Electric Field Applications in Chromatography, Industrial and Chemical Processes
Edited by Takao Tsuda
Wiley-VCH
Electric Field Applications in Chromatography, Industrial and Chemical Processes Edited by Takao Tsuda
4b
VCH
Weinheim . New York . Base1 * Cambridge . Tokyo
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Electric Field Applications Edited by T. Tsuda
VCH Verlagsgesellschaft mbH, D-69451 Weinhelm, (Federal Republic of Germany). 1995 Distribution: VCH. P. 0. Box 10 I 1 h l 1)-69451 ~ Wcinheim (Federal Republic of Germany) Switzerland: VC'H, P 0.Box, CH-4020 Basel (Switzerland) United Kingdom and Irland: VCH. X Wellington Court, Cilmbridge CBI I H Z (United Kingcknii) USA and Canada: VCH, 220 East 23rd Street, New York, NY 10010-4606 (USA) Japan: VCH, Eikow Huilding. 10-1) Flongo I-chorne. Bunkyo-ku, Ibkyo 113 (Japan) ISBN 3-527-28687-X
Electric Field Applications in Chromatography, Industrial and Chemical Processes Edited by Takao Tsuda
4b
VCH
Weinheim . New York . Base1 * Cambridge . Tokyo
Prolessor Takao Twda Nagoya lnstltute of Technology Ciokiso, Showa, Nagoya 466 Japan
This book was carefully produced. Nevertheless. the editor, authors and publisher do not warrant the information contained therein to bc free of errors. Readers are advised to keep in mind that statements, data. illustrations, procedural details or other items may inadvei-lcntlybe inaccurate.
Puhlishcd jointly by VCH Verlagsgcscllschalt mbII, Weinheim (Federal Republic of Germany) VCH Publishers, Inc., New York, NY (IJSA)
Editorial Directors: Dr. Christina Dyllick-Brcnzinger, Karin Sora Production Manager: Peter J. Die1
Library of Congress Card N o . applicd for British Library Cataloguiiig-in-Publication Data: A catalogue record lor this book is available from the British Library. Die I k u t s c h c Bibliothck CIP-Einheitsaufnahinc Electric field applications in chromatography, industrial and chemical processes / cd. by Takao Tsuda. - Weinheim ; New York ;Basel : C:anihridgc ; Tokyo : VCH, 1995 ISBN 3-527-28687-X NE: Tsuda, Tikao [Hrsg.] ~
0 VCI1 Verlag5gc~cllschaltmhH, D-69451 Weinhelm (Federal Republic of Germany), 1995 Printed on acid-ti ee and low-chlorine paper All rights reservcd (including those of translation into other languages). No part of this hook may be reproduced in any form - by photoprinting. microfilm, or any other incans - nor lransmitted or trailslated into a machine language without writtell permission from the publishers. Kegistcrcd narncs. trademarks, etc. used in this book. cvcn when not specifically marked as such, arc not to be considered unprotected by law.
Composition: Mitterweger Werksatz GmhH,D-68723 Plankstadt Printing: Bctzdruck GmbH, D-642 19 Ilarmstadt Hnokhiiiding: Grolibuchbinderei J. Sch2ffer CmhH & Co. KG, D-67269 Criinstadt Printcd i n the Federal Republic of Oernlany
To my parents, Sukesaburo and Tamiko Tsuda, for their encouragement throughout my life.
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Preface
Research and development in both academic and industrial laboratories frequently involves sophisticated methods and techniques often based on the application of electrical, magnetic, centrifugal, or adsorptive forces. Electrical forces are probably the most widely used since they are very flexible in the sense that they can (1) generate a great number of chemical and physical phenomena (including magnetism, heat and light), (2) affect both inanimate and living matter in many different ways and (3) easily be regulated, by means of constant or (extremely rapidly) pulsating direct voltage, or alternating voltage of almost any frequency. If an electric field is combined with a magnetic field the area of application is still larger. Such combinations are not used as widely as they deserve to be; the area is almost virgin soil for truly basic research! Rotation of a liquid or liquid droplet is only one instance of what can be done by this approach to solve several technical problems. This book is divided into three parts: 0 Electrochromatography (Chapters 2-6) , 0 Applications of electric fields in industrial processes (Chapters 7-11), and 0 Applications of electric fields for concentration, immunoassay and molecular
orientation (Chapters 12-14). At first glance, the scope of the book seems very broad, but the unifying feature is that the electric field causes primarily either a displacement of solvent (a phenomenon called electroosmosis) or an orientation andor a migration of solute (electrophoresis). The contents of the book are, from this point of view, relatively limited but are broad on practical topics. This is an attractive approach, since it shows the analogy among and the interplay between various techniques, which gives a creative reader many new ideas. It is quite natural that several chapters (Chapters 2-6) deal with electrochromatography, since the editor of the book, Professor Takao Tsuda, introduced this powerful separation method in 1982. An attractive feature of the method is that the mobile phase moves through the bed by means of electroosmosis; no expensive pump is required. Another advantage is that the electroosmotic flow profile is flatter than the hydrodynamic one and thus causes less broadening of the solute zone. Packed or opentubular capillary columns are commonly used for this purpose. We introduced recently so-called continuous beds and they might be preferable in electrochromatography. As far as detection methods are concerned, one can expect mass spectrometry to become the method of choice in the near future in electrochromatography as well as in many other microchromatographic techniques, as pointed out in Chapter 6. Most chemical syntheses and extraction processes are performed by the mechanical mixing of solutes. To decrease the reaction or equilibration time (i.e., to increase the collision frequency among solute molecules) the experiments are often carried out
VIII
Preface
with stirring. This approach is not always convenient, for example when the reaction volumes are extremely small or large, when dilution of the solutes is to be avoided, or when the reaction vessel must have a certain shape, for instance a thin disk or sheet. In such cases “electrophoretic or electric mixing” may be preferable. Examples are presented in the chapters dealing with immunoassay (Chapter 13), solvent extraction (Chapters 10 and 11) and the manufacture of ceramics (Chapter 8). The latter two are examples of industrial applications. It is fascinating to see how an electric field can increase the reaction (extraction) speed by its capacity to produce small droplets and nonlaminar streaming. It is well-known that free-draining polymers, such as DNA molecules, align themselves in parallel chains in strong electric fields. For a variety of reasons one can expect such oriented structures with specifically adsorbed molecules and lined up carbon particles to be studied in the near future. The chapters on photochemistry (Chapter 14) and electro-rheological fluids (Chapter 9) present these topics. Removal of water by electroosmosis is a method which dates back to 1809, but has recently come back into use for large-scale industrial operations (Chapter 7). Desalting is another industrial application of the electric field (Chapters 11 and 12). By means of a voltage gradient one can also control the viscosity of a liquid, and this technology is useful in car production (Chapter 9). The experiments described in this book refer to outer, artificial electric fields. It is, however, fascinating to consider whether the electric fields in biological systems are of any importance. One should remember that a small voltage over a small distance, such as the dimensions of a living cell, gives rise to a high electric field strength. In preparing this preface I have become more convinced than ever that one should encourage publication of scientific - and popular - books of interdisciplinary nature. Together with interdisciplinary symposia, books such as this serve the important function to stimulate to discussions between people with different areas of expertise. Stellan Hjerttn Department of Biochemistry Uppsala University Sweden
Table of Contents
1
Introduction and Summary 1 Takao Tsuda
Part 1 Electrochromatography 11 2
Electrochromatography in Analytical Chemistry 13 Takao Tsuda, Shinya Kitagawa
2.1 2.2 2.3 2.4 2.5
Theory of Band Broadening 13 Apparent Mean Linear Flow Velocity and Elution Time 14 Processes in Band Broadening 14 Electrochromatography Zones 17 Profiles of Pressurized Flow, Electroosmotic Flow, and Zones of Ionic Solutes 18 Flow Profiles of Pressurized Flow 18 Flow Profiles of Electroosmosis in an Open Tube 19 Flow Profiles for Charged Molecules 27 Pressurized Flow-Driven Electrochromatography on Microlcolumns 29 Instrumentation 29 Features and Operational Factors 31 Chromatographic Behavior in Pressurized Flow-Driven Electrochromatography 32 Chromatographic Variation due to the Application of High Voltage 35 Relation between Elution Time Ratio and pH 35 Variation of Electrophoretic and Electroosmotic Flow Velocities with pH 37 Dependence of Electrophoretic and Electroosmotic Velocities on the Composition of Eluents Containing Methanol 39 Ion-Exchange Chromatography in an Electric Field 40 Voltage-Programmed Electrochromatography 42 References 45
2.5.1 2.5.2 2.5.3 2.6 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 2.6.6 2.6.7 2.6.8 2.6.9 2.7
3
Electroosmosis and Electrochromatography 47 Takao Tsuda
3.1 3.1.1 3.1.2 3.1.3
Electroosmosis 47 Surface Charge of Silica Gel and Packing Support 47 Electrical Potential in the Vicinity of a Solid Surface 48 Origin of Electroosmotic Flow 50
X
Table of Contents
3.1.4 3.1.5 3.1.6
Thickness of the Double-Layer 51 Charge Density on Silica Gel Surfaces 53 Chemical Modification of the Inner Surface by Adsorption in Open-Tubular Capillary Columns 54 3.1.7 Effect of pH on Electroosmosis 55 3.1.8 Electroosmotic Mobility in Open-Tubular Capillary Columns 56 3.1.9 Electroosmotic Flow Velocity in Packed Columns 56 3.2 Electroosmotically Driven Chromatography and Electrochromatography 59 3.2.1 Electroosmotically Driven Electrochromatography 59 3.2.2 Electroosmotically Driven Chromatography 61 3.2.2.1 Open-Tubular Capillary Columns 61 3.2.2.2 Packed Microcapillary (Drawn Packed Capillary) Columns 66 3.2.2.3 Slurry-Packed Capillary Columns 70 3.2.3 Advantages of Electroosmotic Flow for Liquid Chromatography 72 References 73 3.3 4
Electrochromatography with Radial Applied Voltage : Ion Separation by Electrochemical Approach 75 Tsutomu Nagaoka
4.1 4.2 4.2.1 4.2.2 4.3
Introduction 75 Experimental Details 76 Design of the Electrode Column 76 Preparation of Stationary Phases 77 Redox Separation of Electroactive Metals on the Conductive Stationary Phase 79 Direct Electrostatic Interactions for Potential-Dependent Separation of Electroinactive Species 80 Pretreated Carbon for the Separation of Metal Ions 80 Stationary Phase Coated with Crown Ether for the Separation of Alkali Metal Ions 80 Electrosorption for the Separation of Neutral Organic Compounds 82 Indirect Electrostatic Interactions for Potential-Dependent Separation of Electroinactive Species 83 Conducting Polymers for Separation of Anions 83 Summary 88 References 88
