Miniaturization of Analytical Systems: Principles, Designs and Applications

Miniaturization of Analytical Systems: Principles, Designs and Applications ANGEL RIOS University of Castilla-La Mancha,...

Author: Angel Rios | Alberto Escarpa | Bartolome Simonet

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Miniaturization of Analytical Systems: Principles, Designs and Applications ANGEL RIOS University of Castilla-La Mancha, Ciudad Real, Spain ALBERTO ESCARPA University of Alcal a, Madrid, Spain BARTOLOME SIMONET University of C ordoba, C ordoba, Spain

Miniaturization of Analytical Systems: Principles, Designs and Applications

Miniaturization of Analytical Systems: Principles, Designs and Applications ANGEL RIOS University of Castilla-La Mancha, Ciudad Real, Spain ALBERTO ESCARPA University of Alcal a, Madrid, Spain BARTOLOME SIMONET University of C ordoba, C ordoba, Spain

This edition first published 2009 Ó 2009 John Wiley & Sons, Ltd Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com. The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. 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, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. The right hand circular image on the cover design is reproduced from The Essential Guide to Analytical Chemistry, Georg Schwedt, John Wiley & Sons, Ltd, 1997. Original German, Georg Thieme Verlag, 1996. Library of Congress Cataloging-in-Publication Data Rios Castro, Angel. Miniaturization of analytical systems : principles, designs and applications / Angel Rios, Alberto Escarpa, Bartolome Simonet. p. cm. Includes bibliographical references and index. ISBN 978-0-470-06110-7 1. Chemistry, Analytic. 2. Miniature electronic equipment. I. Escarpa, Alberto. II. Simonet, Bartolome. III. Title. QD75.3.R56 2009 543–dc22 2009016260 A catalogue record for this book is available from the British Library. ISBN 978-0-470-06110-7 Set in 11/13pt Times by Thomson Digital, Noida, India. Printed and bound in Great Britain by CPI Antony Rowe Ltd, Chippenham, Wiltshire.

Contents Preface

xi

1 Miniaturization in Analytical Chemistry 1.1 Introduction 1.2 Miniaturization as One of the Critical Trends in Modern Analytical Chemistry 1.3 Evolution in the Field of Analytical Miniaturization 1.4 Classification of Miniaturized Analytical Systems and Definition of Terms 1.5 Theory of Miniaturization 1.6 Features of Miniaturized Analytical Systems 1.7 Incidences of Miniaturization in the Analytical Process 1.7.1 Miniaturization of the Steps of the Analytical Process 1.7.2 Integrated (Micro)systems for the Performance of the Entire Analytical Process 1.8 Outlook References

1 1

2 Tools for the Design of Miniaturized Analytical Systems 2.1 Introduction 2.2 Miniaturized Analytical Processes: The Downsizing and Integrating Phenomena 2.2.1 Transport within Microfluidic Systems 2.2.2 Microsystem Design from Transport Parameter Information 2.3 Microfluidic Devices 2.3.1 Microvalves 2.3.2 Moving Liquids in Miniaturized Systems 2.3.3 Mixers 2.3.4 Volume-dispensing and Sample-introduction Devices 2.3.5 Detection Systems for Analytical Microsystems 2.4 Microtechnology 2.4.1 Computer Simulations in Microfluidics 2.4.2 Micromachining 2.4.3 Packaging of Microsystems

2 8 10 15 17 19 20 30 33 36 39 39 40 42 45 45 45 51 59 63 64 66 66 68 74

vi

Contents

2.5 MEMS and NEMS 2.5.1 Fabrication and Characterization 2.5.2 Functionalization 2.5.3 Detection Methods 2.6 Outlook References 3 Automation and Miniaturization of Sample Treatment 3.1 Introduction 3.2 Simplification of Sample Treatment: Microextraction Techniques 3.2.1 Calibration in Microextraction Processes 3.2.2 Solid Phase Microextraction (SPME) Techniques 3.2.3 Liquid Phase Microextraction (LPME) Techniques 3.2.4 Comparison of Solid and Liquid Phase Microextraction Techniques 3.3 Simplification of Sample Treatment: Continuous Flow Systems 3.3.1 Coupling to Gas Chromatography 3.3.2 Coupling to Liquid Chromatography 3.3.3 Coupling to Capillary Electrophoresis (CE) References

74 75 76 81 84 88 93 93 94 94 95 114 117 119 120 123 127 135

4 Miniaturized Systems for Analytical Separations I: Systems Based on a Hydrodynamic Flow 4.1 Introduction 4.2 The Earliest Example of Miniaturization of a Gas Chromatograph and Some Other Developments 4.3 Capillary Liquid Chromatography (CLC) 4.4 Liquid Chromatography on Microchips 4.4.1 The Agilent HPLC Chip 4.4.2 Other Approaches to Microchip HPLC 4.4.3 Some Selected Applications References

141 144 150 150 155 157 163

5 Miniaturized Systems for Analytical Separations II: Systems Based on Electroosmotic Flow (EOF) 5.1 Introduction 5.2 CE on the Microchip Format 5.3 Modes and Theories of CE Microchips 5.4 Microfabrication Techniques 5.4.1 Microfabrication of Glass CE Microchips 5.4.2 Microfabrication of Polymer CE Microchips

165 165 168 170 175 177 178

139 139

Contents

5.5 Basic Fluidic Manipulation/Motivation: Electrokinetic Injection and Separation Protocols 5.6 Electrochromatography in Microchip Format: Designs and Applications 5.7 Comparison of Hydrodynamic and Electroosmotic Flow-driven Miniaturized Systems 5.8 Analytical Applications 5.8.1 DNA Analysis 5.8.2 Protein Analysis 5.8.3 Small-molecule Analysis 5.9 Outlook References

vii

181 184 187 189 190 196 200 206 208

6 Detection in Miniaturized Analytical Systems 6.1 Introduction 6.2 Laser-induced Fluorescence (LIF) Detection 6.2.1 Lamp-based Fluorescence Detection 6.2.2 Fluorescence Excited by Light-emitting Diodes 6.2.3 (Electro)chemiluminescence Detection 6.3 Electrochemical Detection (ED) 6.3.1 End-channel Detection 6.3.2 In-channel Detection 6.3.3 Off-channel Detection 6.3.4 Electrode Materials 6.3.5 Modes of Detection 6.4 Microfluidics–MS Interfacing 6.4.1 Microfluidics-based Electrospray Ionization (ESI) Sources 6.4.2 Microfluidics–MALDI-MS Interfacing 6.5 Unconventional Detection Methods 6.5.1 Absorbance Detection 6.5.2 Surface Plasmon Resonance (SPR) Detection 6.5.3 Thermal Lens Detection 6.5.4 Other Detection Methods 6.6 Outlook References

213 213 214 218

234 243 248 248 249 251 253 254 254

7 Miniaturization of the Entire Analytical Process I: Micro(nano)sensors 7.1 Introduction 7.2 Evolution of Sensors with Nanotechnology

263 263 264

218 219 220 221 224 225 228 230 234

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Contents

7.3 Micro(nano)sensors 7.3.1 Optical Sensors 7.3.2 Electrochemical Sensors 7.3.3 Magnetic Sensors 7.3.4 Mass Sensors 7.4 Nanoprobes for In Vivo Bioanalysis References 8 Miniaturization of the Entire Analytical Process II: Micro Total Analysis Systems (mTAS) 8.1 mTAS, Microfluidics and Lab-on-a-Chip: Concepts and Terminology 8.2 Basic Concepts of Microfluidics: The Design of Analytical Microsystems 8.2.1 Types of Transport 8.2.2 Laminar and Turbulent Flows 8.2.3 The Design of Microfluidic Systems 8.3 The Basics of Downscaling in Microsystems 8.4 Microfluidic Platforms: Types, Principles and Classification 8.4.1 Capillary-driven Test Strips 8.4.2 Microfluidic Large-scale Integration (LSI) 8.4.3 Centrifugal Microfluidics 8.4.4 Electrokinetic Platforms 8.4.5 Droplet-based Microfluidic Platforms 8.5 Microfluidic Devices for Analytical Lab-on-a-Chip Applications 8.5.1 DNA Analysis Integrated on Microfluidic Devices 8.5.2 Real Clinical Sample Analysis on Microfluidic Devices 8.5.3 Real Environmental and Related Sample Analysis on Microfluidic Devices 8.5.4 Real Food Sample Analysis on Microfluidic Devices 8.6 Outlook References 9 Portability of Miniaturized Analytical Systems 9.1 Introduction 9.2 Portable Gas Analysers 9.3 Portable Electrochemical Analysers 9.4 Portable Optical Analysers 9.5 Portable Lab-on-a-Chip Analysers References

266 266 270 272 273 275 278

281 281 283 284 285 286 290 293 294 296 297 299 300 302 305 310 323 328 338 339 345 345 346 351 351 352 354

Contents

ix

10 Analytical Performance of Miniaturized Analytical Systems 10.1 Introduction 10.2 Quality Control in Miniaturized Systems 10.3 Validation of Microsystems 10.4 Qualification of Microsystems 10.5 Robustness of Microsystems Further Reading

357 357 358 360 361 362 363

Index

365

Preface Without question, the main drivers of modern analytical techniques are the simplification of procedures and the improvement of measurement quality. To reach these goals, modern analytical techniques try to reach lower detection limits, improve selectivity and sensitivity, and achieve faster analysis time, higher throughput and less expensive analysis systems with ever-decreasing sample volumes. These very ambitious goals are exacerbated by the need to reduce the overall size of the device and the instrumentation. These items are termed ‘analytical miniaturization concepts’. The miniaturization of analytical systems is a rapidly growing area. It is associated with performing the analytical process on a small scale (sometimes, a very small scale). Different terms have been used to represent this idea: ‘miniaturized analytical systems’, ‘analytical micro(nano)systems’ and, more ambitious, ‘micro total analysis systems’ (mTAS), also called ‘lab-on-a-chip’. Originally, microsystems came from the need to perform online control and monitoring of industrial processes. This mainly resulted in the development of (bio)chemical sensors. However, after this initial phase, society’s needs increased and sensors became insufficient to respond to new specific problems, as they suffer from poor selectivity for many applications. As a consequence, the concept of the total analysis system appeared in analytical chemistry. The idea of such a system is to integrate all the steps of analytical process (sampling, sample treatment, separation of analytes and detection) in the same device. More recently, the interest in portable instruments, allowing field tests to be carried out with ease, has increased the practical usefulness of miniaturized analytical systems. Between sensors and lab-on-a-chip devices, a wide range of microsystems, which can affect either the entire analytical process or only a part of it, have been described. This exciting challenge has guided our effort to offer a book with a general approach to miniaturization in analytical chemistry, including the principles, designs and applications of miniaturized systems. Through ten chapters, the different issues characterizing such systems are critically discussed. The first two chapters include the basic concepts behinds miniaturization in analytical chemistry, as well as the mechanical and electronic tools needed for designing and fabricating these systems. It is very important to give an integrated classification of the systems and to define the different terms associated with miniaturization, in order to provide a systematic view of both the different levels of miniaturization and the main objectives of the downsizing developments.

xii

Preface

Chapters 3 to 6 represent the solid core of the book. Taking as their basis the analytical process, these chapters deal with: the miniaturization of sample treatment (including the consequent automation), with sections devoted to the problems associated with sample introduction in micro(nano)systems; miniaturized systems for analyte separation, divided into two chapters according to the forces involved in moving the flow; and detection in micro-size environments. Through these chapters, practical aspects such as the representativeness of the portion of sample analysed, the analytical potential of micro- and nanochromatography, the advantages of miniaturized capillary electrophoresis with special attention to microchip format, and both well-established and new approaches to detection in miniaturized systems, are comprehensively studied. Chapters 7 and 8 deal with the miniaturization of the entire process: from the sample introduction to the generation of the corresponding analytical results. Thus, when possible, Chapter 7 considers the use of sensors and biosensors in an online approach, or as micro(nano)probes, very useful for in vivo analyses. The objective of this chapter is not to give a wide report on the sensor field (different books address this topic), but to cover the integration of micro(nano)sensors in miniaturized technology. Chapter 8 covers the miniaturization of the entire analytical process under the philosophy of the mTAS approach. From a practical point of view, it is clear that mTAS entails many different challenges, but this trend in analytical chemistry is very attractive and will be the reality of future analytical work (both inside and outside the lab). Microfluidic concepts and lab-on-a-chip systems will make up the content of this chapter, in which a rich discussion of real samples is offered. The last part of the book (Chapters 9 and 10) deals with two aspects directly connected to the usefulness of miniaturized analytical systems: the design of portable miniaturized systems (very interesting for the performance of field tests) and how to assure the practical reliability of micro(nano)systems (quality control tests, performance and validation activities, as well as the robustness of the miniaturized depicted systems). The ruggedness of micro(nano)systems is briefly discussed and related to the tools used for the design and fabrication described in the first chapters of the book. Finally, the authors wish to thank the broad group of researchers who have contributed to analytical miniaturization developments over the past two decades. They have been cited throughout this book, where their works have been selected, studied and strategically related between them in order to give the reader a novel textbook on analytical miniaturization as a whole. The authors hope that the reader enjoys this book and finds it useful in their own teaching and research developments.

1 Miniaturization in Analytical Chemistry 1.1

Introduction

Miniaturization is rapidly growing, with novel ideas emerging in recent years. Like other fields, analytical systems have been affected by this new technology. Concretely, the capacity to carry out laboratory operations on a small scale using miniaturized devices is very appealing. Micro total analysis systems (mTAS), also called lab-on-a-chip, have renewed interest in scaling laws in the last 10–15 years. A small scale reduces the time required to synthesize and analyze a product, as greater control of molecular interactions is achieved at the microscale level. In addition, reagent cost and the amount of chemical waste can be very much reduced. Now, at the beginning of the twenty-first century, it is clear that the lab-on-a-chip approach is starting to be considered as a potential analytical tool in many application fields. Nevertheless, additional efforts must be addressed to two main points: (i) the laws at nanometre scale must be established, as basic physical and chemical fundaments cannot be applied; and (ii) more applications demonstrating the real use of these systems must be developed, particularly in the area of complex samples analysis. There is no doubt that miniaturized chemical analysis systems have a tremendous potential. For instance, it is foreseeable that such devices will allow the study and analysis of complex cellular processes, facilitate the development of new diagnostic abilities that could revolutionize medicine, and have applications in environmental monitoring, food analysis and industry. Some miniaturized analytical systems, such as capillary gas chromatography, microliquid chromatography and microcapillary electrophoresis – which can be Miniaturization of Analytical Systems: Principles, Designs and Applications and Bartolome Simonet 2009 John Wiley & Sons, Ltd

Angel Rios, Alberto Escarpa

2

Miniaturization of Analytical Systems

considered as intermediate levels of miniaturization – have been consolidated in routine laboratories for the analysis of complex samples. There is no doubt that the majority of real analysis requires an appropriate sample treatment step. In this way, important efforts have been made to reduce the sample volume and its feasible manipulation in a miniaturized environment. The development of flow-processing devices for analyte purification/preconcentration, both on-column and on-fibre solid phase microextraction, hollow-fibre liquid phase microextraction, sorptive stir bars and so on are clear examples of miniaturization of the sample treatment step. The direct coupling of these methodologies (using at-, in- or on-line modes) to the instruments even allows an additional reduction of sample volume. At the same time, these coupled approaches reduce human manipulation and increase the degree of integration of the different analytical steps. The extension of this concept results in the modern concept of miniaturization: a miniaturized instrument integrating sample input, pre- and post-column reaction chambers, separation columns and detection units on to a single and small device. In this chapter, an overview of miniaturization in analytical science is presented. In addition to preliminary aspects dealing with miniaturization, other issues such as the classification and definition of terms, the features and main challenges of miniaturized analytical systems, the incidence of miniaturization in the different steps of the analytical process and present trends in miniaturization are covered in this chapter.

1.2 Miniaturization as One of the Critical Trends in Modern Analytical Chemistry Modern analytical chemistry is more and more a scientific discipline connected to the real world. International standards require a certain quality of client service from analytical laboratories, such as the norms ISO 9001 and, more specifically, ISO 17025. Hence, laboratories are encouraged to obtain client feedback in order to identify their real information needs. As Figure 1.1 represents, the developments and trends of modern analytical chemistry must be coherent with this feedback. Trends toward simplification (‘ease of implementation or use’), automation (‘electromechanical self-operation’, involving a feedback loop to control the system without human participation) and miniaturization (‘small scale’; ‘construction to a very small scale’) are well recognized. A common interface between these three characteristics can be identified as an attractive area for interesting developments, bringing about the so-called (re)-evolution of analytical chemistry from the end of the twentieth century onwards. This trend has a strong influence on present analytical chemistry. On the other hand, a high degree of simplification and automation is intrinsically involved in miniaturized systems. The simplification, mechanization/automation and miniaturization of the analytical process must assure the fitness for purpose of the analytical information generated with respect to customer information needs.

Miniaturization in Analytical Chemistry

MODERN ANALYTICAL CHEMISTRY (trends)

3

CLIENT INFORMATION NEEDS

SIMPLIFICATION AUTOMATION

ANALYTICAL PROCESS

FITNESS FOR PURPOSE

MINIATURIZATION

TECHNOLOGICAL DEVELOPMENTS

ANALYTICAL INFORMATION

Figure 1.1 General trends in modern analytical chemistry and their connection with the analytical process and client information needs

Transportantion to the detector

SAMPLE

Calibration

Sub-sampling SAMPLING

Sample treatment and conditioning

DATA ACQUISITION AND PROCESSING

Introduction

DETECTION

REAL WORLD

INFORMATION NEEDS

Analytical microsystems, when used to provide this analytical information in analytical laboratories, are no exception (Chapter 10 deals with issues related to the performance and reliability of the information provided by analytical microsystems). Following the scheme shown in Figure 1.1 and the idea of implementing different degrees of miniaturization of the analytical process, Figure 1.2 shows the number of steps connecting the real world (the client information needs) with the final results generated by the application of the measurement process. In this figure,

RESULTS

ANALYTICAL PROCESS ANALYTICAL VALIDATION REPRESENTATIVENESS

Figure 1.2 Scheme of the analytical process noting the different steps and activities, with special attention to miniaturization aspects

4


the main steps and activities requiring especial attention for miniaturization purposes have been identified. Thus, among these activities must be considered the sampling (and the corresponding subsampling), calibration and validation of the entire analytical process; steps such as introduction of samples, treatment and conditioning of samples, transportation to the detection point, measurement, and data acquisition and processing; as well as final goals such as quality and representativeness of the analytical results. The possibilities and difficulties inherent in the miniaturization of each of these steps and activities are quite different. Even the level of miniaturization is an important issue. By considering the entire analytical process (fortunately, miniaturization implies reduction or elimination of some of the mentioned steps), the main problems for miniaturization affect the preliminary operations (sampling– sample introduction and sample treatment steps). Conversely, detection and signal transduction, as well as data acquisition and processing, are steps which can achieve a high degree of miniaturization. It seems clear that the final objective of miniaturized systems in the analytical domain is represented by mTAS [1]. In fact, ideally a TAS performs all the analytical steps (sample preparation, analyte separation and analyte detection) in an integrated instrument. The philosophy of TAS has been the enhancement of online and automated analysis, as well as of the analytical performance; however, significant drawbacks still exist, which should be the subject of future work in this field (for example, sample introduction and the successive analysis of a set of samples, slow sample transport, and the necessity of fabricating interfaces between the different components). The mTAS concept was developed from the modification of the TAS by downsizing and integrating its analytical multiple steps (sample preparation, separation and detection) on to single monolithic devices [1]. In essence, a mTAS is a device that improves the performance of an analysis by virtue of its reduced size. But not just analytical tasks can be performed in a miniaturized system; other chemical functions such as synthesis can also be performed. For this reason, today the mTAS concept is also known as ‘lab-on-a-chip’. After almost two decades since the concept was introduced, now is a very exciting time for mTAS, due to the large bulk of advances and challenges that appeared in the last revision published in Analytical Chemistry [2]. Some guidelines may be stated concerning the approach of miniaturization as a whole: (i) Miniaturization implies a micro-size environment. It is very important to keep in mind that miniaturization is not only a decreasing of scale, but that other forces and phenomena are present in micro-size environments [3–5]. (ii) Miniaturization requires technology facilities. Thus, to understand what the revolution of miniaturization in analytical chemistry means, it is necessary to consider the synergic marriage (relationship) between some part of the technology and the objectives of modern analytical chemistry. In other words,


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in their fundamental sense, the objectives or demands of analytical chemistry should answer to some questions related to miniaturization, such as why, when and how miniaturization is performed. Obviously, the answer to the last question comes from the existing technology facilities [6]. Figure 1.3 shows the outlines of this complementation. From an analytical point of view, traditionally chemical analyses have been performed in central laboratories since they require skilled personnel and specialized analytical instrumentation. However, the trend today is to move chemical analyses close to the ‘customer’ or the bulk sample (in-situ analysis). Miniaturization plays a prominent role in the decentralization of chemical analysis. As a consequence of this, miniaturized analytical devices should be portable, easy to operate and reliable. The automation of chemical analysis also requires the real implementation of mTAS in analytical laboratories. In fact, analytical microsystems could ultimately be represented as a black box, where the role of the users basically consists in providing the samples and pushing a start button. From a technological point of view, several potential benefits of analytical microsystems can be observed (see Figure 1.3): very well-understood knowledge about microfabrication techniques and the chemistry of materials; the possibility for fabricating sophisticated microcircuits according to analytical demands/objectives; and high compatibility with mass production. Another advantage lies in the possibility of opening new directions in microfluidics, such as the presence of laminar flow. On the other hand, one weak point of analytical microsystems comes

Technologycal tools

Analytical demands

G

D OO

L MP CO

IM O PR M VE TS EN

- Automation - Simplification: No skilled personnel No sophisticated instrumentation - Decentralization of analyses - Samples: Very low volumes High throughput - Reagents: Low consumption

TY RI TA N E EM

- Microelectronic developments - Microfabrication techniques - Microfluidics - Chemistry of materials - Cleanroom facilities

E ED NE D

-Real-world interfaces: Sampling and sample introduction Incomplete miniaturization Conditioning of the microsystem Change of sample between analyses

Figure 1.3 Relationships between present analytical demands and technological developments for miniaturization

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from the present technology; sometimes the complete miniaturization of all the electronics and mechanical parts of the system is not allowed. Moreover, these elements are expensive and the required technology is not always available (for instance, cleanroom facilities). The microtechnology used in the miniaturized environments could also be understood as the integration between the possibilities offered by microfabrication and microfluidics. Both microfabrication (micromachining) and microfluidics are inversely connected with the grade of complexity in the analytical chemistry process. Indeed, while microfabrication plays the prominent role in the separation and detection systems, the physical control of the process has a prominent role in all steps of sample preparation. In addition, the required microfabrication is very sophisticated in the sample preparation step. Chapter 2 deals with the tools for designing miniaturized analytical systems. Two other intrinsic characteristics of analytical microsystems have clear connections with technology developments: the extremely low sample volumes used, and the presence of laminar flows. Small volumes of both sample and reagent (pl–nl levels) are representative of most miniaturized systems. This characteristic has clear advantages associated with cost and analytical throughput, but it also presents disadvantages, such as the suitability of detection techniques. Consequently, much research effort has been focused on the development of miniaturized and sensitive detection units [7], and today the detection improvements are still one of the most important research focuses [8]. As a consequence of the low volumes required, a very precise handling of sample is crucial in microanalytical systems, and a high dependence of the surface properties of microchannel manifolds and interconnections and dead volumes is observed [3]. Complex fluid manipulation at femtolitre and nanolitre scales is readily achieved without any mechanical valves or external pumps by using the electrokinetic phenomena [9]. In this way, the focus has been centred on the integration of functional components within monolithic systems using both lithography and micromoulding technologies. Micropumps are other typical devices used to propel fluids in microchannels. A recent review has been published by P. Woias [10]. A more sophisticated approach has been proposed by M.S. Anderson [11], who presented a combined atomic force microscope (AFM) and Raman spectrometer as a microfluidic device for sampling and trace chemical analysis. On the other hand, the miniaturization of valves for microfluidics [12], or to set up a miniaturized lab-on-valve system [13], constitutes an additional tool for fluid manipulation in microchips. The main examples of integrated processing within microfabricated devices have been directed at linking analytical principles (i.e. detection/CE separation, reactors, designs of microcircuits). As previously mentioned, miniaturization is more than simply the scaling down of well-known systems. The relative importance of forces and processes changes with the scale. Thus, as a consequence of their miniaturized scale, another feature of the analytical


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microsystems is the presence of laminar flow (Reynolds number is typically very low), where viscous forces dominate over inertia. This means that turbulence is often unattainable and the transport of molecules is only produced by diffusion, which has direct consequences on the design of this type of microsystem. This constitutes one of the most attractive features, since in most analytical microsystems the diffusion process is very fast, as diffusion effects are inversely proportional to the square of the length. This consequence will be especially relevant during the sample preparation step. However, the ability to efficiently process raw samples (as in classical laboratory tests, directly from the body of a person or an animal, or in field tests), and subsequently to perform the required analytical operations on-chip, will be a key aspect in both the definition of the eventual success and the application of microfluidic systems [14,15]. Whereas the previous points are specific for mTAS approaches, other general requirements can be stated, from an analytical point of view, for a successful miniaturized approach: (i) The developed analytical microsystems must be close to real-world demands (the objective is to solve analytical problems). Just one of the main challenges of analytical microsystems is dealing with the real-world interface. That means understanding the information needs of the client (identification of the problem and its translation to an analytical level), which is a general challenge of any analytical work. But, more important for microsystems, it also means ensuring the representativeness of the results through an appropriate sampling plan and a suitable method of analysis at the particular miniaturized level. This includes achieving the selective and sensitivity requirements through sample treatment and separation/detection. In addition, analytical microsystems offer a significant decrease in costs, by dramatically reducing the volume of samples and reagents needed to perform a chemical analysis. This feature also opens up the possibility of processing samples in parallel, which is very useful when the same chemical analysis must be performed many times, as is the case in routine laboratories. This approach is very useful when high-throughput screening is needed. In conventional analyses, handling and processing of the sample is frequently done manually, at least in part, and often in specialized laboratories. However, mTAS allows chemical analyses to be brought close to the place where they need to be performed, independent of both the laboratory and the laboratory personnel. Thus, these integrated analytical systems are very suitable for online measurements. (ii) The analytical microsystems must perform reliably, in order to be coherent with the present quality assurance requirements in analytical laboratories. In this respect, issues related to the calibration and validation of the methodologies carried out by the analytical microsystems must be taken into account. As the

8


final objective will be the analysis of real samples, the validation of such a method has to be performed using the samples for which the method is intended.

1.3 Evolution in the Field of Analytical Miniaturization A. Manz et al. established different periods in the evolution of analytical microsystems in a publication in 2002 [16]. Based on this source, and completed with recent developments, Figure 1.4 shows the schematic history of miniaturization in the analytical field. The most representative milestones are briefly described below. The first period (1975–1989) was characterized by works addressed to the miniaturization of components, such as micropumps, microvalves and chemical sensors, based on silicon technology. The integration of such silicon microcomponents is remarkable for the fabrication of two miniaturized instruments. The first was a miniaturized gas chromatographic analyser [17]. The basic chromatographic device included, in a single silicon wafer, an injection valve and a separation column

S m CONVENTIONAL ANALYTICAL SYSTEMS

Analytical systems partially miniaturized

mm

I

Z

E µm

nm

MICRODEVICES MINIATURIZED DIVICES

1970

Microchips

NANODEVICES

Nanochips

‘The Early Days’ (1975-1989)

Y E A R S

1980

Miniaturized gas chromatographic analyser Fabrication of micropumps and microvalves Chemical sensors Miniaturized coulometric acid-base titration system

1990 ‘The Renaissance’ (1990-1993)

Miniaturized open-tubular liquid chromatograph Introduction of µTAS concept Miniaturization of FIA systems Electroosmotic pumping Electrophoresis in planar chips First applications related to handling biomolecules and cells Microchips for flow cytometry

‘Growing to Critical Mass’ (1994-1997)

2000

Consolidation as a potential analytical alternative for solving real problems Nanotechnologycal approaches

Chip-based analyses Different miniaturized commercial products On-chip liquid chromatography with EC detector Surface chemical modification of the microchannels Miniaturized mass spectrometer Advances in microfabrication, design, separation, biochemical reactors and detection

Molecular or molecular-sized devices Analytical nanotechnology

Figure 1.4 Evolution of miniaturized analytical systems and main milestones (adapted from [16] and completed for years since its publication)


9

1.5 m long. A thermal conductivity detector was fabricated on a separate silicon wafer and mechanically clamped on the wafer containing the column. The second type of miniaturized instrument was a coulometric acid–base titration system, employing a solid-state pH-sensitive sensor to determine the acid or base concentration in the sample [18]. The second period (1990–1993) saw the fabrication of silicon-based analysers in 1990, thanks to new developments producing a miniaturized open-tubular liquid chromatograph fabricated on a silicon wafer [19]. This work presented a 5 · 5 mm silicon chip containing an open-tubular column and a conductometric detector, connected to an off-chip conventional LC pump and valves in order to perform highpressure liquid chromatography. It was also in 1990 that Manz et al. proposed the concept of mTAS [20], in which silicon chip analysers incorporating sample pretreatment, separation and detection played a key role. According to this author, the main reason for this miniaturized approach was to enhance the analytical performance of the existing sensors, due to the poor results in terms of selectivity and lifetime showing at this time. Therefore, the main objective of mTAS, initially, was not the reduction of size, although the advantages of miniaturization were recognized. The miniaturization of a flow-injection analysis (FIA) system, based on stacked modular devices in silicon and plexiglass, was also reported in this period [21,22]. This key device (less than 1 cm3) conformed to a 3D structure in which more than 10 chips were integrated. Despite the new developments in micropump systems and microvalves for microflow arrangements, the high pressures necessary for transporting in microchannels complicated their practical use. In this way, electro-osmotic pumping was an attractive and feasible tool for the movement of aqueous media in mTAS, particularly when separation was needed. In fact, electro-osmotic pumps are characterized by the absence of mechanicallymoving parts and the lack of a specific location of the pump in the manifold. Moreover, the flow in the interconnected channels can be controlled by switching voltages, without the need for valves. These new strategies were critical to the development of electrophoresis in planar chips, used for the first time in 1992 [23,24]. It was also in this period that applications related to the reaction and handling of biomolecules and cells started; for instance, the use of microfabricated chambers to carry out DNA amplification (PCR) or flow citometry. An important increase in the number of publications related to mTAS took place in the third period (1994–1997). A great variety of chip-based analyses were reported, and different miniaturized commercial products were offered by important instrumentation companies. The modular concept of mTAS was revisited, involving both electrochemical and optical detection, and at the same time, surface chemical modification of the microchannels opened interesting possibilities for miniaturized chromatography. Thus, Cowen and Craston developed an on-chip liquid chromatographer with an electrochemical detector [25]. Another interesting development was carried out by Feustel et al. [26], consisting of a miniaturized mass spectrometer

10


incorporating an integrated plasma chamber for electron generation, an ionization chamber and an array of electrodes acting as the mass separator. Additionally, in this period, significant contributions to microfabrication (covering a broader range of applications and using new materials), separation modes, detection devices and new applications (focussed on biological species, mainly) were made [16]. The last 10 years, since 1998, can be considered a period of consolidation of miniaturized developments, which can be seen as potential analytical alternatives for solving real problems. The wide number and variety of publications is decisive proof of this, although in many cases the developments remain in the research world, with limited implementation in control or routine laboratories. As microfabrication technologies, and particularly the design and production of microfluidic systems, constitute one of the milestones of analytical miniaturization, the important developments in flow-control devices (micropumps and microvalves), interconnections and interfaces, together with bonding techniques and surface modifications, have played a key role in the present analytical miniaturization scene. This is the subject matter of part of Chapter 2 of this book. The application fields of analytical miniaturized devices have been clearly expanded, with a particular impact in the bioanalytical area. Thus, many uses deal with protein and peptide analyses, DNA separation, PCR and performance of inmunoassays or clinical diagnosis. A rise in publications related to cellular applications can also be observed. Selected examples have been reported throughout the various chapters of this book.

1.4 Classification of Miniaturized Analytical Systems and Definition of Terms Miniaturized systems can be classified according to different criteria. Figure 1.5 presents a classification distinguishing between general criteria and specific criteria applied to analytical work. Thus, from a general point of view, miniaturization is clearly associated with the reduction of size, although the term can be somewhat confusing, and it does not always involve a ‘small scale’. It is a relative term, depending on what is understood by the ‘normal scale’. This conflict can be resolved by giving to the prefix ‘mini-’ a broad meaning, representing devices, instruments and systems at dimensions greater than 1 mm (even at the cm range). This is the starting point in Figure 1.6, where the basic classification of the miniaturized systems according to the intrinsic size is represented at two fields: the general and the specific analytical field. From a general point of view, the three basic levels of miniaturization are established by using the prefix mini- (higher than 1 mm, or at the cm scale), micro- (lower than 1 mm) and nano- (lower than 1 mm). Due to the frequent use of these systems with liquid (aqueous) media, the corresponding volumes associated with these levels are included in Figure 1.6, as well as the type of technology involved in the fabrication of the corresponding

Miniaturization in Analytical Chemistry ‘NORMAL’(MACRO)SYSTEMS

11

GENERAL STUDIES

MINISYSTEMS SYNTHESIS MICROSYSTEMS

SIZE

OBJECTIVE ANALYSIS

NANOSYSTEMS

GENERAL CRITERIA FOR CLASSIFYING

MINIATURIZED SYSTEMS ANALYTICAL CRITERIA FOR CLASSIFYING

ANALYTICAL PURPOSE

COMPLEXITY OF SAMPLE AND INFORMATION NEDEED

SAMPLE HANDLING AND TREATMENT DIRECT MEASUREMENT OF ONE COMPONENT

(BIO)CHEMICAL REACTIONS SEPARATION OF ANALYTES

MEASUREMENT OF FEW COMPONENTS WITH SOME SAMPLE TREATMENT

DETECTION COMPLEX SAMPLES REQUIRING THE SEPARATION OF THE SAMPLE COMPONENTS

Figure 1.5 Different criteria used for classifying miniaturized systems

systems. Thus, conventional technology has commonly produced the wide variety of minisystems existing so far, whereas micro- and nanotechnology were used to produce micro- and nanosystems, respectively. Microsystems are microstructured devices, integrated as structures in the micrometric range, produced by using microfabrication techniques; the term ‘nanosystem’ refers to a system that has its main structures in the 1–100 nm range. MEMS (microelectromechanical systems) and NEMS (nanoelectromechanical systems) are representative devices often used in micro- and nanoscale works, as well as micro- and nano-fluidic structures. Analytical miniaturization could be viewed as ‘the fact of making to a small scale a part or the whole of the analytical process (see mTAS), or of reducing the size of the different devices involved in the analytical process (see micropumps, microsensors) or the analytical technique itself (see microgravimetry)’. Consequently, when the general-sized concepts are applied to analytical science, the corresponding three levels of miniaturization can be called: (i) Analytical minisystems or minitechniques, involving some specific devices such as minireactors or minicolumns; or the application of minitechniques, for which commonly the prefix mini- has been replaced by micro- (although not appropriately, according to the general classification). This is the case for

12


MINIATURIZATION

smaller scale

Mini-

Micro-

Nano-

>> 1 mm (µL)

< 1 mm (nL)

< 1 µm (pL, fL, aL)

CUASI-CONVENTIONAL FABRICATION TECHNIQUES MINISYSTEMS

MICROTECHNOLOGY

NANOTECHNOLOGY

MICROSYSTEMS

NANOSYSTEMS

MEMS Fluidic microstructures

NEMS Nanofluidics

MINIATURIZATION IN ANALYTICAL SCIENCE ANALYTICAL MINISYSTEMS or MINITECHNIQUES Minireactors Minicolumns (Micro)gravimetry (Micro)titration

Micro-GC Micro/nano HPLC

ANALYTICAL MICROSYSTEMS Microreactors Micropumps Microvalves Capillary columns Microsensors Microactuators Array microsystems µFIA µCE µTAS

ANALYTICAL NANOSYSTEMS Nanoparticles Nanotubes Nanofibers Quantum dots Etc.

(longer scale)

Figure 1.6 Classification of miniaturized systems based on size criteria

microgravimetry and microtitration. Other, longer-scale equipment (chromatographic instruments, mainly) has been named micro-GC or micro-/nanoHPLC, not due to the size of the ‘miniaturized’ equipment, but for the use of minidevices (such as pumps, valves or capillary columns) for handling microor nanovolumes, and for introducing separation advantages against normalsized equipment. From this point of view, the commercially-available capillary electrophoresis (CE) equipment (fabricated at high scale size) could be associated with micro-/nano-CE. Just in this case, the term ‘micro-CE’ is correctly used when the electrophoretic separation is performed in microchips. This fact is not so frequently used for GC and LC in microchips. (ii) Analytical microsystems are microstructured devices, integrated as analytical structures in the micrometric range, produced by using microfabrication techniques. They include microdevices (microreactors, micropumps, microvalves, capillary columns, microsensors and microactuators, array microsystems), microtechniques (mFIA, lab-on-a-chip valve, mCE), and the complete analytical process (mTAS). (iii) Analytical nanosystems are, in fact, systems at nanometer size, built with atomic precision by using nanotechnology facilities. Nanotechnology is a


13

multidisciplinary field of applied science and technology working at approximately 1–100 nm and dealing with the fabrication of devices at this size. Useful materials, devices and systems at this size range may be obtained by two different approaches. In the ‘bottom-up’ approach, materials and devices are built from molecular components which assemble themselves chemically by principles of molecular recognition. In the ‘top-down’ approach, nanoobjects are constructed from larger entities without atomic-level control. Examples of analytical nanosystems are nanoelectromechanical systems, nanoelectrodes, etc. In addition to the proper size classification of the miniaturized systems, from a chemical point of view, miniaturization can be addressed to three main objectives (Figure 1.5): (i) synthesis of compounds; (ii) analysis of samples; and (iii) biochemical studies of cells, microorganisms, etc. In this context, the article by D. Belder presenting the integration of chemical synthesis and analysis on a chip as a potential trend is interesting [27]. This author recognizes that while complex integrated lab-on-a-chip systems have been described for many biochemical applications, similar achievements remain to be made in synthetic chemistry. The main reason for this, besides the often more challenging reaction conditions for synthetic chemistry, is the lack of appropriate detection techniques. Thus, fluorescence detection, which is commonly applied for on-chip detection in bioanalytics, is often not applicable to classical chemical reactions and corresponding products of interest. A promising approach to carrying out chemical reactions and analysis is the use of micro-CE. An example of an integrated reaction/separation device applied to synthetic chemistry and catalyst development has been presented by D. Belder et al. [28]. Figure 1.7 shows a scheme of the microfluidic chip, combining a microfluidic reactor with microchip electrophoresis. The chip layout is basically a merged design of the common cross-channel design for on-chip electrophoresis and a typical meandering channel for mixing and reaction. On the other hand, miniaturization technology provides facilities for creating tools with feature sizes matching the dimensions of cells, and enables integration of cell-handling and fluidmanipulation elements [29]. Therefore, microsystems create new opportunities for the spatial and temporal control of cell growth [30]. The additional integration with bioanalytical platforms creates multifunctional microdevices showing great promise for basic biomedical and pharmaceutical research, as well as robust and portable point-of-care devices. The analytical purpose of the miniaturization can be focussed on sample handling and treatment, the development of (bio)chemical reactions in miniaturized environments, the separation process or the detection. The different incidences of miniaturization in the analytical process steps are discussed in Section 1.7. Microarrays are probably the most representative example of microanalytical systems based on selective and specific (bio)chemical reactions. Their use normally involves

14


Figure 1.7 Integrated synthesis/analysis chip (channel width 50 mm) showing the different parts used for introduction of educts through microvials (1), merging zone (2), mixing and reaction microcoil (3), injection cross for the introduction of the reaction products to the microelectrophoretic system (4), and microchannel for the electrophoretic separation of the products (fluorimetric detection). (Reprinted from [28] with permission from Wiley VCH)

biochemical reagents such as antibodies or genetic material, or specific chemical reagents such as molecular imprinted polymers (MIPs). The second group is constituted by those systems involving (electro)chromatographic separations. Portable GC equipment is a typical example, which can be used to detect volatile compounds in foods. In the case of liquid chromatography, miniaturization results in a high pressure in the system, which makes its use difficult. For this reason, the major tendency is to apply electrochromatographic separations, and hence electrophoretic chips are probably the most developed and studied miniaturized system. The complexity of samples and the intended information required are two factors taken into account by J.P. Kutter and O. Geschkes [31] for distinguishing between: (i) the direct measurement of one or a few components with no or little sample preparation; (ii) measurement of one or a few components requiring some treatment of sample; and (iii) more complex samples (separation of the components). Undoubtedly, these categories are based on sample complexity and selectivity criteria. Accordingly, different analytical microsystems can be associated with them, for example (bio)sensors (probe-type sensors, flow-through sensors and microsensors in a progressive scale of miniaturization) for the first group. FIA and mFIA are the most appropriate (micro)systems for the second group, whereas m-LC and m-CE are ideal for performing microseparations. There is no doubt that information from objects and systems is a relevant part of the requirement for making well-founded and timely decisions [32]. As a response to this high need of information, analytical sciences have developed rapid, low-cost screening methods


15

that allow the classification of samples as positive or negative [33,34]. Most of the screening methods provide a total index of global response, which permits the classification of samples from the legal, toxicological or quality point of view.

