 142, 147–148, 170, 181, 214, 249, 269, 279, 304, 347–348, 350, 357–358, 412, 417, 420, 530, 598, 605, 608, 629, 674, 688, 701, 703–705, 707–708, 713–714, 724, 734, 749, 752 phosphatidyl choline, 709 phospholipid, 737 phosphomolybdate, 605, 705, 707–708, 731, 734–735 phosphomolybdenum blue, 706–710, 712, 714, 734–735, 740 phosphomolybdic acid, see phosphomolybdate phosphorescence, 343, 345–346, 349–350, 352, 500, 531 phosphorus, 32, 105, 144, 146–148, 153–155, 348, 489, 521, 530, 534, 688, 690, 695, 701–706, 734–735, 737, 752–754 phosphorus-32, 489 photochemical oxidation, see UV photo-oxidation photoluminescence, 97, 343–344, 346, 349, 353, 363 photolysis, 143, 154, 594, 673 photometer, 312–313, 329, 338, 427, 706–708, 712–713, 725, 727 photometry, 33, 83, 97, 101, 176–177, 224, 240, 269, 311–313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337–340, 706–715, 717–719, 721, 724–729, 732–733 photomultiplier tube, 318, 346, 353, 597, 651 photo-oxidation, see UV photo-oxidation photo-reduction, 711 phylloquinone, 351, 530 physical model, 48, 67 phytate, see also myo inositol phosphate piezo valve, 679–680 piezoelectric activated pump, 92 piezoelectric crystals, 599 pipedimic acid, 603–604 piribedil, 603, 605

Subject Index

piston pump, 89, 635, 693 plasma proteins, 596 plate height, 59 plug flow, 48, 50, 54, 59, 74 plutonium, 490–491 pneumatic pumping, 676 Poisseulie flow, 50 polarized electrodes, 443 poly(vinyl alcohol), 468, 505 polyatomic interferences, 378 polycyclic aromatic hydrocarbons, see also PAH polymer industry, 634 polyphenols, 245–246, 357, 519 polyphosphates, 147, 357 polytetrafluoroethylene, 30, 160, 198, 219, 236, 262, 300, 305, 417, 436, 642, 753 potable waters, 698, 700, 706 potassium hexacyanoferrate (III), 178 potassium, 16, 113, 139, 141, 143, 151, 154, 161, 170, 178, 182, 258, 357, 361, 518, 600, 607, 728, 731, 747, 749 potentiometric methods, 443, 462, 744 potentiometry, 5, 16, 32, 71, 72–73, 83, 85, 97, 120, 133, 136, 148, 173–174, 211, 218, 223, 229, 258, 269, 441, 442–444, 447–452, 454, 457, 462, 495, 516, 518, 520–525, 527–528, 531–533, 535, 537, 541, 544, 603, 605, 608, 630, 632, 634, 636, 693, 707, 710, 713, 717, 720, 724–725, 729–731, 744, 747–748 practical dispersion coefficient, 54, 63 prazosin hydrochloride, 134 precipitate collectors, 235, 261 precipitation, 148, 254, 259–261, 368, 375, 378, 391, 487–488, 606, 690, 703, 724, 727 preconcentration, 19, 30–34, 42, 84, 101–103, 142–143, 159–161, 163, 165, 167, 169, 171–173, 175, 177, 179–181, 183, 185–187, 189, 191, 193, 195–198, 204, 208, 210, 213–214, 228, 230, 254, 256, 259–261, 273, 288, 294, 297–298, 338–339, 359, 376, 379–386, 388–394, 396–397, 399–401, 411, 454, 478, 490, 505, 514, 641–643, 650, 665, 685, 687, 694–695, 706, 712–713, 715–723, 727, 729–730, 735, 743, 749 proanthocyanidins oligomers, 525 process analysers, 617, 625, 627–629, 635 process analysis, 47, 299, 594, 617–619, 621–627, 629, 631–633, 635–636 process analytical technology (PAT), 599, 613, 618, 637 proline, 360, 365, 368, 521 promethazine, 115, 601, 603