4.4 4.4.1 4.4.2 4.4.3 4.5 4.5.1 4.6 4.7 5
Electrochromatography in Biomolecular Analysis 91 Daniel H. Shain
5.1 5.2 5.3 5.4 5.5 5.6 5.6.1 5.6.2
Introduction 91 Significance 91 Characteristics of Biomolecules 92 Development of Electrochromatographic Techniques 94 Gel Electrophoresis 96 Electrochromatography in Preparative Columns 97 Sequential Electrochromatography 97 Electrochromatography with Modern Adaptions to Liquid Chromatography 99 Focusing in Columns 104
5.6.3
XI
Table of Contents
5.7 5.7.1 5.7.2 5.7.3 5.7.4 5.8 5.9
Electrochromatography in Microcolumns 108 Electro-Osmotically-Driven Electrochromatography 109 Pressurized Flow Driven Electrochromatography 111 Micellar Electrokinetic Capillary Chromatography 111 Capillary Gel Electrophoresis 111 Conclusions and Future Directions 112 References 113
6
Electrochromatography for Focusing, Counter-Current, Mass Spectrometry, and Tho-Dimensional Separation 117 Takao Tsuda
6.1 6.1.1
Continuous In-Column Sample Focusing 117 Multiple Injection for Accumulating a Charged Component in the Column 117 Continuous Sample Introduction for In-Column Sample Focusing 118 Rotation Locular Counter-Current Electrochromatography 120 Instrumentation 120 Preparative-Scale Electrochromatography with a Counter-Current Chromatographic Column 122 ElectrochromatographylMass Spectrometry 123 Instrumentation 123 Combination of Pressurized Flow-Driven Electrochromatography and Mass Spectrometry 124 Two-Dimensional Preparative Chromatography with Continuous Injection 126 Continuous Preparative Electrochromatography : a Large Cylindrical Column with Rotation 127 Separation of a Test Mixture 128 References 129
6.1.2 6.2 6.2.1 6.2.2 6.3 6.3.1 6.3.2 6.4 6.4.1 6.4.2 6.5
Part 2 Applications of Electric Fields in Industrial Processes 131 7
Electroosmotic Dewatering 133 Masashi Iwata
7.1 7.2 7.3 7.4 7.4.1 7.4.2 7.4.3 7.4.4 7.5 7.6 7.7 7.8
Introduction 133 Electroosmotic Flow through a Capillary 134 Electroosmosis in Porous Media 136 Electroosmotic Dewatering of Compressible Solid-Liquid Mixtures Experimental 137 Theory 137 Physical Properties of the Material 141 Comparison of Theory and Experiment 142 Industrial Applications 147 Conclusions 149 Nomenclature 150 References 151
137
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Table of Contents
8
Electrophoretic Forming of Ceramics 153 Takao Tsuda and Hideki Ishida
8.1 8.2
Electrophoretic Behavior of Inorganic Particles in Aqueous Solution 154 Selection of Organic Solvents for Electrodeposition of Inorganic Particles Dispersed in Organic Solution 155 Electrophoretic and Electroosmotic Forming 156 History 156 Industrial Applications of Electrophoretic and Electroosmotic Forming 158 Control of Particle Diameter for High Productivity and a Low Water Content in Electrical Forming 161 Zeta-Potential of Raw Materials 164 Electrodes 164 Electrophoretic Forming Continuous Ceramic Sheet 166 References 169
8.3 8.3.1 8.3.2 8.4 8.5 8.6 8.7 8.8 9
Control of Viscosity by Electric Fields: Novel Materials for ElectroRheological Fluids and their Applications 171 Yuichi Ishino, Tasuku Saito, Norio Goshima, and Kazuya Takano
9.1 9.2 9.3 9.3.1 9.3.2 9.3.3 9.4 9.5 9.6
Introduction 171 Fundamental Characteristics of ERF 171 Development of the Dispersed Phase 175 Chelate Resin 175 Porous Silica Microspheres 178 Carbonaceous Particulate Phases 179 Applications 181 Conclusion 184 References 184
10
Application of Electric Fields to Solvent Extraction 185 Manabu Yamaguchi
10.1 10.2 10.2.1 10.2.2 10.2.3 10.3 10.3.1 10.3.2 10.3.3 10.4 10.5
Introduction 185 Theory 186 Hybrid Flow and Enhanced Mass Transfer Around Drops 187 Electrostatic Liquid Dispersion 192 Electrostatic Drop - Drop Coalescence 194 Electrostatic Liquid-Liquid Extraction 195 Electrostatic Spray Column 195 Electrically Assisted Mixer - Settler Extractor 196 Electrostatic Pseudo-Liquid Membrane Extractor 200 Nomenclature 201 References 203
Table of Contents
11
XI11
Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions 205 Manabu Yamaguchi
Introduction 205 Fundamental Relationships 206 Dielectrophoresis 206 Computational Simulation of Chain Formation 208 Observation of Chain Formation 209 Electrophoresis 211 Effect of Electric Fields on Resolution of Water-in-Oil Dispersions 212 Electrostatic Resolution of Emulsion in the Oil Industry 213 Dehydration 213 Desalting 215 Electrostatic Phase Separation of Rich Water-in-Oil Emulsions in Liquid Membrane Separation 219 11.4.1 Membrane Recovery in Liquid Membrane Separation 219 Conclusions 226 11.5 Nomenclature 226 11.6 References 227 11.7
11.1 11.2 11.2.1 11.2.2 11.2.3 11.2.4 11.2.5 11.3 11.3.1 11.3.2 11.4
Part 3 Applications of Electric Fields for Concentration, Immunoassay and Molecular Orientation 229 12
Dynamic Electroconcentration Processes in Analytical Chemistry 231 Takao Tsuda
12.1
Collection of Metal Ions from Acid Solutions by Hydrodynamic Recycling 231 Electrodialysis for Desalting or Concentration of Metal Ions by Multiple Ion-Exchange Chambers 233 Counter-Current Electroconcentration 236 Principles 236 Apparatus 239 Examples 240 Operational Stability 241 Electroosmosis and Electrophoretic Mobility for Counter-Current Electroconcentration 241 Electroosmosis for Sample Transfer 242 Electrodeposition of Metal Ions and their Selective Release 244 Electrochromatographic Solid-Phase Extraction 245 Operational Procedure 245 Use of Electrochemical Reactions for Decomposition of Metal Complexes 247 Conclusions 248 References 248
12.2 12.3 12.3.1 12.3.2 12.3.3 12.3.4 12.4 12.5 12.6 12.7 12.7.1 12.8 12.9 12.10
XIV
Tuble of Contents
13
Pulse Immunoassay and Pulse Electrovoltage for Cell Manipulation and Analytical Coagulation Processes 249 Eiichi Tamiya
13.1 13.2 13.2.1 13.2.2 13.2.3 13.2.4 13.3
Introduction 249 Pulsed Immunoassay for Pathogenic Cells 249 Experimental Procedures 250 Effect of Pulse Conditions on Linear Agglutination 251 Agglutination by Immunoreaction in an Electric Field 251 Calibration for C. albicans 254 Alternating Current Field Enhancing Latex Immunoassay for Human Myoglobin 254 Experimental Procedures 255 Agglutination Induced by a.c. fields 256 Time Course of Immunoreactions 258 A Micromachined Reactor for Latex Immunoassay Enhanced by a.c. Fields 260 Experimental Procedures 260 Design of Micromachined Reactors 260 Application to Immunoassay for AFP 261 Conclusions 263 References 263
13.3.1 13.3.2 13.3.3 13.4 13.4.1 13.4.2 13.4.3 13.5 13.6 14
Molecular Orientation of Organic Compounds in Electric Fields: Organized Photochemistry 265 Katsuhiko Takagi
14.1 Introduction 265 14.2 Photochemical Reactions 266 14.2.1 Applied Electrostatic Fields 266 14.2.2 Spontaneous Electrostatic Fields 270 14.2.3 Micelles 270 14.2.4 Energy and Electron Transfer 271 14.2.5 Miscellaneous Reactions 277 14.2.6 Reversed Micelles 280 14.2.7 Vesicles 282 14.2.8 Mono- and Multilayer Membranes 283 14.2.9 Polyelectrolytes 286 14.2.10 Inorganic Solid Surfaces 287 14.3 References 295
Index 301
List of Contributors
Norio Goshima Research & Development Division Bridgestone Corp. 3-1-1 Ogawahigashicho, Kodaira-shi, Tokyo 187 Japan Chapter 9 Stellan HjertCn Department of Biochemistry, University of Uppsala P.O. Box 576, S-75 123 Uppsala Sweden Preface Hideki Ishida Basic Research Center, INAX Corporation Koiehonmachi, Tokoname, Aichi 479 Japan Chapter 8 Yuichi Ishino Research & Development Division Bridgestone Corp. 3-1-1 Ogawahigashicho, Kodaira-shi, Tokyo 187 Japan Chapter 9 Masashi Iwata Department of Industrial Chemistry, Suzuka National College of Technology Shiroko-cho Suzuka 510-02 Japan Chapter 7
Shinya Kitagawa Nagoya Institute of Technology Gokiso, Showa, Nagoya 466 Japan Chapter 2 Tsutomu Nagaoka Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering Yamaguchi University 2557 Tokiwadai, Ube 755 Japan Chapter 4 Tasuku Saito Research & Development Division Bridgestone Corp. 3-1-1 Ogawahigashicho, Kodaira-shi, Tokyo 187 Japan Chapter 9 Daniel H. Shain Department of Biochemistry and Molecular Biology, Colorado State University Fort Collins Collorado 80523 United States Chapter 5 Katsuhiko Takagi Department of Applied Chemistry School of Engineering, Nagoya University Chikusa-ku Nagoya 464-01 Japan Chapter 14
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List of Contributors
Kazuya Takano Original Equipment Industrial Products Division Bridgestone Corp. Kashiwao-cho, Tozuka-ku, Yokohama 244 Japan Chapter 9 Eiichi Tamiya Japan Advanced Institute of Science and Technology (JATST) Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa, 923-12 Japan Chapter 13
Takao Tsuda Nagoya Institute of Technology Gokiso, Showa, Nagoya 466 Japan Chapters I , 2, 3, 6, 8, 12 Manabu Yamaguchi Department of Chemical Engineering, Faculty of Engineering Science Osaka University 1-3 Machikaneyama, Toyonaka 560, Osaka Japan Chapters 10,11
1
Introduction and Summary Takao Tsuda
The applications of electric fields are one of the most exciting topics in Science today. The introduction of electric fields in chromatography, industrial processes, and unit chemical processes offers the prospect of remarkable advances with its application in areas ranging from separation technology to the oil industry. When we apply an electric field to a column or a solution by using two electrodes, a microelectric field is induced at every point. The value of the induced potential gradient depends on the characteristics of the local matrix. Under these potential gradients a charged molecule moves (electrophoretic mobility). The local electric field on a solid surface induces surface charges, and hence electroosmosis [1.11, [1.21. A high local potential gradient imposed on the surface adsorption layer leads to orientation of organic molecules. A droplet of water or oil is deformed and dispersed into a group of small droplets under an applied electric field. The micro particles of solid in a medium line up in the electric field, keeping their energy to a minimum. This book is divided into three sections: I) Electrochromatography 11) Application of electric fields for industrial processes 111) Application of electric fields for concentration, immunoassay, and molecular orientation Electrochromatography is a unique separation method with high selectivity and efficiency and has potential for a wide range of applications. There are a number of remarkable industrial applications, where electric fields induce unique phenomena. Some processes, including the application of voltage have energy-saving potential. The third section presents: a) a unique concentration method based on electrochemistry and electrical phenomena; b) agglutination induced by electric fields, which is very useful for rapid immunoassay; and c) induced molecular orientation including stereospecific alignment within micro cavities. Throughout this book, the electric field, in macro- or micro-spaces plays a key role.