1.5

Theory of Miniaturization

Theoretical considerations can first be established in terms of similarity and proportionality. The similarity approach uses dimensionless parameters to consider similarity, whereas the proportionality approach uses the characteristic length of known systems versus scaled-down systems to consider proportionality [16]. The former can be used to correlate, in an easy way, experimental results when a great deal of variables are involved, and they are defined in terms of parameters that are assumed to be constant through the whole system. The proportionality approach provides very useful information related to the behaviour of a simple flow system when miniaturized. It assumes the miniaturization as a downscaling process in three dimensions. In fact, as the key factor of miniaturization is the size of the devices, the typical device length, d, can be used as the basis of fundamental changes in the characteristics of miniaturized devices [35]. Thus, Table 1.1 summarizes the basic characteristics of devices at three different sizes: 1 mm, 100 mm and 10 mm. The corresponding volumes associated with these typical lengths clearly decrease, with values of 1 mL, 1 nL, and 1 fL, respectively; as do the number of molecules for a particular concentration of the flowing solution. More important are the three final parameters in Table 1.1. Thus, for instance, as the number of units that can be arranged on a given surface increases with 1/d2, for a typical length of 10 mm, 2.5 · 105 devices/cm2 (250 000 units) can theoretically be integrated per cm2. The smaller dimensions have a further impact on analytical standard operations such as mixing, separation and detection, because turbulent flow mixing is avoided. Thus, on the microscale, viscous forces dominate over inertial forces, leading to a laminar flow regime. Under this condition, two liquids can co-flow without turbulent

Table 1.1 Device characteristics for different typical-length d values (extracted from [35]) Typical length:

1 mm

100 mm

10 mm

Volume Number of moleculesa Diffusion time Unit density (devices/cm2) Information density (values/min/cm2)

106 l 6 · 1011 15 min 25 1.5

109 l 6 · 108 10 s 2500 250

1012 l 6 · 105 100 ms 2.5 · 105 2.5 · 106

a

For a concentration 1 mM.

16


mixing. Reynolds number characterizes the type of flow, and can be calculated by the following equation: rrh v Re ¼ ; ð1:1Þ h where r is the fluid density, rh is the hydraulic diameter of the capillary, u is the average fluid velocity and Z is the dynamic viscosity. The lower Re, the closer the behaviour of the flow to a laminar flow. In general, systems are considered to be in the laminar flow regime for Re < 2000. This value is even lower than 1 for microfluidic devices. Therefore, diffusion is an important parameter for these microsystems. Thus, as the time that a molecule needs to travel a length d decreases with d2, for a small molecule (diffusion coefficient 109 m2/s), this results in diffusion times decreasing with the typical length of the device (Table 1.1). For instance, diffusion time is only 100 ms for devices of a typical length of 10 mm. As the diffusion times can be related to the capacity for exchanging molecular information according to the expression: Information ¼

1 ; tdiffusion

ð1:2Þ

when the number of volumes is taken into account, an information density parameter can be calculated as a function of the time and surface area, given the values shown in Table 1.1. These values theoretically increase with the fourth power of d. This is a critical aspect, because it is the basis of the high-throughput capability which characterizes miniaturized analytical systems. The influence of relevant parameters on the behaviour of a miniaturized system can be evaluated from proportionality considerations. For this purpose, the parameter of interest must be defined as a function of space (d) and time (t), which are the key variables of the miniaturization. This allows knowledge of the characteristics of a parameter after downscaling, without having knowledge of other constants associated with the material. Under this model, miniaturization is viewed as a simple 3D downscaling process based on d factor. Depending on whether or not the other key variable, time, is constant, two different situations can be distinguished: (i) Time-constant system, for which diffusion is of lesser importance. In this case, the timescale is the same for the miniaturized system as for the large system. Variables characterizing the time parameters, such as transport time, response time and analysis time, remain the same. This is the situation characterizing simple transportation systems and FIA, where an analyte is injected into a flowing carrier stream for subsequent analysis. According to A. Manz et al. [36], when a time-constant system is scaled down, the linear flow rate decreases by a factor d, volume flow rate by d3 and voltage by d2 (Table 1.2). Hence, the main advantage is the reduced consumption of sample, carrier and reagent solutions.


17

Table 1.2 Scaling factors for miniaturized analytical systems, where d represents a typical length in the system (extracted from [36]) Parameter

Time-constant system

Diffusion-controlled system

Space Time Linear velocity Volume flow rate Pressure dropa Voltageb Plate number

d constant d d3 constant d2 —

d d2 1/d d 1/d 2 constant contant

a

Laminar flow conditions; b For electro-osmotic pumping.

(ii) Diffusion-controlled system, in which the time is regarded as a surface and proportional to d2. Parameters such as molecular diffusion, heat diffusion and flow characteristics are important because they control the separation efficiency. Therefore, in these cases, timescale needs to be considered. As the diffusion time on the travel length is proportional to d2, a 10-fold downscaling produces a 100-fold reduction of related time parameters (for instance, the analysis time). On the other hand, as Table 1.2 shows, the pressure drop scale is proportional to 1/d2, meaning that for a 10-fold downscaled system, a 100 times higher pressure drop is required to generate the same flow. If an electrical potential is applied to produce an electro-osmotic flow (EOF), the generated flow remains the same when the electric field is kept constant. The separation efficiency (expressed through the plate numbers) is also unaffected by miniaturization, but is directly proportional to the applied voltage. Obviously, the applied voltage is limited by the microsystem heating (Joule effect), although for miniaturized systems, lower currents are used, and the higher surface-tovolume ratio allows a good heat dissipation. Based on this fact, ultrafast, highefficiency separations can be achieved in miniaturized diffusion-controlled systems.

1.6

Features of Miniaturized Analytical Systems

The previous section pointed out some of the features and advantages of miniaturized systems. Now, Figure 1.8 summarizes some of the main characteristics of analytical microsystems, establishing their relationship with the quality of the generated information. Automation (human participation reduction) and simplification are two features commonly associated with miniaturization. The other characteristics listed in Figure 1.8 show clear influences on the speed of result generation (high throughput), the amount of information (simultaneous or multiparametric)

18

Miniaturization of Analytical Systems CHARACTERISTICS OF ANALYTICAL MICROSYSTEMS

LOW SAMPLE VOLUME

HIGH THROUGHPUT

RAPID ANALYSIS HIGH EFFICIENCY ROBUST

SIMULTANEOUS DETERMINATIONS OF ANALYTES

LOW COST PORTABLE

MORE REPRESENTATIVE RESULTS

HIGH LEVEL OF INFORMATION

FIELD TESTS

LOW HUMAN PARTICIPATION

QUALITY OF RESULTS Figure 1.8 Main characteristics of microsystems and their effects on the quality of the analytical results provided

and the autonomy given for microsystems allowing field tests (portability). These characteristics represent very advantageous aspects for the corresponding analytical methods using miniaturized systems. The development of such microsystems requires the miniaturization of the electronic and mechanical parts of the system. The microtechnology used in the miniaturized environments could also be understood as integrating the possibilities offered by microfabrication and microfluidics. The term ‘microfluidic’ is used to denote any process that involves the use of small amounts of fluids, such as 109–1018 litres, in channels with a small diameter (tens to hundreds of micrometres). Hence, mTAS are examples of microfluidic systems. From an analytical point of view, these microsystems offer a number of useful capabilities: (i) the ability to use small quantities of samples and reagents; (ii) the possibility of carrying out separations with a high resolution; (iii) the use of low-cost setups; and (iv) the short time taken to perform complete analyses. In addition, microfluidics also exploits characteristics of fluids in microchannels, such as laminar flow. This allows new capabilities in the control of the molecules in space and time. One of the most important characteristics of microfluids is the dramatic difference between the physical properties of fluids moving in large channels and those travelling through micrometer channels, mainly the turbulence or its absence (laminar flow). On large scales, fluids mix convectively, as inertia is often more important than viscosity. However, in microsystems, the fluids do not mix convectively and, consequently, two fluidic streams that merge in a microchannel will


19

flow in parallel, without any turbulence. Therefore, the diffusion of molecules across the interface is the unique phenomenon responsible for mixing. When effective mixing is required, it is necessary to introduce specific devices to accomplish this. Another important characteristic is the presence of electro-osmotic flow, which, as in CE, is a consequence of the ionization or surface charge of the microchannels. Therefore, when an electrical potential is applied across the microchannel, the fluid moves as a plug, with a characteristic planar flow profile.

1.7

Incidences of Miniaturization in the Analytical Process

(Bio)chemical analysis is performed through the so-called analytical process, which integrates a group of steps and substeps connecting the sample with the corresponding results. Miniaturization can affect a single step/substep, various integrated steps/substeps or the entire process. Figure 1.9 illustrates these possibilities, taking the analytical process as the central part, and considering it the ‘analytical black box’, which can be the basic subject of integrated analytical (micro)systems; or the specific sequence of the three main steps: preliminary operations, signal measurement and transduction, and data acquisition and processing. Hence, the upper part of the figure represents a (micro)system performing the whole process, whereas the lower part includes the different analytical standard operations involved in the process. This view allows a description of the incidence of miniaturization in the analytical process as: (i) the partial miniaturization of step(s), devices or equipment;

INTEGRATED MICROSYSTEMS

MINIATURIZATION (simplification)

µTAS

AUTOMATION SIMPLIFICATION INTEGRATED SYSTEMS

TAS

IN VIVO MEASUREMENT DEVICES

PORTABLE EQUIPMENT

S A M P L I N G

SAMPLE

SUBS A M P L I N G

ANALYTICAL ‘BLACK BOX ’

ANALYTICAL PROCESS PRELIMINARY OPERATIONS

SIGNAL MEASUREMENT AND TRANSDUCTION

RESULTS DATA ACQUISITION AND PROCESSING

Sample introduction Sample preparation and conditioning Chemical reaction

Chromatographic & Electrophoretic Separations

DETECTION

Microprocessor (software facilities)

ANALYTICAL STANDARD OPERATIONS

Figure 1.9 Incidence of miniaturization in the whole analytical process or individually in the different analytical standard operations involved in the process

20


or (ii) the miniaturization of the integrated systems performing the entire analytical process, in which miniaturization should be viewed in combination with automation and simplification of the process. 1.7.1

Miniaturization of the Steps of the Analytical Process

The main approaches used for the miniaturization of the steps making up the analytical process are given below, pointing out the present challenges and evaluating the main strengths/advantages and weaknesses/disadvantages existing in each. Taking into account the natural development of the miniaturization devices in analytical works, the logical sequential steps of the analytical process have been inverted, in order to present them in accordance with the increasing difficulty of their miniaturization. Detection Techniques Detection has been one of the main challenges for analytical microsystems, since very sensitive techniques are needed as a consequence of the ultra-small sample volumes used in micron-sized environments. In principle, a wide variety of detection alternatives can be used in microfluidic systems [37]. Laser-induced fluorescence (LIF) was the original detection technique and is the most used detection scheme in CE microchips, due to its inherent sensitivity [38,39], given by the ease of its focusing. This characteristic, together with the fact that molecules of biochemical relevance are fluorescent in many cases, is an important reason for the wide use of LIF in microfluidics. Today, LIF has attained a position as a standard detection technique for microchip separation [39]. However, the high cost and the large size of the instrumental setup of LIF are sometimes incompatible with the concept of mTAS. Also, tedious derivatization schemes are needed to use LIF with nonfluorescent compounds. The most important alternative to LIF detection is, without any doubt, electrochemical detection (ED). ED is very important because of its inherent miniaturization without loss of performance and its high compatibility with microfabrication techniques. Likewise, it has a high sensitivity, its responses are not dependent on the optical path length or sample turbidity, and it has few power-supply requirements, which are all additional advantages. As proof of the prominent role of ED, see the recent publication of excellent reviews [40–43] and others papers [44–48]. The main challenge in CE–ED coupling has been the conflict between the high voltages used in the electrophoretic separation and the potential used for the detection. However, at microscale this drawback is over. Three strategies have been employed: end-channel, in-channel and off-channel electrochemical detections. In end-channel detection the electrode is placed just outside of the separation channel, which involves the alignment of the electrode. Separation voltage has a


21

minimal influence on the applied potential in the electrochemical detector because most of the voltage is dropped across the channel. For in-channel detection, the electrode is placed directly in the separation channel using an isolated potentiostat. Off-channel detection involves grounding the separation voltage before it reaches to the detector by means of a decoupler. Electrode placement in off-channel detection is similar to that in in-channel detection, but the separation voltage is isolated from the amperometric current through the use of a decoupler. Conceptually speaking, the decoupler effectively shunts the separation voltage to ground and a field-free region is created where analytes are pushed past the electrode by the EOF generated before the decoupler. Since no decoupler is necessary, end-channel configurations offer the following advantages: they are simple and rugged, electrode replacement is feasible, and microfabrication facilities are not strictly required. However, the main drawbacks are the alignment of the electrode with the outlet of the channel, and the loss of separation efficiency due to the distance between the end of the channel and the working electrode. This separation distance is also crucial for the signal-to-noise ratio obtained and can lead to a complete loss of the analytical current. In both in- and off-channel configurations, the analytes migrate over the electrode while they are still confined to the channel, thus eliminating the band broadening often observed with end-channel alignments. However, in these configurations, microfabrication facilities are usually needed and in addition the nature of on-chip miniaturized electrodes limits the ability to modify and periodically clean the electrode surface. Conductimetric detection on microfabricated devices has recently been developed. It constitutes an important possibility for detection in analytical microsystems. In comparison to amperometry, conductimetric detection is less sensible, but it is a universal detection technique and has been applied as a detection mode in CE microchips, both in the galvanic (a pair of electrodes is placed in the separation channel for liquid impedance measurement) [49,50] and in the contactless (no contact between the pair of electrodes and separation channel solution) [51–54] mode. Contactless conductimetric detection is preferred for three main reasons: (i) the electronic circuit is decoupled from the high voltage applied for separation (no direct DC coupling between the electronics and the liquid in the channel); (ii) the formation of glass bubbles at the metal electrodes is avoided; and (iii) electrochemical modification or degradation of the electrode surface is prevented. Both contacts and contacless detectors integrated into a microchannel require physical connection to read out electronics placed inside or even outside the microdevice. Conductimetric designs are often very sophisticated and microfabrication facilities are required; however, simple and easy alternatives have also been proposed. These alternatives involve the deposition of conducting electrodes on the cover plate of microchips only, avoiding the clean-room laboratory infrastructure and showing significant advantages over the other approaches due to their simplicity [51].

22


As already mentioned, the inherent strength of ED versus other detection modes is its compatibility with miniaturization (without loss of performance), plus advantages in microtecnology requirements (microfabrication facilities). It also constitutes, itself, an important improvement on LIF implementation, in which only one setup is possible. The possibilities in the implementation of ED can be understood as follows (see Figure 1.10): (i) analytical configurations showing complete integration of the electrochemical cell in the separation system (hard lithography); (ii) a partial integration, in which the separation system is microfabricated in one layer using soft lithography and the electrochemical detection is first deposited in another one; and (iii) the partial integration of the electrochemical cell (reference and auxiliary electrodes) with the separation system, allowing a replacement of the working electrode. In addition to LIF and ED, there are other detection approaches for analytical microsystems, but, from a realistic point of view, they are less developed than those discussed above. All of them are the focus of a recent and very interesting investigation. For instance, integrated UV and visible light absorption detectors have been tried on microchips [55]. Recently, the use of thermal lens microscopy (TLM) in combination with microchips has demonstrated an interesting analytical potential, as T. Kitamori et al. have clearly reported [56]. TLM is one of the most powerful absorption microspectrometries, and its sensitivity is comparable to LIF. TLM can be applied to both fluorescent and nonfluorescent analytes, thus expanding its versatility. Typical applications of microchip TLM include extraction, immunoassay, flow injection,

(a)

CE microchip (Glass)+ Electrochemical cell Complete integration (one piece): hard lithography

(b)

CE microchip (PDMS)

Electrochemical cell

Partial integration (2 layers): soft lithography

(c)

CE microchip (Glass or Polymer)

Electrochemical cell

Electrochemical cell + set up integration

Figure 1.10 Micromachining of electrochemical detectors integrated in microchips. (Reprinted from [101] with permission from Elsevier)


23

enzymatic assay, in vitro bioanalysis and CE methods [56]. Complicated (bio) chemical processes can also be carried out by microchip TLM, as has recently been demonstrated [57]. Although the evolution of TLM instruments has been impressive, the nonminiaturized environment, the sophistication and the cost of such instruments are the main disadvantages of TLM microsystems. Relevant challenges in coupled detection techniques are focused on the interfaces, particularly on making the micro flows from microsystems compatible with the flow requirements of detection units. Mass spectrometry (MS) and ICP are examples of approaches in this area. MS, as one of the most powerful detection and identification techniques, has been successfully interfaced with microdevices, showing significant possibilities in the corresponding microchips from a practical point of view [55]. Some attempts have also been made to devise an interface between a chip and ESI (electrospray ionization) [58]. Some reports described an efficient sample introduction into the mass spectrometer. However, its size and price remained a challenge to its widespread use. A very interesting new interface, microchip capillary electrophoresis with inductively coupled plasma spectrometry for metal speciation, has been also reported [59,60], opening the possibility of new applications involving inorganic analytes. Detection in miniaturized analytical systems is systematically covered in Chapter 6. Separation Techniques The potential benefits of miniaturization were quickly applied to separation techniques. As M. Szumski and B. Buszewski reported, miniaturized separation systems are currently divided into ‘column’ and ‘chip’ systems [61]. The former are related to the miniaturization of column chromatographic systems, while in the latter the separation is performed in the channel of a chip device. The first group is characterized by the presence of micro- and nano-HPLC [62], whereas the second is more related to mTAS. As we mentioned before, diffusion effects are very fast in micron-sized environments. These effects have a direct advantage in separation techniques, since the reduction of the size of microchannels accelerates samplestationary phase equilibria. In general, miniaturized separation techniques such as chromatography, electrophoresis and electrochromatography have great advantages over techniques at conventional scale. The decrease of the scale provides very rapid separations, versatile channel designs, very small sample volumes and low reagent consumption. The increase of the molecular interaction also makes the chemical process highly efficient [55,63–65]. CE was one of the earliest examples of mTAS, and constitutes one of the most representative examples of an analytical microsystem. Using CE microchips, analysis times can be reduced to seconds and extremely high separation efficiencies can be achieved. Over the past decade, the most active field (as judged by publication outputs) of analytical microsystems development has been the transference from

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conventional separation techniques (macro scale) to planar chip formats. Therefore, without any doubt, CE microchips have a prominent role in the miniaturization field. In fact, they can be considered a synergic combination of miniaturized CE and microchip technological developments. On one hand, this combination involves those features derived from the analytical performance itself, such as the ability to simultaneously assay hundreds of samples in a matter of minutes (or less); rapid analysis combined with massively parallel analysis arrays, which should yield ultrahigh throughput; and the low volume of sample needed (at picolitre level), potentially prepared onboard for complete integration of sample preparation and analysis function (i.e., derivatization). On the other hand, the synergic combination conceptually involves those features derived from important aspects involved in their miniaturization. That means the easy microfabrication of a network of channels using materials of well-known chemistry, exhibiting by themselves a good electro-osomotic flow, and the possibility of using the electrokinetic phenomena to move fluid – namely valveless microdevices – and subsequently increasing their analytical possibilities. Since electrokinetics is easily applied (just a pair of electrodes is needed), EOF-driven flow has been successfully implemented using different types of material in the manufacturing channel, with glass the most commonly used. Microfabrication on polymers is faster and cheaper than that on glass, so these materials have great possibilities for mass production. In contrast, glass chip presents the best EOF, and the chemical modification on the surfaces of its microchannels is better understood than that in polymers. These features involve larger versatility in chemical analysis. The two major polymers used on chips, PDMS and PMMA, both present good optical transparency and low EOF. However, if PDMS is oxidized in a plasma discharge it presents similar EOF to glass material. Furthermore, as PDMS is an elastomer, the process of bonding substrate to the cover plate is easier, whether reversible or irreversible bonding (by oxidation in a plasma discharge) is chosen. Nowadays, PDMS is overtaking PMMA in microchip use. Another important advantage it has is the possibility of obtaining disposable CE microchips [66]. In addition, micro-CE has shown distinct advantages when compared to conventional CE, such as reduced analysis times and extremely high separation efficiencies obtained. Indeed, it has been theoretically indicated that analysis times can be reduced through a reduction in channel length or an increase in separation voltage [65]. Many of the benefits mentioned for CE microchips could equally be applied to downsized chromatographic techniques; however, the literature covers relatively few examples of chip-based chromatographic instruments compared with chipbased CE devices, even though the first analytical instrumentation on-chip was a gas chromatograph [67] and later a liquid chromatograph [68]. This is unsurprising as CE is almost perfectly suited to miniaturization, while the miniaturization of chromatographic systems involves some technical challenges, such as the microfabrication of valves and pumps, which are generally not faced in CE. On the other


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hand, the early on-chip liquid chromatography (LC) examples showed the potential advantages that miniaturization could provide [68]. These advantages included superior efficiency compared to conventional LC, facile positing of detection cells and low unit cost. In addition, the use of an opened tubular system instead of a normal packed column was deemed advantageous due to the shorter analysis times and low pressure drops for a given performance. The common conventional-scale alternatives of packed and tubular columns in LC have also been transferred to the microscale. Although packed columns may be desirable, the introduction of stationary phase material into microfabricated channels is not a trivial process. Frits must be fabricated within the channel structure to retain the packing and a high-pressure interface between the chip and an external pump must be applied. In addition, due to the reduced channel, the packing process is difficult to carry out and can often lead to nonuniform density particles at channel walls, reducing separation efficiencies. Consequently, the majority of initial on-chip chromatographic methods employed a tubular approach [65]. The difficulties associated with packing microfabricated channels can be eliminated if the packed bed is replaced by a porous bed formed by in situ polymerization from organic monomers. The process of bed formation is easy, since a low-viscosity monomer solution can be introduced by vacuum or pressure into the microfluidic channel prior to initiation [69]. In this way, it is important to note that electricallydriven chromatographic separations are especially attractive within chip-based systems, due to a lack of pressure gradients and reduced fabrication complexity. Due to the fact that the mobile phase runs by electro-osmosis, the flat flow profile significantly reduces band-broadening when compared to conventional LC methods. Chapter 4 presents the miniaturized systems for analyte separation based on hydrodynamic flow, while the microsystems based on electro-osmotic flow are described in Chapter 5. Since, as stated before, one of the primary advantages of micromachining analytical instrumentation is the ability to facilitate processes or fabricate structures that are extremely difficult or even impossible to recreate on the microscale, future research is focussed in this area. The concept, which can be thought as in situ micromachining, involves the creation of micron-sized particle support structures on the surface of a planar wafer [70,71]. This line of research is now underway, and is opening exciting new analytical possibilities. Sample Preparation One of the first approaches of miniaturization in sample treatment can be identified with the use of microdevices/units for performing nonchromatographic/electrophoretic separation techniques as sample treatment tools [72]. Solvent microextraction (liquid phase micro-extraction, LPME) and solid phase microextraction (SPME) procedures are two examples.

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As stated earlier, most advances have been made in separation and detection schemes. However, many of the necessary analytical steps (such as sampling and sample pretreatment) are still performed outside the chip. An important effort has been made to move them on-chip in the last few years. Thus, some excellent reviews on this topic have been published [73,74]. Although this analytical process step is less developed than the separation and detection steps, potentially it has a very exciting future. The combination of two important aspects underlines this statement: the possibility of designing complex layouts in connection with the presence of fast diffusion (statistic transport), and the simplicity of applying electrokinetics (directed transport). Table 1.3 lists the main strengths and weaknesses in the integration of sample preparation in analytical microsystems, and has been taken as the basis of the discussion of this section. Although microfabrication and microfluidics are very well adapted to many sample preparation steps, several challenges – such as miniaturization of components, direct analysis of raw samples, sampling and sample introduction – are still present. However, the good current knowledge of microfluidics and the possibility of creating sophisticated layouts can be drawn as the main technological strengths. In addition, derivatization schemes in different formats, using the well-implanted detection modes (LIF and ED), have already been successfully introduced [7,41,64]. Sample treatment Filtration, extraction/preconcentration (including clean-up in some cases) and derivatization are the most common analytical operations that can be carried out in sample preparation. Prior sample filtration is, probably, the most essential step for analyses using microfluidic systems. Raw samples and other already-treated samples (in aqueous media) require basic filtration before analysis. Due to the small dimensions of typical microstructures, particulates can cause serious operational problems, providing sites for blockage. The simplest solution is

Table 1.3 Strengths and weaknesses in the integration of the sample preparation in analytical microsystems (A) MAIN TECHNOLOGICAL SUPPORTS Microfabrication (layouts) Microfluidics (fast diffusion, electrokinetics) (B) MAIN STRENGTHS Derivatization schemes successfully introduced (pre- and post- strategies/LIF and electrochemical) (C) MAIN WEAKNESSES (challenges) Miniaturization of some components Direct analysis of real samples Representativeness of the portion of sample analysed Sampling/sample introduction in the microsystem Changes of samples within a set of analysis


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to filter reagents and samples prior to their introduction. Therefore, it is desirable to integrate a sample filtration system on-chip prior to the analysis. Two approaches have been employed: structurally-based filtration (controlled by the manufacturing process) and diffusion-based filtration (controlled with diffusion). Structurallybased or microfabricated filters have been proposed in popular architectures such as frits, pillar structures and flow restrictions within fluidic channels to mimic conventional filters [75]. H-filter structures have been most used in diffusion-based filtration processes [76]. Filtration can be induced by allowing the analytes of interest to migrate across a laminar boundary (between a sample and a solvent stream) while unwanted heavier particulates are retained in the original fluid stream. It is very important to underline that both approaches are principally linked to miniaturization itself: the first because of the well-known technology integrating multiple micropieces into a single microdevice; and the second because it works on the basis of an inherent property in microfluidics: presence of laminar flow. However, the limit of the structurally-based filters is the resolution limit of the manufacturing process, while the great advantage of the diffusion-based filters is that the whole process is controlled by physics, although a very sophisticated technology is also required. Liquid–liquid extraction could play a prominent role in miniaturized systems. The high surface-to-volume ratios and the short diffusion distances typical within microfluidic environments, combined with laminar flow conditions, offer the possibility of performing liquid–liquid extraction within microchannels without the need for agitation. The main challenge is to induce appropriate electro-osmotic flows when common organic solvents are used. The few examples found in the literature have also employed H-filter strategies [76]. Two approaches to performing solid phase extraction in microfluidics have been proposed: first, coating channel walls with a high-affinity stationary phase, and second, packing the microchannels with the stationary phase material [66,72,73]. The advantages and disadvantages are clearly defined in miniaturized environments. Coating-channel approaches depend on the available surface area for interaction, and in micron-sized channels the contact surface is very small. A simple way to increase the surface area is to pack the microchannels with stationary phase; however, the packing process is not easy, and this route is often avoided. A very attractive possibility, compatible with miniaturized dimensions, is to replace conventional stationary phase materials with a continuous porous bed in situ formed from polymerization of organic monomers. The process of bed formation is easy, since a low-viscosity monomer solution can be introduced by vacuum or pressure into the microfluidic channel prior to initiation [66,75]. An excellent paper showing the power of integration filtration, concentration and separation was recently published [77]. In this paper, filtration, concentration and separation are integrated on to microchip. Filtration consists of an array of seven thin channels (1 mm deep) which come together into one channel (5 mm deep). The input of the thin channels is

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communicated with sample reservoir and the sample loading is electrokinetically carried out. Sample concentration is performed by solid phase extraction. The separation channel is coated with C18 (1.5–4 mm) particles. Then, simply by increasing injection times, the analytes are retained and concentrated in the separation channel, and afterwards eluted with the appropriate solvent. In some cases, these basic operations for sample treatment have been seen as micro unit operations (MUO) [78], and used for developing microintegrated chemical systems on the basis of a concept similar to electronics: a chemical process can be constructed like an electronic circuit, but in place of the resistor, capacitor and diode, mixing, extraction, phase separation and other operations are integrated. Field-amplified sample stacking is a common method for sample preconcentration in electrophoretic systems. However, its implementation into a chip platform is not easy. The initial difficulty associated with sample stacking in a microfluidic format is the control of the analyte zone during the stacking and separation procedures. This often requires the use of relatively complex channel networks and voltage programmes to stack the analyte zone [73,74]. Derivatization on microchip is well established in analytical microsystems. The reason for this development can be found in the traditional use of LIF as a sensitive detection system, since the earliest times. Again, the role of microfabrication in the design of complex microcircuits offers a unique route in sample pretreatment and especially in derivatization schemes, where the process is carried out before or after analyte separation and before analyte detection. In other words, the high degree of functional integration (reagent mixing, product separation and post-column labelling) provides an elegant indication of the potential benefits of microfluidic systems. Derivatization schemes carried out prior to the separation (precolumn) or immediately prior to the detection (post-column), have been proposed using LIF and ED detections. Thus, derivatization in fluorescence has been well implemented into microchip in connecting with LIF detection [79]. A group of works uses a very attractive strategy based on the combination of suitable pre- and post-channel layouts with bioreagents such as enzymes and antibodies, along with electrochemical detection [80,81]. It is important to remark that, in some cases, these are truly lab-on-a-chip devices [82,83]. In fact, the generation of electroactive products using selective bioreagents such as specific enzymes, class-enzymes and antibodies in adequate pre- and post-column derivatization schemes has revealed the potential of these strategies in the simplification of treatments of complex samples. This line can be used for developing other sophisticated layouts for sample treatment when real samples are analysed in these microsystems. Sampling and sample introduction Sample introduction and the representativeness of the small size of the sample introduced are two important aspects of microfluidic systems. Most chip-based systems still adopt a discrete and often


29

manual approach to on-chip sample loading and sample changing. Such practice seriously lowers the overall throughput and counteracts the advantage of achieving fast separations. On-chip loading and changing of a series of samples has become a limiting factor in real-world applications. Efficient assays of real-world samples will require the incorporation of a continuous sampling capability (from the external environment) or rapid sampling of multiple discrete samples. Such an ability to continuously introduce real samples into micro-sized environments should make analytical microsystems compatible with real-life applications. Different devices, based on pressure-driven [84–86], hydrodynamic [87] and autonomous polymerloading [88] techniques have been reported for sample injection. Finally, one of the most important aspects to consider is the continuous introduction of samples, which requires an interfacing system between the macroenvironment (mL–mL) and the microchip (nL–pL) in order to achieve an efficient sample change with low carryover. Hence, a suitable design of interfaces, similar to the hyphenated detection techniques, is necessary. Although bibliography is poor in papers dealing with this subject, various approaches have been proposed in excellent works. In one, using a microfluidic matrix device, sample introduction was performed through a wide-bored microchannel, drilling an access hole through the sample reservoir [89]. Very recently, the use of a sharp sampleinlet tip was reported [90]. An excellent report dealing with trends relating to different configurations of sample introduction interfacings was recently written by Fang, showing a critical vision of different strategies used by his research group [91]. This report shows that the trend is to separate the sample-introduction channel (SIC) with hydrodynamic flow from the separation channel with electrokinetic flow, thus avoiding totally pressure-driven flows. This was achieved by introducing a sample-loading channel (SLC) between the SIC and the separation channel. The SIC acts as a flow-through sampling reservoir and the sample is electrokinetically loaded into the SLC from the SIC. The SLC connects with the separation channel. This split-flow strategy is improved by a flow-through sampling reservoir featuring a guided overflow design. This array avoids Poiseuille flows, maintaining equal liquid levels for the sample, the buffer and the waste reservoirs. A fast and simple sample introduction was recently reported [88]. It consists of the use of a sharp sample-inlet tip alternately placed in the sample and the buffer vials. This tip was fabricated by sharpening the inlet side of the chip with a diamond saw. A good reproducibility was obtained (100 repetitive flow injection measurements resulted in a response with an RSD of 3.7%). C.-W. Huang and G.-B. Lee have proposed an interesting contribution consisting of microautosamplers for discrete sample injection and dispensation in microchips [92]. It is a promising approach toward realizing the mTAS concept. All these aspects related to the miniaturization of sample treatment are reported in more depth in Chapter 3.

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1.7.2

Integrated (Micro)systems for the Performance of the Entire Analytical Process

As Figure 1.11 shows, an ideal analytical measurement system should be characterized by: (i) Portability and self-operation, in order to avoid or reduce the sampling step and allow the possibility of performing field tests. (ii) Either the noninvasive measurement approach or the performance of in-line measurements. These features allow the minimum perturbation of the sample. (iii) Facilities for the maintenance of the system and large lifetimes. Alternatively, reusability at low price. (iv) Self-calibration incorporated in the system. (v) The possibility for the incorporation of quality-control activities, in order to assure a reliable response. Sensors were one of the first approaches to performing the entire analytical process. They represent by themselves a miniaturization and simplification of the analytical process, although the initial expectation of a wide field of application was demonstrated to be unfounded by limitations in selectivity and lifetime. But in any case, sensors constitute a growing area of interest with a strong social and industrial impact. In fact, sensors provide fast response with reduced costs. Ideally, a sensor is a device that provides an analytical signal for a specific compound present in a raw sample in a direct, reversible, continuous and reliable manner. From a practical point of view, most of the sensors reported in the literature fall short of the requirements of the above definition. Hence, a more realistic definition of a sensor is that it is a sensitive microzone where a (bio)chemical reaction occurs that is connected to or integrated in a physical (optical, electrochemical, thermal, masssensitive) transducer [93]. Through this connection to an instrument, analytical information can be produced in situ in real time. The problem of interferences is partially solved by the so-called ‘biosensors’, which are defined by IUPAC as selfcontained integrated devices that are capable of providing specific quantitative or semiquantitative analytical information using a biological recognition element (biochemical receptor) in direct contact with a transducer element. Biosensors including transducers based on integrated circuit microchips are known as ‘biochips’. The biological recognition element used in a biosensor can be an enzyme, an antibody/antigen, a nucleic acid, a cellular structure or a biomimetic receptor. The term ‘microsensor’ describes a sensor whose active sensing area is in the micro range, while the sensing device itself is much larger for easier handing. ‘Microelectrode’ and ‘ultramicroelectrode’ are terms used in electrochemistry used instead of microsensor, although electrodes with smaller radii (nanometric size) are currently used [94]. The small size of these electrodes makes diffusional mass

Figure 1.11 Different approaches to the implementation of the entire analytical process, combining various degrees of automation, simplification and miniaturization (see text for details)

Miniaturization in Analytical Chemistry 31

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transport extremely efficient and double-layer capacitance very small, attaining greater signal-to-noise ratios and lower detection limits than those of macroelectrodes [95,96]. Chapter 7 reports the miniaturization of the entire analytical process through micro(nano)-sensors. The TAS approach is addressed to meet these ideal features, integrating the different analytical standard operations of the analytical process. Portable instruments can be considered as a particular case of TAS, designed to operate outside the laboratory (miniaturized portable analytical systems are reported in Chapter 9). Both are integrated analytical systems, with a high level of automation and simplification for the intended analyses. Miniaturization commonly exists in these systems, but in a relative sense, with respect to other laboratory alternatives using conventional equipment to carry out the same application. On the other hand, the following step giving integrated microsystems (mTAS or in vivo measurement devices) is strongly characterized by miniaturization, although simplification and automation are implicit in these microsystems. Chapter 8 is devoted to the miniaturization of the entire analytical process through mTAS approaches. As illustrative examples, Figure 1.12 shows four common categories of integrated systems miniaturizing the entire analytical process. The first one (Figure 1.12a)

Figure 1.12 Examples of different levels of miniaturized analytical systems: (a) portable X-ray fluorescence equipment (Reprinted from [97] with permission of American Chemical Society, Copyright 2007); (b) small analyser for SPR with optical biosensor detection (Reprinted from [98] with permission from Elsevier); (c) portable miniaturized equipment for health care (Reproduced from [99] with permission of IEEE, Copyright 2007); (d) mTAS for micro-ELISA analysis (Reproduced from [78] with permission of IEEE, Copyright 2007)


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is a portable total reflection X-ray fluorescence spectrometer. Despite the macrosize (cm level), it represents a clear miniaturization with respect to a conventional X-ray fluorescence spectrometer operating at laboratory-routine work [97]. The second category (Figure 1.12b) is represented by small-sized laboratory equipment used for the determination of environmental organic pollutants based on a surface plasmon resonance (SPR) optical biosensor device [98]. The third (Figure 1.12c) is a type of portable miniaturized equipment for health care used for home medical diagnosis. It corresponds to a calorimetric three-item measurement chip (triglyceride, total-cholesterol and HDL) combined with an electronic blood-collection system (6 mL of whole blood) [99]. The final category (Figure 1.12d) is represented by mTAS, as the multichannel micro-ELISA chip in the image shows [78]. From a practical point of view, the reliability of the information generated by these integrated microsystems is a key factor. Performance tests for the proper microsystem and the appropriate validation of the whole analytical method must be carefully carried out. Within these activities, calibration must be planned in tandem with the miniaturized equipment work, especially in the case of portable systems. In this context, the integration of the calibration with the particular manifold or scheme of the microsystem should be taken into account. Major difficulties can be found in miniature analysis instruments for measuring trace levels of gases, commonly used for field tests. In these cases, standard gas in situ generation for onsite calibration is a very useful alternative. Thus, recently S.-I. Ohira et al. described one of these systems for on-site checking or onsite calibration of a micro gas analysis system, mGAS [100]. The key part of this arrangement is shown in Figure 1.13. The source reagent (R1 in Figure 1.13) was mixed with the desorbing solution (R2) and reacted in the mixing coil (MC), then introduced into a microchannel gas generator. Generated vapour permeated through the membrane and was extracted into the air stream flowing on the opposite side of the membrane. This microsystem was used to generate H2S, SO2, CH3SH and NH3.