773

proportional valve, 91–93 propulsion, 18, 81–83, 87–89, 92, 247, 249, 281, 302, 304, 718 Propyl orange, 170 protein A, 150, 503 protein G, 352 protein immobilization, 559 proteins, 30, 40, 149–151, 219, 271, 276, 290, 423, 425–426, 470, 478, 480, 484, 488, 494, 498, 521, 559, 592, 596, 599, 701 proteolytic digestion, 149 pseudo first-order reaction, 67, 69 pseudoephedrine, 172–173 pseudo-titration, 132, 632–633 Pt, 146, 151, 188, 604, 677–678, 722 PTFE, see also polytetrafluoroethylene pulsed-flow, 354 pump pulsation, 335 putrescine, 536, 548 4-(2-pyridylazo)resorcinol, 181 pyridinium chloro-chromate, 496 pyrrolidine dithiocarbamate, 753 pyruvate decarboxylase, 348, 351 pyruvate, 348–349, 351, 528, 707 QCL, 417, 435–436 quadrupole mass spectrometer, 479 quantum cascade lasers, 417, 436 quantum yield, 352–353, 356–357 quartz crystal microbalance, 481, 502–505, 544, 599 quinine, 357, 497 quinoneimine dye, 601, 609 radial dispersion, 35, 53, 63, 82, 255 radical scavenging, 595 radium, 490 Raman spectrometry, 407–408, 430–434 random walk model, 59, 68 Rayleigh scattering, 603, 606 reaction rate measurement, 37 reagent injection, 88, 93–94, 99, 259, 333, 337, 628, 694, 706, 713, 736, 738, 740 redox, 221, 444, 447, 452, 457, 486, 522, 690, 695, 703 reference electrode, 3, 16, 218, 364–365, 448, 450–451, 456, 605–606, 747 reference materials, 98, 117, 119, 125, 136, 138, 185, 193, 268–269, 532, 692 reflection mode, 72 reflective detection cell, 736 refractive index, 38, 71, 94, 98–99, 242, 311, 315, 334, 336, 338, 340, 465, 469, 505, 631, 635, 652, 654, 697, 736

774

Subject Index

refractometry, 335, 461, 465 renewable drops technique, 563 renewable separation column, 475, 477, 561 reproducible timing, 7, 25–26, 84, 105, 470, 624 Residence Time Distribution (RTD) function, 48, 74 residence time, 25, 48, 52–54, 68, 74, 143, 228, 393, 455, 475, 625, 693 resonance light scattering, 566 Restricted Access Material, 265, 273, 276, 284 reverse FIA, 87, 360, 462, 694, 719, 726, 729, 736, 740 reverse flow injection analysis, see reverse FIA, or reagent injection reverse osmosis, 212 Reynolds number, 23, 35, 50, 55, 74, 84, 106 Rh, 151, 188, 356, 664 Rhodamine B, 348, 357, 707, 735 robustness, 133, 280, 314, 322, 331, 562, 622–624, 626, 628, 631–632, 635, 705 ruthenium, 3, 6, 23–24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 151, 188, 364, 366, 608, 731, 749 S, 421, 642–643, 667 S1/2, 85, 106 salbutamol, 173, 178, 273, 276, 601, 603 salicylamide, 610 salicylate, 134, 173, 609, 712–713 salicylic acid, 134, 160, 268, 274–275, 277–279, 303, 500, 609–610, 612 salinity-compensation, 337 Salmonella typhimurium, 501, 537, 548 sample conditioning, 693, 751 sample preconcentration, 42, 198, 401, 706 sample preparation, 33, 130–131, 135–137, 204, 231, 258, 358, 361, 367–368, 430–431, 489, 499, 546, 618, 623, 694, 704 sample pretreatment, 23, 42–43, 104–105, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 159–161, 163, 165, 167, 169–171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 210, 250, 267, 273, 277, 287, 291, 297–298, 379, 382, 385, 408, 411, 420, 426, 513–514, 547, 562, 594, 605–606, 627, 717 sandwich cell, 97, 168, 323 sandwich hybridization, 30, 563 Sb, 33, 138, 141, 188, 268, 392–393, 396, 426, 700, 702, 716, 722 Sc, 56–57, 74, 188, 192–194, 281, 476, 648, 653