Part 1 Electrochromatography Liquid chromatography is now commonly used for the separation of mixtures and is well established as a very efficient method [1.3]. Separation relies on the partition between a mobile and stationary phase. The mobile phase, containing the solute, moves through the column by pressurized flow. During this period the solute moves between the stationary phase and the mobile phase by diffusion. The partition ratio of a solute, the distribution of a solute between staionary and mobile phase, depends on its molecular nature.
2
1 Introduction and Summary
Electrophoresis is also used for the separation of biomolecules. The separation obtained with gel electrophoresis has the highest resolution, especially for the nucleotides and proteins. The separation process in electrophoresis is based on the electrophoretic mobility of a solute. Sometimes the solute will temporarily be in contact with or be adsorbed by the matrix i. e., when partition occurs. TGavoid these contacts, salts and/or urea are added to the medium to decrease the adsorptive ability of the matrix. To some extent there is an interaction between solute and matrix in electrophoresis. It is, therefore, reasonable to consider that the separation sometimes proceeds with electrophoretic mobility and partition between medium and gel network. Electrochromatography is a combined form of chromatography and electrophoresis [ 1.41. Its separation processes include both phase partition and solute electrophoretic mobility. Therefore, electrochromatography can be a very efficient separation method [1.5], 11.61. The term was first used by Berraz [1.7] in 1943, in referring to a primitive form of paper electrophoresis. Paper electrochromatography has been applied to the separation of metal cations by Strain and coworkers [1.8], [1.9]. They used the electrophoretic mobilities of metal ions and their sorptive interaction with paper. The sorptive interaction was confirmed by the observation of the effect of sample concentration on elution time, (nonlinear isotherms) [1.81. Polysaccharides have been separated by paper electrochromatography by using an electroosmotic flow as a driving force [1.10]. Amino acids and carboxylic acids have been separated on paper impregnated with stannic arsenate [1.11], hydrated zirconium oxide [ 1.121, and titanium tungstate [ 1.131. In these separations two factors, ion exchange and electrophoretic mobility, both contributed. Thin layer elcctrochromatography has also been attempted [1.14]-[1.16]. The bed was agar gel deposited on a glass plate. Plain agar gel already has cation-exchange properties, and separation of hemoglobins below their isoelectric points have been performed by both electrophoretic mobility and ion exchange in parallel [1.14], y1.151. Yaron and Sober [ 1.161 provided conclusive evidence that ion exchange increases the resolving power of electrophoresis of oligonucleotides and oligopeptides. These authors insisted on the importance of working at a pH close to the pK value of the ionexchange group. Haber [ 1.171employed a cellulose filter (5 x 10 cm) or layers of other adsorbents as a flat bed. He applied a very high voltage, e. g., 10 kV. He separated the mixture of Rhodamine B and 6G in 3 s with a migration distance of 0.2 cm under the following conditions: separation medium: 40 % propylene carbonate, 40 % propylene glycol, 16 % N-methylacetamide, 4 % tetrahydrofurfuryl alcohol; bed: Whatman No. 1 paper; applied voltage 12 kV (1.2 mA) [1.17]. Haber reported very rapid separations at high applied voltage. The method has commercial potential [1.18]. In the very early days of electrochromatography, continuous paper electrochromatography was used for separation of cobaltous and ferric ions with continuous injection. The stabilization medium for the pressurized flow (2-direction) of a medium and concomitant transverse electrical migration (x-direction) is attained in a tapered filter paper [1.19].The separation depends on differences in electrophoretic mobility and ion exchange equilibrium constant. In the present book we have tried to focus on advanced column electrochromatography. Chromatography on columns is generally more reliable than paper or thin layer chromatography. Column chromatography involes elution; the solute is transferred to the end of a column and then detected. Most chromatographic processes are confined by the wall of a column. Therefore, column chromatography generally gives better qualitative and quantitative information, better reproducibility, and better resolution than paper chromatography. In elcctrochromatography, the use of a column also gives enhanced performance. Otsuka and Listowsky [ 1.201 pioneered the set up technique.
1.4 Electrochromatography with Radially Applied Voltage
1.2
3
Electrochromatography in Analytical Chemistry (Tsuda and Kitagawa)
The theory of the separation by electrochromatography is somewhat different from ordinary liquid chromatography. There are two new factors, electrophoretic mobility in the mobile phase and electroosmosis generated by the applied electric field [1.4], [1.21], [1.22]. These new factors can be used to improve separation. The apparent theoretical resolution depends on the flow profiles of pressurized flow, the electroosmotic flow, and the zone profile due to electrophoretic mobility. In electrochromatography, we need to apply high voltage along the column to get high migration of a solute due to electrophoretic mobility. By using a high applied voltage, e. g., 1-20 kV (200-2000 Vkm), we can separate the mixture with high resolution within 30 min, the same as conventional liquid chromatography. However, it is necessary to dissipate the Joule heat from the column. As micro- and capillary columns have high heat dissipation, they are recommended for use in analytical electrochromatography. Electrochromatography with pressurized flow is a fine method since it can be operated with good stability, and it is easy to determine the characteristic points by conventional liquid chromatography. In Chapter 2 the state-of-the-art of pressurized flow-driven electrochromatography is described.
1.3
Electroosmosis and Electrochromatography (Tsuda)
Theoretical consideration of electroosmosis and factors which affect electroosmosis, such as pH and the amount of surface charge are described. Then electroosmosis is used as the driving force for transferring solute in the column without pressurized flow. The characteristic nature of electroosmotically driven electrochromatography is compared with conventional liquid chromatography. Open-tubular capillary, packed microcapillary (drawn packed capillary), and slurry-packed capillary columns are used. The flow velocity of electroosmosis in packed capillary columns depends on the pH of the medium (namely, the number of surface charges of SiO-), and on the tortuous and constricted channels within the packing support. Fine and rapid separations are demonstrated.
1.4
Electrochromatography with Radially Applied Voltage: Electrochemical Separation of Ionic Substances (Nagaoka)
In Chapters 2 and 3, a high voltage is applied along the column. In Chapter 14 the applied voltage is between 1 and -1 V, applied in a radial direction [1.23]-[1.26]. The column is generally packed with carbon or modified carbon powders, and an electrode is inserted inside the column. In a Vycor glass tube wall there are a lot of very fine pores (30 A) passing from the inside to the outside. It is possible to position another electrode outside the column [ 1.231 or to use a stainless steel column so that the wall itself forms the electrode [1.25]. There are two kinds of solute: electroactive and electroinactive. Electroactive solutes can be deposited onto carbon packing powders under
+
4
1 Introduction und Summary
an applied voltage, and then released on removal of the applied voltage. For electroinactive solutes, it is possible to have an electrostatic interaction between the solute and charged powders.
1.5
Biomolecular Analysis by Electrochromatography (Shain)
For the separation of biochemical solutes, such as proteins and DNA fragments, acrylamide or agar gels on a flat bed show splendid separation ability. In liquid chromatography, proteins can be separated by gradient elution, but both separation modes have drawbacks. Chapter 5 introduces several applications of electrochromatography columns filled with gels [1.20], [1.27], [1.28]. Columns filled with acrylamide or agar gels are coupled with modern instrumentation for the fractionation or separation on a semipreparative and analytical scale. Related techniques for the separation of biomolecules are described, in detail.