1.8

Outlook

Analytical microsystems constitute an important trend within analytical chemistry science. The high number of scientific contributions published in this field so far is a good reason to imagine that one of the next objectives will be dealing with application issues and expanding their use to laboratories at bench level. Miniaturization presents different difficulties and problems regarding the different steps involved in the analytical process [101]. Detection schemes, such as LIF and ED (amperometry), are well established in analytical microsystems; nevertheless, novel designs, especially in ED, are being developed. Although MS was studied from earliest times because it is a very powerful technique [102,103], its use in mTAS is nonetheless not widely proposed due to its size and price. Recently, F. Foret and

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Figure 1.13 Liquid flow system for gas generation with microchannel gas desorber. R1: source reagent; R2: desorbing solution; MP: liquid minipums; MC: mixing coil; GD: gas desorber; BP: back pressure tube. (Reprinted from [100] with permission from Elsevier)

P. Kus y published a review on microfluidics for multiplex MS analysis [104]. As the authors recognize, the development of microfluidics–MS coupling is still in its infancy, despite the analytical potential of these microsystems. They pointed out that ‘the future success will depend on the willingness of both the instrument manufacturers and the users to adopt this technology in practice’. This will require a clear demonstration of the technology’s superiority and robustness. Regarding the detection capabilities, LIF and amperometry are not very suitable for small molecules, including inorganic species. Alternative detection schemes, such as conductivity, ICP-AEE or ICP-MS, are necessary and consequently research on new detection systems for analytical microsystems is still open. Additionally, the use of TLM for detection and imaging studies performed in microchips will open interesting new possibilities. Separation on chips is primary developed using CE, which constitutes the most representative example of mTAS. In microseparation, new tools such as the design of new analysis schemes and strategies including the use of nanoparticles, employment of specially designed polymers and new cover materials will be developed. Sample preparation at microscale level has been less developed than both detection and separation techniques. In general, some of the most relevant promises of lab-on-a-chip devices, such as integration of all laboratory functions in order to get practical sample treatments, commercialization, truly handheld operation and ease of use, are coming, but today are not a reality. However, the possibilities of using electrokinetics as directed fluid transport, of creating suitable layouts for


35

problematic needs, and of laminar flow, allow us to think of future developments as a near reality. Total integration [105] and, especially, world-to-chip interfacing are considered the major challenges, particularly in high-throughput applications requiring frequent sample change, such as online continuous process monitoring. More specifically, various world-to-chip interfacing schemes are being developed to meet different requirements of sample and microfluidic chips, in terms of sampling rate, sample introduction, sample consumption, precision, stability and degree of automation. Two clear trends are the development of more integrated, rugged, portable and fully automated sample introduction systems, and systems based on various hydrodynamic principles outside of the electrokinetic environment. On the other hand, a basic trend with respect to analytical applications is the exploration of the possibilities of these microsystems in other important analytical areas, ranging from medical diagnosis and pharmaceutical screening to food and environmental control [106]. This expanded use can only be fulfilled by versatile and highly sophisticated analytical devices. One exciting trend in miniaturization is the use of micro/nanodevices for biological and bioanalytical studies of cells, genes, proteins and individual molecules. Different alternatives are available for cell trapping in microfluidic chips [107], demonstrating that miniaturization technology provides facilities for creating tools with feature sizes matching the dimensions of cells, and enables integration of cell-handling and fluid-manipulation elements and the chemical analysis of single cells [108]. Also, microsystems create new opportunities for the spatial and temporal control of cell growth. Further, as recently reported by J. El-Ali et al. [109], typical unit operations such as cell growth, treatment, selection, lysis, separation and analysis have been demonstrated to be implemented using mTAS. But, as they pointed out, robust approaches to fabrication, integration and packaging (such as communication with the macroenvironment) remain major areas of research. Gene analysis on a single chip is now a possibility, although with some challenges [110]. Microfabricated electrophoresis devices offer several advantages over conventional methods for rapid genotyping [111] and DNA sequencing [112]. Proteomic analysis based on microfluidics is also the subject of interesting developments. Electrophoresis devoted a special issue to this topic in 2006 [113], and other interesting contributions have appeared since. In an exciting article, H. Craighead discussed future lab-on-a-chip technologies for interrogating individual molecules [114]. As he reported, these advances allow the manipulation and measurement of individual molecules, and ‘the adaptation of these approaches to lab-on-a-chip formats is providing a new class of research tools for the investigation of biochemistry and life processes’. It can be said that the first generation of lab-on-a-chip devices is starting to work, and in the future, successive (more sophisticated) generations will introduce themselves as a new reality in analytical laboratories. In this new generation, a further integration of sample collection and sample preparation, improving

36


lab-on-a-chip for world connections in order to reach the goal of ‘total integration’, will be necessary. New approaches to avoid the major shortcomings of chip-based analytical systems, such as the risk of the analyte adsorption on walls and at interfaces (which is especially high in low-volume analytical systems), and the optical interference at the walls of chips in the detection point, will help to solve practical problems. The concept of digital microfluidics in the late 1990s can be addressed in this way, as it involved the manipulation of discrete volumes of liquids on a surface by mechanisms such as electowetting, thermocapillary transport and surface acoustic wave transport [115]. Sample levitation can open the exciting new approach of lab-on-a-drop, representing an alternative for preparing nano-pico sample volumes without contamination from solid walls or other external objects, or between samples [116].

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2 Tools for the Design of Miniaturized Analytical Systems 2.1

Introduction

This chapter deals with the tools needed to design analytical microsystems at micro and nano scale, according to the classification shown in Figure 1.6. Micro(nano) fluidics and micro(nano)electromechanical systems (MEMS and NEMS, respectively) are the characteristic scenarios in which the analytical process occurs at a miniaturized scale. Miniaturization at this scale is completely different from miniaturization at the mini scale, of which some examples were given in Chapter 1. Many of the phenomena that we are used to living with in the macro world, and which we take for granted, have next to no significance for fluids in the micro world. For instance, inertia and turbulent flow play an important role in the macroscale world (transport in conventional pipes and tubes, rivers, oceans, etc.) but mean nothing at micro- and nanofluidic scales. By contrast, diffusion (having practically no transport significance at the macro flow scale) becomes the dominant process at micro(nano)fluidic scales. In addition, surfaces become an ever more important factor as dimensions are reduced. In fact, the ratio of surface to volume increases drastically as this happens. The objective of this chapter is to give the basic principles of the behaviour of microfluidic systems for an analytical intended use, as well as the main microfluidic devices needed to develop the analytical process at this downsizing scale. For further information about the engineering side of microfluids, readers should consult more specialized books and publications (see e.g. [1–4]). Figure 2.1 shows a simplified scheme of the main steps from planning to the final analytical use of Miniaturization of Analytical Systems: Principles, Designs and Applications and Bartolome Simonet 2009 John Wiley & Sons, Ltd


40


DEFINITION OF ANALYTICAL PROBLEM

TECHNICAL SPECIFICATIONS

MICROSYSTEM DESIGN

SIMULATION AND OPTIMIZATION

FABRICATION: (1) PROTOTYPE (2) MASSIVE FABRICATION

ANALYTICAL USE

PERFORMANCE TEST

Computerized software

Cleanroom facilities Microfluidic devices Microelectronic facilities

Verification of technical specifications NO

OK OK

ANALYTICAL VALIDATION

NO

Figure 2.1 The main steps to be followed in the development of an analytical microsystem, and the different tools and devices involved

a specific microsystem. This figure also summarizes the main points to be described in this chapter: (i) design of the microsystem according to some previous technical specifications (connected to the analytical problem to be solved); (ii) simulation and optimization by use of an appropriate computerized software; (iii) the proper fabrication as a previous prototype using cleanroom, microfluidic and microelectronic facilities for micromachining and final integration by packaging; and (iv) technical verification of the prototype and, if it passes, analytical validation for its intended use. If the expected technical and analytical purposes are achieved, massive fabrication can be planned if needed. As stated in the previous chapter, the two most important phenomena characterizing microfluidic devices are the almost exclusive presence of laminar flow and the dependence on diffusion as the major available transport mechanism.

2.2 Miniaturized Analytical Processes: The Downsizing and Integrating Phenomena Fluid behaviour has been studied extensively for several centuries, and an important number of monographs and articles have been published on this subject. At macro

Tools for the Design of Miniaturized Analytical Systems

41

and mini scales, density, the pressure of the liquid and its viscosity are the characterizing parameters. Knowing density and viscosity, other predictions can be made for liquids in motion, particularly about what kind of flow will be found in microsystems (meaning the flow in channels at typical dimensions on the micro scale). The dimensionless Reynolds number equation, Re, establishes the relation between the magnitudes of the inertial and the viscous forces: Re ¼

rrh v ; h

ð2:1Þ

as was introduced in Chapter 1. Numbers larger than about 2300 correspond to turbulent flows. Under this regime, inertial forces are dominant (this is the behaviour we typically know in everyday life). According to Equation 2.1, large Reynolds numbers are attained at higher liquid densities, higher flow velocities, larger typical length scales or lower viscosities. A Reynolds number between 2000 and 3000 is in the so-called regime of transitional flow, and the region below about 2000 is referred to as the laminar flow regime. This type of regime is obtained at lower velocities, smaller dimensions, smaller densities or higher viscosities. It can be demonstrated that flows in capillaries with dimensions smaller than 100–150 mm need a rather impossible pumping velocity, n, value in order to reach the transitional flow regime, and hence the laminar flow regime is the characteristic situation in this microfluidics. For a capillary with a cylindrical cross-section, following the Hagen–Poiseuille theory, the volume flow, Q, is given by the expression: Q¼

DV pR4 DP; ¼ 8hL t

ð2:2Þ

where R is the radius of the capillary, L is its length and DP is the pressure drop across this length (also called hydraulic pressure). The velocity profile (i.e. the velocities n(r) at different radial positions between the centre (r ¼ 0) and the wall (r ¼ R)) is given by the equation: vðrÞ ¼ ðR2 r 2 Þ

DP ; 4hL

ð2:3Þ

clearly describing a parabolic flow profile. The term 8hL=pR4 (the reciprocal appears in Equation 2.2) is also called the fluidic resistance. Therefore, the fluidic resistance dramatically increases as the channel dimensions are reduced, and hence higher pressure drop values are necessary to move liquid through smaller conduits. For channels with noncylindrical cross-sections, similar expressions to Equation 2.2 can be found [5]. Another useful relation for the design of microfluidic channels is based on the continuity equation, describing the behaviour of flow in channels with changing cross-sections. This equation states that the product of the cross-sectional area and the flow velocity is a constant: A1 v1 ¼ A2 v2 ¼ const:

ð2:4Þ

42


On the other hand, Bernoulli’s equation also considers pressure and height differences in flows, and plays a key role as a direct application of the law of energy conservation, relating pressure, kinetic energy and potential energy according to the equation: 1 Pþ rv2 þrgh ¼ const: 2

ð2:5Þ

It is very useful to know the value of n because it can give an indication of the transit time through a microfluidic channel network, and therefore helps to assess whether there is enough time for a chemical reaction or mixing to take place. 2.2.1

Transport within Microfluidic Systems

The motion of a fluid in a channel can be driven by internal forces that are determined by fluid and channel properties, such as interfacial forces, or by an external field (electric, thermal, magnetic, etc.). At the micro and nano scales the relative importance of forces is given by the following order: buoyancy < inertial force or gravitation force < viscous force interfacial force. Therefore, interfacial forces become prominent upon system downscaling. There are two different types of transport within microfluidic systems, depending on the nature of the driving agent behind the transport: directed transport and statistical transport. Directed transport is transport that is controlled by exerting work on the fluid. This results in a volume flow of the fluid (characterized by a direction and a flow profile). When the work is generated by a pump, a pressuredriven flow is produced, whereas if the work is generated by a voltage, the so-called electro-osmotic flow is established. By contrast, statistical transport is not a directly controlled transport, as it is based on an entropy-driven transport. Thus, the transport occurs only if a fluid is more disordered after transport than before. This is the case with transport by diffusion: movement of molecules from more concentrated zones to less concentrated ones in a liquid medium. In practice, both types of transport take place, particularly because commonly directed transport meets with some gradients in the flow, such as a temperature gradient and a concentration gradient. By definition, heat transfer by mass transport is called convention. Free or natural convention is caused by a difference of temperature, but fluids can also be moved with external forces to create a directed flow (forced convention). There are several ways in which forced convention can generate directed flow, such as capillary flow or other forces (gravity, pressurized air, etc., which create a single instant of pressure difference in the microsystem). Migration is the directed transport of molecules in response to an electric field. This is the original basic force involved in (micro)capillary electrophoresis and its different modes. The electrically charged molecules, in a polar solvent medium (aqueous


43

solutions, mainly), experience a Coulomb force, F, due to the electric field: F ¼ qE;

ð2:6Þ

where q is the charge on the molecule and E the strength of the electric field. When the terminal velocity of the ions is reached, the Coulomb force is balanced by the Stokes force: F ¼ 6phrv;

ð2:7Þ

where Z is the viscosity of the liquid, r the hydrodynamic radius of the molecule and n is the speed of the charged molecules. At the terminal speed, both forces are equalized: qE ¼ 6phrn;

ð2:8Þ

q E; 6phr

ð2:9Þ

and hence: n¼

where the mobility of the molecules, m, is given by: q m¼ 6phr

ð2:10Þ

Diffusion is a pure statistical transport process. Thus, in a liquid or gas medium, all molecules move in all directions as long as no external forces are applied. This phenomenon takes place when there is a concentration gradient of one kind of molecule within a fluid. The statistical movement of a single molecule in a fluid is a random movement characterized by the Einstein–Smoluchowski equation: pffiffiffiffiffiffiffiffi x ¼ 2Dt; ð2:11Þ where x is the average distance moved after an elapsed time t between molecule collisions, and D is a diffusion constant characteristic for each molecule. If x is half of the channel width, W, it is possible to estimate the time a molecule takes to cross this distance: tcross ¼

W2 8D

ð2:12Þ

The consequence of this equation is the key influence of the channel width on the mixing of liquids inside it. On the other hand, the overall effect of the random walk of all molecules can be described as temporal and spatial changes in concentration. These changes are governed by Fick’s laws. The fact that molecules travel from areas of high molecular concentration to areas of low concentration is expressed by using the concept of particle flux, j (the number of molecules crossing a certain area during

44


a time t):

@c ð2:13Þ @x This equation means that the molecular flux is stronger when the concentration gradient is steeper. Additionally, it can be stated that when molecules leave a test volume, the number of molecules in the test volume is correspondingly lower. Thus, the continuity equation can be expressed as: j ¼ D

@c @j ¼ ; @t @x

ð2:14Þ

and therefore, by combining Equations 2.13 and 2.14: @c @2c ¼ D 2; @t @x

ð2:15Þ

which is Fick’s second law of diffusion, showing that the variation of concentration with time is directly proportional to the diffusion coefficient of the molecules. D is affected by different variables in a (micro)fluidic environment, according to the Stokes–Einstein equation: kT D¼ ; ð2:16Þ 6phr where k is the Boltzmann constant and T the temperature. The denominator is commonly known as the frictional constant (depending on the viscosity of the medium and the hydrodynamic radius): f ¼ 6phr

ð2:17Þ

It is important to remark that under different circumstances and for different geometries of microfluidic systems, either directed or statistical flow can dominate, or both types may be of equal importance. To evaluate the various flow situations, the ratio between the mass transport due to directed flow and that due to diffusion can be estimated. This ratio gives a dimensionless number called the Peclet number (Pe), which is expressed by: vd Pe ¼ ; ð2:18Þ D where d is a characteristic length of the microfluidic system. Thus, the Peclet number, for a particular microchannel, can be calculated, and the diffusion compared to the directed flow can be evaluated. This is important information for the design of microfluidic systems that need to keep control of diffusion, such as chemical separation systems. If the Peclet number is much smaller than 1 then diffusion dominates the microfluidic flow (directed flow is of secondary importance), whereas if the Peclet number is much larger than 1, the molecules mainly flow according to the externally applied driving force (diffusion has only a minor influence).


45

Another term used for transportation in minichannels is dispersion, commonly applied to flow-injection analysis (FIA) systems. In this case, a sample plug containing a high concentration of certain molecules moves along channels of 300–700 mm (lower for micro-FIAs). The concentration boundaries become vaguer due to diffusion of molecules, and the widening of the sample plug is generally called dispersion. In addition to the band broadening caused by diffusion, there is another bandbroadening causedbythe parabolicvelocityflow profile, whichoccurswhen the flow is pressure-driven. This superposition phenomenon is called Taylor dispersion. 2.2.2

Microsystem Design from Transport Parameter Information

The previously defined dimensionless Peclet number is useful in designing microsystems. In microsystems, the flow velocities are usually small, and the crucial variable that determines the Peclet number, according to Equation 2.18, is therefore the channel length, d. For long enough channels, the Peclet number is always larger than 1, and the flow is consequently directed. On the other hand, if the Peclet number is much smaller than the length-to-width ratio of the microchannel, d/w, then Taylor dispersion is observed. By contrast, if the Peclet number is much larger than this ratio, then diffusion is not the main agent of dispersion. Thus, narrow microchannels allow transverse diffusion to play a significant role, which does not happen in relatively broad channels. Therefore, microsystem designers have to decide the length, width and flow speed. They then have to adjust the microchannel volume by choosing the depth of the channels. As the two most important phenomena found in microfluidic devices are the almost exclusive presence of laminar flow and the dependence on diffusion as the major available transport mechanism, the possibilities for developing microfluidic devices come from the application of these physical principles to the practice. These aspects will be considered in the next section of this chapter.

2.3

Microfluidic Devices

Different devices are used to generate and control the hydrodynamic flow in microfluidics. Among the most important devices are pumps and valves, but other elements such as injection/metering devices, mixers, heaters, etc., are commonly integrated in the microfluidic layout. Devices dealing with the detection are also key elements in microfluidics. Although detection in microsystems is considered in this chapter, it will be systematically described in Chapters 6 and 7. 2.3.1

Microvalves

Valves are designed to manipulate the direction of the motion of the fluid, and they are seldom combined with micropumps in microfluidics. Basically, they introduce

46


directionality into the flow, and according to their type of work can be classified as passive or active valves. The ideal characteristics of microvalves can be summarized by the following parameters: zero leakage, zero power requirements, zero dead volume, high differential pressure capability, zero response time, chemical resistance and not being sensitive to particulate contamination. Of course, any valve meets all these requirements, and the selection of the appropriate valve for a particular use becomes a key factor. A large number of different valves are commonly used in microfluidic systems, as reviewed by K.W. Oh and C.H. Ahn [6]. Following these authors, valves can be classified in two main categories, as Table 2.1 shows. Passive microvalves can use mechanical or nonmechanical moving parts, whereas active microvalves can use both mechanical or nonmechanical moving parts and external systems. Different physical principles are involved in the functioning of both categories of valves, given a variety of types of microvalve, as reported in Table 2.1.

Table 2.1 Classification of microvalves Category

Mode

Base of actuation

Type

Passive

Mechanical

Check valve

Nonmechanical

Capillary

Mechanical

Magnetic

Flag Membrane Ball In-line mobile structure Diffuser Abrupt Liquid-triggered Brust Hydrophobic valve External magnetic fields Integrated magnetic inductors Electrostatic Electrokinetic Piezoelectric Bimetallic Thermopneumatic Shape memory alloy Bistable Electrochemical Hydrogel Sol-gel Parafin Electrorheological Ferrofluids Built-in Rotary Membrane In-line

Active

Electric Piezoelectric Thermal Nonmechanical

Bistable Electrochemical Phase change Rheological

External

Modular Pneumatic


47

Passive valves do not need any external energy for their function. Those with moving parts cannot be opened or closed without changing the geometry of flow paths, such as with check valves, pressure control valves or flap valves. Other passive, nonactuated, valves have no moving parts, and they are the more interesting for combination with autonomous capillary systems [7]. A simple passive valve can be built by using a cantilever in which a thin strip of silicon bends when enough pressure is applied from one side (valve flap type) [8]. The simple work of this check valve is schematically shown in Figure 2.2. It is based on mechanical action and does not require any external energy for its operation (only flow pressure in the microchannel). There are also passive valves not based on mechanical action but using other forces, such as surface-tension or capillary valves [9,10], hydrophobic valves [11] and pH-sensitive valves [12], among others. These types of valve are designed for a single use, because their function depends on an air–liquid interface, which is typically present only when first filling a microchip. Indeed, they influence how a liquid fills the region of the valve, but not how a liquid afterward flows through the valve. The simpler version of this approach is a restriction in the microchannel. Liquid cannot penetrate into the next channel segment because of the increased surface tension around the restriction. Only when a higher pressure is applied will the liquid break through the restriction into the next section of the channel network. Other basically passive valves are based on the use of hydrogels, which are sensitive to some external stimulus. Commonly, they suffer a change in their volume. Depending on the stimulus generated by the solution flowing through the channel, the hydrogel swells or stays in the shrunken state. As many of these

Figure 2.2

Scheme of a simple passive microvalve based on a cantilever

48


hydrogels are pH-sensitive, this chemical stimulus can easily be used. Recently, H.J. Cho and coworkers have developed passive valves for microfluids based on the use of a nanostructured functional polymer surface [13]. They have fabricated and tested two fully integrated microfluidic valves, one with a superhydrophobic polymer surface, and the other with a switchable, thermosensitive polymer surface. The passive valve with the superhydrophobic polymer surface selectively inhibits the flow of water-based reagents and passes aqueous solutions containing surfactants. In the case of the thermosensitive valve, the switchable polymer surface becomes hydrophobic when heated to temperatures exceeding 65 C, thus inhibiting the flow of water, and becomes hydrophilic at room temperature, thus allowing the flow of water. Figure 2.3(a) shows the general scheme of the microvalve, while Figure 2.3(b) is a photograph of the fabricated structure, micromachined from the designed scheme. In these figures, the reservoirs for the inlet and outlet microchannels can be seen, as can the location of the polymer film. The same design was used for both polymers. The microchannels were fabricated by standard

Figure 2.3 Scheme of a passive valve for microfluids based on the use of a nanostructured functional polymer surface (In, inlet; O1, outlet 1; O2, outlet 2) (a) and the corresponding photograph (b). The fabrication of a passive microfluidic valve with a superhydrophobic surface (see text for details) (c) and the fabrication process of a thermosensitive valve (see text for details) (d). (Reprinted from [13] with permission from Elsevier)


49

photolithography and wet-etching techniques, while the polymer surface for both the valves was fabricated using the layer-by-layer deposition technique, in which multiple layers of polyelectrolytes (poly(allylamine hydrochloride), PAH) were coated in a channel wall, followed by silica nanoparticle treatment. For the thermosensitive valve, the polymer surface was further coated with the thermosensitive polymer poly(N-isopropylacrylamide) (PNIPAAm). Figure 2.3(c) shows the steps followed for the fabrication of the passive microfluidic valve with the superhydrophobic surface: (i) A glass slide is lithographically patterned to form the T-junction microchannel shown in Figures 2.3(a) and (b). (ii) The patterned glass slide is wet etched by using the photoresist as an etch mask. (iii) The glass slide is photolithographically patterned to form an opening for the initial polyelectrolytes. (iv) Positively charged PAH is dip-coated on the opening. (v) Negatively charged poly(acrylic acid) (PAA) is then dip-coated. This layerby-layer deposition is continued successively until 101 bilayers of PAH and PAA are formed. (vi) A poly(dimethylsiloxane) (PDMS) slab is bonded to the glass slide after oxygen plasma treatment. For the fabrication of the thermosensitive valve, the stages involved are schematically shown in Figure 2.3(d): (i) An etched glass slide with a lithographically patterned opening for the initial polyelectrolites is dip-coated with positively charged PAH. (ii) Negatively charged silica nanoparticles are then dip-coated. This layerby-layer deposition is continued successively until 40 bilayers of PAH and silica nanoparticles are formed. This is followed by a second run of layerby-layer deposition to decorate the polymer surface with silica nanoparticles. (iii) The glass chip with the rough polymer surface is annealed at 400 C for 2 hours. (iv) After a third run of layer-by-layer deposition to create two bilayers of PAH and PAA, the glass chip is dipped into the initiator solution for 2 hours. Finally, PNIPAAm is grafted on to the initiator-coated surface to create the switchable, thermosensitive polymer surface. Both passive valves were successfully tested for passing/stopping liquid samples depending on their wettability. Active valves, by contrast, require an actuator to provide the mechanical action, allowing or stopping the flow passage. They are normally designed to be in one or other of these states (‘open’ or ‘closed’) without actuation, while energy must be applied to pass or maintain the opposite state. Therefore, actuators are decisive

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Figure 2.4 Illustrations of the actuation principles of active microvalves with mechanical moving parts: (a) electromagnetic; (b) electrostatic; (c) piezoelectric; (d) bimetallic; (e) thermopneumatic; (f) shape memory alloy actuation. (Reprinted from [10] with permission of Elsevier, Copyright 2007)

elements. They can be based on different principles: pneumatic, electrostatic, piezoelectric, electromagnetic, etc., but are typically characterized by four features: (i) the pressure they can build up; (ii) the stroke displacement they generate; (iii) their response time; and (iv) their reliability. Each type of actuator presents better characteristics in one feature or the other. Figure 2.4 illustrates the actuation principles widely employed in active microvalves based on mechanical principles. Most of these active microvalves couple a flexible membrane to some actuation methods (magnetic, electric, piezoelectric, etc.). Traditionally, these active microvalves are accomplished using MEMS-based bulk or surface micromachining technologies. Active microvalves based on nonmechanical operation are of particular interest in terms of their simple device structure and disposability, making them well suited for applications in life sciences. External active microvalves are actuated by the aid of external systems such as built-in modular [14,15] or pneumatic means [16,17]. Many other microvalves can be developed using different designs, materials and actuation principles. Thus, in addition to the examples described, others can be found in the literature using different principles. G.M. Whitesides and coworkers have described torque-actuated valves for controlling the flow of fluids in microfluidic channels [18]. These valves consist of small machine screws (about


51

500 mm) embedded in a layer of polyurethane cast above microfluidic channels fabricated in PDMS. Turning the screws actuates the valve by collapsing the PDMS layer between the valve and the channel, controlling the flow of fluids in the underlying channels. These valves do not require power to retain their setting (on/off), and additionally they allow setting between ‘on’ and ‘off’. They can be integrated into portable, disposable microfluidic devices. J. Wang et al. recently proposed the use of nanowires for the fabrication of switchable microchip devices [19]. They used functional nickel nanowires for switching on-demand operation of microfluidic devices. Controlled reversible magnetic positioning and orientation of these nanowires at the microchannel outlet offers modulation of the detection and the separation processes, respectively. These devices have been used in electrophoretic separations in microchips. 2.3.2

Moving Liquids in Miniaturized Systems

Pumps are devices to set fluids into motion. Many of these devices have been developed based on a large variety of principles. Interesting reviews can be found for wider information about pumping devices in microfluidics [20–22]. In this section we have selected some representative examples, and Chapter 4 covers the miniaturized systems based on a hydrodynamic flow. A special case is pumping fluids by electro-osmosis, which is briefly described in this chapter and developed in more detail in Chapter 5, where the miniaturized systems based on an electro-osmotic flow are reported. Following recent reviews on micropumping techniques, pumping mechanisms can be classified as shown in Table 2.2. Two main categories can be identified. The first is mechanical displacement micropumps, defined as those that exert oscillatory or rotational pressure forces on the working fluid through a moving solid–fluid (vibrating diaphragm, peristaltic, rotary pumps) or fluid–fluid (ferrofluid, phase change, gas permeation pumps) boundary. The second category, nonmechanical micropumps, covers electro- and magnetokinetic micropumps, defined as those that provide a direct energy transfer to pumping power and generate constant/steady flows by the continuous addition of energy (electro-osmotic, electrohydrodynamic, magnetohydrodynamic, electrowetting, etc.) [21]. Micropumps in the above categories can be further divided into subcategories based on their actuation principle. As A. van den Berg et al. have stated [23], the flow of fluids within enclosed channels of glass, silicon or plastic can be actuated spontaneously, nonmechanically or mechanically. As the hydrodynamic resistance increases due to confinement, the most frequently used pumping method (syringe pumping) is increasingly difficult to use in micro(nano)channels due to the reduction in dimension. Mechanical Micropumps These are truly microfabricated pumps, used for moving liquids in microsystems. They need to meet different basic requirements to assure accurate analytical results:

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Table 2.2 Classification of micropumps with different actuation methods Category

Mode of operation

Type

Mechanical micropumps

Diaphragm

Electrostatic Piezoelectric Electromagnetic Thermal Pneumatic Polymer materials Peristaltic Ferrofluid Phase change Gas boundary Rotating gear Viscous force Induction Injection Polarization Ion drag DC AC

Fluid Rotary Nonmechanical micropumps

Electrohydrodynamic

Electroosmotic Magnetohydrodynamic Electrowetting Others

. . . .

Flexural plate wave Optoelectrostatic Bubble type

stability, with little pulsation; controllable flow rate at a preset range; resistance to aggressive reagents and high temperatures; reasonable cost.

Sometimes these requirements are extremely hard to meet, or imply the substitution of the traditional silicon by alternative materials for the microfabricated pumps. Normally, these pumps use the same type of actuators as active valves. The combination of the actuators chosen for the pumping and the valves determines the overall performance, in terms of generated pressure, stroke displacement, response time and reliability. The most common version of a mechanical micropump consists of a pump chamber and two valves: an inlet valve and an outlet valve. The pump chamber is mostly a combination of a diaphragm or membrane and an actuator to displace the membrane or diaphragm. This displacement, together with the action of the valves, results in alternating flux into and out of the chamber (pumping action). Both passive and active microvalves can be used in these arrangements. These types of micropump employ many different actuation mechanisms, as Table 2.2 shows. Figure 2.5 gives some selected examples of mechanical displacement micropumps.


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Figure 2.5 Examples of different types of mechanical micropumps: (a) vibrating diaphragm micropump; (b) peristaltic micropump; (c) dynamic, active diaphragm valve; (d) principles of operation of different rotating microvalves. (Reprinted from [21] with kind permission from Springer)

The first (Figure 2.5(a)) is the scheme of a simple diaphragm displacement micropump, as described above. During the expansion stroke, the pumping chamber expands, resulting in a corresponding decrease in chamber pressure. When the inlet pressure is higher than the chamber pressure, the inlet valve opens and liquid fills the expanding chamber. During the compression stroke, the volume of the chamber decreases with the moving diaphragm, causing the internal pressure to increase, whereby liquid is discharged through the outlet valve. The different types of actuation mechanism reported in Table 2.2 can be used to vibrate the diaphragm. One of these possibilities is a peristaltic mechanism. Pumps based on this principle incorporate the peristaltic motion of actuators in series to generate pumping action (Figure 2.5(b)). Thus, they can be considered a subset of the vibrating diaphragm pumps, utilizing the different types of transducer (piezoelectric, pneumatic, etc.). When the first diaphragm is actuated, it restricts the flow to the inlet of the pump. As the second diaphragm is actuated, fluid is pushed toward the third pumping chamber. Similarly, actuating the third diaphragm in succession pushes the fluid through the outlet of the pump. This sequence is continuously repeated for pumping

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action from left to right. Recently, L.-S. Jang and Y.-Ch. Yu have presented a driving system for a peristaltic micropump that is based on piezoelectric actuation [24]. This micropump with an integrated driving system is portable and has the potential to be integrated with other components, such as biosensors. Y.-C. Hsu et al. have developed an interesting peristaltic antithrombogenic micropump for in vitro and ex vivo blood transportation tests [25], very useful for studies in clinical environments. Dynamic-geometry valves can also be used as pumping microdevices, as they provide flow direction by deformation, motion or deflection. Figure 2.5(c) shows the schematic functioning of such active valves working as pumping devices. Traditional rotary micropumps constitute another category of mechanical micropump, in which a toothed gear rotating in a fluid chamber with an inlet and an outlet port produces the fluid flow. However, there are several additional micropumps in which the fluid is driven with a rotating component (internal or external). Figure 2.5(d) shows different possibilities based on rotating-gear and viscous-force pumping mechanisms. Typically, rotating-gear micropumps drive the finned or toothed gear with an electric motor for rotation. Fluid becomes entrapped between the gear teeth while turning and thereby is transported from the inlet to the outlet position. Fluid displacement using viscous forces generated by a rotating component has been also developed, using different configurations, as Figure 2.5(d) indicates. Eccentric placement of a rotating shaft in a straight channel is one of the possibilities. When the cylinder rotates, a net force is transferred to the fluid due to unequal shear stress on opposite sides of the rotor. Spiral-channel viscous pumps operate in a similar way to Couette flow, in that the movement of the boundary induces viscous stress on the fluid near the wall. As a final example (Figure 2.5(d)), disc viscous micropumps have been developed, in which the flow is generated by the rotation of a disc, which also acts as a boundary to the channel flow. Many other micropumps based on mechanical operation modes have been described. Table 2.3 briefly summarizes the main actuation forces involved in the most common mechanical micropumps. Nonmechanical Micropumps Nonmechanical micropumps require the conversion of nonmechanical energy to kinetic energy to supply the fluid with momentum. This phenomenon is practical only at the microscale level. In contrast to mechanical micropumps, nonmechanical pumps generally have neither moving parts nor valves, so geometry design and fabrication techniques in this type of pumping in microsystems are relatively simpler. These micropumps directly convert electrical and magnetic forms of energy into fluid motion. Since these pumping processes occur in a continuous manner, the resulting flow is generally quite constant. Whereas electrokinetic pumps often utilize an electric field to pull ions within the pumping channel,

Based on the Coulomb attraction force between oppositely charged plates. The membrane is forced to deflect in either direction as appropriate voltage is applied on the two opposite electrostatic plates located on each side. A piezoelectric disc attached on a diaphragm, a pumping chamber and valves. Actuated by the deformation of the piezoelectric materials. The actuation involves the strain induced by an applied electric field on the piezoelectric crystal. A chamber which is full of air inside is expanded and compressed periodically by a pair of heater and cooler devices. The periodic change in volume of chamber actuates the membrane with a regular movement for fluid flow. Consists of soft magnetic cores activated by currents in energized coils, or of permanent magnets. Electromagnetic actuation requires an external magnetic field, usually in the form of a permanent magnet. Based on the differences in thermal expansion coefficients of materials. When dissimilar materials are bonded together and subjected to temperature changes, thermal stresses are induced, providing a means of actuation. The diaphragm is made of two different metals, which exhibit different degrees of deformation during heating. Micropumps make use of the shape memory effect in SMA materials such as titaniumnickel. The shape memory effect involves a phase transformation between two solid phases. Actuated by stress gradient by ionic movement due to electric field. ICPF is composed of polyelectrolyte film, with both sides chemically plated with platinum. Due to the application of an electric field, the cations included in the two sides of the polymer molecule chain will move to the cathode. At the same time, each cation will take some water molecules to move towards the cathode. This ionic movement causes the cathode of the ICPF to expand and the anode to shrink. When there is an alternating voltage signal, the film bends alternately.

Electrostatic

Ion-conductive polymer film (ICPF)

Shape memory alloy (SMA)

Bimetallic

Electromagnetic

Thermopneumatic

Piezoelectric

Foundation

Type of actuation

Table 2.3 Common actuation forces involved in mechanical micropumps

[47,48]

[45,46]

[44]

[40–43]

[37–39]

[30–36]

[26–29]

Selected references

Tools for the Design of Miniaturized Analytical Systems 55

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magnetokinetic pumps typically utilize the Lorentz force on the bulk fluid to drive the microchannel flow. Due to these characteristics, in strict terms these ‘pumps’ are mechanisms to produce the movement of flow in microchannels without the existence of pumping devices as in mechanical micropumps. Electrohydrodynamic pumping This modality utilizes electrostatic forces acting on dielectric liquids to generate flow. The different types of electrohydrodynamic pumping (Table 2.2) are based on the method by which the charged particles are introduced into the fluid. Thus, induction pumping requires a gradient in either the electrical conductivity or the permittivity of the working fluid. This is typically achieved by anisotropic fluid heating or by discontinuities in properties, which occur for layers of nonmixing fluids or suspended particles in the fluid. Alternating voltages are imposed on the electrodes present on the boundary of the fluid channel. These voltages vary in time, creating a travelling wave that moves through the working fluid, perpendicular to the gradient in conductivity. By contrast, in micropumping by injection, electrochemical reactions at the electrodes cause the injection of free ions into the bulk liquid. These ions experience Coulomb forces due to the presence of the electric field. This causes the movement of ions, which in turn carry the bulk fluid with them. In the polarization type of micropumping, a nonhomogeneous electric field is used through the working fluid to create a variation in the fluid electric field density, causing the pumping action. Electro-osmotic flow Electro-osmotic flow (EOF) [49] is the liquid flow originating in the presence of an electric field when an ionic solution is in contact with a charged solid surface. Oppositely charged ions in the fluid shield the surface charge and can be manipulated with a DC or AC electric field. DC electro-osmotic pumping is normally used, particularly for electrophoretic separtions [50], and is described in more detail in Chapter 5. Therefore, only a basic treatment is given in this section. To give a well-known example, in a silica material in contact with an aqueous electrolytic solution the solid surface has an excess of negative charge due to the ionization of the surface’s silanol groups. A high number of counterions of these anions are found on the interphase between the capillary wall and the solution originating the so-called electric double layer, which is formed by a stagnant layer adjacent to the capillary wall (Stern layer) and a mobile diffuse layer (Gouy– Chapman layer) (see Figure 2.6). The cationic counterions in the diffuse layer migrate toward the cathode. As they are solvated, they drag solvent with them, originating the EOF. The linear velocity of the EOF, veo, depends on the potential originated across the double layer and the so-called zeta potential, z, through the following equation: veo ¼

eEz ; 4ph

ð2:19Þ

where e is the dielectric constant of the solution. Since e, z and Z depend on many properties of solids and liquids, a great number of experimental variables control the


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Figure 2.6 Formation of the double layer and generation of the EOF. C, electrical potential; z, zeta potential. (Copyright 2000 Agilent Technologies. Reproduced with permission)

EOF. The extremely small size of the double layer leads to flow at the walls of the capillary, resulting in a flat profile which enables high separation efficiencies with respect to those separation techniques with hydrodynamic flow profiles (pressuredriven techniques). EOF modifies the migration of a species, since if it moves in the same direction as EOF its velocity will increase, and if it moves against the EOF its velocity will decrease. Therefore, it is possible to create a flow by filling a microchannel with a buffer solution and applying a suitable voltage at the channel ends. This is an easier way to move fluids in microchannels than the use of micropumps, but the particular flow profile resulting from EOF is another interesting advantage over hydrodynamic flows. In fact, contrary to hydrodynamic flows characterized by a parabolic distribution of the flow velocities, EOF is generated close to the wall. Therefore, it produces a plug-like profile with a very uniform velocity distribution across the entire cross-section of the channel. Probably the main disadvantage of EOF is its strong dependence on the chemical factors involved in the microsystem. This dependence makes EOF hard to control, as any change affecting zeta potential has a direct effect on the EOF. The use of buffers allows some control of EOF. EOF can also be manipulated by changing the pH and by coating the walls of the capillary. Thus, at low pH, electro-osmotic flow reduces in magnitude, and it can be suppressed totally for lower pH values; the permanent or periodical coating of internal walls of the capillary with nonchargeable compounds cancels the

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electro-osmotic flow. Other major limitations of this pumping technique are the high voltage and the electrically conductive solution required. A wide variety of examples using DC electro-osmotic flow can be found. C.H. Chen and coworkers have described planar electro-osmotic micropumps [51,52], while L. Chen et al. have reported the fabrication and characterization of a multistage electro-osmotic micropump [53]. Other authors have developed microsystems with packed particles or monoliths using high-pressure electro-osmotic pumping [54,55]. Recently, F.-Q. Nie et al. developed a robust, compact, on-chip, electro-osmotic micropump for microflow analysis based on parallel, encased, monolithic silica capillary columns [56]. AC electro-osmotic flow has emerged as a viable microscale pumping mechanism for conductive or electrolytic solutions. Unlike the deprotonation on the channel surface for DC-EOF, electrodes positioned on the channel boundary provide the charge to establish the electric double layer in the AC case. The advantage of AC-EOF pumping is that relatively high velocities can be achieved with less than 10 V [57,58]. In addition, flow reversal can be achieved, making this approach bidirectional [59,60]. B.P. Cahill et al. have used this modality of EOF with interdigitated electrodes for portable lab-on-a-chip systems [61]. Other nonmechanical micropumping techniques Electrowetting involves wettability change due to applied electric potential. In electrowetting, the fluid is transported using surface tension (an interfacial force which dominates at microscale). Voltage is applied on the dielectric layer, decreasing the interfacial energy of the solid and liquid surface, which results in fluid flow. Thus, when applying the electric potential along the interface of a liquid metal (mercury, generally) droplet in an electrolyte, charge redistribution occurs, resulting in a gradient in surface tension at the interface, which causes movement of the droplet to regions of lower surface tension. Switching the direction of the applied potential also changes the direction motion. F. Mugele and J.C. Baret have reviewed electrowetting, including a discussion of the challenges of this technique and a review the state of the art [62]. Furthers works on models of droplet manipulation with electrowetting on flat and rough surfaces have also been carried out by V. Bahadur and S.V. Garimella [63,64]. The pumping effect in bubble-type micropumps is based on the periodic expansion and collapse of a bubble generated in microchannel. This modality always needs to be heated, so the application scope is limited in that heating may not always be desired or allowed. Geng et al. reported a bubble-based micropump for electrically conducting liquids [65], while Zahn et al. reported microneedles integrated with an on-chip MEMS bubble micropump for continuous drug-delivery applications [66]. In another article, Z. Yin and A. Prosperetti reported data obtained on a simple micropump based on the periodic growth and collapse of a single vapour bubble in a microchannel [67].