schlieren effect, 98–99, 322, 335, 337–339, 636, 652, 697, 706, 735–736 Schmidt number, 56–57, 74 scrubber, 188, 198, 639–643, 649–653, 657–659, 662, 665, 676, 736 Se total, 327, 690, 723 Se, see selenium seawater, 116, 119, 143, 175–176, 186–187, 196, 224, 269, 335, 350, 352, 501, 695–696, 701, 716, 723, 726–727, 740, 742–743, 745, 752–753 second moment about the mean, 64, 75 second order reaction, 68–70 secondary flow, 55–57, 84, 86, 96, 255, 391 segmented flow coil, 672 selection valve, 82, 88–89, 95, 102–103, 134, 164–166, 168–169, 185, 190–191, 209, 215, 246, 381, 398, 417, 471, 482, 485, 543–544, 598, 607 selectivity, 8, 44, 98, 104, 133, 154, 185, 211, 214–215, 219, 223, 229, 240, 247, 250, 252, 271, 277, 283, 288, 297, 303, 314, 316, 339, 344, 347, 349–350, 358, 363, 367, 376–377, 379, 386, 397, 401, 407, 462–463, 473, 476, 483–484, 486, 497, 545, 548, 605–607, 642, 658, 695–696, 705, 734–735, 743, 748, 750–751 selenate, 177, 182 selenite, 177, 182 selenium, 9–10, 100, 141–143, 159–162, 165–170, 177, 182, 186, 188, 198, 218, 269, 392, 396, 700, 702, 716, 723, 741–742 Se(IV), 142–143, 177, 357, 396, 723, 742 self assembled monolayers, 369, 576 self calibration, 618, 622 S-enalapril, 602, 608–609 sensing mechanism, 47–48, 60, 63, 71–73 Sephadex, 40, 176, 181, 431, 560, 596, 601 Sepharose, 31, 40, 390, 396, 399–400, 560 Sep-Pak, 172–173, 176 sequential extraction, 19, 105, 398 sequential injection affinity chromatography, 280 Sequential Injection Analysis/Lab-On-Valve, 15, 265, 284, 287, 313, 347, 352, 370, 375, 403, 407, 549, 560, 26–27, 30–34, 593, 618, 619, 637, 651, 82–87, 95, 111, 133, 165, 217, 236, 262 sequential injection analysis, 15, 26–27, 30–34, 81–84, 87, 95, 111, 125, 133, 165, 217, 231, 236, 262, 265, 284, 287, 313, 347, 352, 370, 375, 403, 407, 549, 560, 593, 613, 618–619, 637, 639, 651, 753

Subject Index

Sequential Injection Chromatography, 23–24, 41, 103, 134, 155, 265, 272, 277–278, 284, 339, 593, 609, 613 serial scrubber, 642 serpentine reactors, 96 serum, 9, 32, 117, 132, 173, 214–215, 224, 231, 268–269, 277, 280, 284, 351–352, 362, 368–369, 397, 428, 463, 476, 597–598, 613 sheath-flow cell, 321 SIA-bead injection-lab-on-valve silica, 30, 41, 89, 91, 149–150, 160, 172–173, 178, 180, 182, 220, 267, 271, 278, 283, 289, 301–302, 304, 325, 327, 350, 382, 422, 501, 563, 601, 606, 608, 610–611, 629, 650, 672, 695, 717, 740 silicate, 34, 131, 137, 186, 359, 688, 691, 700–701, 707, 714, 734, 742 silicomolybdate, 714, 734 silicon, 91, 149, 161, 690, 714 silicotungstic acid, 606 simultaneous atomic absorption spectrometry, 376, 403 single bead string reactor, 83 single drop headspace microextraction, 649 smoke, 139 SO2, 101, 211, 224–225, 356, 486, 544, 639, 643–645, 648, 657–659, 664, 667, 669–671, 676 sodium, 4, 41, 141, 144, 147, 152, 155, 168, 182, 268, 278, 282, 352, 362, 370, 392–393, 396, 422, 465, 470, 494, 496, 604, 607–608, 629 soft drinks, 267, 419, 514, 522–525, 633 solenoid actuated diaphragm, 88 solenoid pump, 693–694, 707, 712, 740, 742 solenoid valve, 88, 92, 94, 140, 148, 272, 281, 408, 415, 430, 482–483, 609, 626, 635, 643, 645, 648, 653, 656, 658–659, 666, 694, 740–741 sol-gel layers, 369 sol–gel membranes, 608 solid phase extraction, 33, 159, 171, 173, 175, 177, 180, 183, 266, 485, 490, 499, 505 solid phase spectrometry, 182, 199 solid samples, 19, 97, 116, 130, 136–139, 228–231, 375, 377–378, 397, 426, 548, 622, 633, 710 solid-contact ion-selective electrode, 450 solid-phase extraction, 19, 30, 103, 249–250, 265–266, 268, 273, 284, 351, 361, 375, 378, 382–384, 388, 399, 544, 594, 596, 687, 689, 735–736, 753 solid-phase micro extraction, 339, 340, 597, 649