1.6
Electrochromatography for Focusing, Counter-Current, Mass Spectrometry, and Two-Dimensional Separation (Tsuda)
This chapter includes four unique technologies which are possible only by electrochromatography. First, a charged solute, introduced stepwise or continuously in a liquid chromatographic column can be accumulated in the column when its electrophoretic velocity is sufficiently large and its flow opposes the pressurized flow [1.5], [1.29]. Second, a voltage is applied along a rotation locular counter-current column (12 X 1000 mm), which has 100 loculi. Kabasawa has demonstrated the effective separation of dyes [1.30]. Third, the combination of electrochromatography and mass spectrometry has been demonstrated by van der Greef et al. t1.311. This is a unique method. The electrochromatogram obtained shows a sharper, higher peak compared with conventional liquid chromatography (the solute is concentrated in a narrow zone). This is an important advantage for electrochromatography/mass spectrometry. Fourth, Scott proposed instrumentaion for two-dimensional separation with a continuous introduction of the sample solution [1.32]. The system is composed of an annular column of adsorbent that is slowly rotated past a stationary feed and eluate take-off points. The position of the exit points of the solute are defined by their electrophoretic mobility and distribution coefficient. In electrochromatography the applied voltage has a direct effect on a solute in a mobile phase, not in a stationary phase. However the latter might be also affected by alteration of the nature of the stationary phase or the increased amount of charge. I would like to add a comment on the method of using two different beds in a column for enhancing the separation of a mixture [1.22]. The time taken the solute to run through column is equal to the period given by [(column void volume)/(flow rate due to pressurized and/or electroosmosis)]. Each solute can stay in the mobile phase for a time period to.The electrophoretic mobility of a solute p, does not depend on the column packing material, but is constant. The ratio pJpb is always constant even for
1.8 Electrophoretic Forming of Ceramics (Tsuda and Ishida)
5
different columns, where suffixes a and b refer to different solutes. Therefore, we cannot store the solute in the column just by using a different column bed under a constant flow rate of the mobile phase, in which the column is packed with column support A and B at the upper and lower portion of the column [1.33]. From these considerations we can obtain new ideas for the column focusing of a solute. For example, when we remove a portion of the mobile phase from the centre of the column and recycle it to the top, the flow rate at the bottom of the column becomes less than at the top. Therefore, it is possible to store solute in the middle of the column if its electrophoretic mobility is smaller than the pressurized flow at the top of column and larger than the pressurized flow at the bottom.
Part 2 Applications of Electric Fields in Industrial Processes Electric fields are used in accelerating ion beams and controlling liquid crystals. They can be used for industrial processes, such as dust removal or obtaining salts from seawater by ion-exchange membranes. In Part 2, we focus on several new processes in which it is essential to use an electric field. These “electroprocesses” have advantages over processes which do not use an electric field. The electroprocesses are based on charged molecules or neutral molecules with a partial induced charge, charged particles, charged droplets, or droplets with induced charges, or dispersions of large droplets in many small droplets, or in distorted liquid droplets. Each electroprocess has a unique character, and may suggest further ideas for advanced processes.
1.7
Electroosmotic Dewatering (Iwata)
Electroosmosis is applied for the process of dewatering porous materials, such as a sludge of colloidal particles. Although mechanical dewatering is the conventional method, it is impeded by the hydrodynamic resistance of the sludge. In electroosmotic dewatering, there is no accumulation of pressure resistance during the process, because the phenomenon is a molecular one. In Chapter 7, the principal theory of electroosmotic dewatering for both ideal capillary and compressive porous media are presented, followed by model experiments 11.341. Finally, industrial applications are described. The commercial equipment for sludge treatment based on this principle is characterized by its high performance [1.35]. Electric dewatering is deemed superior to conventional dewatering processes, such as pressure-driven filtering.
1.8
Electrophoretic Forming of Ceramics (Tsuda and Ishida)
In the ceramic industry, pressure forming and slip casting by capillary attraction are generally used as production processes. In Chapter 8, electrophoretic forming is introduced as an alternative method. The brief history of this method is reviewed from the viewpoint of production conditions and raw materials. A new continuous forming method is described 11.361. When natural clay and nonplastic fine materials are mixed with water, a very viscous solution (slip) can be obtained. On application of a voltage,
6
1
Introduction and Summary
the slip is electrophoretically gathered on the surface of an electrode as a thin layer, and at the same time the thin layer is dewatered electroosmotically. By rotating the electrode, a continuous thin film is formed and subsequently fired. This unique process for obtaining a continuous fine ceramic sheet offers maximum energy saving. Physical phenomena encountered during the process are also discussed.
1.9
Control of Viscosity by Electric Fields (Ishino, Saito, Goshima, and Takano)
Electrorheological fluid (ERF) consisting of dielectric powders and insulating oil, shows a large viscosity change when a high voltage is applied [1.37]. ERF has been considered for the application to car production processes [1.38], [1.39]; a number of patent applications reflect this interest. Ishino and colleagues have also publically announced a product which is to be made available commercially. The dispersed dielectric particles line up when a voltage is applied. The selection of particles, the dependence and the rapid response of viscosity to applied voltage, and a possible geometrical design for practical application are discussed.
1.10
Applications of Electric Fields to Solvent Extraction (Yamaguchi)
Solvent extraction technologies in the hydrometallurgy, chemical, and oil industries are vcry important. The extraction processes dealing with liquid drops can be enhanced by producing a large interfacial area of drops for diffusion and a higher degree of turbulence outside the drops for eddy diffusion. The application of an electric field meets these requirements [1.40], and this technique promises become an extremely efficient industrial extraction process. Yamaguchi describes the theoretical treatment of drops in an electric field with photographs and figures which show clearly what is going on [1.41], [1.42]. Several model towers for solvent extraction processes also are discussed.
1.11
Applications of Electric fields to the Resolution of Water-in-Oil Emulsions (Yamaguchi)
This chapter describes the recovery of water from emulsions of water-in-oil or oil-inwater, specially applicable to demulsification processes in the oil industry. The electrical treatment used for these processes is highly successful and operates at lower cost compared with other demulsification processes, and with increased adaptability [1.43]. There are a number of ways of forming oil emulsions in oil production and refining. The application of electrostatic field in the demulsification process increases the rate of collision of water drops, and makes them rapidly coalesce, aiding the phase separation. The method may be used for solvent extraction to accomplish fast recovery.
I.I 4 Orientation of Organic Molecules in Electric Fields: Organized Photochemistry
7
Part 3 Applications of Electric Fields for Concentration, Immunoassay, and Molecular Orientation In chemical unit processes, electric fields can play unique roles. For example, in concentration processes we can use the transfer of molecules directed to an electrode. This movement can be utilized to create new concentration methods. Particles dispersed in the solution can be made to form aggregations. Molecular orientation results from applied electric fields or spontaneous electrostatic fields. These phenomena, resulting from electric fields, are not difficult to visualize. The chemical processes are reviewed and essential experimental conditions are given.
1.U
Dynamic Electroconcentration Processes in Analytical Chemistry (Tsuda)
Analytical chemistry generally concerns the determination of an unknown sample qualitatively or quantitatively. When the sample is very dilute, it may be necessary to concentrate it. The use of electric fields has unique results. We can use the electrophoretic mobility of a solute, the oxidation-reduction potential of a solute, and electrodeposition. They are combined with pressurized flow, electroosmotic flow generated by applied electric field, ion-exchange membranes, silver powders for the bed and chemical reactions, etc. These combinations produce high selectivity and analytical enhancement [1.44], [1.45]. The sample can be of small or large volume. Eight different concentration methods with applied electric fields are described.
1.13
Electric Field Enhancement of Immunoassays (Tamiya)
Often in immunoassays we encounter an antigen-antibody agglutinating reaction governing the rate of the whole process. The application of an electric field is effective for the acceleration of this reaction. When an alternating current or pulsed electric field is applied to a solution containing particles modified with antigens, the dispersed particles begin to form linear linkages, like that of a necklace. This phenomenon promotes the agglutination reaction, because the frequency of the contact between antigen and antibody is increased [1.46]. We can see the same phenomenon in the formation of linked carbon powders in oil (Chap. 9). In Chapter 13, examples of model experiments are rather limited. However, the great possibilities of applied electric fields are presented. The method can be also applied to micro-volume samples.
1.14
Orientation of Organic Molecules in Electric Fields: Organized Photochemistry (Takagi)
An electric field induces molecules to arrange themselves. If the field is confined to a micro-cavity, it can produce stereospecific arrangements. The introduction of photochemical methods gives a unique reaction field for specific synthesis.
8
1
Introduction and Summary
The author describes two electrostatic fields: applied and spontaneous. Under a strong applied field, ionic guest molecules are arranged in parallel. Spontaneous fields are generated in micelles, reversed micelles, vesicles, mono- and multilayered membranes, silica surfaces, clay interfaces, etc. [1.47]. The author gives many examples and offers mechanisms for photochemical reactions under these natural electrostatic fields. Throughout this book, electric fields play a unique role in separations, industrial processes, and molecular processes. Sometimes the introduction of an electric field is quite troublesome, but understanding how to control these fields properly, opens up new areas in which this technology promises great advances in the future. Acknowledgements I greatly thank Mr. Gramham van Zwoll, Mr. D . Boe Hamillton, Jr., HirofumiTanaka, Masakuni Nakashima, Shinya Kitagawa, Chizuko Tsuda and my co-workers for their help, and Mrs. Karin Sora and Dr. Christina Dyllick of VCH for their assistance.