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Other, less common nonmechanical micropumping techniques are based on optoelectrostatic microvortex [68] and the use of flexural plate wave pumps [69]. A recent, more in-depth evaluation and discussion of all these micropumping technologies can be found in the review published by B.D. Iverson and S.V. Garimella [21], which includes exhaustive tables showing the main developments reported in the literature. The review published by A. Nisar et al. is mainly addressed to biomedical applications [22]. A systematic study of multiphase flow in microand nanochannels, taking special interest in multiphase confined fluid manipulation, has been reported [23] (systems in nature are rarely either single-phase or nonconfined). 2.3.3

Mixers

Mixing is one of the challenges in microfluidic devices. The goal is to obtain a homogeneous blend of two solutions in as little time as possible. Passive and active mixers can be distinguished by the strategy used to achieve the homogenous mixture. Passive mixing on microdevices depends solely on diffusion as the transport mechanism. Hence, to improve mixing using this alternative, a reduction of the diffusion distances or the introduction of some kind of extra movement needs to be considered. For active mixers on the other hand, external energy is used to induce turbulence. As C.-C. Chang and R.-J. Yang stated [70], electrokinetic mixing represents one of the most practical alternatives for mixing analytes and reagents in microfluidic systems, due to the difficulties derived from the low Reynolds number flows in microscale devices. Electrokinetic mixing approaches can also be categorized as either active or passive in nature (Figure 2.7). Active mixers use either time-dependent (AC or DC field switching) or time-independent (DC field) external electric fields to achieve mixing, while passive mixers achieve mixing in DC fields simply by virtue of their geometric topology and surface properties, or electrokinetic instability flows. Passive Mixers The simplest examples of passive mixers are T- and Y-junctions, where two different streams meet. At the macroscopic level, the mixture of the two streams is almost instantaneous, because of the presence of a turbulent flow. Nevertheless, in microchannels, the only lateral transport mechanism that allows the mixture is diffusion (Figure 2.8). Although different parameters affect diffusion, the main design parameters for effective mixtures are the width of the main channel (resulting channel) and the linear flow velocity. Thus, the worst-case diffusion distance corresponds to half the width channel, whereas the flow velocity determines the contact time between the two streams and therefore translates into a minimum length of the main channel at any given flow velocity. By an opposite approach, it is possible to select the conditions (channel width, flow velocity and channel length)

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Figure 2.7 Classification scheme for microfluidic mixing based on electrokinetic mixing. (Reprinted from [70] with kind permission from Springer)

in which hardly any mixing occurs and in which the two streams flow almost unperturbed side by side down the channel. Hence, for practical purposes, channel width, flow velocity and main channel length are decisive parameters for designing microfluidic systems. However, the fabrication of narrow and deep channels requires special machining technology, and they introduce a higher fluidic resistance. One possible strategy is to work with standard-sized channels, split each channel into an array of smaller channels and then merge them again in such a way that the split flows of one solution get interlaced with the split flows of the other. Electrokinetically passive mixing is enhanced by the particular geometry topologies, surface properties or instability phenomena which occur naturally under a static (DC) electric field. According to the classification shown in Figure 2.7, electrokinetic passive mixing can be categorized as either lamination or chaotic mixing [70]. In the former case, mixing mainly relies on the molecular diffusion effect between two or more parallel streams, while the latter usually involves the interfacial contact area between two mixing streams. This area is greatly increased and the diffusion length is reduced by means of the repeated stretching and folding


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Figure 2.8 Scheme of two mixer/filtering microdevices. (a) a T-junction where two liquids meet and flow downstream side by side, slowly mixing by diffusion only (laminar flow); (b) mixingfiltering microdevice in which laminar flow and diffusion are involved

of the fluids in a microchannel. The previously mentioned T- or Y-shaped mixing configurations are, in fact, a type of laminar mixing based on multiple parallel electro-osmotic streams (mixing by molecular diffusion) [71,72]. More recent developments based on this principle can be found in the literature [73–75]. In contrast with multistream lamination mixing, split-and-recombine lamination mixing relies on a multistep procedure, in which three basic steps are required [76]: fluid element splitting, recombination and rearrangement. The three steps can be completed when the two mixing streams flow through an out-of-plane microchannel structure. Grooved surface-enhanced electro-osmotic mixing was proposed by Stroock et al. [77]. It consists of a chaotic mixing channel with patterned grooves for a pressure-driven flow system. Transverse flows can be produced in the grooved channels, which results in a helical flow motion at a low Reynolds number regime. On the other hand, a heterogeneous surface charge patterning can produce an enhancement in electro-osmotic mixing. This principle has been used by different authors for electrokinetic passive mixing in a variety of applications [78–81]. Further, the electrokinetic instability phenomenon produced in microfluidics [82] can be used as another passive mixing alternative. Despite limitations found with this modality, several uses have been reported [83–85].

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Active Mixers Whereas passive mixers rely solely on energy from within the system to facilitate mixing, active mixers can be designed in which external energy of some form induces chaotic behaviour to accelerate the mixture. This modality refers to the enhancement of mixing in electrokinetically-driven microfluidic systems using a time-dependent electric field, or in pressure-driven flow systems by means of an externally time-dependent or -independent electrical force. Chaotic mixing can be achieved by several different methods (Figure 2.7). In general, the choice of driving amplitudes and frequencies, and optimization of operation conditions, is a challenge in designing an active micromixer. Under appropriate operation conditions, the majority of electrokinetic active mixing schemes in the literature are able to induce chaotic mixing, with the exception of periodically-switching electro-osmosis mixing enhancement. Dielectrophoretic-based mixing refers to the polarization of a particle or biological element relative to a suspension medium in a nonuniform electric field which results in motion [86]. Positive dielectrophoretic forces move particles to regions with a higher electric field, while the particles are repelled from the electrode edges to a lower electric field by negative dielectrophoretic forces. Positive or negative dielectrophoretic force is only induced by applying the specific range of the frequency of the AC field. Electrowetting-on-dielectric droplet-based mixing is based on the wetting properties of a droplet in contact with an insulated electrode, which can be altered by means an electric field. If an electric field is applied nonuniformly on an electrode array, a surface energy gradient is induced, which can be used to manipulate the motion of the droplet [87,88]. Micromixing by electrohydrodynamic instability generally refers to the force generated by the interaction between electric field and net charge density near the solid–liquid interfaces. A microfluidic mixer has been developed by El Moctar et al. utilizing the electrohydrodynamic instability phenomenon [89]. Oddy et al. first presented an active micromixer in which an AC electric field induces a more chaotic flow field to enhance the mixing of two pressure-driven flow streams [82]. A highintensity AC electric field from side channels was still required to induce electrokinetic instability to enhance mixing in this work. Many researchers have investigated a periodically switching electro-osmotic mixing approach for an efficient active mixing [90–93]. It is performed by in some way pulsing the velocities or pressures of two pressure-driven flow streams working at low Reynolds number. Field-induced electro-osmosis enhanced mixing, presenting various modalities, has also been widely applied to the active control of electroosmotic flows and liquid pumping in microchannels, but only scarcely applied in micromixing [94,95]. A novel strategy for mixing solutions and initiating chemical reactions in microfluidic systems has been proposed by A.N. Hellman et al. [96]. This method utilizes highly focused nanosecond laser pulses form a Q-switched Nd:YAG laser


63

to generate cavitation bubbles within 100 and 200 mm-wide microfluidic channels containing the parallel laminar flow to two fluids. The bubble expansion and subsequent collapse within the channel disrupts the laminar flow of the parallel fluid streams and produces a localized region of mixed fluid. This approach to generating the mixing of adjacent fluids may present advantages in many microfluidic applications, as it requires neither tailored channel geometries nor the fabrication of specialized on-chip instrumentation. 2.3.4

Volume-dispensing and Sample-introduction Devices

A very important function in microfluidics is the ability to dispense very welldefined small amounts or volumes of solutions. In practice, this means introducing a volume whose size is not exactly known but which is always the same, or a volume whose size is exactly known and is given by a particular geometric arrangement of channel segments. The latter case is called ‘metering’. For many analytical techniques it is necessary to inject small, well-defined amounts of sample solutions. Popular implementations of an aliquoting or injection function on microchips make use of a channel cross or a double T arrangement, along with electrokinetic fluid manipulation, to achieve this objective. Section 5.5, in Chapter 5, deals with this method of introducing very small volumes into microchips. An overview of developments in high-throughput microfluidic sample-introduction techniques based on a capillary sampling probe and a slotted-vial array has recently been published [97]. These sample introduction systems have received attention in miniaturized flow injection analysis, sequential injection analysis, capillary electrophoresis and liquid–liquid extraction. Introduction of samples to a microfluidic chip requires an interfacing system between samples with volumes in the ml–ml range from the macro world, and microfluidic systems handling volumes in the nl–pl range. Without any doubt, world-to-chip interfacing is considered a major challenge. Q. Fang et al. [97] summarize the different possible modes of sample introduction for microfluidic chips (Figure 2.9). As these authors explain,

Figure 2.9 Different designs for microfluidic world-to-chip interfacing. (Reprinted from [97] with permission from Elsevier)

64


in mode A, sample and reagent solutions are loaded in the on-chip reservoirs connected with microchannels. This approach has been employed frequently in most reported chip-based analytical systems. It presents a high degree of integration, but the sample-change operation, which usually requires the sample reservoir to be manually emptied, rinsed and refilled with a new sample solution, is timeconsuming and difficult to automate. Mode B (Figure 2.9) is characterized by employing a split-flow interface fabricated on the chip to achieve continuous sample introduction. Different sample solutions are sequentially delivered to the chip via connecting tubing through the split-flow interface. Various designs have been reported based on this interfacing model [98–100], allowing a high throughput, and continuous and automated sample introduction. Mode C (Figure 2.9) involves the use of an on-chip sampling probe for continuous sample introduction, which replaces the on-chip sample reservoir in mode A and the sample-introduction channel in mode B. In this case, a sampling probe is directly connected to the chip microchannel, and sample introduction is performed simply by inserting the inlet of the sampling probe into an off-chip sample vial [101]. The main problem with this mode is that sample-change operations are manually performed by moving the inlet tip of the sample inlet tubing from one sample vessel to the next. Some improvements have been achieved by Q.-H. He et al. with the fabrication of a monolithic sampling probe system for automated and continuous sample introduction in CE microchips [102]. Mode D (Figure 2.9) is characterized by the use of autosamplers based on x-y-z stages to automate sample introduction [103]. Although these approaches are very effective, the cost and size of the systems are significantly increased. Other dispensing devices have recently been developed. C.K. Byun et al. have reported an electro-osmosis-based nanopipettor [104]. This consists of a microfabricated electro-osmotic flow pump, a polyacrylamide grounding interface and a nanoliter-to-picoliter pipet tip. One advantage of this nanopipettor is that it has no moving parts, and it works with good levels of accuracy and precision. M.L. Kovarik and S.C. Jacobson reported an attolitre-scale dispensing unit for nanofluidic channels [105]. This dispenses to the nanochannels volumes at the fl range. The transport was electrokinetically produced by applying up to 10 V directly from an analogue output board. 2.3.5

Detection Systems for Analytical Microsystems

Although detection in miniaturized systems is the subject of Chapter 6, this section exists to complete the list of the basic microdevices involved in analytical microfluidic systems. Section 2.6.3 also includes detection in MEMS. The most common detection systems used in microfluidics are based on optical and electrochemical sensing. B. Kuswandi et al. recently reviewed the optical sensing


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systems for microfluidic devices [106]. They distinguished between the coupling approach (off-chip approach) and the integration approach (on-chip approach). The off-chip approach combines macroscale optical detection with micro-sized detection areas by using pinholes at focus points along the optical path or optical fibre technology. Although very low levels of background signal can be achieved, the reduction of the path length within the device can decrease the sensitivity of the method. Absorbance, fluorescence and chemiluminescence are the most common detection modes. On the other hand, the integration of optical components or functions in a microfluidic platform that should be able to perform all chemical functions and detections in a single device requires increased integration of not just fluidic elements, but also electrical or other types of elements (on-chip approach). MEMS have demonstrated the integration of mechanical and electrical functionalities into small structures for diverse applications (Section 2.6.3). The development of optofluidic technology, as the fusion of microfluidics and optics (the use of optical microscopy to study microorganisms in aqueous solutions, the common use of optical methods to analyse liquids and liquid solutions, etc.), has opened interesting possibilities [107]. The development of a large body of research to accommodate this knowledge at a miniaturized scale has produced a variety of elements allowing real optofluidic integration for microanalysis [108]. Electrochemical detection is another powerful tool for detection in microchips. This offers great advantages in realizing the lab-on-a-chip concept, such as inherent miniaturization, low power requirements, low limits of detection and compatibility with advanced micromachining systems. A wide variety of materials have been described for electrochemical sensing in microfluidic chip platforms [109]. Amperometric, conductimetric and potentiometric modes are the normal detection alternatives in microchips, although the first is clearly the most common. Amperometric detection on a microfluidic chip platform is typically accomplished using DC amperometry, which simply involves holding the working electrode at a particular potential sufficient to oxidize or to reduce the compounds of interest, and measuring the current that results. This method has proved to be sensitive, although a decoupler system must be used [110]. Conductivity detection is a simple and universal detection technique in miniaturized electrophoretic systems. This detection can be accomplished in electrophoretic systems either by direct galvanic contact of the run buffer and the sensing electrodes or by the contactless mode, in which the electrodes do not contact the solution. Potentiometry on a microfluidic chip platform is applied less as an electrochemical technique, most probably because microfluidic chips usually contain an inherent separation step (generally by electrophoresis or electrochromatography), so there is no need for discrimination of one analyte with respect to other components of the sample by ion-selective electrodes (ISEs).

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2.4 Microtechnology Following the description of the different microfluidic devices in the previous section, microtechnology must now be reviewed in order to explain the integration of the different elements, at microsystem scale, to produce different layout designs. The journey from electronics to microelectronics can be seen as following a similar evolution to that from fluids to microfluidics. Thus, the main milestones in the electronics–microelectronics evolution (invention of the transistor, improvements in semiconductor technology, introduction of the integrated circuit, etc.) have been used to inform a general strategy toward fabricating miniaturized systems in other disciplines (mechanics and optics, for instance). Microelectromechanical systems (MEMS), described in Section 2.5, are gaining more and more analytical interest through the development of microcantilevers. Microfluidics is another area of interest, following the introduction of the mTAS concept by Manz and coworkers in 1990, as was described in Chapter 1. It is necessary to briefly describe the manipulation of small amounts of samples and reagents on microchip, which allows the fabrication of microsystems for chemical analysis in silicon, glass and plastics, the packaging of such microsystems and the management of detection. 2.4.1

Computer Simulations in Microfluidics

As the fabrication of microsystems is very complex and requires infrastructure facilities (particularly cleanroom facility) and the participation of experts in different scientific and technological fields, computer simulations are extremely convenient. Simulations do not only provide a more complete understanding of the physical and chemical processes involved in the microsystem, but are very useful for the optimal development of the designs and for minimizing the risk of fabricating unsuccessful materials. G. Goranovic and H. Bruus have systematically described the different aspects (and stages) necessary to appropriately implement simulations in microfluidics [111]. A brief summary is given below. Physical Aspects and Design Different aspects must be taken into account: Dimensions: A microsystem is a network of fluidic channels and other additional components occupying an area of about one square centimetre, with channel widths typically on the order of 100 mm. Geometry: The basic component of a microfluidic network is the channel. Its main features include length, cross-section and surface properties. After a channel is made in a substrate, it is covered with a bonded lid, which can be made of different materials.


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Surface: Chemical groups at the surface of channel walls, such as silanol (SiOH) groups in glass, can react with the ions in an electrolyte solution and create a very thin polarized layer, creating an EOF in an applied electric field (as explained in Section 2.3.2). Fluid Properties: Density, viscosity, electrical and thermal conductivity, diffusion coefficients and the surface tension of the liquids filling the channels are important to take into account. Heating: Joule heating is generated when an electrical current flows through a channel. In some applications, such heating may cause a negative effect in the samples analysed or the systems studied (e.g. biological samples). If this is the case, heating should be taken into account in the simulation. Software and Hardware Requirements Software packages are essential for simulations. Capability and price are the basic guidelines for selection. CFDRC (www.cfdrc.com) offers one package, a patent named Integrated Microfluidic System Design Using Mixed Methodology Simulations. This is a simulation-based method for rapidly designing, evaluating and/or optimizing complex microfluidic systems and biochips. It simulates processes including pressure-driven and electro-osmotic flows, electrophoretic separation, analyte dispersion, mixing of analytes, heat transfer, electric and magnetic fields, and biochemical reactions in order to complete the design of a particular microsystem. Coventor (www.coventor.com) offers another software package specifically oriented toward microtechnology, including MEMS and microfluidics. Coventor 3D MEMS software presents three different products. CoventorWare is used for MEMS R&D, design and product development. MEMulator enables MEMS designers and process engineers to visualize the effects of a design and process modifications in 3D before fabricating an actual device. EM3DS is used for electromagnetic modelling. All these packages describe the hardware requirements for their operation. Any commercial computational fluid dynamics package typically consists of a pre-processor, a solver and a post-processor. The pre- processor is the interface between the user and the numerical solver. Different factors must be considered before the solver can be started. Thus, geometry, grid generation, model, fluid parameters, boundary conditions and initial conditions have to be fixed and/or defined [111]. Numerical parameters must be adjusted so that solvers can solve the problem at hand most efficiently. Solvers are numerical algorithms intended to solve the governing equations. Solver settings include type of solver, numerical schemes, number of iterations, convergence criteria, etc. Both surface boundary conditions and solver setting must be specified for the simulation. Solvers commonly work by following the finite-volume method, based on the concept of transport equations, or the finite-element method, which uses simple piecewise functions (for instance, linear or quadratic functions) to approximate the exact solution. The first method is

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used mostly for fluid dynamics, and the second is more suitable for mechanical stress analysis and simulations of MEMS. Finally, post-processors are used to visualize results, following different techniques. Reliability of the Microsystem It is very convenient, after describing the various stages of the simulation process, to summarize the numerical errors and uncertainties involved. In computational fluid dynamics, the following errors and uncertainties can be identified: Model Uncertainty: Real flow in comparison to exact solution of modelled equations. Discretization or Numerical Error: Exact solution with respect to the numerical solution of the equations. Iteration or Convergence Error: Fully converged compared to not fully converged solutions. Round-off Errors: Parameter values that are below machine accuracy. Application Uncertainties: Lack of available data. User Errors: Mistakes and carelessness. Code Errors: Bugs in the software. Interpretation and Evaluation of Simulations Correct interpretation of numerical results is one of the most important issues, because of the complexity of simulations. In particular, the visualizing capabilities of computational fluid dynamics programs are of special importance, as well as their complete understanding by the user. The definitive test for the simulation results is the experiments: thus, one can see to what extent the simulation results represent reality. The problem is that in microfluidic systems it is rather difficult to make detailed measurements, because of the small size of these systems. One experimental possibility is the use of fluorescent tracers and a CCD camera to monitor the position of the tracer in two consecutive instants by illuminating it with a light source. A nonexperimental alternative is to compare the simulation with well-established theoretical models, such as the equivalent circuit theory. This theory describes a fluidic network as an equivalent electrical network by expressing a linear relationship between a pressure drop (equivalent to a voltage drop) and the corresponding flow rate (equivalent to a current) [112]. 2.4.2

Micromachining

After the design, to particular specifications, and the revision of the computer simulation, fabrication of the microsystem is carried out. Two main aspects have to be considered: the material for the fabrication and the specific facilities required (mainly the cleanroom processing with the appropriate tools). Without any doubt,


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silicon is the most common substrate because it is a well-known material, it can be obtained in the highest purity (99.99999%) and it is of great interest to the semiconductor industry. Nevertheless, other materials such as glass and polymers are also used to fabricate analytical microsystems. Silicon and Silicon-compatible Materials Silicon substrates are used in many of the devices fabricated so far for analytical microsystems, and probably will continue to be used to a large extent in the future. It is the most-studied material and its micromachining is at a very advanced state. The substrates used are almost exclusively single-crystalline silicon, in the format of disc-like wafers (normally with a thickness of between 200 and 650 mm and a diameter between 75 and 150 mm). Wafers are usually cut to give top surfaces that roughly correspond to the main crystal planes in silicon, given by the Miller indices (100), (110) and (111). After cutting, the wafers are subjected to a lapping step, to give a surface roughness of about 0.5 mm and to remove about 50 mm of material from both sides of the wafer. Then one of the sides is further polished, using finer mechanical procedures, to produce a surface roughness commonly between 10 and 100 nm. Wafers are characterized by several parameters, conforming to different types of specifications of interest for the particular application to be developed. After the substrate preparation, a set of steps has to be followed to produce the silicon wafer at the packaging level [113]: . . .

optical lithography; deposition; etching/removal.

Lithography is the technique used to transfer a computer-generated pattern on to a substrate, such as silicon, but also glass or other materials. Although photolithography is the most common lithography technique in microelectronic fabrication, electrobeam (e-beam) and X-ray lithography are in progressive use in the MEMS and nanofabrication areas. Figure 2.10 summarizes the main steps in a photolithography process. Most of the fabricated microstructures contain materials other than that of the substrate, which are obtained by various deposition techniques or by modifying the substrate. Most of the thin films deposited have different properties to those of their corresponding bulk substrate. Different thin-film deposition and doping strategies have been reported, such as surface oxidation (film of SiO2 from Si), doping with certain impurities (for instance, boron to form p-type regions; phosphorus or arsenic to form n-type regions), chemical vapour deposition, physical vapour deposition (evaporation and sputtering), electroplating and pulsed laser deposition [114]. Thin-film and bulk-substrate etching is another fabrication step that is of fundamental importance to both the very large-scale integration (VLSI) processes and micro/nanofabrication. Thus, various conducting and dielectric films deposited

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(a)

(b)

1 – Oxidize the substrate SiO2

1

Silicon substrate

2

Deposit thin film

3

Spin photoresist

Substrate 2 – Spin the photoresist and soft bake Photoresist Substrate 3 – Expose the photoresist Light

4

Soft bake

5

Align the mask

Photomask

Substrate

6

Expose the waver

7

Develop the resist

4 – Develop the photoresist and hard bake

Substrate 5 – Etch the oxide

8 9

Hard bake

Substrate 6 – Strip the photoresist

End of lithography Substrate

Figure 2.10 (a) Main steps in a photolithography process, and (b) scheme of a photolithography with a positive PR

for passivation or masking purposes need to be removed at some point. The different etching techniques can be grouped into wet and dry categories. J.L. Perry and S.G. Kandlikar have reviewed the fabrication methods of nanochannels for single-phase liquid flow [115]. They distinguish four alternatives: (i) blulk nanomachining and wafer bonding; (ii) surface nanomachining; (iii) buried channel technology; and (iv) nanoimprint lithography. In bulk nanomachining and wafer bonding, features are created of the bulk of a silicon wafer. This can be done by reactive ion etching (RIE) or by a wet anisotropic etchant with aqueous KOH- or ethylenediamine-based solutions. Figure 2.11(a) describes this process, where wet anisotropic etching of one-dimension nanochannel is performed [116]. A (110) silicon wafer is used, which has a thin native oxide. The wafer is then lithography patterned and the oxide mask is etched with an HF solution. The silicon is anisotropically etched at an elevated temperature by a developer solution, which is essentially a water-dilute solution of TMAH (tetramethyl ammonium hydroxide). Next, the oxide mask is stripped and bonded to a borofloat glass wafer. Nanochannels can also be fabricated by surface nanomachining. This involves embedding the structures in a layer of appropriate sacrificial material on the surface of the substrate. The sacrificial material is dissolved, leaving a complete nanochannel.


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Figure 2.11 (a) Fabrication process for bulk nanomachining with wafer bonding (left side, reproduced from [115] with permission from Springer, Copyright Springer) and cross-section of a silicon wafer with a 50 nm deep channel bonded to a borofloat wafer (right side, reproduced from [116] by permission of IOP) following this fabrication technique. (b) Schematic cross-section of an a-Si nanochannel array fabricated by Stern et. al [117] (left side) and the corresponding picture showing the surface nanomachined channels of 0.5 mm wide 100 nm high and 1 mm wide 100 nm high (right side), Copyright Springer.

Figure 2.11(b) is a schematic cross-section of an amorphous Si (a-Si) nanochannel array made by Stern et al. [117]. First, a thick layer of thermal oxide is grown for the electric isolation of electronic devices. Then a set of 0.05 mm microlayers (tetraethyl ortho-silicate, TEOS; Si3N4) are put down to form the lower-channel dielectric layer. These alternating layers help maintain the channel dimensions because each imposes an opposite thin-film stress (the TEOS layer is compressive, while the nitride layer is tensile). Afterwards, a thin a-Si film of nanometer thickness is grown in a furnace. The a-Si is patterned lithographically to define the nanochannels. The Si3N4 film below the a-Si is used as an etch stop during chemical wet etching. The top-channel dielectric layers are then deposited over the patterned a-Si layer and capped with a thick phosphosilicate glass (PSG) to protect the structure during channel etching. Additionally, reservoir regions are created at the ends of the nanochannels. Buried-channel technology consists of a set of steps, as Figure 2.12 shows [118]. In step 1, a bare substrate is covered with a suitable masking material and

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Figure 2.12 Fabrication sequence for conduit using buried channel technology (left side) and a picture of microchannels formed with this technology [118] (right side). Channels are closed with silicon nitride, which is buried underneath the silicon surface. Figures selected from reference [115]. SPRINGER-VERLAG

lithographically patterned. An isotropic etchant is then used to make a rounded-out feature (step 2). Then a trench is etched in the substrate by deep RIE (step 3). In step 4, the trench is conformally coated with a material to prevent lateral etching of the sidewalls in step 6. In step 5 (Figure 2.12), the coating is removed only at the bottom of the trench, and the structure is etched in the bulk of the substrate again with an isotropic etchant (step 6). After stripping the coating in step 7, the structure is closed by filling the trench with a suitable material, as step 8 shows. For nanoimprint lithography, the fabrication of a previous mould (by lithography, mainly) is necessary. Then two basic steps are followed (Figure 2.13(a)). In the first (the imprint step), a mould with nanostructures on its surface is pressed into a thin

Figure 2.13 (a) Schematic of nanoimprint lithography process and (b) comparison between the conventional and the template approaches. (c) Pictures of nanoimprinted fluidic channels with template used to enclose the nanochannels [120]. Figures selected from reference [115]. SPRINGER-VERLAG


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polymer on a substrate. This step duplicates the nanostructures on the mould in the polymer film. The second step is the pattern transfer, where an anisotropic etching process is used to remove the residual polymer in the compressed area. This step transfers the thickness contrast pattern into the entire polymer. As was pointed out by J.L. Perry and S.G. Kandlikar [115], who presented an interesting comparison of these nanochannel fabrication methods, one last important aspect necessary for the fabrication of working nanofluidic systems is the enclosing of the channels. This is not an easy task, and several different sealing techniques to close up nanochannels have been developed. Thus, for instance, Cao et al. sputter silicon dioxide over the nanochannels [119], whereas Guo et al. have developed a more practical solution [120]. Their technique simply imprints a channel template into a thin polymer film while on a glass substrate. This makes it easy to control the nanochannel dimensions by a simple relationship involving the initial polymer layer thickness and the mould pattern configuration. Figure 2.13(b) compares the typical nanoimprint lithography process with the template approach. This fabrication process can be controlled to give predictable channel heights, as shown in Figure 2.13(c). Other Substrates Used in Analytical Microsystems Glass and polymers can form alternative substrates to silicon for microsystems fabrication. Glass is a monolithic noncrystalline solid consisting mainly of silicon dioxide (SiO2). Glass often contains additives or impurities that modify properties such as mechanical stability and the glass transition temperature. Glass can be used instead of silicon in analytical microsystems in chip formats because of its unique properties: it is resistant to many chemicals, optically transparent (optical detection and visual inspection are allowed) and a dielectric material (useful for creating electrokinetic-driven flows and separations). In addition, glass presents other advantages such as its hardness, high thermal stability and relative biocompatibility (applications in bioanalysis). Borofloat glass and quartz wafers are most commonly used as they are compatible with many cleanroom processes. Some traditional silicon microfabrication techniques, such as photolithography and wet chemical etching, have been adapted to glass processing. On the downside, micromachining of glass is less versatile than that of silicon, due to its noncrystalline structure and our limited experience with glass as a material for microsystems. In some cases, polymers are better suited to fabricating microsystems than is silicon. Many types of polymer show better resistance to chemical treatment and better biocompatibility. Moreover, they can be produced at significantly lower cost than silicon microsystems. There are a wide variety of plastics, with very different characteristics, including transparency in the ultraviolet (PDMS), visible (polymethylmethacrylate, PMMA; polycarbonate, PC) and infrared (polyetherimide) regions of the electromagnetic spectrum. The two major methods for machining polymers are replication from a master and direct machining. Replication methods

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often produce a microstructure by allowing a polymer workpiece to form an inverse copy of a mould. Direct machining methods remove small amounts of polymer in places where microstructures, such as microchannels or microwells, should be located. 2.4.3

Packaging of Microsystems

The functions of packaging are to protect the devices form the environment and the environment from the devices. The appropriate package is essential for an efficient operation of the microsystem. Aspects such as microfluidic interconnects (connections between microdevices, but also the interface between a macrocomponent and a microdevice), electrical connections, optical interconnections, and mounting and encasing devices that provide an appropriate interface to other devices or to the external environment, must be taken into account. Packaging also includes the protection of the microsystem from humidity and the possible introduction of foreign species, as well as the sealing techniques to assure hermeticity and structural integrity. A systematic treatment of microsystem packaging has been reported by G. Perozziello [121]. D. Erickson and D. Li reviewed the integrated microfluidic devices for a broad range of application areas [122]. More recently, G.T. Roman and R.T. Kennedy have published a review of fully integrated microfluidic separations systems for biochemical analysis [123]. In this review, the authors systematically discuss the integration of detection transducers into microfluidic devices, of pumps and power supplies for fluid manipulation, and of complete systems for biological analysis. In same cases, control of the assembly of microsystems is carried out by image processing [124]. A new fluidic interconnection approach for packaging of microsystems has recently been proposed by O. Geschke and coworkers [125]. They proposed a reversible, integrated fluidic interconnection composed of custommade cylindrical rings integrated in a polymer house next to the fluidic network. R. Lo and E. Meng have also described integrated and reusable in-plane microfluidic interconnects [126].

2.5 MEMS and NEMS The fabrication of micro- and nanoelectromechanical systems (MEMS and NEMS, respectively), has clearly been growing during the last decades. A wide variety of MEMS devices have been produced, and some are commercially used for different industrial, consumer and biomedical applications. From an analytical point of view, several types of sensor have been developed. Perhaps biosensors (BioMEMS and BioNEMS) and gas sensors have been given the most attention. MEMS and NEMS are an integration of mechanical elements, sensors and actuators, and electronics on a common silicon substrate through microfabrication technology [127]. The


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invention of scanning tunnelling microscopy (STM) in 1981, and later atomic force microscopy (AFM) in 1986 by Binning et al., were the first milestones in the development of nanoscience and nanotechnology, and examples of the use of MEMS and NEMS. In fact, STM and AFM are not simply microscopy tools, but are also extremely useful techniques for chemists and biochemists. In AFM, a microcantilever, which is the most simple MEMS-based device, measures the small force acting between a sample surface and a sharp tip at the apex of a thin beam clamped at its side. The force on the tip bends the cantilever, which acts as a force transducer. Cantilever-based devices have been shown to be highly versatile sensors using mechanical, optical, electrostatic and electromagnetic methods to actuate or sense cantilever motion in order to detect gases, chemicals or biological entities. They also have wide applications in the field of medicine, specifically for the screening of diseases, detection of point mutations, blood glucose monitoring and detection of chemical and biological warfare agents. Different issues must be taken into account to develop microcantilevers as miniaturized analytical sensors. These are briefly discussed below. 2.5.1

Fabrication and Characterization

Silicon, silicon nitride, silicon oxide and polymers are the main materials used in the fabrication of microcantilevers. Cantilevers are batch-fabricated using wellestablished thin-film processing technologies that provide low cost, high yield and good reproducibility. The fabrication techniques generally include thin-layer deposition, photolithographic patterning, etching, and surface and bulk micromachining. Usually, a sacrificial layer is deposited on a prepatterned substrate before the structural material of the cantilever is deposited. This structural layer must be free of a stress gradient, otherwise the cantilevers will have problems in initial bending. It is also possible to fabricate arrays of thousands of identical cantilevers on one wafer [128]. In most cases only Si is used as a starting/base material and for the large-size passive structures/components such as the pedestal of the cantilever, but the material of the components can be replaced by other materials with even lower cost that are easily machined and differently functioned. Thus Wakayama et al. [129] proposed a new type of cantilever consisting of Si beams (with a small slab) and a ceramic pedestal for AFM and gas-sensor applications. This new cantilever has a lower cost and better quality, by which they mean improved homogeneity and reproducibility, especially for the tip/stylus part [129]. Young’s modulus is a key factor that affects the spring constant of a cantilever, which is directly related to the properties of the cantilever material. Silicon or related materials were traditionally used to make cantilevers, but they have a high Young’s modulus. If cantilevers were made of a softer material, they would be more sensitive for static deflection measurements; hence, polymers with a much lower Young’s modulus than silicon have been used as a substitute material for the fabrication of

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cantilevers [130,131]. Among polymers, SU-8 has been shown to be very sensitive, exhibiting a Young’s modulus about 40 times lowers than that of silicon. Theoretically, SU-8 cantilevers can reach the same sensitivity as previously fabricated Si cantilevers, and metallic resistors are known to have a very low electrical noise. 2.5.2

Functionalization

The development and application of chemical and biochemical cantilevers has been one of the fastest growing fields over the last decade or so [132,133]. A key step in developing a sensor is the immobilization of sensing agents. A good immobilization method should meet the following requirements: (i) be simple and fast; (ii) be nonspecific, i.e. the method should be usable for the immobilization of various sensing agents; (iii) produce immobilized reagents that are stable and do not leach from the substrate; and (iv) produce immobilized reagents that retain their chemical and biochemical activities. There are three widely used methods for immobilization [134]: (i) adsorption of sensing agents on to a solid substrate; (ii) covalent binding, which involves the formation of permanent chemical bonds between sensing agents and a support; and (iii) encapsulation or entrapment of sensing agents within a polymeric matrix. The receptor layer deposited on the cantilever surface directly affects the selectivity, reproducibility and resolution. We want to deposit a thin (to avoid changes in mechanical properties of the cantilever), uniform (to generate a uniform stress) and compact (to avoid interactions with the solid substrate beneath) layer of receptor molecules. This layer should be stable and robust: hence the receptors should be covalently anchored to the surfaces; on the other hand, receptor molecules should have enough degrees of freedom to freely interact with their specific ligand in the environment. The receptor activity should last over time and possibly stand regeneration of the sensing layer if the sensor has to be reused several times. Most of these requirements are common to other sensors. In fact, the proposed coating techniques and procedures are shared with other transducing principles. Self-assembled Monolayers (SAMs) The use of the self-assembling properties of alkane chain molecules with thiol (-SH) groups on gold substrates [135] or silane (-SiOX) groups on silicon substrates [136] is an easy and popular method for creating monolayers on cantilever surfaces. They spontaneously form uniform, densely packed, robust (covalent binding) monolayers. They can be synthesized with different chain lengths and end groups with specific chemical properties. They are therefore ideally suited to act as cross-linkers to anchor the receptor molecule to the substrate. SAMs on gold substrates The most frequently used technique to prepare organosulfur SAMs is self-assembly from organic or aqueous solvents. The process


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simply involves immersion of a clean, gold-coated substrate into a solution of thiol, sulfide or disulfide. It is focussed on the use of the x-substituted alkanethiols, since they have been used most frequently. The effects of single-component x-substituted alkanethiolate SAMs on gold depend primarily on the temperature, the solvent used, the purity of the thiol, the immersion time and the quality of the gold substrate. The purity of the compound may at first be considered important when dealing with thiols since it is well-known that thiols readily dimerize to form a disulfide when exposed to oxygen. However, a small fraction of the corresponding disulfide in the solution is not critical for the formation of single-component monolayers, because disulfides are known to undergo S–S bond reductive cleavage and adsorb as thiolates on gold. As an example, a biotinylated silicon nitride cantilever has been reported [135]. One side was coated with 3 nm of chromium and 40 nm gold layer. The thin layer was used to increase the adhesion of gold to silicon nitride. The cantilever was incubated in a mixture of simple thiol alcohol and complex biotinylated analogue. A stable, dense and highly structured thiol monolayer on the freshly coated gold was then formed, while on the opposite side, physisorbed thiols were removed by rinsing the cantilever in ethanol. In this case, incubation of the whole cantilever structure in a thiol solution was suitable. If the deposition has to be on one side only, spraying or other multistep procedures can be employed. Ji et al. developed microcantilevers modified with a SAM to respond sensitively to specific ion concentrations [137]. The authors report the detection of trace amounts of CrO42 using microcantilevers modified with a SAM of triethyl12-mercaptododecylammonium bromide. The SAM was prepared on a silicon microcantilever coated with a thin layer of gold on one side. The predicted surface structure of the modified microcantilever used in this experiment is shown in Figure 2.14(a). When CrO42 was passed through the flow cell housing the modified cantilever, it was adsorbed on to the surface, displaced the halide ion and combined with the cationic ammonium group, forming a stable ionic association that altered the surface stress, causing a cantilever deflection. In other work [138], the same group demonstrated that the 1,6-hexanedithiol SAM on a gold coated cantilever acts as an unusually specific recognition agent for CH3Hgþ. Zuo et al. reported a novel SiO2 microcantilever-based sensor operated in static mode and functionalized with a self-assembled bilayer of Cu2þ/11-mercaptoundecanoic acid (11-MUA), which can specifically adsorb organophosphorus compounds [139]. This composite layer can specifically recognize P¼O-containing compounds with the formation of P¼O-Cu2þ coordination structure on the surface. SAMS on silane substrates Silanes are a group of molecules that consist of a silicon atom covalently attached to four variable groups. One or more of these groups can be readily substituted by the oxygen of a hydroxyl group at a silicon oxide surface. Thus, these molecules are capable of forming covalent bonds with a variety of glasses and silicon substrates. As stated, some of the most thoroughly

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Figure 2.14 Schematic representation of the functionalization of a microcantilever with (a) SAM on the gold surface; (b) SAM on the glass surface of the microcantilever; and (c) nanotubes on the microcantilever surface

studied and well-characterized SAMs in this category are prepared from alkyltrichlorosilanes (or chlorosilanes), e.g. octadecyltrichlorosilane (OTS). Other silanes commonly used include the aminosilanes, e.g. aminoethylaminopropyltrimethoxysilane (which is also known as trimethoxylsilylpropylethylene diamine). Maraldo et al. describe a silanization procedure with 3-aminopropyl-triethoxysilane (APTES) prior to the immobilization of activated antibody [140]. The procedural steps used to functionalize the cantilever sensor surface and activate the target antibody are schematically represented in Figure 2.14(b). The glass surface is sequentially cleaned with methanol–hydrochloric acid solution (1:1, v/v), concentrated sulfuric acid, hot sodium hydroxide and finally boiling water (step 1). After cleaning, the glass surface is silanylated with APTES in deionized water at pH 3.0 (adjusted by hydrochloric acid, 0.1 M) and 75 C for 2 hours (step 2). Covalent coupling of the stable intermediate with the amine functionalized glass surface is carried out by flowing the activated antibody past the glass surface (step 3). Sol-gel Functionalization Sol-gel glass is an optically transparent glass-like material produced by the hydrolysis and polycondensation of organometallic compounds at a low temperature.