775

solvent extraction, 100–101, 159–160, 164, 198, 375, 378–382, 629 solvent solid-extraction, 606, 159, 160, 171, sorbent beads, 383 sorbent column, 383, 476, 490 sorbent, 32, 173–177, 179, 181, 186, 205, 210, 230, 247, 251, 254, 259, 262, 271, 276–277, 332–333, 339, 379, 383, 386, 388, 396, 400, 430, 476–477, 483, 490–492, 504, 561, 611, 641, 672 SPE, see also solid phase extraction speciation, 31, 105, 112–113, 124, 142–143, 171, 182, 196–197, 251–252, 377, 389, 397, 401, 455, 491, 547, 685–686, 690–691, 694, 697, 704–705, 736–737 species analysis, 377 spectroelectrochemical FIA cell, 71 spectrophotometry, 31, 33, 39–40, 97, 118, 131, 133, 151, 153–154, 160, 172–174, 177–178, 181–182, 184, 187, 198, 268–270, 311–318, 332, 339, 563, 594, 597–598, 603, 622, 693, 743 spectroscopic interferences, 376, 382 S-pentopril, 603, 608–609 split-flow interface, 565 Sr-90, 475–477, 490–491 S-ramipril, 603, 608–609 stability testing, 592 standard reference materials, 117, 125, 185, 268–269 statistical moments, 64–66, 70, 73 steel samples, 633 stop-flow mode, 99 stopped-flow, 8, 19, 34, 37–38, 42–44, 98, 135–137, 143, 208, 268, 270, 334, 337, 347, 354, 359, 389–390, 396, 402, 427, 432, 607, 639, 652, 654–657, 673 straight open tubes, 47, 49, 53, 57, 59 streptavidin, 261, 479, 502 streptomycin, 531, 597 s-Triazines, 731 stripping analysis, 454–455, 462 stripping techniques, 441, 454 strontium, 490–492 submersible FIA, 698, 740 suction, 88, 95, 187, 300, 656 sugars, 218, 267, 412–413, 497, 519–520, 523, 531, 538 sulfacetamide, 154 sulfadiazine, 154 sulfaguanidine, 154 sulfamerazine, 154 sulfamethizole, 154 sulfamethoxazole, 154, 431, 599, 611

776

Subject Index

sulfate, 146, 172, 259, 293–294, 420, 428, 468, 470, 487–488, 494, 521, 597, 629, 669, 725, 734, 744 sulfide, 131, 154, 224, 364, 473, 501, 634, 667, 676, 709, 725, 727, 736, 744 sulfite, 117, 297, 347, 350, 352, 357, 359, 361–362, 389, 420, 520, 523, 543, 604, 665, 676, 712–713 sulfonamides, 115, 132, 431, 608 sulfon-urea herbicides, 431 sulfur dioxide, 117, 196, 224, 230, 357, 486, 520, 523, 544, 604, 667 supported liquid film, 645 supported liquid membrane extraction, 205, 247–248, 252, 262 surface activity, 478, 493 surface detectors, 60, 71 surface enhanced Raman scattering, 431, 435–436 Surface enhanced resonance Raman scattering, 433, 436 surface plasmon resonance, 340, 483 surface renewal, 383, 562 surfactants, 147, 161, 164–165, 172–174, 240, 347, 350, 382, 420–421, 428, 470, 478, 487, 493–494, 496, 634, 702, 713, 730, 751 synchronous spectrofluorimetry, 133 synephrine, 530 syringe injection, 4 syringe pump, 18, 29, 42, 83, 88–89, 91, 95, 102, 133–134, 150, 152, 166, 185, 190–191, 197, 252–253, 272–273, 280, 283, 300–301, 304, 381, 384, 388–389, 408, 415, 422, 433, 477, 482, 562, 597–598, 652, 694, 746 tangential flow filtration, 101–103, 106, 740 tanks-in-series model, 48, 74–75 tanning, 270, 634 tannins, 488 tartaric acid, 412, 518 m-TAS, 16–18, 89, 124–125, 150, 319, 322, 586, 636, 657, 676, 751–752 Tc-99, 475, 477, 491 technetium, 491–492 Teflons AF2400, 316, 735 temperatures effects, 69 terbutaline, 597, 603–604 tetracaine, 603, 607–608 tetracycline, 30, 115, 174, 180, 531, 537, 542, 597 textile, 634, 700 theobromine, 525, 542