1.15
References
S . Hjcrtcn, Chromatogr. Rev., 9, 122 (1967). R. J. Hunter, Zeta potential in colloids science, Academic Press, New York, 1981. J. C. Giddings, Dynamic of Chromatography, Dekker, New York, 1965. E. Heftman fed.), Clzrornatography,3rd ed., Van Norstand, New York, 1975, Chaps 2 and 10. [lS] T. Tsuda, Anal. Chem. 9, 521-523 (1987). [1.6] T. Tsuda, LC-GC Intl. 5 (9), 26-36 (1992). [1.7] G. Berraz, Anales Asoc. Quim. Arg., 31, 96 (1943). [1.8] T. R. Sato, W. P. Norris, H. H. Strain, Anal. Chern., 27, 521-525 (1955). 11.91 G. H. Evans, H. H. Strain, Anal. Chem. 28, 1560-1563 (1996). [1.10] D. L. Mould, R. L. M. Synge, Biochem. J . , 58, 571-585 (1954). [1.11] S. S. Sandhu, P. S. Thind, J. Indian Chem. Soc., 55, 1002-1007 (1978). [1.12] A. K. Misra, D. K. Misrd, V. K. Maheshwari, J. Liq. Chrornatogr., 14, 1469-1481 (1991). [1.13] S. D. Sharma, S . Misra, J. Planer Chromatogr., 3, 399-401 (1989). [1.14] R. Consden, A. H. Gordon, A. J . P. Martin, Biochem. J., 40, 33 (1946). [1.15] W. B. Gratzer, G. H. Beaven, J. Chrornatogr., 5 , 191 (1960). [1.16] A. Yaron, H. A. Sober, Anal. Biochern., 12, 173 (1965). [1.17] N. Haber, Proc. Nad. Acad. Sci. USA, 79,272-276 (1982). [1.18] Chem. Eng. News, April 22, 36 (1991). [1.19] H. H. Strain, Anal. Chem., 30, 228-231 (1958). [1.20] S. Otsuka, L. Listowsky, Anal. Biochern., 102, 419-422 (1980). [1.21] T. Tsuda, Anal. Chem., 60, 1677-1680 (1988). [1.22] P. H. O’Farrell, Science, 227, 1586-1589 (1985). [1.23] T. Nagaoka, M. Fujimoto, Y. Uchida, K. Ogura, J. Electroanal. Chem., 336,45-55 (1992). [1.24] T. Fujinaga,T. Nagai, S. Okazaki, C. Takagi, Nippon Kugaku Zasshi, 84,941-942 (1963). [1.25] R. Antrim, R. A. Schemer, A . M. Yacynych, J. Chrornatogr., Anal. Chirnica Acta, 164, 283-286 (1984). [1.26] H. Ge, P. R. Teasdale, G. G. Wallace, J . Chrornatogr., 544, 305-316 (1991). [1.27] D . H. Shain et al., Anal. Biochern., 200, 47-51 (1992). [1.28] L. Saso, T3. Silverstrini, C. Y. Cheng, Anal. Riochern., 212, 315-369 (1903). [1.29] T. Tsuda, Y. Muramatsu, J . Chrornatogr., 515, 645-652 (1990). [1.30] Y. Kabasawa, J. Cliem. Soc. Jpn, Chem. Ind. Chem., 1355-1359 (1990). [1.1] [1.2] [1.3] 11.41
I .I5 References
9
[1.31] E. R. Herheij, U. R. Tjaden, W. A. Niessen, J. van der Greef, J . Chromatogr., 554, 339-349 (1991). [1.32] C. D. Scott, Sep. Sci. Technol., 21, 905-917 (1986). [1.33] See also comments in the section of column focusing (Chapter 6). [1.34] M. Iwata, H. Imagi, T. Murasse, H. Yoshida, J . Chem. Eng. Japan, 24, 4.5-50 (1991). [1.35] Shinko Pantec Co, Ltd., Catalog No. 8902-1. [1.36] H. Ishida, J . Mineralogicul Soc. Japan, 22 (2), 79 (1993). [1.37] W. M. Winslow, US Patent 2417850 (1947). [1.38] R. Pool, Science, 247, March. (1990). [1.39] T. Ushima, K. Takano, T. Noguchi, Annual meeting of SOC.Automobile Eng., Technical Paper No. 880073 (1933). [1.40] J. D. Thronton, J. E. Porter, Symp. Electrochem. Eng., 3,3-38 (1971). [1.41] T. Takamatsu, M. Yamaguchi, T. Katayama, J. Chem. Eng. Japan, 15 (1982). [1.42] M. Yamaguchi, H. Sugaya, T. Katayama, J . Chem. Eng. Japan, 21, 179-183 (1988). [1.43] M. Yamaguchi, A. Kobayashi, T. Katayama, Kugaku Koguku Ronbunshi, 11, 729-734 (1985). [1.44] Yu. A. Zolotov, N. M. Kuz’min, “Preconcentration of trace elecments”, Comprehensive Analytical Chemistry X X V Elservier, Amsterdam, 1990. [1.45] A. Hori et al.,Anal. Chem., 65, 2882-2886 (1993). [ 1.461 E. Tamiya, I. Kaube, “Electric Pulse Accelerated Immunoassay” in Electrochemical Sensors in Immunological Analysis T. T. Ngo (ed.) Plenum Press, 1987. [1.47] K. Takagi, K. Aoshima, Y. Sawaki, J . Chem. Soc., Perkin Trans, II, 1771 (1986).
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Part 1 Electrochromatography
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2
Electrochromatography in Analytical Chemistry Takao Tsuda, Shinya Kitagawa
2.1
Theory of Band Broadening
Chromatography is one of the main tools for separating mixtures, and it is widely accepted as the most important. There are many types of chromatography (partition, adsorption, affinity, size-exclusion, ion-exchange, etc.) in each of which equilibria between the mobile phase and the stationary phase are attained. In this book, we introduce an additional method, the application of an electric field to a column. This leads onto using the electrophoretic mobility of a solute and the influence of the electric field on the stationary phase. We call this method electrochromatograph y. Electrochromatography introduces two new factors, electrophoretic mobility and electroosmotic flow, that do not exist in ordinary liquid chromatography; the band broadening of a solute in electrochromatography is different from that in ordinary chromatography [2.1]-[2.8]. Sample movement in the column under applied voltage is shown schematically in Figure 2.1. The direction of the flow velocity depends on the sign of the surface charge of the packing material, and its magnitude depends on the amount of surface charge, pH, the concentration of electrolyte in the effluent, the composition of the effluent, etc. [2.91-[2.14].
Figure 2.1. Movement of solute in a packed column under high voltage applied along the column.
14
2
2.2
Apparent Mean Linear Flow Velocity and Elution Time
Electrochromatograplzy in Analytical Chemistry
The apparent mean linear flow velocity of a solute, vaPp, in electrochromatography under pressurized flow is: vapp
= Vprcs
+ vmub + V o m
(1)
where vprc5, vmCthr and v,,,, are mean linear flow velocities due to pressurized flow, mobility of solute (electrophoretic velocity), electroosmotic flow (electroosmotic velocity), respectively [2.4]. Elution time, te, is: t,
=
L [R vdpP]-'
(2)
where t and R are column length and the retardation factor by (1 + k')-', where k' is the capacity factor. Vmnh
v,,
=
L ( R k)-' - Vprea - vosm
is estimated from a nonretained peak ( R vosm
=
(3) =
1, v,,,,h
(L/G,) - vprcs
=
0) as: (4)
where to is the elution time of the nonretained peak.
2.3
Processes in Band Broadening
In general chromatography, four processcs contribute to the band broadening of component zones as they migrate through the column; axial molecular diffusion; resistance to mass transfer in the stationary phase; resistance to mass transfer in the mobile phase; and eddy diffusion. The plate height is given by Giddings [2.15] as: H
=
Blv
+ C,V+ C,,V + (Z/A + l/C,V)~'
(5)
where v is the mean linear velocity and A , B, C,, and C, are the coefficients for eddy diffusion, axial molecular diffusion, resistance to mass transfer in the stationary phase, and resistance to mass transfer in the mobile phase. Only the Cmvvalue in Equation (3) is affected by the shape of the velocity profile in the column. Considering flow profiles in electrochromatography, the flow of vpreqoperates with a Poiseuille flow profile, and we suppose that the flow profile of v,& is a plug flow profile. The flow profile of v,,, is also a pseudo-plug flow. (These flow profiles are discussed below.) Therefore, the velocity inequality in the radial direction is due only to laminar flow, vpres;the flow pattern in electrochromatography is assumed to be mixed Poiseuillelplug flow (see Fig. 2.2). If the solute and eluent diffusion coefficients are assumed zero, the flow profile at time t , will resemble F ( t , , , ) in the absence of an applied voltage. As every point on F (tl,,)receives an additional contribution from vmOh, the actual electrochromatographic flow profile is F (t,,2) at time tl. The process of mass transfer in the mobile phase is a hybrid of lateral diffusion and velocity inequality of the flow pattern. The application of a random walk model for
-i
v(mob)
15
Processes in Band Broadening
2.3
Figure 2.2. Addition of a flow velocity due to electrophoretic mobility to a Poiseuille flow profile.
+
estimation of band broadening in general chromatography has been well by Giddings [2.15]. With a random walk model, the contribution of mass transfer in the mobile phase, the third term in Equation (9,is calculated as follows. A molecule must diffuse a distance wad, to reach one extreme velocity from another. The multiplying term w, takes values ranging from well below to well above unity [2.15]; dpis the particle diameter. Giddings [2.15] considered five or six different w values for various eluent nonequilibrium conditions, not all of which are equally important. For simplification, we will not discuss them individually. The average time t, needed to diffuse the distance at one of the velocity extremes is:
(6)
t, = w;d,2/2Dm
where D, is the diffusion coefficient in the mobile phase. The length of step 1is the distance gained or lost with respect to the mean (peak center) because of temporary residence at one of the velocity extremes.
I
= W/3Vpres
4
(7)
where ws = Avpres/vpres and A v is the difference between the extreme and the mean. The number of random steps while a solute passes through the total pass length (column length L ) is: n
=
L',f
+ V,,JL
(vpres
(8)
On combining these expressions with 3 and H tion of the zone length, we obtain H3:
f&
= vprw w:
where Cm= (w, wp d:)' H
=
wj (d:
vpreq)
(2DJ'.