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The organometallic compounds commonly used are silicon alkoxides, e.g. Si(OCH3)4, tetramethylorthosilicate. Silica glass prepared by the sol-gel method is a porous matrix that contains interconnected ‘bottleneck-like’ pores (or so-called cavities and cages) formed by a 3D SiO2 network. The pores are usually in the size range of 1.5 to 10 nm, depending on the composition of the precursor and the conditions of preparation. It is found that the large sensing agents can be securely trapped, but small analytes can diffuse readily into and out of the pores of the sol-gel matrix. The process that is used to produce sol-gel glass is called the sol-gel process. It can be divided into the following steps: mixing (to form a solution), gelation, aging and drying. In a typical procedure to prepare a silica glass by the sol-gel technique, one starts with an appropriate alkoxide precursor, e.g. Si(OCH3)4, which is mixed with water, an acidic catalyst such as HCl and a mutual solvent such as methanol to form a solution (the sol) by stirring or sonication. Hydrolysis results in the formation of silanol groups (Si-OH). Linkage of silanol with siloxane occurs as a polycondensation reaction and eventually leads to the formation of an SiO2 network (with silanol groups on the surface). The resulting amorphous gel contains water and methanol. Aging of a gel involves maintaining the gel immersed in liquid for a period of time, from hours to days. During aging, polycondensation continues and the strength of the gel thereby increases. Finally, in the drying process, the solvents (water and methanol) are removed from the interconnected pore network. A sensing agent is added to the mixture at some time during the formation of the sol or gel. A sol-gel is a viscous solution of colloidal silica particles in a liquid. The sol-gel solution containing a sensing agent can be coated on to cantilever sensors as a sensing element. In the sol-gel glass, the reagents are trapped inside the pores of the matrix, where they may move freely or interact with silanol groups on the inner surfaces of the pores. The analytes to be determined, usually smaller, can diffuse into the pores and react with the sensing agents. On the other hand, the sol-gel glass has a large porosity (about 30%) and a very large specific surface area (>300 cm2/g) [134]. Therefore, a substantial fraction of the entrapped reagents may be exposed to a neighbouring phase and intrapore volume and can react with the analytes therein. The use of sol-gel materials for the development of chemical sensors and biosensors is of intense current interest [141]. In most applications, the sol-gel material functions as a porous support matrix in which analyte-sensitive species are entrapped. The sol-gel process is attractive because gelation can occur at low temperatures, which allows for the encapsulation of a variety of temperaturesensitive molecules, including biological reagents. A glucose sensor based on the entrapment of the protein glucose oxidase within a sol-gel matrix has been developed [142], as has an optical sensor based on the entrapment of high pKa dyes within a sol-gel matrix, which is used to determine pH in highly acidic and highly alkaline environments [143,144]. Raiteri et al. have used thin films of sol-gel

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coated on silicon nitride microcantilevers via a dip-coating process to show the change in response of the microcantilever to different mixtures of ethanol and water [145]. Fagan et al. modified and evaluated silicon microcantilevers with thin films of sol-gel [141]. Thin films of sol-gel were deposited on to microcantilevers using a spin-coating procedure. The method of film formation requires less equipment and is less expensive than conventional techniques such as CVD, evaporation or sputtering [146]. The authors have also studied the ability to modify the selectivity of the microcantilevers by modifying the chemical nature of the surface of the sol-gel film through reaction with organosilanes. Biofunctionalization Many bioanalytical methods, in particular biosensor microcantilevers, require a high density of functional molecules, low nonspecific protein adsorption, long-term stability and durability. Proteins – including antibodies and enzymes – and cells are immobilized on the sensor surface. This immobilization process should be uniform, avoiding any change in the mechanical properties of the cantilevers, and allow accessibility by the target molecule. Adsorption, entrapment and covalent attachment are the leading techniques employed for immobilization of biomolecules on to sensor surfaces. Carrascosa et al. give a good discussion of DNA and protein immobilization on cantilevers and array systems [128]. To ensure long-term stability, a chemical surface functionalization has to be applied to deposit a receptor layer on the cantilever surface. The ideal case for surface functionalization is a closed monolayer, in which the receptor is covalently anchored to the surface with high density but with enough space between the receptors to interact with the specific ligand. The receptor activity should be maintained over time and resist several regeneration cycles. If one wants to use the deflection detection method for cantilevers, only one side has to be functionalized. By using thiol monolayers, this can be done by evaporating gold on one side of the cantilever only or by incubating only one side of a double-sided, gold-coated cantilever in the thiol solution. Incubating only one side causes stress, which has to be taken into account [147]. Raiteri et al. reported a procedure for coating each side of the cantilever with a different thiol [148]. Nevertheless, surface functionalization is as easy as it sounds; however, if the deflection detection is used, the experimenter has to ensure that the specific interaction takes place at one side of the cantilever only and, even more importantly, that unspecific interaction is blocked. Not all biomolecules which may be used as recognition sites are commercially available as thiols. Yan et al. reported a method to apply a nanoassembly layer-by-layer technique on one side of a cantilever only [149]. This method produces ultrathin organized polymer films to which the recognition sites can be attached either electrostatically or by chemical reaction.


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Functionalization Using Carbon Nanotubes By integrating nanotube growth into batch-fabricated microsystems, NEMS and MEMS are able to create sensors and develop methods and devices for direct and reliable measurement of nanotube transducer properties. Hierold et al. recently published a good review concerning MEMS and NEMS sensors based on carbon nanotubes (CNTs) [150]. This article presented the fabrication techniques and methods used for the integration of CNTs in these sensors. Jungen et al. describe a series of processing steps required to achieve successful integration of single-wall nanotubes (SWNTs) directly into a MEMS chip [151]. The process is schematically shown in Figure 2.14(c). An e-beam lithography resist (PMMA) of 400 nm thickness is spin coated on top of the 2 · 2 mm chip (step 1). The written structures are openings, typically 2 mm in feature size, defining placeholders for the catalyst material and resulting in a locally defined spot for catalysts for spatial control of nanotube growth. After development, a droplet of a catalytic solution based on iron nitride dissolved in methanol is placed on the chip and evaporated using a hotplate (40 C) (step 2). In order to grow tubes that span a 2 mm trench, the catalyst coating should come with a high surface area presenting nonaggregated nanoparticle-sized sites for the assembly of carbon molecules into nanotubes. The liftoff process is completed by stripping the PMMA in acetone under ultrasonic agitation (step 2). The chip is placed into a 4 inch quartz-tube LPCVD reactor. The furnace is heated under argon to 900 C. Methane fed at 1000 SCCM and kept at 100 mbar is provided as a carbon feedstock for 10 minutes. The reactor is evacuated and cooled to at least 300 C before venting to atmosphere using nitrogen (step 3). A second e-beam lithography and liftoff step are used immediately after this to deposit a Cr/Au layer for electrical connectivity (step 4). Since the growth occurs on the surface of the Poly 2 layer, most of the CNTs are freestanding prior to the HF release of the MEMS. The second spin coating has been proven not to destroy the CNTs. After a standard HF release the MEMS chip is ready for actuation. 2.5.3

Detection Methods

In general there are three different methods for transducing the recognition event into micromechanical motion: first, the frequency change due to additional mass loading or changing in the force constant can be measured (i.e. the cantilever is used as a microbalance); second, the binding of the bimetallic cantilever can be used as a temperature sensor (e.g. to sense calorimetric effects upon adsorption); and third, cantilevers can work as stress sensors by measuring the bending due to changes in the surface stress at one side of the cantilever. Adsorption of molecules, when they are restricted to one of the cantilever surfaces, produces differential surface stress that bends the cantilever. At the same time, the resonant frequency of the cantilever also varies due to mass loading. The bending and the changes in resonant frequency can be monitored by several techniques, using optical reflection, capacitive and

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piezoresistive/piezoelectric techniques. Changes in resonant frequency can be detected by measuring the thermal noise of the cantilever. However, to achieve great sensitivity, especially when working in liquids, it is necessary to pre-energize the cantilevers by using alternating electric, magnetic or acoustic fields. Optical Detection The method most often used to measure cantilever bending is the optical reflection technique. In this technique, deflection of the cantilever is measured by reflecting a beam from a solid-state laser diode on to the free apex of the cantilever and then off the back of the cantilever. The reflected laser beam from the cantilever surface is collected by a position-sensitive detector (PSD) consisting of two closely spaced photodiodes whose output signal is collected by a differential amplifier. When the cantilever bends, the reflected laser beam moves on the photodetector surface, and the distance traveled is proportional to the cantilever deflection. The advantage of this detection system is that it is capable of detecting deflection in the subnanometer range and can be implemented easily. But it also has its disadvantages. First, the presence of a focused laser beam in a liquid cell environment can result in additional thermal management issues, giving rise to extraneous readings. Second, the alignment system is expensive and involves great precision, which can ultimately raise the cost of the whole diagnostic kit. In addition, it also reduces the kit’s portability. Further, the implementation of an optical method for readout of arrays is technologically challenging, as it requires an array of laser sources with the same number of elements as the cantilever array. This technique is employed in the optically-based commercial array platforms, but sequential switching, on and off, of each laser source is necessary to avoid overlap of the reflected beams on the photodetector. This problem can be elegantly solved using a scanning laser source, where the laser beam is scanned along the array in order to illuminate the free ends of each microcantilever sequentially [152]. Lechuga et al. introduced a new type of optical waveguide cantilever that does not need an array of laser sources as the cantilever itself acts as the waveguide in conducting the light [153]. At the exit of the optical cantilever, light can be collected by another waveguide or by a photodetector. This new device has been shown to have a good performance, and it offers an interesting approach to further integration in lab-on-a-chip microsystems. Another implemented optical deflection method is interferometry detection. This is based on interference of a reference laser beam by the one reflected by the cantilever. The cleaved end of an optical fibre is brought close to the cantilever surface. One part of the light is reflected at the interface between the fibre and the surrounding media, and the other part is reflected at the cantilever back into the fibre. These two beams interfere inside the fibre, and the interference signal can be measured with a photodiode. Interferometry detection is highly sensitive and provides a direct and absolute measurement of the displacement. The light has


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to be brought close to the cantilever in order for enough to be reflected. For this purpose, Rugar et al. positioned the cleaved end of an optical fibre a few micrometers away from the free end of the cantilever [154]. By doing this, they could measure deflections in the 0.01 A range. Bonaccurso et al. have proposed an interferometric system based on two cantilevers for parallel surface-stress monitoring of two cantilevers only 200 mm apart [155]. However, a few technical problems arise. Positioning the fibres is a demanding task, and interferometry works for only small displacements: absolute deflection is defined only within a single wavelength, and measurements in liquids are, in general, less sensitive. An alternative approach is to use interdigitated cantilevers as an optical diffraction grating [156,157]. In this case, the reflected laser light forms a diffraction pattern whose intensity is proportional to cantilever deflection. An interferometric sensor for the AFM with a cantilever micromachined into the shape of interdigitated fingers to form a diffraction grating has been presented [156,157]. Cooper et al. demonstrated a promising type of microfabricated accelerometer based on the optical interferometer, and this interdigitated cantilever can also be used for infrared imaging and chemical sensing [158]. Piezoresistive/Piezoelectric Detection The piezoresistive method involves the embedding of some piezoresistive material near the top surface of the cantilever to record the stress change occurring there. As the microcantilever deflects, it undergoes a stress change that applies strain to the piezoresistor, thereby causing a change in resistance that can be measured by electronic means. Piezoresistive detection shows several advantages compared to the optical sort: no expensive macroscopic optical components and no timeconsuming laser alignments are needed and read-out electronics can, in principle, be integrated on to the same silicon chip supporting the cantilevers. Optical techniques can also be affected by artefacts due to changes in the optical properties of the medium surrounding the cantilever (e.g. a change in the refracting index when exchanging two different solutions), which can move the laser spot on the photodetector surface. Piezoresestive/piezoelectric detection does not suffer from this problem and can work in nontransparent solution. A disadvantage is that the deflection resolution for the piezoresistive readout system is only one nanometre, compared with one angstrom with the optical detection method. Another disadvantage with this method is that a piezoresistor has to be embedded in the cantilever. Fabricating such a composite cantilever is complicated. Capacitive Detection Although the most common detection mechanism reported in the literature is the optical beam deflection method, capacitive sensing can also be used. This does not require a complex adjustment and alignment system, as the optical beam does, and

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provides an integrated readout, which is very convenient for portable low-cost systems. The capacitive method is based on the principle that when the cantilever deflection takes place due to the adsorption of the analyte, this deflection is detected by the capacitance variation between the cantilever itself, acting as a moveable electrode, and the fixed electrode placed underneath. This deflection technique is highly sensitive and provides absolute displacement, but is not suitable for measuring large displacements. Moreover, it does not work in electrolyte solutions due to the faradic currents between the capacitive plates. Therefore, it is limited in its sensing applications. Interferometric Detection Interferometric detection of cantilever deflection is based on constructive and destructive interferences that occur when a collimated beam of light reflects off two surfaces displaced from one another [159]. The intensity of light is measured by a photodiode array. Although this is not a very common detection technique, it is capable of measuring very small deflections, but has a very limited dynamic range. Interferometric detection is used for high-temperature vibration sensors [160], as well as for studying the different resonance modes of cantilevers in arrays [161].

2.6 Outlook Fully integrated microfluidic devices have been widely developed during the last few years. A variety of platforms have been proposed for different applications. Without doubt, biochemical analysis has been the area that has received most attention. In fact, as P. Abgrall and N.T. Nguyen recently reported [162], the proximity of the dimension of nanochannels and the Debye length, the size of biomolecules such as DNA or proteins, and even the slip length, added to the excellent control of the geometry, give unique features to nanofluidic devices. These devices not only find applications wherever there is less well-defined porous media (electrophoresis gels, mainly) but give new insight into the sieving mechanisms of biomolecules and the flow of fluid at the nanoscale. Moreover, the control of the geometry allows smarter design, resulting in new separation principles (among other things) through taking advantage of the anisotropy. This fact connects with the trend of bioanalytical chemistry to analyse the profiles and dynamics of molecular components and subcellular structures in living cells. For these purposes, microfluidic devices have come to be of marked interest, because of their advantages in performing analytical functions such as controlled transportation, immobilization and manipulation of biological molecules and cells. Further, their ability to carry out separation and mixing processes, as well as the dilution of chemical reagents in a controlled way, allows the analysis of intracellular parameters and detection of cell


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metabolites (even on a single-cell level). Therefore, microfluidics technology is an excellent tool for the manipulation and analysis of biological cells [163]. Cell manipulation on chips commonly uses different forces. In magnetic manipulation, magnetic particles are selectively attached to cells. This is a common method for cell separation or purification in microfluidic devices. The optical manipulation of biological species has received increasing interest due to its noncontact and contamination-free manipulation process. On the other hand, the separation of target cells on microfluidic structures for culture and assay purposes is an important application of on-chip mechanical manipulation. Different microstructures have been developed for this goal [163]. Electrical manipulation through dielectrophoresis is another alternative which has been used as a microfluidic cell trap. Cell treatment is crucial for the integration of cell treatment steps on-chip to develop microfluidic devices for cell analysis. In this context, cell lysis is absolutely necessary for the analysis of cell constituents. The ability to integrate both steps (lysis and analysis) greatly increases the power and portability of many microfluidic devices [164]. Moreover, microfluidic devices are especially suitable for cell culture, as the scale of these devices allows the creation of locally formed stable microenvironments for this purpose [165]. Compared to traditional culture tools, microfluidic platforms provide much greater control over cell microenvironment and rapid optimization of media composition while using relatively small numbers of cells. For other cell-based assays, in which the introduction of hydrophilic or membrane-impermeant molecules into cells is necessary, electroporation, electrofusion and optoporation processes have been successfully applied in microfluidic systems for cell treatment (e.g. [166–168]). Cell analysis on microchips introduces numerous benefits, such as reduced cell consumption, automated and reproducible reagent delivery, and improved performance. Consequently, the progressive development of microfluidic devices for specific bioanalytical applications of cell processing has become a reality. M. Yang and coworkers have reviewed numerous publications dealing with these applications [163]. Within these applications, cytometry has received much attention. Cytometry refers to the measurement of the physical and chemical characteristics of cells. This technique is commonly restricted to specialized laboratories, and is generally expensive. The development of readily adaptable chip technology allows the flow cytometer unit to be adaptable to a wide variety of potential uses, with portability and low cost. Differing from conventional flow cytometers, which usually employ fluorescence detection, microfluidic flow cytometers can be mounted on a variety of detection instruments. In particular, chemical cytometry, referring to the use of highly sensitive analytical tools to characterize chemical composition of single cells, is essential for understanding the molecular mechanisms of many fundamental biological processes. But it is also crucial for the study of serious health disorders, because these studies require simultaneous analysis of a large number of chemical species within single cells. These chemical species

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cover small molecules generally related to cell metabolism, large molecules such as proteins, and compounds of genetic interest such as nucleic acid. Using this technology, S.R. Quake and coworkers developed a microfluidic chip for automated nucleic acid purification from small numbers of bacterial or mammalian cells [169]. Microfluidic systems also show great potential for the analysis of intracellular parameters and the detection of the presence of cell metabolites, even on the singlecell level. Thus, a representative number of applications, including monitoring of ATP, NAD, FAD, IgE, pH and Ca2þ, among other compounds, have been reported by M. Yang et al. [163]. One interesting use of microfluidic systems is as reactors for DNA amplification [170], particularly via polymerase chain reaction (PCR). This enzyme-catalysed reaction allows any nucleic acid sequence to be generated in abundance in vitro, and has become a fundamental tool in molecular biology. Using this process with conventional thermal cyclers is slow and inefficient due to the large thermal masses associated with instrumentation. To avoid this problem, many microfabricated devices for PCR have been reported, showing a clear reduction in the thermal mass of the system. Thus, different heating mechanisms have been used for effective and rapid PCR in volumes ranging from 1 pL to 50 mL. As an example of the progress made in this crucial area, Figure 2.15 shows the integrated microfluidic bioprocessor for PCR assays developed by R.G. Blazej et al. [171]. This demonstrates that

Figure 2.15 Nanolitre-scale microfabricated bioprocessor integrating thermal cycling, sample purification and capillary electrophoresis for complete Sanger sequencing from 1 fmol of DNA template. [171]. Reprinted with permission of Nature Publishing Group


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complex biological processing can be performed at higher speeds and better efficiencies than previously reported. Microfluidic systems also allow miniaturization and integration of complex functions, which could move sophisticated diagnostic tools out of the developedworld laboratory. Thus, microfluidic diagnostic technologies for public health can be seen as a reality, as T. Edwards et al. report [172]. These authors present an example of an integrated disposable diagnostic card, which is reproduced in Figure 2.16. Without doubt, microfluidics opens a wide field of applications and possibilities in a variety of different contexts, as previous examples and references demonstrate. But, as G.M. Whitesides states [173], ‘the field is still at an early stage of development,’ and despite these developments, other issues must be addressed. Real problem-solving and commercialization are two important challenges to date.

Figure 2.16 Integrated disposable diagnostic card: (a) image of the card, where the O-rings are for interfacing with off-card components (valves, pumps, etc.); (b) scheme of the card. This card accepts filtered saliva from the syringe and contains an H-filter for further sample conditioning, a herringbone mixer for mixing antibodies with the sample, and channels with gold-coated surfaces for detection of analyte in the sample using an SPR imaging-based immunoassay. (Reprinted from [172] with permission of Nature Publishing Group)

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It is important to identify exactly which applications will need these types of miniaturized formats, because of their advantages or, even more importantly, because alternatives cannot be used (much reduced size of samples, in vivo monitoring, etc.). As A. Escarpa et al. report [174], ‘real (or non-ideal) sample analysis performed on microfluidics conceptually involves the complete integration of sample preparation, analyte separation, and detection on these platforms’. Transition from the behaviour of analytical microsystems with synthetic standard solutions (academic research) to natural (‘real’) samples is not a simple change of the inlet vial of a microfluidic device; in many cases it is a complete change of behaviour. From this applied-practical side of analytical microsystems, it is clear that the main achievements have been reached in the bioanalytical field. Despite the excellent reviews in the fields of environmental [175,176], explosives [177] and food [178] analysis, many challenges remain for the routine implementation of microfluidics in these areas. At the end of the day, as usual in the analytical method, analytical validation, involving not only target analytes but also objective samples, will be the definitive demonstration of the true usefulness of miniaturized analytical systems.

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3 Automation and Miniaturization of Sample Treatment 3.1

Introduction

In recent years, analytical instrumentation has evolved exponentially. A wide range of instruments capable of performing highly accurate determinations based on a variety of physical and/or chemical principles currently exists as a result. Such instruments include molecular and atomic absorption and emission spectrophotometers, vibrational and mass spectrometers, and electrochemical detectors such as conductimeters, to name a few. These advances in instrumentation have run in parallel with substantial improvements in separation science, particularly as regards liquid chromatography and capillary electrophoresis (CE). Also, the growing trend to miniaturization has facilitated the development of analytical equipment, enabling accurate, expeditious separation of a variety of components by using reduced amounts of samples and reagents. Despite the above advances, work at routine analytical laboratories continues to be slowed down by the preliminary operations of the analytical process. In fact, pretreating complex samples is often labour-intensive and time-consuming. Therefore, automating or integrating such operations could help simplify routine analyses. In this context, miniaturization of sample treatment devices provides an elegant, effective way of automating sample treatment. This chapter is concerned with sample treatment simplification through miniaturization and automation. Miniaturized sample preparation methods have been regarded as the most attractive techniques for the pretreatment of complex sample mixtures prior to the determination process. Miniaturization of Analytical Systems: Principles, Designs and Applications and Bartolome Simonet 2009 John Wiley & Sons, Ltd


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The effective online coupling of the miniaturized sample preparation and the instrumental separation technique (chromatographic or electrophoretic) enables several advantageous features. These advantages can be itemized as follows: . . . . .

low cost of operation due to the extremely low solvent consumption; high-speed analysis due to the simplification of the process; high efficiency; high selectivity when tailored systems are designed; development of environmentally friendly analytical procedures.

3.2 Simplification of Sample Treatment: Microextraction Techniques There is no doubt that miniaturization in the field of sample treatment has led to a simplification of this tedious step of the analytical processes. The miniaturization of sample treatment devices has resulted in the use of a lower sample volume, low sample processing time and low consumption of reagents [1]. In this context, it is important to present two trends in microextraction techniques: solid phase microextraction (SPME) and liquid phase microextraction (LPME). Microextraction techniques were developed to address the need to facilitate rapid sample preparation both in the laboratory and onsite at the system under investigation. In microextraction techniques, a small amount of the extracting phase (solid or liquid) is exposed to the sample for a well-defined period of time. A partitioning equilibrium is then reached between the sample and the extraction phase. This approach requires long times to reach equilibrium but is not affected by the convection conditions. Another interesting approach is based on utilizing a short partitioning time (pre-equilibrium conditions). This approach reduces the sample treatment time but requires exhaustive control of the convention/agitation conditions; in this way the amount of analyte extracted is related to time and can be quantified [1–3]. This extraction process generally follows the profile shown in Figure 3.1. 3.2.1

Calibration in Microextraction Processes

Calibration based on equilibrium extraction is very simple and has been the method most used to date. However, the fact that equilibrium times can range from seconds to months must be taken into account. When the equilibrium time is long, the calibration can be performed in the linear range (see Figure 3.1) by using kinetic calibration methods. Calibration based on equilibrium extraction is widely used in onsite sampling. The initial concentration of the target analyte can be calculated according to the

Extracted amount of analyte

Automation and Miniaturization of Sample Treatment

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Near equilibrium

Kinetic regime

linear regime

t50

t95

Time

Figure 3.1 Typical extraction profile for microextraction techniques

following equation: CS ¼

Ce n ¼ ; Kes Kes Ve

ð3:1Þ

where Cs and Ce are the concentration of the target analytes in the sample and of the extraction phase respectively, n represents the total amount of analyte extracted, Ve is the volume of the extraction phase and Kes is the distribution coefficient of the analyte between the phases. This calibration only requires that the distribution coefficient is known. It is recommended for rapid equilibrium times. When the analysis is performed in the linear range (see Figure 3.1), quantification can be performed by assuming that the rate of mass transfer remains constant during the extraction process. Under this condition, the following equation is verified: nt Cs ¼ ð3:2Þ RS As can be seen, the concentration of the target analytes in the sample, Cs, is related to the total amount extracted, n, through the sampling rate of the transfer between phases Rs and the extraction time t. Typically, the calibration methods in microextraction techniques are limited to either the equilibrium regime or the linear regime. Recently, kinetic calibration methods that can be used for the entire extraction profile have been proposed [4–7]. However, these calibration procedures are not commonly used in methods involving a microextraction step. They are most used when the microextraction step is used for both preconcentration of the target analytes and to perform the sampling step, for example in environmental matrices [7,8]. 3.2.2

Solid Phase Microextraction (SPME) Techniques

In SPME processes, a small amount of extracting phase dispersed or bounded on a solid support is exposed to the sample. After a well-defined period of time,

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an amount of the target analyte is preconcentrated on the solid phase. Quantification, as was indicated in Section 3.2.1, can be performed based on equilibrium or on timed accumulation of analytes in the solid phase. Today, there are several procedures to implement an SPME, namely: Beds: Extraction of analytes on dispersed microparticles. Fibres: Extraction of analytes on coated fibres. Vessels: Extraction of analytes on the wall of chemically modified vessels. Stir Bar: Extraction of analytes on the wall of stirrers. In Tubes: Extraction of analytes on the wall of capillaries. Microextraction in Packed Syringe. Discs/Membranes: Extraction of analytes on the surface of disc or membrane filters. Figure 3.2 illustrates several implementations of SPME. In SPME techniques it is possible to distinguish between liquid and solid coatings. As shown in Figure 3.3, these coatings have resulted in two types of extraction mechanism, called absorptive and adsorptive extraction. In liquid coatings the target analytes penetrate on to the whole volume of the coating thanks to the salvation of the analytes by the coating molecules [9]. The penetration of the analytes is limited by the diffusion coefficient in the liquid coating. This type of extraction is called absorption. In the case of solid sorbents, the extraction occurs only on the surface. This is called adsorption [9].

Figure 3.2 Configurations of SPME systems. (Adapted from [2] with permission from Elsevier, Copyright 2000)


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Support (fibre core) Coating

Analyte molecule

Absorption

Adsorption–large pores

Adsorption–small pores Figure 3.3 Schematic representation of the extraction processes of analytes on solid phases. Two mechanisms can be distinguished: adsorptive and absorptive extraction. (From [9] reproduced by permission of the Royal Society of Chemistry)

Extraction Modes with Coated Fibre Three basic types of extractions can be performed using SPME, namely direct extraction, headspace configuration and a membrane protection approach. Figure 3.4 illustrates the differences between these modes. In the direct extraction mode, the coated fibre is directly inserted into the sample and the analytes are transported from the sample matrix to the extracting phase. To facilitate extraction, it is important to agitate the solution in order to transport the analytes from the bulk solution to the vicinity of the fibre [10,11]. In the headspace mode, the analytes need to be transported through the barrier of air before they can reach the coating. The main advantage of this mode is the protection of the fibre from high molecular mass and other nonvolatile interferences present in the sample matrix [12–14]. Another important advantage is that it allows modification of the matrix, for example to an extreme pH, without damaging the fibre. The amounts of analyte extracted into the coating from the sample vial at equilibrium using direct and

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Figure 3.4 Modes of SPME operations: (a) direct extraction; (b) headspace configuration; (c) membrane-protected configuration

headspace sampling are identical so long as the sample and gaseous headspace volumes are the same. This is because the equilibrium concentration is independent of fibre location in the sample/headspace system. If the above condition is not satisfied, a significant sensitivity difference between the direct and headspace approaches exists for very volatile analytes only [15]. The choice of sampling mode has a very significant impact on extraction kinetics, however. When the fibre coating is in the headspace, the analytes are first removed from the headspace, then indirectly extracted from the matrix. Overall, mass transfer to the fibre is typically limited by mass transfer rates from the sample to the headspace. Therefore, volatile analytes are extracted faster than semivolatiles since they are at a higher concentration in the headspace, which contributes to faster mass transport rates through it [16]. Temperature exerts a significant effect on the kinetics of the process by determining the vapour pressure of the analytes. In fact, the equilibrium times for volatiles are shorter in the headspace SPME mode than for direct extraction under similar agitation conditions. This outcome is produced by two factors: (i) A substantial portion of the analyte is in the headspace prior to extraction. (ii) Diffusion coefficients in the gaseous phase are typically four orders of magnitude larger than in liquid media. Since concentrations of semivolatiles in the gaseous phase at room temperature are typically small, however, overall mass transfer rates are substantially lower and result in longer extraction times. They can be improved by using very efficient agitation or by increasing the extraction temperature [8,17]. Extraction Modes with In-tube Solid Phase Extraction Figure 3.5 shows that there are two fundamental approaches to in-tube SPME: active or dynamic, when the analytes are passed through the tube; and passive or

Automation and Miniaturization of Sample Treatment (a)

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(b)

attachment hub

sealing septum

septum-piercing needle

microtubing

coated fused silica fibre

sample

sample flow

Figure 3.5 Comparison of (a) passive versus (b) dynamic modes of in-tube extractions

static, when the analytes are transferred into the sorbent using diffusion. In each of these approaches, the coating may be supported on a fused-silica rod, or on the inside of a tube or capillary [18]. In the dynamic configuration, we can assume for example the use of a piece of fused-silica capillary coated with a thin film of extracting phase. In these geometric arrangements, the front of analyte migrates through the capillary with a speed proportional to the linear velocity of the sample, and inversely related to the partition ratio [19]. For short capillaries with a small dispersion, the extraction time can be assumed to be similar to the time required for the centre of the band to reach the end of the capillary. The extraction time is proportional to the length of the capillary and inversely proportional to the linear flow rate of the fluid [20]. The extraction time also increases with an increase in the coating/sample distribution constant and with the thickness of the extracting phase but decreases with an increase in the void volume of the capillary. An increase in the coating/sample distribution constant produces an increase in the absolute amount extracted. It has been shown that increases in the amount extracted can be achieved, in many cases, by preconditioning the capillary with methanol or some other appropriate solvent prior to extraction. Enhancement has even been observed when a plug of methanol is aspirated into the capillary before the sample is drawn in, and following the use of a sample plug in the capillary during the extraction aspirate/dispense steps. This is analogous to the solvent preconditioning used to enhance solid phase extraction (SPE).

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Figure 3.6 Schematics of different implementations of in-tube SPME. (From [24] with permission from American Chemical Society, Copyright 1997)

In practice, in-tube SPME is implemented by replacing a section of the tubing in a commercially available autosampler and then programming the autosampler to pass sample in and out of the extraction capillary until equilibrium or a suitable extraction level is reached [21–24]. Several options for implementing in-tube SPE are summarized in Figure 3.6. The removal of analytes from a tube is an elution problem analogous to frontal chromatography and has been discussed in detail [24,25]. In general, if the desorption temperature of a GC is high and thin coating is used, all the analytes will be in the gaseous phase as soon as the coating is placed in the injector, and the desorption time corresponds to the elution of two void volumes of capillary. For liquid desorption, for example into liquid chromatography equipment, the desorption volume can be even smaller since the analytes can be focused at the front of the desorption solvent [26–28]. In addition to the measurement of analyte concentration, it is possible to obtain an integrated sampling with a simple SPME system [29,30]. This is particularly important in field measurements, where changes of analyte concentration over time and space must often be taken into account.


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When the extracting phase is not exposed directly to the sample, but is contained in a protective tubing (needle) without any flow of the sample through it, the extraction occurs through the static gas phase present in the needle. The integrated system can consist of the extraction phase coating the interior of the tubing, or it can be externally coated fibre withdrawn into the needle. These geometric arrangements represent a very powerful method able to generate a response proportional to the integral of the analyte concentration over time and space (when the needle is moved through the space). In these cases, the only mechanism of analyte transport to the extracting phase is diffusion through the gaseous phase contained in the tubing. During this process, a linear concentration profile is established in the tubing between the small needle opening, characterized by surface area and the distance between the needle opening and the position on the extracting phase. Fibre Coatings As was indicated above, the efficiency of the extraction process is dependent on the distribution constant. This is a characteristic parameter that describes the properties of a coating and its selectivity toward the analyte versus other matrix components. Specific coatings can be developed for a range of applications. Coating volume determines method sensitivity. Therefore, it is important to use the appropriate coating for a given application. This is clearly demonstrated in Figure 3.7, which

Figure 3.7 Total ion current GC/MS chromatogram of bencene, toluene, ethylbenzene and o, m, p-xylene (BTEX) and phenol in water extracted with (a) a poly(dimethylsiloxane) coating and (b) a poly (acrylate) coating. (From [31] with permission from American Chemical Society, Copyright 1994)

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compares the performance of two different coatings for analysis of polar and nonpolar compounds from aqueous matrix. The distribution constant and the sensitivity of the method drop over two orders of magnitude for o-Xylene, and increase by an order of magnitude for 2,4-dichlorophenol when the film is changed from nonpolar poly(dimethylsiloxane) to polar poly(acrylate) polymer [31]. Coating selection and design can be based on chromatographic experience. To date, several experimental coatings have been prepared and investigated for a range of applications. In addition to liquid polymer coatings such as PDMS for general applications, other more specialized materials have been developed. For example, ion-exchange coatings were used to remove metal ions and proteins from aqueous solutions [32–34], liquid crystalline films to extract planar molecules and carbowax for polar analytes [35,36], metal rods to electrodeposit analytes, pencils to extract pesticides [37] and Nafion coating to extract polar compounds from nonpolar matrices [38,39]. Polypyrole coatings have recently been developed to extract polar or even ionic analytes and possibly to explore the conductive polymer properties of the polymer [40]. This might involve applying a charge to the polymer during extraction in order to selectively extract analytes of interest and then reversing the charge to facilitate desorption. Coating polymers are versatile materials in which molecular/analyte recognition can be achieved in different ways, including: (i) the incorporation of counterions that introduce selective interactions; (ii) the utilization of the inherent and unusual multifunctionality (hydrophobic, base–acid and p–p interactions, polar functional groups, ion-exchange, hydrogen bonding, electroactivity, etc.) of the polymer; (iii) the introduction of functional groups to the monomers; (iv) the co-deposition of metals or other monomers within the polymer; and (v) the application of appropriate electrochemical potentials. A very pronounced difference in selectivity toward target analytes and interferences can be achieved by using surfaces common to affinity chromatography. Using the method of polymer imprinting [41], antibody mimics can be generated with specifities to an analyte of choice. Briefly, the desired affinity can be introduced by adding an amount of the compound of interest to the polymerization reaction. This ‘pattern’ chemical may be removed after polymerization, leaving vacant sites of a specific size and shape, suitable for binding the same chemical again from an unknown sample. In this technique, we have observed that nonspecific binding should be controlled for, but that enhancements in sensitivity are seen, particulary at low analyte concentrations. Commercial Devices The first commercial version of the laboratory SPME device was introduced by Supelco in 1993 (see Figure 3.8). This was later improved by the inclusion of


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Figure 3.8 Design of the first commercial SPME device made by Supelco

an adjustment for the depth of the fibre with respect to the end of the needle, which allows control of the exposure depth in the injector and extraction vessel. The device incorporates such useful features as colour marking of the fibre assemblies, to distinguish between various coating types. In addition to standard PDMS coatings of various thicknesses and polyacrylates (PAs), Supelco developed new mixed phases based on solid/liquid sorption, such as Carbowax–DVB and PDMS–DVB. Supelco also introduced a high-performance liquid chromatography (HPLC) interface (Figure 3.9) that integrates the original concept with the injection valve (Figure 3.10). Varian has developed an SPME autosampler based on its 8000 GC autosampler system, taking advantage of the facts that the SPME device is analogous to a syringe in its operation and that after desorbtion the coating is cleaned and ready for reuse [42]. The major challenge is to incorporate agitation and temperature control, as well as other enhancements such as fibre internal cooling or dedicated injectors. One improvement is an SPME system that incorporates an agitation mechanism consisting of a small motor and a cam to vibrate the needle. The fibre in this design works as a stirrer. The vibration causes the vial to shake and the fibre to move with respect to the solution; the result is a substantial decrease in equilibration times compared to a static system. This mode of agitation simplifies fibre handling because it does not require the introduction of foreign objects into the sample prior to extraction. Recently, Varian has reached an agreement with CTC Analytics of Switzerland to incorporate SPME sampling on its CombiPal autosampler. This is a robotic system with a great deal of flexibility for programming SPME analyses. Samples are loaded

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Miniaturization of Analytical Systems SPME fibre holder

Septum-piercing needle Needle guide Double tapered ferrule

Compression union

SPME fibre Solvent desorption chamber To interface

Static desorption (no flow) HPLC column

Solvent from syringe Waste

Valve

Mobile phase from pump

From interface

To interface From interface

Sample injection HPLC column

Solvent from syringe

Waste

Valve

Mobile phase from pump

Figure 3.9 Schematic diagram of the Supelco SPME–HPLC interface

on to trays accomodating five vial sizes, and samples are heated and agitated during extraction, using a separate sample-preparation chamber. To facilitate agitation, the extraction chamber is rotated at a programmable rotation speed during extraction. Additional vials/stations are present to accommodate wash solutions, derivatizing agents, temperature control, derivatization and fibre conditioning, in order to facilitate operation of SPME at optimal conditions. While the built-in software can be used to perform basic SPME analyses, extra programming flexibility is provided by the Cycle Composer software, available as an accessory. We expect that this new instrument, with its greatly enhanced flexibility, will significantly expand the range of SPME methods amenable to automation. New coatings and devices are expected to follow, as interest in SPME grows along with the unprecedented number of new applications appearing in the literature. SPME coupled to high-speed gas chromatography is a good combination for the performance of rapid and cost-effective investigations in the field, even of complex organic samples. As discussed above, SPME is particularly suited to fast GC as it is solvent-free and the thin coatings can provide very fast desorption of analytes at high temperatures. Some instrumental modifications were recently performed in order to achieve successful fast separations. A portable system was optimized for SPME–fast GC field investigations and was commercialized by SRI Instruments [43]. The instrument was tested in combination with a flame ionization detector (FID), a photoionization detector (PID) and a dry electrolytic conductivity detector (DELCD). A dedicated injector, presented in Figure 3.11, was mounted on the portable system in order to use SPME for


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Figure 3.10 SPME–HPLC interface: (a) 1/16 in. stainless steel (SS) tee; (b) 1/16 in. SS tubing; (c) 1/16 in. PEEK tubing (0.02 in. ID); (d) two-piece, finger-tight PEEK union; (e) PEEK tubing (0.005 in. ID) with a one-piece PEEK union

Figure 3.11 Design of dedicated injector. 1, modified Swagelok fitting; 2, stainless steel tubing; 3, moulded septum; 4, nut; 5, needle guide; 6, nut; 7, blind ferrule; 8, stainless steel tubing; 9, 0.53 mm ID fused-silica capillary; 10, contact

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high-speed separation. The injector guarantees very fast thermal desorption of the analytes from the SPME fibre [43]. The injector for high-speed GC should produce as narrow an injection band as possible. Internal volumes of regular injector ports are too large (e.g. split/splitless injector), since they have been designed to accommodate large volumes of gaseous samples or vapours produced by solvent injection. Thermal focusing for separation improvement is not convenient for fast separations, since temperature programming is impractical for high-speed GC, hence an injector port with a small internal volume was required for this application. Also, very fast thermal desorption from the SPME fibre was required to produce a narrow injection band and achieve effective separation. In the dedicated injector for SPME–fast GC, the injector port was maintained cold during needle introduction and was rapidly heated only when the fibre was exposed to the carrier gas stream. Small custom modifications of the commercial devices can open new possibilities. For example, the addition of input and output connections to the autosampler vial allows the system to be used to continuously monitor flowing streams, as shown in Figure 3.12. The flow-through design facilitates agitation of the sample. Alternatively, when a connection is added directly to the needle of the autosampler syringe, the system can analyse samples present in the vial without needing to expose the fibre. The fibre containing the extracted analytes in its coating can then be introduced to the instrument for desorption. SPME has been introduced as a modern alternative to traditional sample preparation technology, and it is able to address many of the requirements put forward by analytical researchers. This technique eliminates the use of organic solvents, and substantially shortens the time of analysis and allows convenient

SPME-FIBRE INLET

OUTLET

FLOW-CELL

Figure 3.12 Representation of an automated SPME device. Inlet and outlet to be connected through the flow cell can be selected from the carousel.