theophylline, 115, 601, 603 thermal or microwave digestion, 105 thermistor sensors, 476 thermodynamics, 18, 376–377, 402 thermometric, 97, 461, 476, 492–493 thermopneumatic, 91 thiabendazole, 527 thiamine, 174, 178, 434, 609, 665, 708 thiochrome, 178, 665, 708 thioglycolate, 347, 350 thiouracil, 368 thiourea, 425, 716–717, 722–723, 742 time at peak maximum, 63, 74–75 time-based injection, 92, 94 Time-of-flight mass spectrometry, 754 time-resolved fluorescence, 577 total acidity, 633 total CO2, 725 total dissolved nitrogen, 690, 754 total dissolved phosphorus, 147, 690, 754 total inorganic carbon, 754 total Kjeldahl nitrogen, 688, 144 total nitrogen, 144–146, 690 total particulate nitrogen, 690 total phenols, 732 total phosphorus, 146–148, 155, 690, 754 total reactive phosphorus, 690 toxicity testing, 592 toxins, 527, 546, 596 trace metals, 143, 186–187, 193–194, 251, 399, 685, 687–690, 695, 701–702, 705, 715, 742 transient effects, 19 transverse capillary cell, 323 travel time, 63 triamcinolone acetonide, 268, 277, 609 tricyclic antidepressants, 601 trifluoperazine, 601, 603 trihalomethanes, 700 triiodide, 726, 747 trimecaine, 278 trimethoprim, 154, 603, 608, 611 trimethylamine, 425–426, 436, 536–537, 548 triplet excited state, 345 tris(2,2u-bipyridyl)ruthenium(III), 355, 360, 363, 365, 609, 748 Triton X-100, 137, 183–184 trypsin, 149–151, 502, 599 turbidimetric methods, 744 turbidimetry, 461, 467–468, 487–488, 521, 528, 539, 603, 725 two-channel fountain cell, 451

Subject Index

Ultra Performance Liquid Chromatography, 270, 284 ultrafiltration, 212, 223, 597–598 ultrasonic pump, 90 ultrasound-assisted continuous liquid–liquid extraction, 244 underwater vehicles, 698 urea, 113, 152, 224, 347, 350, 413, 420, 428, 430, 446, 463, 493, 521, 534, 543–545, 713, 720 urine, 34, 116–117, 119, 132, 139, 141–143, 153, 173, 175, 186–187, 215, 223, 250, 252, 268, 270, 273, 280, 298, 350, 360–362, 420, 430, 463, 496, 501, 608–609 UV photodegradation, 605 UV photo-oxidation, 104–105, 144, 148, 151, 339, 687, 693–695, 708–711, 728, 734, 737–738 UV spectrophotometry, 598, 693 V, see vanadium valproate, 603, 608 vanadate, 475 vanadium, 169, 181, 363, 475, 489 vapour generation, 212, 375, 392, 394, 403, 423, 426, 547 vapour phase FTIR, 423, 426 variance, 64–65, 68, 75, 130, 137 vefuroxime axetil, 601, 602 verapamil, 603, 606 vibrating wire amperometric electrode, 73 vibrational spectrometry, 97, 407–411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435

777

vinegar, 117, 464–465, 467, 486, 538, 540, 548, 633 viscous forces, 50 vitamin, 139, 151, 486, 543 voltammetric flow-through cell, 451 voltammetry, 71, 83, 97, 120, 142, 269, 442–443, 451, 454, 462, 519, 521–523, 525, 528, 531, 537, 603, 633, 636, 693, 731, 747 volumetric injection, 92 wall-jet configuration, 444, 451, 454 waste generation, 124, 416, 422, 435, 618, 623, 697 wastewater, 144–146, 148, 151, 172, 187, 241, 269–270, 455, 640, 703, 735, 740, 751 water content, 630 wetting film extraction, 101, 159, 165, 169, 236 wood pulping, 634 xenon arc lamp, 346 yttrium, 490 zeroth moment, 64, 75 zigzag microchannel, 657 zinc, 113, 131, 137–139, 143, 175, 181, 186, 393, 518, 525, 529, 533, 536, 539, 689, 701–702, 704, 716, 721, 723, 743 ZnS, 131 zone fluidics, 89, 111, 125