(2 D
m vpres)-'
=
d I L , where
=
Cm
CT
is the standard devia-
~ p r e ? Vap;'
(9)
Therefore, H in electrochromatography is:
(Blvapp)+ C, vapP+ H 3 + (1IA
+ 1/H3)-'
(10)
Equation (10) is valid in the region vapp> 0. H as given by Equation (10) is different from that in general chromatography, given by Equation ( 5 ) , in the following respects. First, the flow velocity of a solute is (vpres v,,, vmoh).Second, H3 in electrochromatography is completely different from H3 in general chromatography, given by C, vpres. When vapp > vpres, flow velocities due to electroosmosis and electrophoretic mobility
+
+
16
2
Electrochrornutogrccyhy in Analyticul Chemistry
0.01
-3 u
(
Figure 2.3. Relation between H a n d “am.
reduce the values of the first and third terms of Equation (10) compared with general chromatography. When vdPp< vprc,,owing to the negative value of v , , ~ the solute will remain in the column longer than in general chromatography. Hence the second term in Equation (10) becomes smaller and the other terms larger compared with the corresponding terms when ( v , , ~+ v , , ~ ) = 0. Variations of Equation (10) with different vpresand vapp are shown in Figure 2.3 [2.16]. Curve 1 is for (v,,,, + v,,,) = 0; curves 2 and 3 are for vpres= 0.1 and 0.2 cmh, respectively. As the application of a voltage along the column generates v,,,b and vOsm, curves 2 and 3 correspond to the H-v relations in electrochromatography. The values of B, C,,C,, andA are as 2.0 x lo4, 5.0 x lo-?, 5.0 x lo”, and 7 x lo-’, respectively. These values for curve 1 are approximated from the H-v relation given for conventional liquid chromatography by Reese and Scott in the following conditions: Partisil as packing material; particle diameter 10 km; ethyl acetateln-hexane (5 :95) as effluent; benzyl acetate sample [2.17]. In the region vdPP> vpreq,the value of H (height equivalent to a theoretical plate) is always lower than for conventional chromatography, (curve 1).The contributions of v,,, and vmOhto band broadening are less than vprcSper unit time, mostly because of their flow profiles. However, in the region vaPp< vpres,H increases because retarded flow velocity prolongs the stay of solute in the column, and the effects of both diffusion and pressurized flow cause band broadening. The differcnce between curves 2 and 3 is due to the different sum of v,,,,, and v,,,,. From Figure 2.3, H depends on vapp,like vpresin ordinary chromatography, but H in electrochromatography is generally less than the H value at the same value of vpresin ordinary chromatography.
2.4 Electrochromatography Zones
17
-v(mo b)=v(pres)
T 0
-v(mob)=2v(pres)
7 0
z -v(mob)=3v(pres)
0
2.4
Figure 2.4. Assumed flow profile for pressurized flow-driven electrochromatography at time tl (unit period after injection) with zero diffusion coefficient and zero electroosmotic flow [2.15]. Z” indicates direction of flow.
Electrochromatography Zones
A charged solute is transported under an applied voltage. The movement of a solute zone in a column is dependent on the value of vaPp.The assumed flow profile and its position in the column is shown in Figure 2.4..These assumed flow profiles are obtained under the assumption that: a) the diffusion coefficient of solute in the medium and the flow velocity due to electroosmosis are assumed zero; b) the profile of injected solute is a plug; c) the profile of a solute due to electrophoretic mobility is also a plug [2.5]. If vpresIv,,,b, the solute is partially or completely trapped in the column. Therefore we can continuously introduce the dilute sample solution which contains a charged solute, and concentrate the solute at the column head, see also Section 1 of Chapter 6. The greatest advantage under an applied voltage is that the flow velocity in the very vicinity of the inner wall has a finite speed. This will reduce the band broadening of a zone in the mobile phase, which is mainly due to the different values of the extreme flow velocities at the edge and central region.
18
2
2.5
Profiles of Pressurized Flow, Electroosmotic Flow, and Zones of Ionic Solutes
Electrochromatography in Analytical Chemistry
It is well known that the flow profile of electroosmosis is very different from that of pressurized flow. This leads to a very narrow zone in capillary zone electrophoresis. The charged solute is transported owing to its electrophoretic mobility under an applied voltage. If the electric field strength is the same at any local position in a column cross-section, the flow velocity due to electrophoretic mobility under an applied field may be the same at every location in the column. Thus, in electrochromatography these additional flow velocities can decrease the zone broadening compared with the zone broadening in ordinary liquid chromatography. For a discussion of zone broadening, it is essential to know the zone profile of these flows.
2.5.1
Flow Profiles of Pressurized Flow
Zone Front Profile. For the analysis of zone broadening under pressurized flow, Taylor [2.18] used a method in which a colored solution was introduced continuously into a narrow capillary tube at several different velocities. The concentration of the colored solute at each axial point of the tube was measured after the zone had passed the central part of the capillary tube. A glass tube, 0.5 cm internal diameter and a length of 152 cm was used, and 1% potassium permanganate aqueous solution was used as the colored solution. The diffusion coefficient for potassium permanganate in water is 7 x 10“ cm2/s. The colored solution was poured continuously into the glass tube at a different travel period, at linear flow velocity u,,,,. The mean linear flow velocity is given by 0.5 vmax,where v,, is the flow velocity in pressurized flow. The travel periods of the colored solution are 4, 12, 240, and 11220 s, with mean linear flow velocities 8.25,6.67,0.263, and 0.00283 cm/s, respectively. The observed values of C/Cn are plotted against Z”, [Z” = (0.5 vnlaxt)-’] (Fig. 2.5). Z”, vmax,t, C , and C o are 2”-axis (flow direction), maximum flow velocity at the central line of the open tube due to Poiseuille flow, travel period (s), concentration of solute, and its initial concentration, respectively. Figure 2.5 demonstrates the effect of molecular diffusion on the concentration distribution in the transition zone between clear water and solution. In other words,
* rn
t 0
: 11 220 : 240 : 12 :4
0604 02-
0
04
08
16
12
20
2Figure 2.5. Distribution of concentration about point Z ”
=
0.5 vprcs,maxt[2.15].
2.5 Profiles of Pressurized Flow, Electroosmotic Flow,and Zones of Ionic Solutes
19
pressurized flow broadens the sample zone considerably. At high linear velocity of pressurized flow, the zone is broadened almost equally from 26 (original point) to the point v,,, t = 2. The concentration profile corresponds directly to the value of the local flow velocity, v, = v,,, (1-?/R2), where R and r are the radius of the circular tube and the distance from its central line.
Dispersion of Sample Injected as a Plug Under Pressurized Flow and Molecular Diffusion. When a sample is injected as a plug into a laminar flow, the concentration of the sample Ci along the z-axis and r can be calculated by solving Equation (ll), based on diffusionkonvection flow: aC/dt
= - v,,,
+
(1 - r2/R2)( d ~ / a z ) (DJr) d ( r X / a r ) / d r
(11)
The followin! parameters are introduced for the calculation: K = v,, R2/LD,; Co = 1; z’ = I.’,,,t L- , where L is the length of the plug; the contour line of concentration A Ci is given by ACi = Co 2” (n is an integer). The contour lines a-p in Figure 2.6 correspond to 1, %,I 1/2, t/4, %, %, 5/16, 3/16, N6, 5/32, 3/32, 1/32, 7/64, 5/64, 3/64, and v64 times c,, respectively. The calculated result is shown in Figure 2.6 for K = 30 [2.19], [2.201. This K value means that, for example, v,, = 0.5 cm/s, L = 0.5 cm, D, = 1 x 10“ cm /s and R = 55 pm. These values are not appropriate for capillary liquid chromatography. In capillary liquid chromatography the K value might be 0.25, under the following conditions: v,, = 0.1 cm/s, L = 0.1 cm, D,= 1 X 10“ cm2/s, and R = 5 pm. Therefore, the K value in Figure 2.6 is about 100 times larger compared with capillary liquid chromatography [2.16]. The arrows in Figure 2.6 depict the front position given by v,, t/L. The most concentrated part in the dispersion of the zone is located at the half-value of z’ for each zone. The band broadening of Figure 2.6 clearly shows the contribution of convection flow (pressurized flow) and mutual molecular diffusion. In Figure 2.6, B travels the distance L from A . The back of the B zone is broadened to the region of -Z”, owing to molecular diffusion, and its front is also more advanced than 2L, since, in this case, the solute concentration gradient is high. From B to F in Figure 2.6, the contribution of v,, bends the zone convexly, so that the central region of the zone is forced to advance more, compared with the edge region (near the inner wall). As vmg, is too fast in Figure 2.6, molecular diffusion cannot attain the zone profile of a Gaussian distribution. However the position of 2” of the central high concentration of the zone is located half-way along the zone front, owing to mutual molecular diffusion during the zone migration. If we look at the leading edge of the band, we might expect that the zone front profile for continuous introduction of samples to be similar, i. e., the front zone profile of pressurized flow is due to convection flow and diffusion, and differs greatly from the flat profile of plug flow. In chromatography, we are always very careful to reduce the contribution of convection. We try to use very fine packing materials to reduce the radius of the channels between them, in which diffusion can reduce band broadening.
2.5.2
Flow Profiles of Electroosmosis in an Open Tube
The electroosmotic flow profile has been discussed for electroosmotically driven opentubular liquid chromatography [2.8], [2.21], [2.22], capillary zone electrophoresis (CZE) [2.24]-[2.26], and electroosmotically driven electrochromatography (EO-EC) [2.26].The electroosmotic flow profile differs from the parabolic laminar velocity pro-
20
2 Electrochromatography in Analytical Chemistry
I
I
I
I
I
I
I
I
32
28
24
20
16
17.