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automation of the sample preparation step. It can integrate sampling with sample preparation, which makes it suitable for onsite analysis and process monitoring. The configuration and operation of SPME devices are very simple. For example, for the coated fibre implementation of the technology, anyone who knows how to use a syringe is able to operate the SPME device. In the case of automated in-tube extraction for HPLC, fitting a piece of the GC capillary into the system and then turning on the autosampler is all that is required to start its operation. The technology is designed to greatly simplify sample preparation. This feature, however, creates the false impression that the extraction is a simple, almost trivial process. This misunderstanding frequently results in disappointments. It should be emphasized that the fundamental processes involved in SPME are similar to those in more traditional techniques and therefore the difficulty of developing successful methods is analogous. The nature of target analytes and the complexity of the sample matrix determine the level of difficulty in accomplishing a successful extraction. The simplicity, speed and convenience of the extraction devices primarily impact the costs of practical implementation and automation of the developed methods. The potential savings in analysis time, reduced solvent use and apparent simplicity of SPME techniques will continue to attract interest among analytical chemists searching for improved analysis methods. So long as analysts have a sound understanding of the theory and principles behind this technique, good accuracy and precision will follow. Stir Bar Sorptive Extraction (SBSE) Stir bars are coated with a layer (typically 0.5–1 mm thick) of polydimethylsiloxane and then used to stir aqueous samples, thereby extracting and enriching solutes into the polydimethylsiloxane coating. After extraction, either thermal desorption or liquid desorption can be used. This technique is named stir bar sorptive extraction (SBSE) [3]. The basic principles of SBSE are thus identical to SPME using polydimethylsiloxane-coated fibres, but the volume of extraction phase is 50–250 times larger. Early SBSE applications have been reviewed by David et al. [44], and more recently Kawagushi et al. reviewed SBSE applications in environmental and biomedical analysis, mainly focusing on SBSE in combination with in situ derivatization [45]. In this review article, the principle of SBSE is discussed, some practical issues are considered and an overview is given of SBSE in different application areas, including environmental analysis, food analysis and life science and biomedical analysis. Figure 3.13 shows the influence of Ko/w and phase ratio on extraction efficiency. For a given phase ratio (sample volume/PDMS volume), an ‘S-shape’ curve is obtained, where the position of the curve depends on the b ratio. For SPME, the volume of polydimethylsiloxane is approximately 0.5 ml. For a sample of 10 ml, the

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Figure 3.13 Theoretical recovery (%) in function of solute logKo/w for SPME (100 m fibre, 0.5 ml PDMS) and SBSE (1 cm 0.5 mm df, 25 ml PDMS) and 10 ml sample volume. Equilibrium sampling is assumed. (Reprinted from [3] with permission of Elsevier, Copyright 2007)

phase ratio is thus 20 000. This results in poor recoveries for solutes with low Ko/w values. A solute with logKo/w ¼ 3 (e.g. naphthalene, Ko/w ¼ 3.17) is only recovered at 4.8%. The increased recovery obtained by SBSE in comparison with SPME has been demonstrated by different groups, using PAHs [46,47] and pesticides [48] as test solutes. Stir bars of 1 or 2 cm length coated with a 0.5 or 1 mm layer have been made commercially available. A magnetic rod is encapsulated in a glass jacket, on which a polydimethylsiloxane coating is placed. Direct contact between metal and PDMS has been found to catalyse polymer degradation during thermal desorption. Most applications have been performed using pure polydimethylsiloxane coatings. Recently, other phases have also come under development and been tested. Liu et al. [49,50] described the use of sol-gel technology to obtain thin (30 mm) layers of PDMS on stirring rods. Lambert et al. [51] coated restricted-access material (RAM) on stir bars for the extraction of caffeine (logKo/w ¼ 0.1) and metabolites in biological fluids. The RAM-coated stir bars were used in combination with liquid desorption, followed by liquid chromatography. Bicchi et al. [52] described the use of a dual-phase stir bar both in SBSE (immersion) mode and in headspace (HSSE) mode. These new stir bars consisted of an outer PDMS coating with a carbonadsorbent material inside. Magnetic stirring is possible, with two small magnets placed at the ends of the stir bar. This dual-phase device, whereby sorption is combined with adsorption (on the carbon material), showed increased recovery of very volatile compounds emitted from plant material and of more polar solutes in water samples. Alternative designs have also been used [53–55]. Large ‘PDMS rods’ or glass tubes (without magnet) with dimensions up to 8 cm long and coated


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with 250 ml PDMS have been used, in both SBSE (using shaking) and HSSE mode. Zhao et al. [55] used a PDMS rod for passive sampling, and time-weighted average (TWA) concentrations of polycyclic aromatic hydrocarbons were measured in a water stream. SBSE of a liquid sample is performed by placing a suitable amount of sample in a headspace vial or other container. The stir bar is added and the sample is stirred, typically for 30–240 minutes. The extraction time is controlled kinetically, determined by sample volume, stirring speed and stir bar dimensions, and must be optimized for a given application. Optimization is normally accomplished by measuring the analyte recovery as a function of the extraction time. The highest recovery is obtained under equilibrium conditions, where no additional recovery is observed when the extraction time is increased further. However, as with SPME, selected sampling times are often much shorter than the time needed to reach full equilibrium. These non-equilibrium conditions often result in sufficient sensitivity and good repeatability, while the extraction time is not excessively long. Normally, SBSE is applied to the extraction of aqueous samples containing low concentrations of organic compounds. Samples containing high concentrations of solvents, detergents, etc. should be diluted before extraction. For the extraction of highly apolar solutes, such as PAHs and PCBs from water samples, an organic modifier is often added to minimize wall adsorption. The organic modifier will affect the PDMS–water partitioning, but for these applications the overall effect is higher recovery. For the extraction of solutes covering a broad polarity range, optimization of the organic modifier concentration is necessary, and even dual extractions (with and without modifier) are used. SBSE is also used, in combination with in situ derivatization. Polar solutes with low Ko/w values will normally result in very low recoveries. In general, derivatives have higher Ko/w values, and consequently higher recovery and higher sensitivity are obtained. Typical derivatization reactions that can be performed in aqueous media include acylation of phenols using acetic anhydride, esterification of acids and acylation of amines using ethyl chloroformate, and oximation of aldehydes and ketones using pentafluorobenzyl hydroxylamine (PFBHA). Solutes can also be derivatized after extraction in order to improve chromatographic performance and/or detectability. This can, for instance, be performed by silylation. Silylation cannot be performed in aqueous media, but is possible in combination with thermal desorption. Several examples of in situ derivatization and post-extraction derivatization are discussed in a recent review by Kawaguchi et al. [56]. After extraction, the stir bar is removed, dipped on a clean paper tissue to remove water droplets, and introduced into a thermal desorption unit. In some cases, it is recommended that the stir bar be rinsed a little with distilled water to remove adsorbed sugars, proteins and other sample components. This step will avoid the formation of nonvolatile material during the thermal desorption step. Rinsing does not cause solute loss as the sorbed solutes are present inside the

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polydimethylsiloxane phase. Alternatively, liquid desorption can be used. Typically, the stir bar is placed in a small vial (2 ml, or a vial with insert) and desorption is performed with apolar solvents (hexane), followed by GC analysis, or with polar solvents (methanol, acetonitrile), followed by LC analysis. It has been demonstrated [57] that loaded stir bars can be stored at 4 C for 1 week without loss of solutes. This opens interesting prospects for onsite sampling and extraction. The loaded stir bars are sent to the laboratory for analysis, not the samples. After thermal or liquid desorption, the stir bars can be reused. Typically, the lifetime of a single stir bar is 20 to more than 50 extractions, depending on the matrix. For headspace sampling, the stir bar can be placed above a liquid or solid sample, and special devices are available to hold it. PDMS-coated stir bars have also been applied for passive air sampling or for continuous monitoring of persistent organic pollutants in water. Paschke et al. [58] used a stir bar inside a semipermeable membrane. The solutes were enriched based on the diffusion through the membrane, then sorbed into the PDMS material. SBSE was first applied in environmental analysis. The main advantage of the technique is that it can be applied to volatile organic compounds (VOCs) and semivolatile compounds [59]. Most applications, however, deal with semivolatile compounds, typically eluting after decane on an apolar column in GC. For the determination of PAHs in water samples, sensitivities at 1 ng/l can be obtained with RSDs below 15% (at 10 ng/l) [60]. An important aspect in the extraction of these very apolar solutes (logKo/w of benzo[a]pyrene ¼ 6.11) is the reduction of wall adsorption in the extraction vessel. For this purpose, methanol (5–10%), sodium chloride or hyamine (ionic tenside) are added prior to extraction. Leon et al. [61] also demonstrated that a high desorption flow (100 ml/minute) is needed for efficient thermal desorption of the high-molecular weight PAHs (dibenzo[ah] anthracene, benzo[ghi]perylene). SBSE has also been successfully applied to the extraction of pesticides and polychlorinated biphenyls (PCBs) [62]. Popp et al. [63] demonstrated the analysis of PCBs including coplanar congeners (PCB 77, PCB 126 and PCB 169) in water at sub-ng/l levels. For this analysis, 20% methanol was added to the sample to reduce wall adsorption and interaction with humic acids. A multiresidue method for PAHs, PCBs and pesticides was validated by Leon et al. [64] according to ISO/EN 17025. A 100 ml sample was saturated with sodium chloride and extracted (overnight) over 14 hours using a 2 cm stir bar coated with a 0.5 mm thick PDMS film (0.5 mm df). Using thermal desorption combined with GC–MS in scan mode, LODs were in the order of 0.1–10 ng/l (lowest for apolar solutes with high MS response, highest for more polar solutes such as simazine) and repeatability was in the order of 7% at 50 ng/l. The method was used in an interlaboratory trial and the results showed excellent agreement with those obtained by classical methods [65]. Recently, the analysis of odorous compounds in drinking water has also received lots of attention. Compounds such as 2-methylisoborneol (MIB), geosmin and


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chlorinated and brominated anisoles, originating from the corresponding phenols, have odour thresholds of less than 10 ng/l. Using SBSE, these compounds can be extracted with high recovery from drinking water samples. In comparison to labourintensive techniques such as closed loop stripping, the SBSE method provides a much higher sample throughput with better sensitivity, reproducibility and accuracy [65–67]. Nakamura et al. [65] demonstrated that for the target compounds, the sensitivity was 10–50 times better than that of SPME. The method was validated according to an official norm (AFNOR XP T 90–210) by Benanou et al. [67]. A 100 ml sample was extracted using a 2 cm · 0.5 mm df stir bar over 120 minutes. After extraction, thermal desorption and GC–MS in SIM mode were used. Limits of quantification were below odour threshold for all target solutes (0.5 ng/l for geosmin, 1 ng/l for methyl-isoborneol and 0.1 ng/l for trichloroanisole), while the repeatability was better than 15% at 2 ng/l. Parallel sniffing was also used to detect other compounds responsible for off-odours in water [68]. SBSE can be combined with in situ derivatization. Although some papers have described the extraction of phenols without derivatization [69], better recoveries are obtained after in situ derivatization. This can be achieved by adding the derivatization reagent (acetic anhydride) to the sample after pH adjustment (potassium carbonate). The extraction can be performed simultaneously with derivatization. The acylated phenols also perform better during GC analysis, and detection limits down to ng/l levels can be obtained [70,71]. The extraction was performed by SBSE using a 10 mm · 0.5 mm df PDMS stir bar on a 10ml sample after addition of 0.5 g K2CO3 and 0.5 mL acetic anhydride. After extraction, the phenyl acetates were thermally desorbed in splitless mode and analysed on a 30 m · 0.25 mm inner diameter (ID) · 0.25 mm df HP-5MS column using MS detection in selected ion monitoring (SIM) mode. The chromatogram shows the extracted ion chromatograms for the mono-chlorophenols (as acetates) (ion 128, 3 isomers), dichlorophenols (ion 164, 6 isomers, 2 isomers not separated), trichlorophenols (ion 196, 5 isomers), tetrachlorophenol (ion 232, 1 isomer) and pentachlorophenol (ion 266). The excellent sensitivity is clearly illustrated. The same derivatization method was used by Itoh et al. [72] for the analysis of hydroxy-PAHs. Applications of SBSE in food analysis can be divided into three categories: the analysis of minor constituents (volatiles, additives), the determination of trace compounds responsible for off-odours (aldehydes, haloanisoles) and the analysis of trace contaminants (pesticides). In food analysis, SBSE is used for the analysis of volatiles in plant material [73–81] and in fruit, including strawberries [82], grapes [83], snake fruit [84] and raspberries [85]. Both SBSE (immersion) mode and headspace (HSSE) mode were used. SBSE and HSSE were also used for the analysis of volatile constituents and aroma compounds in coffee [86], beer [87], wine [88–91] and whiskey [92]. Volatiles in vinegar [93], volatiles emitted by fungi (including mycotoxin-producing fungi) [94] and decomposition products of peptides and amino acids [101] were measured using SBSE. In general, sorptive

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extraction allows the detection of important flavour and fragrance compounds below olfactory threshold values, as demonstrated by the analysis of compounds released by oak into wine [95]. These compounds included furfural, guaicol, vanillin and whiskey lactone. Unlike in other techniques, no artefact formation, such as transformation of vanillin into the corresponding acetals, was observed. Alves et al. [91] used chemometric data treatment on the results obtained by SBSE–TD–GC–MS to classify Madeira wines according to variety and age. The information obtained on trace levels of monoterpenes in particular was found to increase the discriminating power. Bicchi et al. [96] studied the impact of phase ratio, PDMS volume and sampling time, and temperature on the recovery of volatile organic compounds in the essential oils. It was found that by using HSSE, a concentration factor of 1000 in comparison to static headspace could be obtained, and limits of detection were in the nmol/l range. Besides the analysis of relatively volatile compounds, preservatives such as benzoic acid, sorbic acid and parabens were also measured. For benzoic acid (pKa ¼ 4.21), the sample was adjusted to low pH (pH 2). At pH > pKa, the ionic form results in very low recoveries (logKo/w < 0). Analysis was performed by thermal desorption in combination with GC–MS. Under these conditions, excellent sensitivities (8 ppb) and repeatability (RSD < 5%) were obtained using a 1 cm 1 mm df stir bar in a 25 ml sample [97]. Nonvolatile compounds have also been analysed, such as hop bitter acids in beer, using SBSE followed by liquid desorption and micellar electrokinetic chromatography (MEKC) [98]. A special application was the use of a stir bar, placed in a special holder, for the detection of aroma compounds released in the mouth during wine tasting [106]. Besides the analysis of minor constituents, SBSE has also been used for trace analysis of off-odours. The most important application is the analysis of trichloroanisole (TCA) in wine. This compound is responsible for cork taint. Using SBSE, TCA can be detected at sub-ng/l concentrations, below the odour threshold values [99]. Trichloroanisole can also be detected in cork, using HSSE and thermostating the cork samples at 100 C [100]. As for food applications, SBSE can be applied to biological samples, including urine, plasma, saliva, etc., for the analysis of both volatiles and semivolatiles. In the biochemical/life-science application area, special attention should be paid to the possibilities of in situ derivatization combined with SBSE, since often the target compounds are quite polar (e.g. metabolites). A very interesting application area is described by Soini et al. [101]. SBSE was used to characterize chemical signal compounds in animal urine samples and gland excretion. Based on the detailed profiles obtained, gender and age could be differentiated using chemometric data processing. Wahl et al. described the analysis of barbiturates in urine [102]. Other applications of SBSE for the determination of environmental contaminants in biological samples include the determination of PCBs in sperm [103] and the determination of phenols and chlorophenols in urine [104]. For the determination


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of pharmaceuticals and drugs of abuse (including the metabolites), both direct SBSE and SBSE combined with in situ derivatization are used. More recently, SBSE with in situ derivatization was used by Stopforth et al. in various diagnostic analyses, including the determination of tuberculostearic acid in sputum (a marker of tuberculosis) [105], the determination of the oxidative stress marker 4-hydroxynonenal in urine [106] and the determination of estrone and estradiol (as markers in female urine) [107]. For this application, 1 ml urine was extracted by SBSE using in situ derivatization with PFBHA (oximation of aldehyde). The extracted oxime was further derivatizated by placing the loaded stir bar in the headspace of the acylating reagent (acetic anhydride), thereby derivatizing the hydroxyl function. Finally, thermal desorption–GC–MS in SIM mode was used. The PFBoxime-acetate derivative of hydroxyl-nonenal could be detected in urine at pg/ml levels [108]. Microextraction in a Packed Syringe Microextraction in a packed syringe (MEPS) [108–116], or in a packed pipette tip, is a relatively new technique [108–116]. A small amount (1 mg) of the SPE material is inserted into a syringe (100–250 ml) or pipette tip as a plug, which is secured by frits at either end. The sample is withdrawn through the solid phase plug, and the analytes adsorb on the SPE material. The SPE material can then be washed before being eluted with a suitable organic solvent. The procedure can be performed automatically by an autosampler and even connected online with a GC injector if large-volume injection (LVI) techniques are used. Most current MEPS applications involve online connection with LC rather than GC, since with the automated procedure it is not easy to dry the SPE material before elution and small amounts of water get injected into the GC instrument. In addition, the elution is typically performed with relatively polar solvents such as methanol, which in GC can cause problems. Online connection with GC is possible, but a very careful optimization of injection parameters is then required. Packed pipette tips are typically used in offline mode. The selection of SPE material is the most critical parameter in the optimization of the extraction. In the case of offline procedures, more than one washing step can be carried out, and drying can be done by placing the pipette tip into vacuum or by using a drying agent to remove water from the final extract. The elution solvent, type of injector and injection conditions are critical if MEPS is to be coupled online. In an automated system, where drying typically is not possible, the eluent should be miscible with water. It should also be sufficiently volatile. Methanol is used in most applications, though it cannot be considered a very good choice, especially for LVI to GC. The programmable temperature vaporizer (PTV) interface is used in most applications, usually with solvent split technique. The choice of injection rate, injection temperature and flow rate is particularly critical for volatile analytes.

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3.2.3


Liquid Phase Microextraction (LPME) Techniques

A miniaturized version of traditional liquid–liquid extraction (LLE) has been developed. It is called either liquid phase microextraction (LPME), single-drop microextraction (SDME), solvent microextraction or liquid–liquid microextraction. In this technique, a microdrop of solvent is suspended from the tip of a conventional microsyringe and then either exposed to the headspace of the sample or immersed in a sample solution in which it is immiscible. The operation of SDME has been described in several literature reviews [117]. Headspace SDME is similar to traditional headspace sampling in that volatiles are sampled from the vapours above the sample, so that interference from the sample matrix is avoided. A variety of methods and specialized pieces of equipment are available for this purpose. Another type of microscale LLE is dispersive liquid–liquid microextraction (DLLME). In this method, the appropriate mixture of extraction solvent and disperser solvent is injected rapidly into an aqueous sample, resulting in a cloudy solution. The cloudy solution consists of minute droplets of disperser solvent and extraction solvent in aqueous solution. In fact, this technique can be regarded as multiple-drop microextraction. In DLLME, the instantaneous mixing of the three components ensures equilibration within a few seconds due to the infinitely large interface between the fine extractor droplets and the aqueous solution. Thus, transfer of analytes from the aqueous phase to the organic phase is very short compared with typical equilibrium extraction times for SPME and LPME. To separate the phases, centrifugation is required. Miscibility in organic phase (extraction solvent) and aqueous phase is the main criterion for the selection of the disperser solvent. Typical disperser solvents are acetone, acetonitrile and methanol [117–119]. Liquid phase extraction with microdrops can be carried out as a two-phase or three-phase system. The three-phase system can be regarded as a micro LLE-back-extraction system which exploits the acid–base character of the analytes to achieve simultaneous enrichment and cleanup. The simplest mode of LPME is the SDME, in which analytes are extracted from a stirred aqueous sample into a drop of organic solvent (1–3 ml) suspended from the needle of a microsyringe. Once the extraction is complete, the drop is retracted into the syringe and injected into the chromatographic system for analysis. The main limitation of LPME is the drop instability. The droplet may be lost from the needle tip of the syringe during the extraction, particularly when samples are stirred vigorously, or emulsion may form, particularly in the extraction of biological samples. The advantages of the single-drop technique include the suitability of a wide range of solvents, low solvent consumption and low cost. In addition, no preconditioning is required, unlike in SPME, where the fibre must be pretreated for efficient extraction. Also, memory effects are avoided. However, because solvents must have a relatively high boiling point, the method is not suitable for the extraction of highly volatile analytes.


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Parameters that have been considered for SDME include solvent type, size of drop, shape of needle tip, temperature of sampling, equilibration and sampling (extraction) time, effect of stirring and ratio of headspace volume to sample volume [120,121]. Naturally, pH and salt addition and other parameters that affect the LLE process must be considered as well. The choice of solvent is critical. In headspace analysis, the solvent should not be too volatile, because otherwise the drop will evaporate during the extraction. At the same time, the solvent should not elute together with analytes, and it should not, therefore, be too nonvolatile. Neither should it be insoluble in the sample solvent. Naturally, the solvent in the drop should dissolve well to analytes. Typically, n-octyl acetate, isoamyl alcohol, undecane, octane, nonane and ethylene glycol are used as solvents. Drop volumes are typically 1–2 ml, as solvent drops of these volumes are relatively stable. In most cases, the extraction is done at room temperature, because even though increase of temperature usually increases the extraction efficiency, it also leads to drop instability and thereby to lower reproducibility. Stirring must also be done at low speed because otherwise it affects the stability of the drop. One way of minimizing the problems of drop instability in LPME is to add a polymeric membrane to serve as a support for the extracting solvent. This not only enables the use of larger volumes but also acts as a physical barrier between the phases. The membrane is usually made of polypropylene or other porous hydrophobic material. Polypropylene is highly compatible with a broad range of organic solvents and, owing to a pore size of 0.2 mm, the membrane strongly immobilizes the organic solvent in the pores. A further benefit of the use of a membrane is that, owing to the pore structure, the concentration of high-molecular mass compounds in the sample extract is reduced. Membrane-assisted liquid extraction can thus be considered a subtechnique of LPME. Membrane-assisted liquid extraction can be performed in a hollow fibre, a flat-sheet membrane module or a membrane bag [124,125]. The membranes can be microporous, where the pores are filled with an organic solvent, or nonporous. In the latter case, the extraction mechanism is different, as analytes must diffuse through the membrane before extraction to the acceptor phase. Figure 3.14 shows different systems utilized in membrane-assisted liquid extraction. The extraction can be static or dynamic, depending on the membrane module. As in LPME, two-phase and three-phase systems are possible. Membrane extraction systems based on hollow fibres closed at both ends are also available, and can be used for onsite sampling [126]. The two-phase mode is typically utilized in microporous membrane LLE (MMLLE) and the three-phase mode in supported liquid membrane LLE (SLM). The MMLLE version is mostly used in combination with GC, while SLM, in which the final extract is typically aqueous, is used with LC and electro-driven separations. In two-phase extraction, the extraction is based on diffusion of the analytes from the aqueous sample (donor) to the organic acceptor solution. The extraction process depends on the partition coefficients of the analytes. Static membrane-assisted extraction typically employs

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Figure 3.14 Different solutions for membrane-assisted liquid extraction: (a) a U-shaped hollow fibre; (b) a rod-like hollow fibre; (c) a membrane bag; (d) dynamic MMLLE. (Reprinted from [122] with permission from Elsevier, Copyright 2008)

hollow fibres or membrane bags, both normally disposable. The acceptor volume, i.e. the volume of the organic solvent, is from 5 to 25 ml, while sample volumes range from 0.5 to 4 ml. In dynamic MMLLE, the membrane unit is either a planar membrane or a hollow-fibre membrane. In the planar configuration, the membrane is clamped between two blocks and separates two flow channels: the donor and acceptor channels. In the hollow-fibre membrane module, the acceptor phase flows inside and the donor phase outside the membrane. Membrane-assisted solvent extraction offers a means to combine conventional LLE online with GC. The interface between the extraction unit and the GC is typically an on-column interface or a PTV. From an instrumental point of view, online MMLLE–GC is straightforward. The MMLLE is virtually a microsystem for continuous LLE and is used for the extraction of nonionizable compounds with nonpolar organic solvents. Also, the solvent volume is typically small and well suited for online transfer. Miniaturized membrane extraction systems have also been reported [127], where the extraction is performed in a system installed directly to a GC injector (Figure 3.15). The interfacing of membrane extraction and GC can be done directly or via a sample loop located in a multiport valve. The main disadvantage of all membrane extraction techniques is that the extraction tends to be non-exhaustive. The recoveries are usually at a similar level as those in SPME, and calibration must be carefully carried out. Quantitative recoveries are obtained


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Figure 3.15 Miniaturized online MMLLE–GC system, installed to a GC injector port. The parts numbered are: 1, sample pump; 2, sample pipette; 3, sample tray; 4, pipette needle port; 5, extraction card holder, called a card guard (CG); 6, waste valve; 7, waste bottle; 8, solvent pump; 9, 4 ml solvent vial; 10, GC needle; 11, solvent cup; 12, washing fluid

for highly hydrophobic analytes, and also when either the flow rate of the donor is kept sufficiently low or the sample is circulated across the extraction system. If quantitative recoveries are not required, the extraction time can be decreased by increasing the flow rate of the sample, or the sensitivity can be increased by using an excessive amount of sample. Selection of the solvent is critical, but the literature data on conventional LLE can be utilized in the selection. The solvent should be immiscible with water and strongly immobilized in the pores of the membrane. It should also be sufficiently volatile, though too volatile a solvent (e.g. pentane) can cause problems in the extraction. Selective extraction can be achieved by a careful choice of the material and the pore size of the membrane, as well as the extraction solvent. Pore size is important because in addition to the LLE process, size exclusion takes place in the extraction, increasing the selectivity. 3.2.4

Comparison of Solid and Liquid Phase Microextraction Techniques

Choosing a suitable extraction technique requires consideration of a range of factors, including the efficiency of the extraction, the sample throughput time, the

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complexity (cost) of equipment, the complexity of method development, the amount of organic solvent used and the range of applicability. SPME and SBSE instrumentation is commercially available, and SPME in particular has been widely applied to the analysis of several types of volatile compound. SPME instrumentation is simpler, as the injection in SBSE requires a special interface for thermal desorption. However, the extraction efficiency is clearly better in SBSE than in SPME, and SBSE is thus more suitable for trace analysis. Both techniques are solvent-free and easy to use, and the enrichment factors are typically high. In addition, SPME and SBSE devices are easily stored and transported, and they can be applied even in onsite sampling. SPME has also been used for in vivo sampling. Field sampling is effective because only the fibre or stir bar with the absorbed analytes needs to be brought back to the laboratory. Transportation of large sample volumes is avoided, and no sampling accessories – such as pumps or filters (as found in onsite SPE) – are required. SBSE is the more rugged system for onsite sampling because SPME fibres are quite fragile. In addition, if trace amounts are to be extracted, SBSE gives higher recoveries and thus better sensitivities. Both techniques are very useful for samples where the sample volume is limited, as for example in the determination of the composition of pore water in marine sediments. Although derivatization can improve the extraction of polar analytes, both SPME and SBSE are best suited to the extraction of relatively nonpolar and reasonably volatile analytes. Online SPE–GC and MEPS and pipette-tip SPE are adsorptive extraction techniques and they give quantitative recoveries, unlike the abovementioned sorptive extraction techniques, and are therefore well suited to trace analysis. Awide range of SPE materials are available, and not only nonpolar but also polar analytes can be extracted efficiently. These SPE techniques tend to be less selective, and matrix components are often extracted as well. On the other hand, the rather low selectivity is an advantage for a profiling type of analysis, i.e. when all or most of the sample components are of interest. SPE–GC can be performed in a fully automated and closed system, where the whole extract is transferred for the GC analysis. This improves the sensitivity and reliability of the analysis, as problems with sample loss and contamination are minimized. Although use of the instrumentation is fairly straightforward, the system is complex, particularly in the method development. Pipette-tip SPE is simple to use, but as an offline technique is performed manually and is user-dependent and less repeatable than the online system. Also, because only part of the extract is analysed, the sensitivity is not as good as in the online system. The optimization is easy and flexible, only small volumes of solvents are needed, and as a new pipette tip can be used for each extraction, there are no problems with memory effects. MEPS can be employed as an automated online technique, but the lack of a drying step makes the interfacing with GC demanding. Unlike the other techniques described here, LPME does not require any special instrumentation. It is not particularly rugged, however, and the repeatability is very much dependent on the user. Although it is often said that


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LPME has been developed as an alternative to SPME, the applications are not quite the same. In LPME, the solvent must be relatively nonvolatile, and thus highly volatile compounds cannot be analysed because of coelution with the solvent peak in GC. In contrast, (relatively) nonvolatile analytes can be extracted and desorbed without problem, unlike in SPME and SBSE. Thus, this technique is best suited for semivolatile to (relatively) nonvolatile analytes. The membrane-assisted extraction techniques, which apply the same extraction mechanism as LPME, are likewise best suited for semivolatile to (relatively) nonvolatile analytes. The choice of solvent is less critical than in LPME. The repeatability is typically better than in LPME because there are no problems with drop stability as there are in LPME, and larger volumes can be extracted. Static membrane-assisted extraction, with either hollow fibres or membrane bags, is inexpensive and simple to use, and since the membranes are typically for single use, there are no problems with cross-contamination. The flat-sheet modules are best suited to dynamic extraction of larger sample volumes and to online connection with GC. In the flat-sheet modules the membranes are normally used for several extractions, and careful cleaning between extractions is required to minimize the risk of cross-contamination. The main advantage of the miniaturized SPE methods over SPME, SBSE and LPME is that because the extraction is typically quantitative, the calibration is straightforward. This is in contrast with the other techniques, where various calibration methods are employed, including classical calibration relying on equilibrium extraction, or more novel kinetic calibration.

3.3

Simplification of Sample Treatment: Continuous Flow Systems

Flow systems provide an elegant, effective way of automating sample treatment. This section is concerned with how sample processing can be improved by using a flow system connected to a discrete sample introduction system. As stated in other chapters, flow systems are especially relevant to sample treatment and automated method calibration. Online combinations of automated sample pretreatment units and discrete sample introduction devices are highly attractive in this context as they allow the whole analytical sequence to be developed in a single instrumental assembly while avoiding the typical problems associated with conventional sample treatment approaches. Simplification also influences the quality of the information delivered by analytical laboratories. Flow analysis systems can help consolidate this as a trend. In fact, the intrinsic advantages of flow systems, which typically include low costs, high flexibility and throughput, and the ability to easily minimize errors, make them especially suitable for developing fast-response analytical systems. A flow analyser provides binary information that can be used for sample classification and qualification in accordance with preset threshold values imposed by either clients or

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legislation [128]. To this end, the flow system can be online coupled to a detector (vanguard configuration) or a high-performance instrument (vanguard/rearguard configuration), the flow-processing unit being used to treat samples [28]. Vanguard/ rearguard configurations are the more flexible inasmuch as the sample treatment unit and the high-performance instrument can either share the same detector or use different ones, which facilitates alteration of the system as required to perform additional or alternative confirmation measurements. Depending on the degree of human participation and the particular hardware used, samples can be treated for analysis by using batch procedures, integrated devices or coupled devices. In the latter case, devices can be coupled at-line, online or in-line. At-line coupling involves the use of a robotic interface to link the flow system to the measuring instrument. Usually, the robotic interface operates either by placing a fraction of treated or conditioned sample into an autosampler vial or by inserting the sample directly into the injector of the analytical instrument. Unlike at-line coupling, online coupling involves direct contact between a flowing stream coming from the flow system, a valve (or an appropriate interface) and a part of the instrument, usually the injection region. The flow system and the instrument are usually linked via a flow element such as a valve, T-connector or split-flow interface. Finally, in-line coupling involves complete, close integration of the flow system and the instrument. This is accomplished by incorporating a critical element of the flow system (e.g. an extraction unit) into the instrument. Manufacturers have favoured at-line coupling in commercial instruments. One typical example is the device used to couple SPME in fibres with liquid or gas chromatographs. Because the commercial instrument is of the closed type, the analyst has no access to specific parts, which restricts the potential for linking to other devices. This is probably why flow systems and instruments are most often coupled by inserting a fraction of sample into the injector of the measuring instrument. 3.3.1

Coupling to Gas Chromatography

The main difficulty in coupling flow systems to gas chromatographs arises from the different aggregation states of the fluids involved and the relatively low sample volume that needs to be inserted (a few microlitres) in order to maintain chromatographic resolution [129]. This problem can be avoided by using the LVI technique, which affords injections of hundreds of microlitres with a negligible effect on resolution. This section deals with the online coupling of flow configurations of the low- and high-pressure type to gas chromatographs. Most existing configurations have evolved from previous offline arrangements, which can be deemed at-line systems when an autosampler is used to inject samples.


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Properly coupling a flow-processing device to a gas chromatograph entails consideration of the following: (i) the volatility and thermal stability of the target analytes; (ii) the compatibility of the medium to be used with liquid samples; (iii) the extract volume, which should be as low as possible in order to minimize analyte dilution and avoid unduly decreasing the sensitivity as a result; and (iv) the transfer of the analytes from the automated pretreatment module to the gas chromatograph, which should be quantitative. A number of interfaces have been used for this purpose, the three most popular being based on on-column injection, solvent vaporization and multiport valves. In the on-column interface, a continuous high-pressure flow system based mainly on SPE is coupled to a gas chromatograph via a six-port injection valve controlled by the GC software; the valve transfers the analyte, in an appropriate medium, via a metal or fused-silica capillary typically 20–30 cm long · 0.1 mm ID that is inserted into the septum of the on-column injector. Because this type of system is usually employed with the LVI technique to inject large volumes of solvent (typically 100 ml), the on-column injector must be connected to a retention gap (a 3–5 m long · 0.53 mm ID deactivated fused silica capillary) followed by a 1–2 mm · 0.25 mm ID retaining precolumn and finally the analytical column. Usually, the precolumn and the analytical column contain the same stationary phase. Also, a solvent vapour exit (SVE) kit is installed between the two columns in order to remove most of the injected solvent and minimize the loss of the most volatile compounds. Alternatively, a programmable temperature vaporizer (PTV) can be used instead of the on-column injector, the SVE being kept even though it is unnecessary as excess organic solvent can be removed in the split valve. When a multiport injection valve is used, the organic phase that leaves the flow-processing device, which contains the highest analyte concentrations, is used to fill the loop of a high-pressure injection valve (typically 5 ml volume). A desiccating column is placed in front of the valve in order to prevent water traces from reaching the chromatographic column. The carrier gas of the chromatograph is also used to transfer the analytes from the loop to the injector via a PTFE or stainless steel tube furnished with an injection needle, which is inserted into the septum of the split/splitless injector. Depending on the particular instrument and injector configuration, the carrier gas should either be split (between the injection valve and the chromatograph) or not in order to maintain peak resolution. In some cases, additional heating of the interface is required. The following section describes the uses of nonchromatographic separation techniques for sample treatment in flow systems and discusses the most salient advantages and disadvantages of the previous interfaces. The main purpose of coupling a flow-processing device online to a gas chromatograph is usually to facilitate the development of a fully automated sample pretreatment process. Obviously, a continuous nonchromatographic separation technique will often be required for this purpose. SPE is the most common choice. This is hardly surprising since SPE is compatible with gas chromatography, because

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the analytes, which are isolated on a packed sorbent material, are eluted with a few microlitres of an organic solvent that can be directly transferred to the instrument for separation prior to detection. Water traces can be removed from the sorbent by drying prior to elution. This online trace enrichment operation can be performed using various flow configurations. The earliest approaches used a low-pressure continuous flow system in which the sorbent was packed in a laboratory-made miniaturized column accommodated in the loop of an injection valve, the sample and reagents (conditioning solvents, eluent and derivatizing reagents, if needed) being directly aspirated into the manifold. A wide variety of applications have been developed following this basic configuration, including the determination of pollutants, vitamins, fatty acids and drugs in samples of environmental, clinical, toxicological and agrifood interest [129]. The flow manifold can also be constructed by using a combination of highpressure injection valves and pumps in order to deliver the sample and organic solvents required to condition the columns, along with a syringe pump for the eluent. The sorbent column is hand-packed (usually with a polymer) and placed between the high-pressure injection valves for the sample and eluent. Typical determinations with these systems include pesticides [130] and endocrine disruptors [131] in water. The extraction selectivity can be increased by using an immunosorbent material, such as that employed in the determination of s-trizine in aqueous samples, with the Prospekt (Programmable Online Solid Phase Extraction Technique) system (commercially available from Spark Holland) for sample preparation, a Midas autosampler (also from Spark Holland) and an HPLC pump for analyte elution. A PTV interface can be used in combination with the LVI technique for the determination of pesticides in water, basically [132]. Dynamic microwave-assisted extraction [133] and dynamic sonication-assisted solvent extraction [134] can be coupled online prior to SPE– LVI–GC in order to determine organophosphate esters in air samples. Analytical pervaporation has proved a reliable alternative to headspace techniques for the direct analysis of volatile fractions when coupled online to gas chromatography [135]. Usually, the pervaporation module consists of a lower unit for sample introduction and an upper part insulated by a membrane permeable to the target analytes. A spacer is usually needed to facilitate formation of the headspace. For coupling, the upper chamber is placed in the loop of an injection valve in order to allow the acceptor gas (namely the chromatographic carrier gas) to pass through and drive the analytes to the injector. LLE can also be implemented in a continuous-flow manifold. However, it is not the first choice as it provides poorer enrichment factors due to the need to maintain the organic-to-aqueous phase ratio as close to unity as possible in order to ensure efficient phase separation, and also because of its lack of robustness, the weakest element in the system being the phase separator (a T-piece, membrane or sandwich connector). In any case, online coupled LLE has been used with or without


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derivatization to determine phenols, fatty acids, pesticides and cholesterol in waters, fats and dairy products. Emerging nonchromatographic separation techniques have also been online coupled to gas chromatography with a view to circumventing the shortcomings of some well-established extraction techniques. Such is the case with MMLLE, which has some advantages over LLE such as the formation of zero emulsions, the use of smaller sample volumes and the obtainment of clean extracts. Also, selectivity can be increased by choosing an appropriate membrane material and pore size. Reported applications use planar flat-sheet polypropylene membranes sandwiched between two blocks 10–100 ml in volume. The extraction unit and the GC instrument are interfaced via a multiport valve accommodating the sample loop. The membrane should be replaced every 50–100 uses, depending on the particular application, in order to minimize potential adsorption problems. Configurations typically use LVI and SVE. These systems have been employed to determine organochlorine pesticides in water [136] and red wine [137]. Hollow-fibre membranes, which provide increased extraction surfaces – and improved extraction efficiency as a result – have been used for the extraction of polycyclic aromatic hydrocarbons and pesticides from soils and grapes, respectively, following online pressurized hot-water extraction. The water phase, containing the analytes, was driven to the donor side of the membrane and the analytes were extracted to the acceptor solution, the concentrated extract then being online transferred to the GC instrument via an on-column interface. Flow-processing devices for coupling to gas chromatographs use a low- or highpressure mode in combination with split/splitless injection or LVI. In general, they afford the introduction of untreated samples, which simplifies the analytical process and results in improved productivity-related analytical properties. The most common nonchromatographic techniques can easily be coupled to GC via an appropriate interface; alternatively, a tandem flow-system interface can be used for the online coupling of a previous auxiliary energy-based extraction system. 3.3.2

Coupling to Liquid Chromatography

The combined use of flow-processing devices and liquid chromatographs has proven a powerful tool for solving a number of problems in analytical science. The nature of the two partners facilitates their coupling, which produces a synergistic effect that enhances the analytical properties of the ensuing methods. The similarities between the two partners are apparent from their associated equipment. Flow-processing devices and liquid chromatographs can be coupled into two main types of configuration, namely: (i) Precolumn arrangements, where the flow system is used mainly to improve sensitivity and selectivity by preconcentration or cleanup, but can also be

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employed for other purposes such as saving reagents or introducing problematic or hazardous samples. (ii) Post-column arrangements, which are intended to facilitate detection of the target analytes by derivatization in the flow system following separation on the chromatographic column. This section focuses on precolumn arrangements, where the flow device is used to introduce treated sample aliquots into the chromatographic system. The highpressure injection valve is the central element of the combined system as it acts as the interface between the low-pressure (flow) and high-pressure (chromatographic) lines. The autoinjector of a liquid chromatograph constitutes the simplest example of a precolumn arrangement (in the at-line mode); an automatic syringe similar to those employed in sequential-injection (SI) analysis is used to fill the sample loop of the injection valve. In practice, coupled flow-processing/liquid chromatography systems depart markedly from the above-described example. In fact, the flow-processing device is used not only to introduce samples into the chromatographic system but also for sample pretreatment. The pretreatment, which is mainly intended to adapt the sample to the chromatographic separation conditions, involves one or more operations including a nonchromatographic procedure. Treated sample aliquots are then driven to the high-pressure injection valve, which is actuated to introduce the plug into the column (see Figure 3.16). Reproducible injection into the chromatograph relies on accurate timing and synchronized operation of the whole system. A wide variety of sample-treatment protocols can be used to increase the sensitivity and selectivity of determinations. Using an SPE protocol for analyte preconcentration and cleanup allows a different type of coupling to be implemented (see Figure 3.17). A precolumn packed with sorbent material is accommodated in the loop of the high-pressure injection valve to retain the target analytes. The flow system allows the initial steps of the SPE protocol (conditioning, equilibration, sample loading and sorbent cleanup) to be developed in an automatic manner. Elution is carried out by switching the valve to its injection position. Fully automated SPE-based configurations such as the Prospekt [138] or Aspec [139] can also be online coupled to a liquid chromatograph by using a high-pressure injection valve as an interface between the two.