Plate 1 Manifold employed for the determination of phosphate by the molybdenum blue method as used by Jarda Ruzicka in Brazil in 1974, where Lego building blocks for the first time were used to arrange and affix the various components. The sample solution was injected into the molybdate stream (entering at top, right), which was subsequently merged with a stream of reducing agent (ascorbic acid, top, left), and then via the reaction coil at the bottom left guided to the detector.

Plate 2 Comparison of a mTAS manifold (at left) and an LOV (at right). While the channel diameters in the mTAS generally are of the order of 10–100 mm, the corresponding dimensions in the LOV system are around 0.5–1.6 mm.

Plate 3 Top (A–E). Principle of sequential injection analysis. (A) Metered volume of sample solution is injected via multiposition valve upstream. (B) Sequential injection of a metered volume of reagent forms a well-defined zone. (C) Injection of carrier solution (or of a second reagent) pushes the stack further upstream. (D) Flow reversal carries reaction mixture downstream and towards detector. (E) Reaction product reaches detector where it is being monitored. Below (a–e). Principle of bead injection. (a) Metered volume of bead suspension is injected into the system and captured within the flow channel. (b) Solution of target analyte is injected and transported towards microcolumn of captured beads. (c) Analyte molecules react with ligands immobilized on bead surfaces. (d) Reaction product is either detected on bead surfaced or eluted to be detected downstream. (e) Beads are discarded. Adapted from Ref. [20] with author’s permission.

Plate 4 Top. Experimental setup for mSI–LOV system. A multiposition valve integrates a flow cell, associated with port #2, flow through port (#5) that allows access to sample to be analysed and reagent ports. A high precision stepper motor driven syringe pump provides bidirectional solution metering and transport within the LOV channels. The holding coil is used to insulate working channels from the pump, in order to prevent sample and reagent solution entering into the barrel of the pump. Typical pump volume is 1 mL, injected volumes are on microlitre scale and working channel diameter 0.8 mm. Bottom. Tools for breaking laminar flow: (A) Tubing (0.8 mm I.D., 1.6 mm O.D. inserted into LOV channel (I.D. 1.6 mm) can be recessed in order to disrupt laminar flow. (B) Flow channel through stator and rotor of a multiposition valve has been designed to have sharp bends and wider (stator) and narrow (rotor) sections. (C) A narrow tube, inserted into LOV channel at a corner efficiently breaks laminar flow pattern due to ‘‘jet and wall’’ effect. (D) Narrow channel section effectively breaks the laminar flow pattern during flow reversals (see Section 6). Adapted from Ref. [20] with author’s permission.

Plate 5 Configurations of a flow cell integrated into an LOV module. (A) Absorbance (1 mm light path). (B) Absorbance (10 mm light path). (C) Absorbance (Garth’s Extended Light Path cell; 100 mm long). (D) Fluorescence. (E) Combined fluorescence and absorbance. The vertical arrow indicates the length of light path (see also Figure 6).

Plate 6 (A) Experimental setup for bead injection spectroscopy and microaffinity chromatography (for details see text). (B) Configuration of a flow cell for capturing and monitoring of beads by UV–VIS spectrophotometry. (C) Configuration of a flow cell for microaffinity chromatography. The beads are retained as a microcolumn by sheath of optical fibre, designed to allow mobile phase to pass freely into the flow cell (light path 10 mm). For details see text and Ref. [20]. Reproduced from Ref. [20] with author’s permission.

Plate 7 Separation and sample pretreatment by peripherals adjacent to LOV module. (The primary syringe pump has been omitted from the graphic design as compared to Figures 3 and 6). (A) External low-pressure chromatographic column as used for affinity chromatography. (B) Sample pretreatment by dialysis using stopped-flow acceptor stream. (C) Sample cleanup achieved by capture of matrix components on external disposable column. (D) Sample preconcentration and cleanup by external column designed to capture target analyte. The captured analyte is eluted from the column by eluent supplied via the LOV module in a stop flow period and subsequently aspirated into the LOV by flow reversal for further processing.

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