8
4
z ' - a x i s ,
0
L
Figure 2.6. Zone broadening under pressurized flow [2.16]. Arrow indicates the front position of v,,,tlL.
of pressurized flow. The former flow profile is much flatter, so that very narrow peaks are obtained in electroosmotically driven chromatography and CZE. In early experiments, Pretorius et al. [2.21] and Tsuda et al. [2.8] obtained 10- und 30-fold less band broadening in electroosmotically driven liquid chromatography than expected with pressurized flow. Pretorius et al. [2.21] suggested that the profile is flat except in the region of the diffuse double-layer near the column inner wall, where the flow has a quadratic velocity profile owing to friction between the viscous liquid and the wall. The diffuse double-layer of fused silica capillary (50-100 pm) is supposed to be 30-300 nm [2.12], [2.27]. Jorgenson and Lucacs [2.24] assumed that the zone broadening in CZE was generated only by axial molecular diffusion of solute and medium. Tsuda et al. [2.8] found it difficult to explain the experimental results purely by axial molecular diffusion, and proposed that the flow profile might be a combination of plug and Poiseuille flow. Rice et al. [2.29] and Guiochon et al. [2.27], [2.28] also assumed that electroosmotic flow is a combination of plug and Poiseuille flow, and proposed an equation to calculate the contribution of electroosmotic flow profiles by using experimental data. Guiochon et al. [2.27], [2.28] proposed equations describing the electroosmotic flow profile as a combination of plug and Poiseuille flow under an applied voltage in a capillary tube. The average flow profile u can be computed as:
2.5 Profiles of Pressurized Flow, Electroosmotic Flow, and Zones of Ionic Solutes
u (vOs,J1 = 1- (2/3) p - (1/6)e2
21 (12)
Guiochon et al. use an adjustable parameter e which can be related to the thickness of the double-layer relative to the column radius. The boundary value p = 1 corresponds to the usual Poiseuille flow (parabolic flow) and e = 0 to plug flow. One may estimate e as: p = (3/xr) 11
+ 1/(4xr) + 1/[4 (xr)’] + 19/[64(~r)~]}
(13)
where r and x are the column radius (channel radius) and the reciprocal of the diffuse double-layer. Their result (dashed curve) is shown in Figure 2.7, with xr = 10 and e = 0.3083. One typically uses as fused silica capillary of 50-100 pm inner diameter and 10 mM buffer solution. Under these conditions, the diffuse double-layer is assumed to be 30-300 nm; the value of m is 83-830 for a 50 pm inner diameter capillary column. Zone Front Profiles in Rectangular Capillary lhbes. n u d a et al. [2.30] used a method similar to Taylor’s experiment [2.18], in which a fluorescent solution (Rhodamine 590 in methanol) is continuously introduced into a capillary, with the front profile being observed through a microscope charged-coupled device (CCD) video recording system. A rectangular capillary tube, 50 x 1000 pm, which has been used for CZE, is used because the flat sides cause fewer distortions in the observed zone front [2.30], [2.31]. Since Rhodamine may be neutral in methanol, the electroosmotic flow profile obtained by using a Rhodamine 590-methanol solution will give a general answer. The arrangement of the experiment is shown in Figure 2.8. The procedure for the introduction of the sample solution is as follows. One end of the empty rectangular capillary (height 50 pm,width 1 mm, length 164 mm, these dimensions defined as the Y-, X- and Z-axes), is inserted into reservoir A, and the medium in the reservoir is introduced via capillary action. Then the other end is inserted into another reservoir B. Subsequently, the end of the rectangular capillary is inserted into the outlet of the syringe containing sample solution (reservoir A) with gentle smooth motion of stage 2. This procedure is very important for producing a sharp zone front. A voltage is then
.8
.6
x/r
.4
c
X
2
z
Figure 2.7. Velocity profile versus relative distance from column axis. Solid curve, electroosmotic flow; dashed curve, partially parabolic flow; xr = 10 and e = 0.3083; dotted curve, Poiseuille parabolic profile (@ = 1) [2.25].
22
2 Electrochromatography in Analytical Chemistry
t
///
U
Figure 2.8. Schematic diagram of the instrumentationfor visual observation of zone front [2.31].
applied. Immediately after the application of the voltage, the colored solution travels into the rectangular capillary and its zone front is followed under the microscope system, by continuous or stepwise operation of stage 1. The sample solution is continuously introduced into the rectangular capillary in a similar fashion to frontal analysis in chromatography [2.32]. With the dye zone front illuminated, its fluorescent image was recorded. The zone front was observed through the 1000 pm wide section of capillary (the XZ surface of the capillary). The current is low (ca. 0.12 pAper 97 Vkm) and very stable during the run; there is only a small difference in conductivity between pure methanol and lo4 M Rhodamine-methanol solution. Thc use of a rectangular capillary for the observation of flow profiles in electroosmosis has several advantages. The flow velocity in electroosmosis depends on the amount of charge on the wall [2.11], [2.12]. The medium in a rectangular capillary is surrounded by two sets of parallel plates, two X Z plates and two YZ plates. At the center of the X-axis of the rectangular capillary the medium is affected mainly by the two parallel XZ plates. The medium near the YZ plates is affected by both the two 1-mm parallel plates ( X Z ) and a 50+m plate (YZ). Therefore, the geometric difference between the edge and center of the 1-mm plate shows the effect of the wall on the flow profile. The difference in flow at the edge of the 1-mm width (X-axis) compared with the center is caused by the 50-pm plates (YZ), because the effect of charges on the 1mm plate and its diffuse double-layer influence the flow profile equally at all points in the X Z plate. Photographs and computer images of the zone front taken from video tapes are shown in Figures 2.9 and 2.10. The distance between each line on the X-axis in Figure 2.10 corresponds to 59 km. The time interval between the pictures is 1 s. The advance of the zone between two pictures can be measured from photographs by matching the positions of the stationary marks on the rectangular capillary. The zone front shown in Figure 2.9 is flat at the center with small bends at both ends of 1000 pm axis, near the 50+m plates. The progress of the zone front recorded on video tapes was analyzed by computer software. The resulting images of the advancing zone front are shown in Figure 2.10. The four images are successive 4-s intervals, with the 1-mm axis divided into seventeen sections. Both edges in the Z-direction correspond to zone fronts.
2.5 Profiles of Pressurized Flow, Electroosmotic Flow,and Zones of Ionic Solutes
23
Figure 2.9. Photographs of the zone front in elcctroosniotic flow 12.311. Colored sample solution: 0.1 mM Rhodaniine 590 in methanol; Applied voltage and current, 1.59 kV and 0.12 PA. The period between photographs is 1 s.
The advance of the zone front along the Z-axis is summarized numerically in Table 2.1. The average numbers at each X-section were similar. The flow profile is pluglike in the center of the capillary from section 1 to 15. Unfortunately, the numerical values of the progress at sections 0 and 16 were not stable enough to estimate. To examine the profile in the vicinity of the 50-pm plate, we focused on the movement of the zone front at high magnification. One of the photographs obtained is shown in Figure 2.11. The zone front near the wall, near sections 0 and 16 of the X-axis, has traveled further than the zone front in the central portion of the capillary. It was concluded that the advances of the central portion are almost equal to the results of Figures 2.9, 2.10 and Table 2.1. The zone front at the edges of the 1000-pm axis in Figures 2.9 and 2.10 is ahead of the central portion. This phenomenon was observed in every experiment which we recorded.
24
2 Electrochromatography in Analytical Chemistry
A
P
C
D
Figure 2.10. Computed images of the zone front on the Z-axis over a l-s span [2.31].
Zone Front Profile in Fused Silica Capillary lhbings. The flow profile of electroosmosis in 50-75 pm fused silica capillary tubings were also recorded. Photographs of the zone front are shown in Figure 2.12. The experimental conditions were as follows: fused silica capillary, 65 pm inner diameter, 16 cm long; applied voltage 1 kV (62.5 V1 cm); ca. 17 nA. The image obtained is reflected at the region focused by the microscope; the focal plane is ca. 2.7 mm long (Z) and less than the depth of 5 pm (Y). From Figure 2.12 the total advance is 420 pm per 2.69 s. We can see the flow profile in a round capillary tubing in Figure 2.12, and it is concluded that the frontal zone profile is similar to those of rectangular shape shown in Figure 2.10, i. e., the central portion of the zone is retarded compared with its edge, and generally the profile is very flat. From these experimental results, we proposed that the zone front profile in a narrow straight tubing of 100-50 pm internal diameter would appear as shown in Figure 2.13. The central portion of the zone front is retarded compared with the edge, as with a rectangular capillary. We therefore suggest the following: the edge portion of the zone front advances at the very start, immediately after application of the voltage. From then on the zone will maintain equal velocity at every point along the Z-axis.
2.5 Profiles of Pressurized Flow, Electroosmotic Flow, and Zones of Ionic Solutes
25
Table 2.1. Advance of zone front along the X-axis over A 1-s period
Section (y-axis)
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Experiment
Average value
1
2
3
4
7.8 7.2 8.0 8.1 8.0 7.6 7.0 7.7 8.1 7.5 8.0 7.4 7.8 6.8 6.0
8.3 7.6 8.3 6.9 7.8 8.3 8.0 8.2 8.0 8.1 8.4 7.7 7.6 7.5 8.0 8.6 8.0
8.0 7.6 8.4 8.0 6.7 7.4 7.8 6.5 7.4 8.0 7.6 8.5 8.2 8.3 7.6
7.5 7.0 7.2 7.8 7.1 8.5 7.8 7.4 8.5 7.6 7.5 7.7 7.0 7.5 6.9 7.7
7.6 7.58 7.78 7.75 7.88 7.7 7.63 7.68 7.8 7.85 7.75 7.63 7.75 7.5 7.48
Mean of average value and its standard deviation are 7.69 and 0.12, respectively, unit of X-axis: 57.8 wrn
To our knowledge, this is the first time that the zone front pattern in electroosmosis has been directly observed. The central part is retarded compared with the edges. This finding is unique. The flow profile observed is a reverse parabolic and very different from that suggested by Pretorius et al. [2.21]. This unique result could be explained in two ways. First, when the osmotic flow exits from the capillary outlet into the reservoir, some pumping power is required. Since there is not enough potential gradient to accept the flow coming from the column, pumping power due to the electroosmotic flow is necessary. This causes the zone front in the central portion of the capillary to be forced back, owing to the hydrostatic backpressure of the reservoir. A second explanation might be as follows. The distribution of positive and negative ions is such that positive ions are extremely abundant in the region of the double-layer
26
2 Electrochrornatography in Analytical Chemistry
Figure 2.12A. Zone front profiles in a fused silica capillary tube [2.14]. Profiles taken from 40.6 to 41.8 sec.