HPP IV FPD

Chromatographic column

D

waste

Figure 3.16 General flow device–liquid chromatographic coupling. FDP, flow-processing device; HPP, high-pressure pump; IV, high-pressure injection valve; D, detector


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HPP IV IV


FPD FPD

D D

waste SPE column

Figure 3.17 Continuous SPE coupled to liquid chromatography. FDP, flow-processing device; HPP, high-pressure pump; IV, high-pressure injection valve; D, detector

Other coupled systems use two or three injection valves. Thus, a configuration with two valves was used to implement sample screening and confirmation methods in the same manifold; this operational mode has been referred to as the dual use of flow configurations in liquid chromatography. To this end, an SPE column is placed in the first valve and the chromatographic column in the second, the two being online connected as shown in the general scheme of Figure 3.18. The second valve is essential for switching between the screening (load position) and confirmation (inject position) modes. Essentially, the flow-injection (FI) system is first employed to screen all samples for the target analytes or analyte families, and then those with total concentrations close to or above the preset cutoff level are individually analysed by liquid chromatography. In the confirmation step, the FI system can be used either to preconcentrate samples or as a post-column derivatization system. Before analytical injection techniques were developed, continuous segmented flow configurations were used in a precolumn combination with liquid chromatography. This allowed samples to be processed in an automatic manner. Such a combination can thus be considered a precursor to current flow approaches based on sample injection. FI techniques evolved from a straightforward operational basis, used inexpensive hardware, were easy and convenient to operate and highly flexible, and provided a high sample throughput and cost-effective performance. Sequential-injection (SI) techniques evolved from FI and shared some of their basic features. Both FI and SI manifolds have been widely used to design flow-processing devices coupled online to liquid chromatographs. Both techniques can be used in combination to assemble complex manifolds for specific applications.


HPP

IV

IV FPD

D

waste SPE column

Figure 3.18 Flow-processing device coupled online to liquid chromatograph involving both a screening and a confirmation method. FDP, flow-processing device; HPP, high-pressure pump; IV, high-pressure injection valve; D, detector

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Miniaturization of Analytical Systems Sample Waste D Mobile phase

CC HC

SV

SP

Figure 3.19 Sequential-injection chromatography. SP, syringe pump; HC, holding coil; SV, selection valve; CC, chromatographic column; D, detector

The recently developed technique of sequential-injection chromatography (SIC) [140] testifies to the highly symbiotic nature of flow manifolds and liquid chromatographs. The SIC technique (see Figure 3.19) uses an SI manifold to introduce samples and deliver the mobile phase, in addition to a monolithic chromatographic column to separate analytes at a low pressure. This combination has been used to determine naphazoline nitrate and methylparaben [141], and lidocaine and prilocaine [142], in pharmaceuticals. The flexibility of FI and SI techniques has been exploited to efficiently pretreat samples for chromatographic separation, implementing several nonchromatographic techniques. Membrane-based separation techniques include dialysis and gas diffusion. These have been used for the determination of anions, and of ammonia and methylamines in natural water, respectively. A typical sandwich diffusion cell is placed in the low-pressure line and the acceptor stream is driven to the loop of the high-pressure injection valve. ASTED systems (automated sequential trace enrichment of dialysate), which perform dialysis and preconcentration, have been successfully used to determine tetracycline in eggs, and ioxidanol in human, rat and monkey plasma. Online membrane extraction, both alone and in combination with pervaporation, has been used to preconcentrate analytes prior to HPLC processing. In recent years, microdialysis coupled online to HPLC has proven effective for the analysis of some species in complex matrices, showing operational ease, expeditious isolation of components from the sample matrix, potential enrichment and the use of little or no organic solvent. The determination of sulphonamides in milk and of organic acids in milk fermentation products constitute two salient examples [143]. The SPE/HPLC couple is a consolidated choice that has been applied to the determination of pesticides in water, lanthanides in rocks, and caffeine and selected anilines and phenol compounds in aquatic systems. This operational approach is in continuous development, particularly as regards sorbent materials and protocols.


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Some authors have used molecular imprinted polymers (MIPs) [144] and carbon nanotubes [145] as sorbents for selective analyte retention. Similarly, new protocols such as renewable SPE are being automated and combined with HPLC. The synergetic hyphenation between flow-processing devices and liquid chromatography has been extensively exploited in analytical sciences, since it is useful and easy to implement, taking into account the nature of the partners. Flowprocessing devices provide an evident selectivity and sensitivity improvement to the developed methods. Several nonchromatographic techniques have been automated and coupled to liquid chromatography, proving the evident versatility of the described arrangement. 3.3.3

Coupling to Capillary Electrophoresis (CE)

CE is a highly efficient, flexible separation technique that has become a serious competitor to other separation techniques, including chromatographies. However, sample requirements are more stringent in CE than in other separation techniques. Appropriate sample treatment is a critical step toward obtaining accurate, reproducible results; this entails avoiding clogging of the capillary and adsorption of macromolecules on its walls, among other things. Electro-osmotic flow is one of the driving forces in CE. This phenomenon arises from the presence of surface charges on capillary walls. The result is a net flow of buffer solution in the direction of the negative electrode. Electro-osmotic flow is quite robust and occurs at a rate of 0.5–4 nl/s depending on the buffer pH. One other important factor is the small inner diameter of the capillary, which affords the use of very low volumes of samples and reagents, and also effective miniaturization. The ability to couple flow-processing devices to CE equipment is limited by the following factors, all of which warrant careful consideration: (i) Compatibility of hydrodynamic flow in the processing device with electroosmotic flow in the capillary. (ii) Compatibility of the high flow rates typically used in processing devices with the low rates of electro-osmotic flow in a CE system. (iii) Compatibility of the sample plug coming from the processing device with the small sample volume to be introduced in the CE capillary. (iv) Compatibility of the sample composition with the electrophoretic system. (v) Decoupling of the high voltages and currents applied to the electrophoretic separation system and the flow-processing device. The following sections describe selected coupled systems and their interfaces, and also some of their more salient uses. Flow-processing devices have been coupled in-, at- and online to CE equipment [146]. Overall, online coupled systems are the most commonplace. Interested readers can find a review of sample treatment devices used in combination with commercially available CE equipment in [147].

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Coupling a flow-processing device at-line with CE equipment entails the use of a robotic arm interface to place the sample coming from the former in an empty vial in the latter [147]. This combination is subject to several restrictions, the most significant of which are the accessibility of the CE autosampler and the minimum volume that can be delivered to the vial. Also, it requires the use of an electronic interface and appropriate control software in order to synchronize the movements of the robotic interface and autosampler. As can be seen in Figure 3.20, the needles of the programmable robotic arm can be positioned in two ways with respect to the autosampler vials. While the flow-processing device is working, the needles are down and the sample is prepared and transferred to the CE vial. When the flowprocessing device stops, the needles are lifted and CE analysis is started by moving the previously filled sample vial to the position where the capillary end and electrode are located. As shown in Figure 3.20, the programmable arm can be fitted with one or two needles. In the latter case, the needles can be identical or different in length, the longer one being used to fill the autosampler vials and the shorter one to drain them in order to maintain a preset liquid level. When a single needle is used, a constant, preset volume is delivered to each vial (e.g. by using air as carrier). As an alternative to a programmable robotic arm, processing devices and CE equipment can be coupled at-line via the replenishment system, which is used to empty vials and fill them with fresh buffer in some commercial instruments [148]. This entails disconnecting the replenishment needles from the Teflon tube coming from the replenishment bottles and reconnecting them to one coming from the flowprocessing device [147]. The most salient advantage of at-line coupled systems is that the CE equipment is run in its normal operation mode. This allows samples to be introduced hydrodynamically or electrokinetically into the capillary. It also facilitates other CE operations such as capillary conditioning. Coupling a flow-processing device online to CE involves inserting the capillary end of the latter in a continuous stream of the former. Because the flow-processing device and the electrophoretic system operate at different flow rates, the two require a split-flow interface for coupling. The split-flow interface was developed simultaneously in a vertical configuration by Fang’s group [149] and in a horizontal configuration by Karlberg’s [150]. Figure 3.21 compares the two types of interface, which possess a low dead volume and are electrically grounded. Samples and electrolyte solutions are introduced in the electrophoretic capillary by the effect of electroosmotic flow and electrophoretic mobility. Hydrodynamic flow should be avoided by ensuring that the liquid level at the interface (the capillary inlet) coincides with that in the capillary outlet. A split-flow interface can be readily constructed from dielectric materials such as Teflon, methacrylate or Plexiglas. Alternatively, the electrophoretic capillary can be inserted into a piece of Tygon tubing and attached to it with glue.

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Figure 3.20 Schematic depiction of the at-line coupling of flow-processing devices to CE equipment. Configuration of robotic interface in (1) sample-collection mode and (2) sample-injection mode for electrophoretic analysis. Description of flow system manifold for (a) robotic interfaces with two needles and (b) robotic interfaces with one needle

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Figure 3.21 Schematic depiction of the online coupling of flow-processing devices to CE equipment. Detail of split-flow interface (a) in vertical design and (b) in horizontal design. Description of systems for performing hydrodynamic injection mode for electrophoretic analysis

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130 Miniaturization of Analytical Systems


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The flow-processing device is made compatible with the electrophoretic system by grounding the interface. This avoids voltage differences between the interface and the flow system. Electrophoretic separation is accomplished by applying a voltage difference to the capillary end, as shown in Figure 3.20. This configuration is highly recommended when an optical detector is used with the electrophoretic system, but hardly useful with other types of detector (e.g. mass spectrometers). According to Valcarcel’s group, the split-flow interface must be subjected to a voltage difference in order to obtain one in the electrospray needle of the electrospray ionization interface of the mass spectrometer [151]. Figure 3.22 shows two configurations with mass spectrometers, one with a grounded electrospray needle and one with an electrospray chamber. The current arising from the voltage difference between the grounded flow-processing device and the split-flow interface – which is connected to a voltage source – must be interrupted in order to avoid arc discharges in valves and pumps in the flow system. Alternatively, the current can be interrupted by using a plug of a substance with a high electrical resistance, such as pure water or air. It is also advisable to insert a safety line consisting of a grounded electrode immediately in front of the pumps. In principle, the use of online coupled systems linked via a split-flow interface is limited to the electrokinetic introduction of samples. However, this injection mode

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Figure 3.22 Schematic design of the online coupling of a flow-processing device to CE–mass spectrometry equipment through a split-flow interface. This configuration requires voltage isolation through a plug of air or a large plug of pure water

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provides biased results in some cases. This has prompted the use of hydrodynamic injection instead. Thus, Pu et al. [152] used a split-flow cell affording hydrodynamic injection of samples into the CE capillary by electro-osmotic flow. The interface included a Nafion joint to connect the CE capillary to the tube of the flow system (see Figure 3.23(a)). An electronic time-relay system was used to control switching of the high voltage. Kuban et al. [150] inserted a valve at the end of a horizontal splitflow interface. Switching the valve off caused the sample to be forced into the capillary. The greatest limitations of this approach arise from the need to control the time during which the valve is on and off, and also the pressure generated in the system. Santos et al. [151] proposed controlling the pressure by using a valve loop capable of withstanding higher pressures. With commercial CE instruments, one can also connect the pressure system [147] (see Figure 3.20). It should be noted that commercial CE equipment affords injection at a low pressure (0.5 psi) with a high precision (0.01 psi). The split-flow interface can be used for the online coupling of both FI and SI systems. Following a common approach, split-flow interfaces, microsequential injection systems and integrated lab-on-a-valve systems have all been coupled to CE. SI systems have also been online coupled via a microvalve allowing the insertion of a constant volume of sample into the capillary. However, the loop volume is high relative to those typically used in CE work and must be reduced by switching the valve or flushing the sample from the interface in order to facilitate electrokinetic insertion of a portion of sample (see Figure 3.23(b)). A modified version of the split-flow interface was recently used to accommodate a typical SPME fibre precisely at the capillary end [153]. Although samples were treated on the fibre and the flow system was only used to couple SPME and CE, they could also be processed at the interface connecting the fibre to the flow system. In-line coupling a flow-processing device to CE involves either inserting the capillary end (inlet region) into the treated sample or vice versa. This can be done by using hollow fibres to perform LPME. As shown in Figure 3.24, the electrophoretic capillary can be inserted into the lumen of a hollow fibre, or the extraction unit into the capillary. In the latter case, the fibre can be fitted to the capillary by heating. Flow systems have also been coupled to microchip electrophoretic systems [154], using a capillary as the interface between an SI system and the microchip; for this purpose, one end of the capillary is attached to the microchip via a Teflon fitting. The principal shortcoming of this combination is the presence of residual hydrodynamic flow in the microchip separation channel [154]. The use of flow-processing devices constitutes one of the most reliable ways of improving performance in analytical methods through automation, miniaturization and simplification of the preliminary operations of the analytical process – which are critical in CE work. Coupling flow-processing devices to CE equipment facilitates three types of task, namely: automatic calibration, automatic screening of samples and sample preconcentration and cleanup [146].

Figure 3.23 Alternatives to automatic hydrodynamic sample introduction for electrophoretic analysis in online flow system–CE combinations: (a) use of Nafion membranes; (b) use of injection valve

Automation and Miniaturization of Sample Treatment 133

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Schematic depiction of the in-line coupling of flow-processing devices to CE

Throughput in this context is limited by electrophoretic time. In fact, the high applied voltage must be interrupted and the capillary rinsed before each new run. Kuban et al. [150], however, have demonstrated that consecutive runs are feasible. The coupled equipment described in the literature includes extraction, filtering, dialysis, membrane, gas extraction and gas diffusion units; hollow fibres; and even auxiliary devices. The principal conclusion is that choosing an appropriate flowprocessing device can facilitate the treatment and analysis of virtually any type of sample (biomedical, pharmaceutical, environmental, food). Readers interested in a more detailed description of the types of device coupled to CE so far are referred to [146,147]. One of the most critical factors in selecting a flow-processing device is the compatibility of the chemical composition of the conditioned sample with the electrophoretic analysis to be performed. A number of sample-treatment techniques (e.g. SPE, LPE) require the use of organic solvents such as methanol, direct insertion of which into an electrophoretic buffer can result in current interruptions. This problem can be avoided by changing the buffer pH, adding a modifier (e.g. a surfactant to the organic solvent) or supplying the buffer with a small amount of solvent. In this way, at-line coupled systems afford slight modification of treated or conditioned samples by collection into vials containing a modifier. Alternative approaches involve modifying samples within the flow system. Other influential factors to be considered include the sample volume and dilution by effect of diffusion phenomena occurring in the flow system. Coupling flow-processing devices to microelectrophoretic chips or electrophoretic systems is of special interest when developing in vivo determinations; this is


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facilitated by the ability of electrophoretic systems to use small volumes of sample and provide rapid analyses. For example, a microdialysis needle online coupled to CE enabled the in vivo monitoring of the evolution of a drug [155]. In fact, this approach allows the characteristic pharmacokinetic response to the drug of a simple animal to be recorded. One other useful advantage is the ability to analyse special samples such as brain tissue [156]. In general, the use of flow-processing devices improves the overall efficiency, selectivity and sensitivity of CE methods. In addition, it facilitates special determinations such as in vivo pharmacokinetic tests. No doubt, miniaturization of flow-processing devices will help further expand the potential of this combination.

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4 Miniaturized Systems for Analytical Separations I: Systems Based on a Hydrodynamic Flow 4.1

Introduction

Analytical separation techniques play a prominent role in analytical science. Without the participation of these techniques, very few reliable analyses could be carried out. Separation techniques are involved in both the treatment of samples (use of the so-called nonchromatographic separation techniques, meaning techniques without instrumental detection) and the detection of analytes after chromatographic or electrophoretic separation. Aspects related to the automation and miniaturization of separation techniques used for sample treatment have been reviewed in Chapter 3. Chapters 4 and 5 deal with miniaturized systems for analyte separation, using either a hydrodynamic flow (Chapter 4) or an electroosmotic flow (Chapter 5). Therefore, these two chapters cover the miniaturization tendencies of both chromatographic (liquid chromatography (LC), basically) and electrophoretic separation techniques; they also connect with Chapter 8, which is devoted to the miniaturization of the entire analytical process through the mTAS approach. The miniaturization of analytical separation techniques can be reviewed in parallel with the evolution of these techniques. Figure 4.1 summarizes this evolution, from the classic modes to their current alternatives. LC and electrophoresis basically involve the movement of fluids and/or analytes for the performance of separation. Planar and column chromatography, performed at atmospheric pressure (open- and macrocolumns), have given way to more efficient approaches such as Miniaturization of Analytical Systems: Principles, Designs and Applications and Bartolome Simonet 2009 John Wiley & Sons, Ltd


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Miniaturization of Analytical Systems ANALYTICAL SEPARATION TECHNIQUES ‘miniaturization’

Planar & Column Chromatography -Paper -Think layer -(Macro)column

Contemporary modes Present modes / tendencies Column Chromatography HPLC COLUMN HPTLC

High pressure Flow rate: ml/min Sample volume: µl

Capillary Liquid Chromatography µHPLC

nl/min nl

CAPILLARY Capillary Electrophoresis CE

Conventional Electrophoresis

HPLC chip

nano-HPLC

Flow rate: µl/min Sample volume: µl

CLC CEC MEKC CZE

Mic r Nan ofluidi c oflu idic

Classic modes

CE-Chip CE chip

High potential Sample volume: nl to fl

G GC

Micro-GC

Figure 4.1 A general view of analytical separation techniques and the tendency to miniaturization

high-performance think-layer chromatography (HPTLC) in the planar mode and high-performance liquid chromatography (HPLC) in the column mode. The improvements were based on the stationary phases, the detection system and the use of closed columns (reduced size with respect to classical LC) working at high pressure. Microcolumns were also needed in ulterior developments, and particularly for the development of new alternatives addressing the use of capillary columns. The replacement of microcolumns by capillaries has led to the development of very interesting modern alternatives in analytical separation science. Thus, capillary liquid chromatography (CLC), through so-called micro- and nano-HPLC, and capillary electrophoresis (CE), have introduced a true revolution in instrumental separation techniques. Capillaries constitute a significant size reduction, but more importantly have allowed the merging of chromatographic and electrophoretic techniques in interesting analytical modes. Modes such as micellar electrokinetic chromatography (MEKC) and capillary electrokinetic chromatography (CEC) are hybrid methodologies with electrophoretic and chromatographic foundations, providing a wide analytical potential. Thus, it is possible to cover everything from pure CLC to pure electrophoresis using the capillary zone electrophoresis (CZE) mode – a wide variety of separation alternatives with an impressive scope of application. Despite the clear improvements and possibilities these modes represent, miniaturization of the equipment as a whole is not truly evident. Miniaturization affects many of the components and devices integrated in the commercialized equipment, but the equipment itself is not considered miniaturized at all. The true pass to miniaturization is given when chromatographic or electrophoretic separations are carried out in chips. In these cases, all the principles of micro- and

Miniaturized Systems for Analytical Separations I

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Table 4.1 Common characteristics associated with LC techniques Name of technique

Column i.d.

Flow rate

Conventional LC Microbore LC Micro-LC Capillary-LC (micro) Nano-LC

3–6 mm 1–3 mm 0.2–1.5 mm 150–500 mm 10–150 mm

0.5–2.0 ml/min 100–500 ml/min 10–100 ml/min 1–10 ml/min 10–1000 nl/min

Injection volume level ml ml ml ml Nl

nanofluidics described in Chapter 2 are involved. Thus, today HPLC chips and CE chips are truly miniaturized systems connecting to the lab-on-a-chip approach. Based on the evolution shown in Figure 4.1 toward the miniaturized hydrodynamic separation techniques, in this chapter liquid chromatographic tendencies focussed on capillary HPLC and HPLC chip are reported. The common characteristics associated with LC techniques are summarized in Table 4.1.

4.2

The Earliest Example of Miniaturization of a Gas Chromatograph and Some Other Developments

Because of its foundation and equipment requirements, gas chromatography (GC) is an exceptional case, as was noted in Chapter 1. Although the objective of this and the following chapter is to describe the miniaturization of hydrodynamic analytical systems, in order to give a complete view of miniaturized chromatographic separations, gas phase separations must first be briefly commented upon. There is not much in the literature about gas phase separation on chips, despite the fact the first working microchip-based chromatographic system was a miniaturized gas chromatograph in 1979 [1]. As J.P. Kutter said [2], this development was hardly pursued afterwards, probably because the analytical community was not yet ready to embrace this new technology. Unsatisfactory results, due to difficulties in producing homogeneous stationary phases, may have contributed to this feeling. However, miniaturized gas chromatographs are of great interest in several application fields. These microsystems could potentially be used for breath analysis, indoor air-quality monitoring and warfare agent detection, allowing the onsite and portable monitoring of gases in a fast and reliable way. Miniaturization of a GC system requires the miniaturization of the gas transportation system that is necessary for the efficient extraction and manipulation of the analytes in the gaseous samples. Due to the ineffective transportation produced by gas micropumps, micro-GC (mGC) systems have had to rely on off-chip, highvolume and high-power gas transportation systems, such as syringe pumps and large mechanical pumps. Such dependence on off-chip systems has introduced difficulties for the creation of a truly portable miniaturized GC analyser. H. Kim et al. have

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developed a micropump-driven high-speed MEMS GC system for volatile organic compound (VOC) separation and detection [3]. Micropump operation is based on the use of a four-stage micropump with integrated microvalves, working as a peristaltic micropump. Commonly, micromachined GC channels are coupled with devices for preconcentration, injection and/or detection of gas phase analytes [4–6]. But, obviously, microcolumns are the heart of the GC technique, and will be for mGC systems too. The first works were concentrated on columns arranged with a spiral geometry, which produce lower dispersion than serpentines in CE microchips [7,8]. More recently, in several published articles, MEMS have been considered as columns for mGC [9–12]. R.I. Masel et al. have compared microcolumns fabricated with different geometries and have demonstrated that, for mGC, serpentine columns give the lowest dispersion, since the small spiral columns generate on overall higher Dean vortice when integrated over the entire column [13]. On the other hand, an efficient stationary phase contained within a microfabricated channel is crucial to the development of a complete mGC. The deposition of the stationary phase into the microchannel can be a complicated task. The most common types of deposition reported for open-tubular microfabricated columns use traditional static and dynamic coatings [14,15] and vapour deposition of a polysiloxane phase [16]. Fullerenes and carbon nanotubes have been demonstrated to have very interesting properties as stationary phases in GC [17]. In particular, multiwall carbon nanotubes have been shown to perform well as a GC stationary phase, in both packed and open-tubular approaches. In fact, compared to traditional stationary phases, they have the higher surface-to-volume ratio characteristic of nanostructurated materials, as well as better thermal and mechanical stability [18,19]. Because of the high thermal stability, this material is ideal for temperatureprogrammed separation. In addition, carbon nanotube stationary phases can be deposited easily by lithography. In this way, O. Bakajin and coworkers have developed an ultrafast GC methodology based on the use of single-wall carbon nanotube stationary phases in microfabricated channels [20]. They used an integrated heater for temperature programming and a synchronized dual-valve for rapid injections. The authors see this development as a field-test sensor microsystem that can be used to continuously monitor the gas composition of atmospheric environments. The integration of all these elements allows the fabrication of mGC microsystems. Researchers from the Engineering Research Center for Wireless Integrated MicroSystems (WIMS ERC) have developed an integrated microanalytical system for complex vapour mixtures, which is a good example of a microfabricated gas chromatograph [21]. This particular mGC is illustrated in Figure 4.2. These authors developed a WIMS mGC prototype, which includes the following parts: (i) An inlet particle filter, consisting of a macroporous silicon membrane with tortuous pores, which exhibits a high filtering efficiency.


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(ii) A calibration vapour source, using a dual-layer structure whose base contains a reservoir for retaining the volatile-liquid calibrant. This microdevice produces a constant vapour generation for several days. (iii) Smart latching valves, manipulating transportation into the system. (iv) Dual-separation microcolumns, based on a convolved square–spiral geometry (3.0 m length each). Wafer-level low-pressure anodic bonding is used to seal the channel with a Pyrex cover plate, and fused-silica capillaries are epoxied into recessed side ports for fluidic connections. The compounds of the sample are separated according to their reversible partition equilibriums between the mobile phases and the thin polymeric stationary phases lining the walls of the columns. (v) An integrated chemiresistor sensor array coated with thin films of different gold-thiolate monolayer-protected nanoparticles (MPNs), whose responses vary with the nature of the analyte vapour. (vi) Commercial microspectrometers, which can be interfaced to the microcolumn. (vii) A distributed vacuum pump for micropumping. (viii) A preconcentrator.

Figure 4.2 Scheme and specific images of the analytical components of the WIMS mGC prototype. (From [21] with permission of IEEE, Copyright 2007)

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Figure 4.3 Photography of the WIMS mGC prototype (top) and separation of a set of common air contaminants (FID detection) (bottom). (From [21] with permission of IEEE, Copyright 2007)

Figure 4.3 shows a view of the WIMS mGC prototype, as well as an example of a chromatogram in which 19 air contaminants were separated in 4 minutes. Microfabricated GC has the potential to achieve superior performance to traditional GC. Other advantages lie in its capability to perform parallel analysis with low cost, low power consumption, small thermal mass (which allows fast temperature programming rates), portability and short analysis time.

4.3 Capillary Liquid Chromatography (CLC) ‘Capillary liquid chromatography’ (CLC) is a general term that includes, in many cases, mLC and nano-LC. This definition is accepted in this chapter, because the separation is carried out into capillaries in both cases. Table 4.1 reports the features of CLC modes. The high resolution intended in CLC with the downsizing of the separation element containing the stationary phase (which follows the sequence shown in Figure 4.4) has the limitation of the pressure drop across the initial packed columns. Although open-tubular capillary columns and packed microcapillary columns present a higher permeability, and have allowed the development of basic CLC separations with a higher resolution, the problem of the pressure still remains.


COLUMN

CAPILLARY

145

CHIP (microchannels)

High-pressure pumps and micropumps Electrically-driven pumping and electro-osmotic flow

Conventional LC

---

CLC (micro- and nano-) - - - Electrochromatography modes -- - - Conventional CE (CZE) ----

LC chip CE chip

Figure 4.4 Key factors for downsizing LC systems and main analytical techniques

As T. Takeuchi recognized [22], one possible reason for the limited development of CLC (which began with Ishii’s group, Japan, in 1974 [23]) lies in the use of electrically-driven pumping (Figure 4.4) for separation methods such as CZE and the other electrochromatographic modes (described in Chapter 5). In fact, these electrochromatographic techniques have the potential to produce much higher theoretical plates than pressure-driven separation methods. The features of CLC are attributed to the use of smaller-diameter columns and lower eluent flow rate. The latter results in the saving of solvents, reagents and packing materials, in comparison with conventional LC. Mass sensitivity is generally improved due to the small volume detection in the capillary, which is of particular interest when only very low sample size is available (biological samples, for instance). The low flow rate in CLC is another advantage for the wider manipulation of the mobile phases, as well as for connection to detectors requiring low flow of sample. This is the case with CLC–MS coupling, presenting a higher compatibility than conventional HPLC–MS systems. Finally, the low heat capacity of the capillary columns facilitates the control of column temperature and hence more effective and easier temperature programming. All these features and advantages are summarized in Figure 4.5, adapted from reference [24]. As stated before, bioanalytical applications are one of the biggest potential uses of CLC, particularly when it is coupled to a mass spectrometry (MS) detector. Recently, J.M. Saz and M.L. Marina have reviewed the application of CLC to the determination and characterization of bioactive and biomarker peptides [25]. The combination of CLC with MS is very selective and sensitive, enabling the analysis of new (even not-yet-discovered) peptides and permitting the simultaneous analysis of a great number of peptides. Both CLC and MS are frequently interfaced with micro- or nano-electrospray ionization (ESI). Miniaturization of the ESI interface improves sample ionization efficiency in the MS system. Additionally, miniaturized ESI devices work with low flow rates of the same order of magnitude as those provided by the CLC system. Here, the use of capillary columns improves separation efficiency and, hence, increases the overall selectivity

146

Miniaturization of Analytical Systems Low consumption of mobile phase

LOW FLOW RATE

Easier manipulation of mobile phase Better compatibility with MS detectors

SMALLER COLUMN DIAMETER (CAPILLARY)

SMALLER PARTICLE SIZE (stationary phase)

Lower stationary phase, solvent and sample volume consumption

Improved mass sensitivity

LOW HEAT CAPACITY

Temperature programming

Figure 4.5 The main effects, and the corresponding advantages, produced by the reduction of the column (capillary) diameter

of the CLC–ESI–MS arrangement. In this review [25], an interesting example was selected to show the potential of CLC–ESI–MS for the analysis of succinylated or trimethylammonium (TMAB)-labelled endogenous peptides extracted from the pituitaries of mice [26]. To detect and quantitate the labelled peptides, a microHPLC (300 mm inner diameter (ID), C18 3 mm particles)–ESI–MS system was used. To identify the peptides, a trapping column (5 mm · 300 mm ID, C18 5 mm, 100 A) to preconcentrate and desalt the sample and a nano-HPLC (150 mm · 75 mm ID, C18 5 mm, 300 A)–ESI–MS system were used. As expected, due to the larger mass difference between the heavy and light forms, peptides labelled with the TMAB reagent generally showed much better resolution in the MS than the succinylated peptides, as Figure 4.6 illustrates. In each of these CLC–ESI–MS systems it is very common to use online preconcentration steps following the scheme shown in Figure 4.7, as already described by A.J. Oosterkamp et al. [27]. Particularly at nano-level LC, the use of precolumns is highly recommended (in addition to the cleanup and preconcentration objectives), since capillaries can easily be blocked at the inlet when real samples have to be analysed. The special compatibility of CLC with particular detectors has been studied by P. Chaminade and coworkers [28]. They compare three commercial universal detectors that allow a direct detection of lipids. These detectors are: the charged aerosol detector (CAD), the evaporative light-scattering detector (ELSD) and the ion trap (IT) mass spectrometer with atmospheric pressure chemical ionization (APCI) and ESI (see above) sources. The detectors are compared in terms of response intensity, linearity and limit of detection, while working at high temperatures. CLC offers interesting possibilities for the determination of lipids, as these compounds are not very sensitive in other instrumental separation techniques.


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Figure 4.6 Mass spectra of labelled peptides obtained from CLC–ESI–MS analysis of succinylated or TMAB-labelled endogenous peptides extracted from the pituitaries of mice. Left-column spectra correspond to a phosphorylated fragment of chromagranin B. Middlecolumn spectra correspond to joining peptide-Gly-Lys-Arg. Right-column spectra correspond to VS-Gly-Lys-Arg. The upper panels correspond to succinylated peptides and the lower panels to TMAB-labelled peptides. From [26], reproduced with permission of John Wiley & Sons, Ltd

Figure 4.7 Typical setup for online SPE preconcentration CLC–ESI–MS (adapted from [27])

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ELSD is the primary detector for the analysis of lipids because of its compatibility with a large range of solvents and elution gradients. ELSD can detect all solutes that are less volatile than the mobile phase. Recently, CAD has been developed as a new detector. It is a universal hybrid detector useful for HPLC applications, initially presented by Dixon and Peterson in 2002 [29]. CAD works in two steps: the first involves nebulizing the LC column effluent and evaporating the solvents (similarly to ELSD); the second is the ionization of the aerosol particles by impact with the positively charged nitrogen cation obtained by corona discharge. The amount of ion charged is afterwards detected by an electrometer (similar to the principle of APCI in MS). It is interesting to compare the sensitivity (through the limits of detection, LOD, estimated on the basis of three times the signal-to-noise ratio; S/N ¼ 3) for representative lipid compounds using the different detectors [28]. Table 4.2 reproduces these results. Without any doubt, these less popular detectors such as CAD and ELSD open very interesting possibilities when used for CLC separations. Thus, ELSD has been coupled to CLC since 1999 [30], is already commercialized and, particularly for lipids, presents analytical advantages with respect MS detection (as Table 4.2 demonstrates). Again, biological samples not available in large amounts can be analysed by CLC–ELSD. An interesting application has been developed by L. Quinton et al. [31]. These authors propose a microanalytical system, based on CLC–ELSD, for the separation of stratum corneum ceramides. Stratum corneum lipids were obtained from the volar side of forearm skins of healthy subjects. A sample injection volume, as low as 1 ml, was used for the chromatographic analysis. Figure 4.8 shows the separation of the different lipid classes of a stratum corneum sample. The main groups of lipids and the chromatographic region for the analysis of ceramide classes can be seen. Table 4.2 LOD a for three representative lipids (in ng) obtained with different detectors coupled to CLC. (Reprinted from [28] with permission from Elsevier) Detector ELSD

CAD

ESI-MS (full scan)

API-MS (full scan)

a

Temperature ( C) 100 125 150 100 125 150 100 125 150 100 125 150

Not detected with this detector.

Cholesterol

Ceramide III B

Squalene

16.00 1.10 10.00 1.00 5.00 0.20 15.00 1.20 7.50 0.80 2.50 0.10 N/Aa N/Aa N/Aa N/Aa N/Aa N/Aa

30.00 2.12 20.00 0.87 10.00 1.00 40.00 1.90 36.00 0.30 30.00 1.69 4.00 0.12 1.50 0.06 1.00 0.01 0.03 0.01 0.04 0.02 0.05 0.01

1.20 0.08 1.00 0.01 0.60 0.02 1.00 0.10 0.70 0.02 0.18 0.01 N/Aa N/Aa N/Aa 0.50 0.10 0.80 0.40 0.84 0.30

Miniaturized Systems for Analytical Separations I 375

mV

149

Sq

300 Cho

Free fatty acids

Triglycerides

225 150

Ceramides classes

75 min

–15 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

Figure 4.8 Separation of lipid classes of a stratum corneum sample. Sq, squalene; Cho, cholesterol. (Reprinted from [31] with permission from Wiley VCH)

Separation capabilities are not only associated with the reduction size of the chromatographic column. The manipulation of the stationary phase can significantly improve the selectivity of many applications. Basically, three main dispositions of stationary phase in chromatographic columns can be identified for CLC: open-tubular capillary columns [32], packed microcapillary columns [33] and monolithic silica capillary columns [34]. Additionally, fused silica capillaries [35] can be used for electrochromatographic methods. Among these, columns of packed particles are still the most popular for HPLC because of their great utility, excellent performance and wide variety. Recently, Q.-S. Qu et al. have proposed the use of gold microspheres (AuMSs) modified with octadecanethiol for CLC [36]. Gold nanoparticles (AuNPs) are becoming increasingly attractive in many scientific fields because of their long-term stability, high surface area-to-volume ratio and ease of chemical modification. With this background, AuMSs have great potential to become substitutes for silica-based materials as stationary phase for CLC. This is due to their stability at high pH, reasonable rigidity and ease of manipulation from a physicochemical point of view. This pure AuMS-based C18 stationary phase may be a promising alternative to conventional C18 material for CLC. F. Fanali and coworkers have reviewed recent applications of CLC (nano-LC for these authors; they refer to the use of capillaries of 10–100 mm ID) [37]. As they report, the main areas of application of CLC are proteomic, pharmaceutical and environmental. One of the most exciting applications of CLC is in proteomics, especially in peptide mapping, protein sequencing and the study of proteins. Nanoscale proteomics studies commonly require CLC–MS technology, employing the interfaces reported above (ESI or APDC) as well as CLC–tandem mass spectrometry (CLC–MS/MS) [38]. Even the use of nano-HPLC–ICPMS for the quantification of sulfur-containing peptides has been described [39]. Figure 4.9 shows the high capacity for separating 24 different peptides using a combination of nano-HPLC–ICP IDMS with nano-HPLC–ESI MS/MS.

150


Figure 4.9 (a) Sulfur mass chromatogram obtained from human serum albumin (HAS) tryptic digest by precolumn isotope dilution analysis with nano-HPLC–ICP–MS. (b) Table with the assignments of the peaks. (From [39] with permission, Copyright ACS)

4.4

Liquid Chromatography on Microchips

The miniaturization of the column inner diameter (Table 4.1 and Figure 4.5) and volumetric flow rates in LC (HPLC) is an ongoing trend that is mainly driven by the need to handle small volumes of complex sample, particularly in the context of highthroughput screening technologies. Recent progress toward HPLC in lab-on-a-chip includes the integration of individual operations (basically, reaction, preconcentration, separation and the corresponding detection) into mass-produced and low-cost device development. This recent progress toward separations in microchip HPLC has the potential to become a more powerful tool than nanoLC for the analysis of complex samples [40]. This is of particular interest for LC–MS or LC–tandem mass spectrometry (LC–MS/MS) analyses, because of its compatibility with the flow-rate requirements of a nano-electrospray interface for the online coupling of the microchip HPLC to MS. This coupling facility is one important advantage over microchip CE, which has clear difficulties with interfacing to MS. Agilent Technologies now markets a microfluidic chip that integrates a trapping column, separation column and electrospray source within a single device. 4.4.1

The Agilent HPLC Chip

H. Yin and K. Killeen, from Agilent Technologies, have recently reviewed the fundamental aspects and applications of the Agilent HPLC Chip [41]. This is a polyimide device which is simultaneously a nano-electrospray interface to a mass spectrometer, an analytical chromatographic column of appropriate size to the nano-electrospray flow rate, and an enrichment column for online sample concentration in advance of the analytical column. In this device, there are no


151

Figure 4.10 View of the commercial Agilent HPLC Chip (top right) and traditional nano-LC components (around the image of the chip). Equivalent locations are indicated by arrows. Details of the electrospray tip are shown at the top of the figure as the HPLC Chip–MS interface. The photography of the Agilent nano-CLC–MS equipment shows the Agilent HPLC ChipCube where microchip is loaded. (Reproduced by permission of Agilent Technologies)

fittings, adapters, connectors or any other dispersive flow elements negatively affecting the performance in nano-LC. The commercial Agilent HPLC Chip is shown in Figure 4.10; traditional nano-LC components are displayed around this image, indicating their location in the chip. The figure is completed with the overall Agilent equipment in which the chip must be assembled, and details of the electrospray tip. Polyamide was chosen as the chip substrate because it is an extremely heat- and cold-stable material and has good dimensional stability (coefficient of thermal expansion of 20 ppm/ C). It is quite insoluble in most organic media and is not appreciably basic or acidic, which is a fundamental requirement for reversed phase LC. In contrast to glass or fused silica, channels can be formed by a laser ablation process, as this material absorbs the light. Thus, according to the information given by these authors [41], the microfabrication process consists of laser ablation of the channels, holes, chambers and columns. The sample-enrichment column and the analytical column are slurry packed with a variety of chromatography media. The basic HPLC Chip has an LC channel of 50 mm (d) · 75 mm (w) · 50 mm (l), with a 40 nl enrichment channel. Particles conforming the stationary phase are retained in the column space by the ‘keystone effect’, avoiding the need for frits, under operating pressure of over 120 bar [42]. The chip-packing process is adapted and modified from methods originally developed for the packing of fused silica capillaries. Both the enrichment and the chromatographic channels are packed using an input capillary that is connected to the chip by a chip-valve interface [43]. Transfer

152


volume between the enrichment column and the analytical column (or other on-chip functions) is minimized by installing the HPLC Chip within a two-position rotary valve. Thus, the chip is sandwiched between the stator and the rotor of the valve, establishing a micro-to-macro interface. The valve assembly, the support plate and the attached valve are mounted on a two-axis stage via a rotating bracket that allows the chip to be loaded into the valve assembly (Figure 4.11a and Figure 4.11b), and then to be rotated into place such that the chip extends into a spray chamber mounted on the mass spectrometer (Figure 4.11c). The two-axis stage allows vertical and axial adjustment of the chip spray tip with respect to the cones on the mass spectrometer inlet (Figure 4.11d). The chip is interposed between the rotor and the stator with enough precision so that three basic running steps can be performed. In the initial position, mobile phase is pumped to the chip, passing through the enrichment unit and separation column

Figure 4.11 (a) Scheme of the chip with clamp in open position, including the stator, clamp and valve assembly. (b) Photography showing the chip in the clamped position ready for rotation into the spray chamber. (c) Details of the chip and valve assembly in operational position on the MS interface. (d) Photography of the spray formed in the nano-LC–MS interface after the flow exits by the electrospray tip. (Reproduced from [43], Copyright 2007 American Chemical Society, and by permission of Agilent Technologies)


153

Figure 4.12 Scheme of the chip–rotor interface in the LC run mode (a) and in the loading process: the sample-loading configuration (b) and the LC-running configuration (c) of the rotor channels. The upper-right part of the figure shows the scheme of the LC run mode with the sixway positions of the rotary valve. (Reproduced from [43], Copyright 2007 American Chemical Society, and by permission of Agilent Technologies)

and then reaching the spray (base-line signal); whereas sample flow is driven to waste (Figure 4.12a). In the enrichment position of the valve, sample is introduced to the chip flow from the autosampler, driven through the enrichment column, and then to waste (Figure 4.12b). When the rotor is again switched 60 (injection of the preconcentrated analytes), the flow from the nanopump enters the enrichment column and sweeps the analytes into the analytical column (Figure 4.12c). At the end of this column the flow passes electrical contacts which allow the biasing of the effluent for electrospray. Agilent has developed the HPLC ChipCube to optimize the performance of the entire system. The valve is designed to form a face seal with the HPLC Chip with minimum mechanical wear on the valve rotor. Figure 4.13 shows details of the entrance of the chip into the cube through the sandwiched position between the stator and the rotor of the valve. The chromatographic performance of the analytical column on an HPLC Chip was studied by G. Rozing et al. [44]. The results obtained for a group of peptides showed that chromatographic peaks resulting from HPLC Chip were narrower and more symmetrical than those obtained from a packed fused-silica capillary column. This fact is in agreement with the recordings shown in the article published by S. Ehlert and U. Tallarek [40], reproduced from a presentation by Vollmer and Miller [45]. These are given in Figure 4.14. As can be seen, the HPLC Chip offers increased resolution and superior peak shape compared to conventional nano-LC

154


Figure 4.13 Introduction and sandwiched position of the chip in the stator–rotor unit. (Reproduced by permission of Agilent Technologies)

Figure 4.14 Comparison of microchip LC–MS with nano-LC–MS by analysis of a yeast gel band. (Reproduced from [40] with permission from Springer)


155

using the same adsorbent particles and similar packed-bed dimensions. The worst results were obtained using a longer nano-LC column packed with smaller adsorbent particles. 4.4.2

Other Approaches to Microchip HPLC

Other microchip LCs have been described in the literature. T.D. Lee and coworkers developed a microfluidic chip that integrates all the fluidic components of a gradient LC system [46], designed as a platform for an LC–MS/MS microsystem. This chip was batch fabricated on a silicon wafer using photolithographic processes, with Parylene as the main structural material. The chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed phase support; integrated frits for bead capture; and an electrospray nozzle. Figure 4.15 shows a photograph of the chip and the corresponding schemes. As Figure 4.15b illustrates, the chip is basically conformed on a 500 mm silicon wafer layer with various layers on top deposited for the fabrication of the different chip elements (50 mm thicknesses). In this top layer is packed the chromatographic column (RP column), the mixer, the exit for the electrospray and the chamber for the pump or the sample. The chip is mounted into a polyetherimide jig that couples a port at the front of the column to Teflon tubing via a poly(dimethylsiloxane) (PDMS) gasket. A slurry of the stationary phase material is forced into the column through an access port between it and the mixer until its entire length is filled. Once packed, the chip is mounted in a different holder that utilizes a 5 mm-thick polyetherimide cover. Chambers are machined into this cover piece to form reservoirs for the sample and solvents. The PDMS gasket allows the

Figure 4.15 (a) Photograph of the LC chip. (b) Diagram showing the placement of the different elements. (c) Chip-holder assembly for the inlet in an Agilent MSD ITMS. (Reproduced from [46] with permission of American Chemical Society, Copyright 2005)

156


sealing between the cover and the chip. For the analyses, the completed chip assembly is mounted on an Agilent electrospray ion source housing using a modified probe that incorporates a 3D positioner (Figure 4.15c). A. Ishida and coworkers have developed a microchip for reversed phase LC using porous monolithic silica [47]. The chip consists of a double T-shaped injector and a 40 cm separation channel (in a serpentine configuration). Figure 4.16 shows a photograph and details of this LC chip. This microchip is fabricated using glass wafer as the substrate and the cover plates. Standard photolithography, wet chemical etching and bonding techniques are used in the different steps of the fabrication process. The layout and the dimensions of the different sections of the chip are shown in Figure 4.16b. Two photomasks are prepared for the fabrication. The first defines the double T-type injector and separation channel pattern, and the second defines the grooves which will serve as connectors. The overall dimensions of this chip are 35 mm · 35 mm. The typical channel width is about 400 mm, and the channel depth is about 30 mm. The connection grooves are etched to a depth of 150 mm. After rinsing with water, the etched glass chip is aligned to the cover plate

Figure 4.16 (a) Photograph of the LC chip. (b) Schematic layout of the chip with details of the channels and inlet/outlet positions. (c) Overall configuration including the LC chip. S, sample; MP, mobile phase; ED, electrochemical detector; W1, mobile phase waste; W2, sample drain; V1, mobile phase inlet valve; V2, mobile phase outlet valve; V3, sample inlet valve; V4, sample outlet valve. (Reprinted from [47] with permission from Elsevier)


157

and then thermally bonded in a programmable oven. Then fused silica capillaries are inserted into the grooves. Monolithic stationary phase in the microchip is created by introducing reactant solutions by syringes. Figure 4.16c shows the schematic configuration of the chip LC system. The ends of the capillaries inserted in the microchip are connected to two-way valves (V1, V3 and V4 in Figure 4.16c) via heatshrinkable tubes. Valves V1 and V3 are connected to syringes allowing the introduction of the mobile phase (V1) and the sample (V3), whereas valves V2 and V4 drive to waste. The end of the separation channel in the microchip is connected to an electrochemical detector (ED) using conventional amperometric detection. Different catechin compounds can be separated in this chip with good resolution, although long analysis times are required. The sample-loading capacity of microchip LC systems can be enhanced by introducing parallel multichannel flow pass, analogous to multicapillaries or fibrepacked capillaries, as T. Greibrokk and coworkers pointed out [48]. This possibility will increase the sample throughput and the speed of the analysis because of the better resolution of separation in microchips. 4.4.3

Some Selected Applications

LC analysis using the microchip approach is practically addressed to the analysis of very small sample sizes, in which appropriate resolutions and sensitivity must be achieved. As the rest of the auxiliary elements, including the detector, are commonly not miniaturized, portability is not the objective of such analytical microsystems. Therefore, biological or related bioanalytical applications have received the main attentionofmicrochip LC, mainlythe use ofMSdetectionfor reliablescreening ofthe numeroustargetcompoundsin thesesamples.Inthissection, some selectedexamples illustrate the practical potential of microchip LC systems. In the pharmaceutical field, for example drug metabolism and pharmacokinetic studies, it is common to use small animals for testing prior to human assays. The animal size sets the limit on the blood volume that can be drawn per unit time, and serial bleeding of a few or even only one animal, which reduces the variability in the pharmacokinetic profile, results in even lower volumes per bleed. Consequently, studies with small animals set extreme demands on the analytical system with respect to both the capacity to handle small sample volumes and the detection sensitivity. For these studies, LC–MS is often used for the analysis of samples. Here, the HPLC chip/MS system fulfils these requirements. In this context, S. Buckenmaier et al. have used the HPLC Chip interfaced to a triple quadrupole (QQQ) mass spectrometer for the monitoring of atenolol, atropine, metroprolol and imipranine in blood plasma samples [49]. M. Vollmer and S. Buckenmaier have coupled the HPLC Chip with a time-offlight (TOF) mass spectrometer detector for the determination of dextromethorphan (DEM) and its metabolites in human plasma and urine samples [50]. The

158


metabolism of DEM has been extensively investigated and has been used as a model drug to distinguish between ‘poor’ and ‘efficient’ cytochrome P450D6 metabolizers. During the metabolism (Figure 4.17a), DEM (m/z ¼ 272) is either demethylated to 3MM (m/z ¼ 258) or converted to the isobar dextrorphan (DOR). The latter compound may be further metabolized to 3OM (m/z ¼ 244) or directly glucoronidated to dextrorphan glucoronide (DORGlu, m/z ¼ 434). 3OM is further metabolized to 3-hydroxymorphinan glucuronide (3OMGlu, m/z ¼ 420). The samples are analysed with two different gradients. Figure 4.17b shows the typical elution profile for the fast gradient. Proteomics is a field in which the LC microchip is paid more attention. Initially, proteomics had the objective of identifying the proteins of biological systems, but more and more this discipline is moving to targeted strategies aiming at identifying key proteins (biomarkers, for instance) that can provide reliable diagnostic and prognostic indicators of disease progression or treatment effects. The problem is that biological samples are complex mixtures of proteins and the detection of biomarkers (existing at very low abundance) in these mixtures needs sensitive and robust analytical methods. One of the conventional and most powerful proteomics approaches involves combining high-resolution separations with high-accuracy MS. Since LC is complementary to MS/MS with respect to the population of peptides that may be detected, an interesting approach is to combine different pieces of information obtained by these two techniques. Thus, for instance, the retention time (RT) obtained by CLC clearly defines a peptide, as does its mass; the characterization of a peptide by a couple of pieces of data (mass measurement

Figure 4.17 (a) Metabolism of DEM by citochrome P4502D6. (b) Analysis of DEM metabolites by HPLC Chip–TOFMS (100 nM in vitro assay; concentration on column 100 fmol; gradient length 2.5 minutes; total run time 9.5 minutes). (Reproduced from [50] with permission from Agilent Technologies)


159

and the corresponding RT, noted as mass/retention time, MRT, for m/z-RT) is very attractive. In this way, a protein will be characterized by different MRT values, which constitute a specific peptide map. Therefore, both the RT and the m/z values must be as accurate and reproducible as possible. The replacement of the CLC equipment by a HPLC Chip system can be of great value in these cases, as microfluidics chromatographic separations provide high separation efficiency and deliver precise and repeatable flow rates. Using these advantages of HPLC Chip–MS systems, J. Hardouin et al. have investigated different biomarkers signatures [51]. In this case, an ITMS was used. These authors carried out the identification of human autoantigens after obtaining autoantigen mixtures by affinity separation (in order to simplify the complexity of the sample) of Caco-2 cell proteins on the immunoglobulins from a group of healthy volunteers. These mixtures were digested by trypsin, and just the tryptic digest-resulting solutions were then analysed by HPLC Chip–MS coupled with an IT. In each sample, around 20 proteins were identified using the software package MASCOT. The measurement of MRTs revealed the identity of a peptide in different situations, such as the lack of fragment information, measured m/z above the authorized mass tolerance, or the presence of different peptides sequences with close m/z ratios. Anti-doping laboratories use a test to detect the misuse of recombinant erythropoietin (rhEPO) based on its different migration pattern on isoelectric focusing (IEF) gel compared to endogenous human erythropoietin (hEPO), which can be explained by structural differences. While there is definitely a need to identify those differences by LC–MS/MS, the extensive characterization that was achieved for the rhEPO was never performed on hEPO because its standard is not available in sufficient amount. Taking this problem into account, P.E. Groleau and coworkers developed an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone (used as a model), using a nanoflow HPLC Chip–ESI–ITMS [52]. The diagnostic ion at m/z 366 of oligosaccharides was monitored in the product ion spectra to identify the four theoretical glycosylation sites, Asn24, Asn38, Asn83 and Ser126 on glycopeptides 22–37, 38–55, 73–96 and 118–136, respectively. The method described by these authors provides a means to detect glycopeptides from commercially available pharmaceutical preparations of rhEPO with the sensitivity required to detect pmol amounts of hEPO, which allows the identification of structural differences between the recombinant and the human forms of the hormone. Proteins in general do not act individually in biological processes. Most cellular events are controlled by multiple proteins, involving – in many cases – noncovalent interactions between proteins and other molecules. Traditional methods used to study noncovalent protein interactions include ultracentrifugation, calorimetry and various types of spectroscopy. Commonly, these methods require large amounts of sample, are relatively slow and can be fairly nonselective. For these reasons, ESI–MS has recently been used to study noncovalently-associated

160


complexes. Despite the advantages of technique for this purpose, care must be taken to ensure the protein complex does not denature in the solvents used for ESI. Moreover, other instrumental parameters (temperature, voltage and pressure) must be controlled to avoid fragmentation of the complex. For identification purposes, high-resolution, accurate-mass and high-mass scanning are critical features in the analysis of macromolecular assemblies. TOFMS posseses all of these capabilities, and hence is a good choice for the study of noncovalent associations. In addition to these advantages of TOF detectors, HPLC Chip provides an extremely stable nanospray and significantly improves upon the ease of use and reliability of nanoflow ESI. G.W. Kilby demonstrated this suitability for the study of apomyoglobin and myoglobin noncovalently-associated complexes using an HPLC Chip–ESI–TOFMS arrangement [53]. The analysis was carried out in less than five minutes. N. Tang and P. Goodley carried out the characterization of protein phosphorylation using HPLC Chip electron transfer dissociation (ETD) ITMS [54]. Protein phosphorylation is a type of post-translational modification which plays an important role in the regulation of many cellular functions. The precise determination of phosphorylation sites within a protein is crucial to the understanding of cell regulation mechanisms. Collision-induced dissociation (CID) and ETD can be used in the same LC–MS run to analyse samples for protein phosphorylation studies. Figure 4.18a depicts the scheme for the ETC process. The ETC reactant is generated by a small negative chemical ionization (NCI) source that is mounted directly upon the inlet section of the second octapole ion guide on the ITMS. The NCI source is filled with fluoranthene, which is sublimed and combined with methane gas in the presence of electrons emitted from a filament. The electrons are slowed by collision with the methane gas and captured by the fluoranthene molecules to make fluoranthene radical anions. From the ESI chamber, the peptide ions are generated from the nanospray chip tip. All the positive ions are allowed to enter the MS ion inlet, through the ion optics and ultimately into the IT. The positive multiplycharged peptide ions are isolated and all other ions are ejected. During this process, the flow of the reactant radical anions is closed by a voltage-gating process. Alternatively, the NCI source can be gated to allow negative ions to enter the IT as a packet. The two packets of ions (negative and positive) exist in the ITat the same point in time. After a few milliseconds, the positive peptide ions and the negative ions become interactive and the electron from the fluoranthene radical anions is transferred to the positive peptide ion. The electron transfer is rapid and sufficiently energetic, yielding a series of positive amino acid residue fragments. The IT is subjected to the scan process in the same manner as CID and the positive ETD product ions are scanned out of the trap, yielding the ETD MS/MS spectrum [54]. As an example of the results obtained by this methodology, Figure 4.18b illustrates the differences between CID and ETD fragmentation for the synthetic phosphopeptide TTHyGSLPQK using HPLC Chip nanospray infusion. As can be seen, CID has very


161

Figure 4.18 (a) Diagram of the Agilent 6340 ITMS with ETD module. (b) Comparative spectra obtained with CID and ETD for the phosphopeptide TTHyGSLPQK (m/z ¼ 404.7) using the HPLC Chip. (Reproduced from [54] with permission from Agilent Technologies)

few fragment ions, while ETD produces nearly complete sequence coverage, with indication of the phosphotyrosine location. The majority of the protein drugs in existence are glycoproteins. It has been demonstrated that the efficacy of a glycoprotein drug (commonly used to treat and prevent diseases) critically depends on the type and extent of glycosylation of the protein. Therefore, it is of great interest to characterize glycoproteins as an essential part of drug quality control. P.D. Perkins has explored the ability of HPLC Chip coupled to a trap XCT ultra-MS system to detect and identify oligosaccharides and N-linked glycans cleaved from two commercially available glycoproteins [55]. The HPLC Chip used in this case contains a porous graphitized carbon column

162

Miniaturization of Analytical Systems BPG, 700-2200 m/z Glycans from 1 pmol starting glycoprotein

Gal Man GlcNAc NeuAc Fuc

or G1

G0

G2

A B

C A: Isoform of Go B: Isoforms of G1 C: Isoform of G2

10

12

14

16

18

20

22 Time (min)

Figure 4.19 Base peak chromatogram of released reduced N-linked glycans from human serum IgG. (Reproduced from [55] with permission from Agilent Technologies)

Table 4.3 Relative percentage amounts of the IgG N-linked glycans identified by HPLC Chip/Trap XCT Ultra MS system from Agilent Technologies. (Reproduced from [55] with permission from Agilent Technologies) Glycan (including isoforms)

Structure

m/z value Peak height of Relative of alditol [Mþ2H]2þ averaged spectrum percent

G0

733.4

46886

14.3

G1

814.4

103172

31.4

G0 þ bisecting GlcNAc

835.2

30969

9.4

G2

895.4

35483

10.8


916.0

25222

7.7

G2 þ bisecting GlcNAc - fuc

924.0

7623

2.3

G1 þ NeuAc

959.9

16101

4.9


997.0

24573

7.5

1041.0

38886

11.8

G2 þ NeuAc


163

specifically designed for oligosaccharide separation applications. The combination of the chip with the sensitivity and scan speed of the trap MS detector provide the appropriate platform to characterize N-linked glycans. After a reasonable reduction of the number of plausible candidate compounds, base peak chromatograms, such as that shown in Figure 4.19, were obtained. As can be seen in this figure, the known major glycans in IgG, designated by G0, G1 and G2, were found. The two linkage isoforms of G1 were separated, due to the high resolving power of the porous graphitized carbon column for oligosaccharide separation. Some minor isoforms were detected whose MS/MS spectra also matched those for G0, G1 and G2. Additionally, the data produced by this system enabled the calculation of the relative distribution of glycans in the sample. Thus, Table 4.3 shows the identities and relative percentage distributions of the IgG N-linked glycans.

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[26] F.Y. Che, L.D. Fricker, J. Mass Spectrom., 40 (2005) 238. [27] A.J. Oosterkamp, M. Carrascal, D. Closa, G. Escobar, E. Gelpı, J. Abian, J. Microcolumn Sep., 13 (2001) 265. [28] A. Hazotte, D. Libong, M. Matota, P. Chaminade, J. Chromatogr. A, 1170 (2007) 52. [29] R.W. Dixon, D.S. Peterson, Anal. Chem., 74 (2002) 2930. [30] S. Heron, A. Tchapla, J. Chromatogr. A, 1140 (1999) 95. [31] L. Quinton, K. Gaudin, A. Baillet, P. Chaminade, J. Sep. Sci., 29 (2006) 390. [32] K. Hibi, D. Ishii, I. Fujishima, T. Takeuchi, T. Nakanishi, J. High Resolut. Chromatogr. Commun., 1 (1978) 21. [33] T. Tsuda, M.V. Novotny, Anal. Chem., 50 (1978) 632. [34] N. Ishizuka, H. Minakuchi, K. Nakanishi, N. Soga, N. Tanaka, J. High Resolt. Chromatogr., 21 (1998) 477. [35] R.D. Dandeneau, E.H. Zerenner, J. High Resolut. Chromatogr. Chromatogr. Commun., 2 (1979) 351. [36] Q.-S. Qu, X.-X. Zhang, Z.-Z. Zhao, X.-Y. Hu, C. Yan, J. Chromatogr. A, 1198–, 1199 (2008) 95. [37] J. Hernandez-Borges, Z. Aturki, A. Rocco, S. Fanali, J. Sep. Sci., 30 (2007) 1589. [38] Y. Shen, N. Tolic, C. Masselon, L. Pasa-Tolic, D.G. CampII, M.S. Lipton, G.A. Anderson, R.D. Smith, Anal. Bioanal. Chem., 378 (2004) 1037. [39] D. Schaumioffel, P. Giusti, H. Preud’Homme, J. Szpunar, R. Lobinski, Anal. Chem., 79 (2007) 2859. [40] S. Ehlert, U. Tallarek, Anal. Bioanal. Chem., 388 (2007) 517. [41] H. Yin, K. Killeen, J. Sep. Sci., 30 (2007) 1427. [42] M. Mayer, E. Rapp, C. Marek, G.J.M. Bruin, Electrophoresis, 20 (1999) 43. [43] H. Yin, K. Killeen, R. Brennen, D. Sobek, M. Werlich, T. van de Goor, Anal. Chem., 77 (2007) 527. [44] G. Rozing, T. van de Goor, H. Yin, K. Killeen, J. Sep. Sci., 27 (2004) 1391. [45] M. Vollmer, C. Miller, Poster Presentation in the ABRF Meeting, Savannah, GA, USA (2005). [46] J. Xie, Y. Miao, J. Shih, Y.-C. Tai, T.D. Lee, Anal. Chem., 77 (2005) 6947. [47] A. Ishida, T. Yoshikawa, M. Natsume, T. Kamidate, J. Chromatogr. A, 1132 (2006) 90. [48] Y. Saito, K. Jinno, T. Greibrokk, J. Sep. Sci., 27 (2004) 1379. [49] S. Buckenmaier, M. Vollmer, L. Trojer, C. Emotte, The Column, May, (2008) 20. [50] M. Vollmer, S. Buckenmaier, Agilent Application Note, 5989-5938EN (2006). [51] J. Hardouin, R. Joubert-Caron, M. Caron, J. Sep. Sci., 30 (2007) 1482. [52] P.E. Groleau, Ph. Desharnais, L. Cote, C. Ayotte, J. Mass Spectr., 43 (2008) 924. [53] G.W. Kilby, Agilent Application Note, 5989-5596EN (2006). [54] N. Tang, P. Goodley, Agilent Application Note, 5989-5158EN (2006). [55] P.D. Perkins, Agilent Application Note, 5989-5157EN (2006).

5 Miniaturized Systems for Analytical Separations II: Systems Based on Electroosmotic Flow (EOF) 5.1

Introduction

The most commonly used separation techniques in analytical microdevices are chromatography and electrophoresis. Chromatography is suitable for the separation of both charged and uncharged molecules. The separation makes use of physicochemical differences between the components, such as different affinities for a stationary phase or different distribution coefficients between a stationary phase and a mobile phase. Chromatographic techniques require the presence of a stationary and mobile phase. Electrophoresis, on the other hand, can be used for charged molecules (ions), which, in the simplest mode of electrophoresis, are separated in an electric field according to their charge-to-mass ratios. Besides this electrophoretic motion, an additional movement is often imparted by electro-osmotic flow (EOF) phase, through which the mobile phase and the mixture to be separated are percolated. Movement of mobile phase is induced by pressure (chromatography) or electro-osmosis (capillary electrophoresis). With the separation technique selected, three steps must be executed in order to perform a successful, useful separation in a miniaturized format: (i) injection, in which a defined portion of sample is introduced into the separation system by pressure or by electrokinetic means; (ii) separation, during which the different components in the sample are moved through the separation system and separated into individual bands or zones; and (iii) detection, by which the individual bands are Miniaturization of Analytical Systems: Principles, Designs and Applications and Bartolome Simonet 2009 John Wiley & Sons, Ltd


166


registered at the outlet of the separation system based on various physical principles. Though the first analytical microdevices were chromatographic systems [1,2], it was soon realized that it would be easier from an engineering point of view to create electrophoretic systems first [3–5]. In Chapter 4 we discussed the miniaturization of systems for analyte separation based on hydrodynamic flow; in this chapter we are going to study the miniaturization of capillary electrophoresis. The miniaturization of capillary electrophoresis (CE microchips) was one of the earliest examples of a mTAS system [6]. This technology emerged as an important new analytical technique in the early 1990s with the introduction of the mTAS concept [6] and the seminal work of Manz, Harrison, Verpoorte and Widmer [7]. This new technique was a result of the marriage of the ability of conventional CE to analyse ultrasmall volumes (nl) with microfabrication techniques perfected in the semiconductor industry to produce very small structures in silicon. CE microchips employ channels etched into a planar substrate, which are based upon microfabrication techniques developed in the semiconductor industry. Early applications of CE microchips employed glass or quartz for chip substrates because they are optically transparent and exhibit EOF properties similar to those of fused silica. A number of other materials, including polymers/plastics, have now been investigated [3,4,8]. Moreover, CE microchips have the potential to simultaneously assay hundreds of samples in a matter of minutes or less. This rapid analysis combined with massively parallel analysis arrays should yield an ultrahigh throughput. These microchips typically consume only picolitres of sample and they may potentially be prepared onboard for a complete integration of sample preparation, separation and detection. Also, a key advantage of the use of these planar devices/substrates is the ease of manufacture and the fact that microfabrication techniques can be adapted for mass production. Therefore, these features make CE microchips an attractive technology for the next generation of CE instrumentation [8]. The key features of CE microchips have been widely stated in the literature and are summarized in Table 5.1 [9]. Very fast analysis without loss of performance (high efficiency) and the possibility of performing several assays simultaneously (parallelization of analysis) are among the most attractive characteristics. Very low sample consumption and waste generation are additional important features, sometimes connected with the low cost, which is mostly due to the use of inexpensive materials (plastics) in single-use microsystems (disposability). A comprehensive overview of these features will be given in the following sections. However, one of the most unique characteristics is the possibility of integrating different analytical steps into the single device (highly-multiplexed systems or lab-on-a-chip). Although this will be touched upon in this chapter, it will be discussed in more depth in Chapter 8. In addition, in this chapter we will study the designs, fundamentals and applications of CE microchips, giving a concise but in-depth overview of the

High efficiency

Ultrafast analysis

Very low sample/reagent consumption

Extremely low waste generation Portability

Disposability

Highly multiplexed systems Lab-on-a-chip

Ultrahigh throughput

High efficiency

Rapid analysis

Low sample

Low waste generation

No disposability

Integration

High sample throughput

No portability

Analytical features

Analytical features

Conventional CE

CE microchip

Miniaturization of CE conceptually offers portability, as a consequence of the size of the analytical instruments Single-use polymeric/plastic microchips can be readily produced at low cost, so that they can be disposed of after use System integration of components of various functions on a single chip enables the construction of a multifunctional analytical laboratory (see Chapter 8) Example for CE microchips: parallel separation channels can be fabricated on a single chip device, enabling high-throughput assays on microchips (see Section 5.8.1)

The miniaturized feature of microchip devices reduces the consumption of samples and reagents, and in consequence extremely low wastes are generated

Applicability of high field strength results in high efficiency per unit length of separation channel (see Section 5.3) The efficient heat dissipation of microchip channels allows application of high electric field strength, which provides high-speed electrophoretic separation (see Section 5.3)

Remarks

Table 5.1 Relevant keys for conventional CE and CE microchips

Facility to use the electrokinetic phenomena (valveless microsystems)

Existence of EOF (glass and polymers)

Easy fabrication of network microchannels with zero dead volume interconnections (microchip format concept) The chemistry of glass and polymers (PDMS, PMMA) is well known

Technical features

Miniaturized Systems for Analytical Separations II 167

168


heterogeneous aspects involved in them, both technological and analytical. For more details, see the excellent reviews [3–5,8] and some informative books [10–12].

5.2 CE on the Microchip Format CE miniaturized into microchip format is an analytical microsystem constituted at least by an injector (where a sample plug is critically loaded) and one separation microchannel (where electrophoretic separation of analytes is performed), interfaced suitably to reservoirs (where different solutions/samples are deposited). Microchannels and reservoirs are fabricated in microchips using photolithography or micromolding to form channels for sample injection, CE separation and analyte detection. Once all solutions, including those of the samples, are loaded, the samples are typically transferred electrokinetically into an injector region. Then their components are separated by application of a high voltage, and afterwards detected using a suitable detection system. The system-integration concept on the microchip platform eliminates the necessity of most fluidic connections that otherwise link microfluidic components. Avoiding such connections greatly reduces sample dispersion, delay times and dead volumes between the different microchip compartments, significantly increasing the separation power of such integrated miniaturized systems. The typical layout of a CE microchip (both a simple cross-T injector (a) and a twin-T injector (b)) is depicted in Figure 5.1. It has a network of channels with (a)

(b) Reservoirs (100–250 µl)

RB

S

RB

SW

SW S

L=3–10 cm Separation channel

10–100 µm

20 µm 50 µm

W

Simple Cross-T Injector

Detection cell

W

Twin-T injector

Figure 5.1 Common microchip layouts

Highvoltage supply

Miniaturized Systems for Analytical Separations II

169

widths varying from 10 to 100 mm, with typical straight separation channels between 3 and 10 cm. In a typical setup, buffer solutions are introduced on sample (S), running buffer (RB) and waste (W) reservoirs. The reservoir volume is often defined by their capacities (100–250 ml). Electric fields are applied to the reservoirs by high-voltage power supplies (1–5 kV) and platinum electrodes are placed in the reservoirs for injection and separation as samples are normally electrokinetically driven into the network channels. In microchip electrophoresis and related separation techniques based on EOF, the primary interface between the regular laboratory environment and the microworld is the buffer reservoirs [13]. Their accessibility and volume are key issues in microchip design and development. One of the considerations is the reduction of buffer separation/reagent volume and sample consumption. Early microchip designs had very low-volume self-contained reservoirs, and increases in glass cover-plate thickness increased the reservoir volume [14]. The two main reasons for increasing reservoir volume were to counter solvent evaporation and to suppress the effect of electrolysis, as the latter can cause unpredictable pH changes in the buffer reservoirs. To keep the pH stable during the electrophoresis process, higher buffer capacities were also considered; however, this led to higher current and concomitantly higher Joule heat generation. To reduce these effects, reservoirs are now designed and fabricated with highly reproducible sizes and shapes and with chemical incompatibility. Also, drilled reservoirs vary somewhat in size and shape in terms of both reservoir-to-reservoir reproducibility and consistency of the inner diameter throughout the reservoir depth. Thus, a robust and reproducible manufacturing process for these connections is crucial to the wide applicability of microseparation devices. With respect to microchip layouts, in general the design of microchips for CE has undergone significant development from simple single-channel structures to increasingly complex ones [3,4,13]. Current designs of CE chips allow reactions on-chip and separations in multiple channels. Microreactors can also be added to perform online reactions such as (bio)chemical reactions and post-column derivatizations (see Section 5.8). Separation channel length and geometry must also be properly designed. The change of the CE column format from long capillaries to shorter microfabricated channels on microchips brings new opportunities, limitations and challenges. Miniaturization, smaller injection plugs and shorter separation paths enable rapid separations without significant peak-broadening due to diffusion. With respect to channel geometry, as mentioned above, CE microchips have channel depths of 15–40 mm and widths of 60–200 mm. The small cross-section of separation channels and the large thermal mass of microchips allow Joule heat to be dissipated efficiently. Thus, high electric fields (over 2 kV/cm) can be applied on microchips. These issues will be discussed in the next section.

170


5.3 Modes and Theories of CE Microchips The transfer of the knowledge gained in conventional CE to microchip format theory is not new. Basically all electrophoretic modes have been successfully transferred to microchip format. Capillary zone electrophoresis (CZE) is the most commonly used separation mode in CE microchip due to its versatility, ease of operation and separation power. However, in principle, it has the limitation of being useful only for separation of charged species (neutral species cannot be separated). In CZE the channel contains only a buffered medium and the eletrophoretic mobilities of the analytes depend on their charge/mass ratio. Indeed, the separation mechanism of the components of the sample is based on these differences (charge/mass ratios), which allow them to have different electrophoretic mobilities and therefore different velocities, and it is possible to separate cations and anions if the EOF is strong, in order to lead anionic substances to the detector located at the cathode (direct polarity). Figure 5.2 shows the separation principle in connection with the microchip design. In general, this mode has been preferred in small molecules analysis (see Section 5.8). Capillary gel electrophoresis (CGE) combines the good resolution of classical gel electrophoresis with the simple instrumentation used in CZE. The separation principle is: a capillary is filled with a gel (with pores of controlled size) that acts as a molecular sieve. A scheme of the separation mechanism is shown in Figure 5.3(a). It is based on the different mobilities of the components of the sample through the pores of the gel due to their different molecular sizes (molecular sieving). The compounds of small size will traverse the gel matrix

µep + anode

< µep

< buffer µeo

µep

Detection – cathode

Figure 5.2 Principle of CZE on microchip. (Reprinted from [15] with permission and adapted)


171

DETECTOR

(a) GEL

anode

cathode –

+

pl1

(b)

pl2

pl3

H3PO4

NaOH

anode

cathode

pH gradlent

+

– Small pH bajo

(c)

High pH alto

pl

µec

µPG

anode +

O P

A A

cathode –

Figure 5.3 Principles of CE modes on microchip. (Reprinted from [15] with permission and adapted)

easier and will have shorter analysis times than those substances of large size, which traverse the gel more slowly. DNA analysis is the golden example of this electrophoretic mode in microchip (see Section 5.8). Capillary isoelectric focusing (CIEF) is employed to achieve separation of proteins, peptides, amino acids and other substances of amphoteric character. In CIEF the separation is not based on the differences in electrophoretic mobilities but on the differences in pI of solutes. Separations by CIEF are based on the electrophoretic migration of amphoteric substances in a pH gradient, as shown in Figure 5.3(b). The pH gradient needed though the capillary is obtained by using a solution of ampholytes (substances of zwitterionic character with different pKa values (carrier ampholyte)). The sample can be added to the carrier electrolyte. When the electric field is applied, a pH gradient is established within the channel from the anode (low pH) to the cathode (high pH). Sample components will move until they find a pH value equal to their pI, since at this pH they lose their charge and then stop (this phenomenon is called focusing). In isotachophoresis (ITP), a volume of analyte is placed between a leading electrolyte (LE) and a terminating electrolyte (TE). The LE is composed of highmobility ions and so has a higher mobility than the sample ions and the TE. Conversely, the TE is composed of low-mobility ions and so has lower mobility than the LE and sample ions. During the electrophoretic separation, analytes in the

172


sample are arranged into discrete bands in order of mobility. The velocity and concentration of these bands are adapted to a value governed by the leading ion. Consequently, both separation and concentration adaptation occur simultaneously. Electrokinetic chromatography has also been transferred to microchip format. Micellar electrokinetic chromatography (MEKC) is characterized by the use of micelles as pseudophase. Micelles are aggregates that are formed by adding surfactants to the separation buffer at a concentration above their CMC. Ionic surfactants form micelles that move at a different velocity to the EOF. In this case, the separation of neutral molecules is produced by their distribution between the aqueous and the micellar phases. When the micellar phase moves to the detector, the elution range is between the time corresponding to EOF and the migration time of the micelles, and all analytes will migrate between these two limits depending on their distribution between the two phases (see Figure 5.3(c)). Part of CZE, this has often been used on microchip format. Capillary electrochromatography (CEC) combines the advantages of HPLC and CE, such as the high efficiency of CE (movement of solutes by electrical forces) and the high selectivity of HPLC (chromatographic interactions). Analytes are separated by the combined action of partitioning an LC-type stationary phase and mobile phase and, if charged, by differences in electrophoretic mobility. In CEC the solutes are transported through the column by the EOF of the solvent and/or by their electrophoretic mobility. This approach will be examined in Section 5.6, since it involves specific technical challenges and deserves its own discussion. On the other hand, the theory and mechanisms of action for microchip CE are also based on fundamental knowledge gained in the development of conventional CE [11,16]. Two electrokinetic phenomena – electro-osmosis and electrophoresis – can contribute to the movement of species in a microchannel as a consequence of the application of an electric field between the ends of the channel. Electro-osmosis can be described as ‘the movement of liquid relative to a stationary charged surface,’ while electrophoresis is ‘the movement of a charged species (i.e. dissolved or suspended material) relative to a stationary liquid’ [11]. While electro-osmosis results in nonselective transport of charged or neutral molecules, electrophoresis is effective in separating ions of differing charge-to-mass ratios. The directed transport of molecules in response to an electric field is called migration. In most cases, the moving molecules are ionized in a polar solvent such as water. These electrically charged molecules experience a Coulomb force due to the electric field. This phenomenon is termed electrophoresis. The charged molecules first accelerate toward one of the electrodes, but then slow down and reach a terminal velocity, because they experience a drag caused by friction with the liquid. The Coulomb force (F) is given by: F ¼ qE;

ð5:1Þ


173

where q is the charge on the molecule and E the strength of the electric field. Once the molecules reach their terminal velocity, the Coulomb force is balanced by a drag force, the Stokes force: F ¼ 6phrn;

ð5:2Þ

where Z is the viscosity of the liquid, r is the so-called hydrodynamic radius of the molecule, which indicates its size, and v is the velocity of the molecule. The terminal velocity of the molecule is reached when both forces are equal and opposite, so that: qE ¼ 6phrn

ð5:3Þ

From Equation 5.3, the terminal velocity is calculated as: n ¼ mE;

ð5:4Þ

where m is the mobility of the molecules, given by: m¼

qE ; 6phr

ð5:5Þ

where q is the charge of the molecule, E is the applied field strength (V/m), Z is the viscosity of the mobile phase and r is the radius of the molecule, which is related to its mass. On the other hand, electro-osmosis generates the EOF. EOF is a bulk solution flow phenomenon that occurs in capillaries filled with mild ionic solutions (typically 9), dielectric properties (the possibility of working with the high voltages used in CE) and well-developed microfabrication methods (adapted from the silicon microfabrication industry). Other advantages of glass are its hardness, high thermal stability and biocompatibility (wide range of applications: DNA separations, enzyme/immunoassays, cell biology and small molecules). Polymeric substrates have mainly consisted of poly(methylmethacrylate) (PMMA) (by injection moulding or hot embossing) and poly(dimethylsiloxane) (PDMS) (by casting). These materials have found favour due to their ease of fabrication. PMMA is an example of a thermoplastic material (because a wide range of microfabrication methods are available for this compound), and PDMS is an example of an elastomer. PDMS has been widely discussed due to several characteristics: (i) it is suitable for optical detection down to 320 nm; (ii) it cures at low temperatures and moulding can easily be replicated through the process of prototyping, master formation and soft lithography; (iii) it can seal reversible to itself and other materials by van der Waals contact with a clear smooth surface at room temperature; (iv) its surface chemistry can be controlled to form EOF using a plasma technique. Polymers also show good resistance to chemical treatment and good biocompatibility. It is possible to find a polymer that has the optical qualities desired for any given application (e.g. PDMS is transparent to the UV region of the electromagnetic spectrum, while most thermoplastic polymers, such as PMMA and PC (polycarbonate), are transparent in the visible region). The large EOF in glass is partly due to the high surface charges because of the deprotonation of silanol groups. However, many polymers do not contain ionizable functional groups and thus would be expected to produce much smaller EOFs, a clear disadvantage. Indeed, under controlled microfabrication techniques, it is possible to generate an EOF very acceptable for PMMA, of 1.2 · 104 cm2/Vs. Although native PDMS exhibits a very small EOF value, subjecting it to oxidation in a plasma discharge changes the surface characteristics. OSi(CH3)2O groups are transformed to OSi(OH)4n producing SiOH groups, which leads to surfaces that can be readily deprotonated as glass or fused silica. Also, polymer microchips are of increasing interest because their potentially low manufacturing costs may allow them to be disposable. As PDMS is transparent in the visible light range, LIF detection is readily applicable. Among the few disadvantages of PDMS are poorly defined EOF and adsorption of nonpolar hydrophobic samples. A comparison between microchip materials is given in Table 5.2 (adapted from [21]).


177

Table 5.2 Comparison of materials in CE microchip. (Adapted from [21]) Feature

Glass/Fused silica

Polymera

Electronic conductivity Thermal conductivity (cal/cm-s- C) Bioassay compatibility

none: OK for CE 2 · 103

none: OK for CE 4.5 · 104b

fair

Optical detection

glass: very good fused silica: excellent isotropic wet etching only

fair to very good (varies according to polymer choice) poor to very good (varies according to polymer choice) Si or glass mastering plus replication techniques; direct methods (ablation, dry etching) dependent on mastering and replication methods:

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