A
6
C
D
Figure 2.l2B. Sketches of zone front profiles in round capillary tubing [2.14]. Profiles A-D taken at 39.22, 40.22, 41.71, 41.91 s.
up to 3 / x (x is the reciprocal of the length of the diffuse double-layer), with the abundance of positive ions decreasing toward the center of the capillary tubing. In other words, negative ions, molecules which have a partial negative charge, or partially polarized molecules, are concentrated at or near the center of the capillary, and their abundance in the region of the double-layer is extremely small or zero. Charge balance must be maintained over the whole XYZ space. Some of the molecules in the central part have a flow direction opposite to that of the wall regions, causing the apparent flow velocity in the central part to be reduced somewhat compared with that at the edges. Therefore, the central region will lay behind the edge.
2.5 Profiles of Pressurized Flow,Electroosmotic Flow,and Zones of Ionic Solutes
27
1 Figure 2.W. Proposed zone front in open-tube capillary [2.14].
Flow Profiles for Charged Molecules
2.5.3
Charged solutes are forced to move by their electrophoretic mobility under an applied electric field. If the electric field is uniform at every local cross-section of the column, and is not affected by the charges on the inner wall, the flow velocity of each solute should be equal. Thus, they form a sharp zone front, with a plug flow profile. As the inner wall of the flow channel may have a charge on its surface and in the vicinity of its wall, a diffuse double-layer will be formed; the charged solute near the wall will be affected by the charged inner surfaces and a radial potential gradient generated.
1mm
1mm
0
1
2
3
4
5 I
s
e
c.
1
Figure 2.14. Photographs of zone fronts of a positively charged solute under electroosmosis [2.17].
28
2 Electrochromatogruphy in Anulyticul Chemistry
Figure 2.15. Photograph of the zone front of a negatively charged
solute [2.31]. We observed the zone front flow profile by using the same apparatus, shown in Figure 2.8. Colored charged solutes Rhodamine 590 and disodium fluorescein were used. In 5 mM phosphate buffer (pH 6.8) with 5 % ethylene glycol, Rhodamine 590 has one positive charge and disodium fluorescein two negative charges, although the former is neutral in methanol. The zone front of these two ionic solutes in the buffer is shown in Figures 2.14 and 2.15. From Figure 2.14, it is clear that the advances at each time interval do not affect the pattern of the zone front. Almost similar zone fronts of positively charged solutes under electroosmotic flow are observed. The charged color molecules migrate with almost equal speed at every point on the X-axis. Therefore, both profiles of electroosmosis and the zone formed by charged solutes are almost plug over a short period, such as a few seconds. If we overlap two front profiles for more precise observation of the advance, we find that the advances in 2 at local points along X are not equal, and we can observe the advances in the neighborhood of both edges, where they are a little larger compared with the central part of X [2.16], [2.30]. A negatively charged ionic solute may experience an electrostatic repulsion from the surface charge on the inner wall in its vicinity, while the positively charged ionic solute is attracted to the wall. The zone fronts of both ionic solutes are different from those of neutral solutes. The difference is particularly pronounced at the ends of the l-mm axis (X)of the rectangular capillary. The zone front at both ends of the X-axis moves forward or back, owing to the positive or negative charge of the solute. Therefore, the zone front profile might be induced by electrostatic forces between ionic solutes and the charge on the surface of the inner wall under a potential gradient. The zone front profile of ionic solutes is not flat [2.30]. The zone fronts of ionic dyes depend on their charges. The front zone profiles in electroosmosis of ionic dyes each have a specific pattern. These findings are not explained by the general equation proposed for electroosmosis. Although the diffuse double-layer and the amount of surface charge on the inner wall are important, they may not be sufficiently strong to affect molecules over distances of several micrometers. The change in the nature of the medium under a potential gradient may be a key factor.
2.6 Pressurized Flow-Driven Electrochromratographyon Microcolumns
2.6
29
Pressurized How-Driven Electrochromatography on Microcolumns
There are two methods of generating a pressurized flow. The first uses a modern LC pump [2.3]-[2.5], [2.7], [2.33], [2.34], and the second uses gravity [2.1], [2.2], [2.35]. A high-flow velocity is easy to control with an LC pump, and an analysis time of 10-30 min can be obtained. Analysis time for the gravity method is much longer (1-10 h), owing to the slow velocity [2.1], [2.2], [2.35]. High efficiency can be obtained at high voltage, providing effective heat dissipation is achieved. The advantage of using microcolumns or capillary columns (50-500 pm inner diameter) is their ability for high heat dissipation and relatively low electric current compared with columns of 2-6 mm inner diameter.
2.6.1
Instrumentation
A schematic diagram of the instrumentation used for electrochromatography with pressurized flow is shown in Figures 2.16A and B (instruments with nonsplit and split injection). In Figure 2.16A, one electrode is placed in front of the injector and the other electrode is placed between the analytical column and a resistant column (not shown). To prevent the evolution of bubbles, 15 cm of 50 pm fused silica capillary tubing was connected in-line after the electrode and UV detector. A microcolumn (0.5 mm inner diameter, Teflon tubing column body) was used [2.4]. In Figure 2.16B, a pump, an injector (60 nL or 0.5 pL), a home-made column with a frit, a UV detector, and a high-voltage power supply (maximum 50 kV) were used. As a resistance tube, a fused silica capillary (100 pm internal diameter) was used [2.7], [2.34]. The column used had a 50 pm inner diameter and a length of 7.5-18 cm. The polyimido coating of the fused silica capillary, located just after the center frit, was removed with a fine blade for 1 mm of its length for UV detection. A split-sample injection method was used. High voltage was applied between a split and a reservoir in Figure 2.16B. A frit 0.1 mm in length was prepared at the center part of the fused silica capillary (total capillary tube length 12.5 cm, frit positioned 7.5 cm from the inlet) [2.36]. Silica gels modified with octadecylsilane (ODS-silica, particle diameter 7 pm, Capcell 120 SG, Shiseido, or TSKgel IC-Anion-SW, particle diameter 5 pm, Toso) was packed using ethanol or methanol-water as solvent under high pressure. Figures 2.16C and 2.16D show the micro-LC (gradient) system used by Tjaden et al. [2.36]. A pre-injector split was applied, as the generation of reproducible and smooth flow rates appeared possible only at flow rates above 250 pL/min with a conventional pump. The flow was split in the desired ratio by a T-piece fitted to a 100 x 3 mm inner diameter packed column as a dummy column for a restrictor. A 100 pm inner diameter fused silica capillary from the gradient system was passed through the splitting T-piece and a stainless steel tube, and ended 5 mm short of the injector. Samples were injected with an injector fitted with a 0.5 pL loop. The capillary column, 200 mm x 220 pm inner diameter, packed with Nucleosil 100-5 C18 was directly connected to the injector by means of a nut, a Vespel ferrule, and a Teflon linear tube (4 mm x 1/16 in outer diameter X 0.35 inner diameter) to fill up the void space [2.37]. The column effluent was monitored at 260 nm by a UV detector equipped with a home-made capillary flowthrough cell, using 50 pm fused silica with a volume of about 4 nL.
30
2 Electrochromatography in Analytical Chemistry
resistance
Electrical resistance
-
Pump
Figure 2.16A. Electrochromatographic system for a microcolumn t2.101.
3
I filter
UV-detector
injector
reservoir
-
r e s i s t a n t tube -
0
0
A
restrictor
4
Figure 2.16C. Electrochromatographic system with a gradient device.
2.6 Pressurized Flow-Driven Electrochromatography on Microcolumns
1
\
\6 7
31
Figure 2.16D. End-fitting device [2.35]. 1) Capillary column, 220 pm inner diameter; 2) 1/16 inch female nut; 3) Drilledthrough union; 4) PTFE union insert; 5) 50 pm inner diameter fused silica capillary; 6) silcanized quartz-wool; 7) Vespel ferrule.
For electrochromatographic experiments, the injector was grounded in order to allow injection during high-voltage operation, while the high voltage was applied to the end of the packed fused silica column by means of a drilled-through union used as an electrode. It is preferable to have a device to exclude bubbles from the end-frit connector, as shown in Figure 2.16D.
2.6.2
Features and Operational Factors
The instrumentation for electrochromatography should have the following features: 1) Any heat generated should be effectively reduced or dissipated: this is a key factor. If a capillary column with small internal diameter (50-500 pm) is used, most of the heat generated will be effectively dissipated from the outside surface of the column. 2) The detector and pump must be protected from damage that may be caused by the application of a high voltage. For example, a long fused silica capillary is used as an electrical resistance between the pump and the capillary column. If a column of large internal diameter (2-4 mm) is used, design geometry should be such as to ensure effective heat dissipation, or a low-potential gradient should be used. Other alternatives include reducing the current by using an eluent of low conductivity (e. g., an eluent containing an organic buffer and/or a weak acid, or a weak alkali, such as ammonium acetate). 3) An amphoteric electrolyte is the preferred reagent for buffer components. When the pH of the effluent is varied, the sign of the charge of an amphoteric electrolyte is changed, which reduces the variation of effluent pH at or near the electrode. This buffering action stabilizes the electric current in the column. 4) To reduce the analysis time and to increase the contribution of mobility to the separation process, a high applied voltage is a key factor. 5 ) Noise caused by bubbles generated at the electrodes must be minimized, or the bubbles must be eliminated from the system. When bubbles form in the system, the current is often stopped, and the system shuts down. In pressurized flow-driven electrochromatography the problem can be overcome by using pressurized flow. This either carries the bubbles out of the system or because the system is kept under relatively high pressure, it facilitates dissolution of the hydrogen and oxygen generated at the electrodes. In electroosmotically driven electrochromatography (electroosmosis as the sole driving flow) it is very difficult to remove bubbles from the system. In this case, it is common practice to use pressurized flow to exclude them. In electrochromatography, care should be taken to control the gases generated at the electrodes to ensure continuous operation of the system [2.3]-[2.5], [2.7], [2.33], [2.34].
32
2 Electrochromatography in Analytical Chemistry
ov
1 OkV
Figure 2.17A. Chromatograms obtained by pressurized flow-driven electrochromatography at different currents and voltages [2.13]. Solutes uracil and ck-Nrnethyl-4-/%styrylpyridium iodide (CSI), eluted in that order.
-4P-
1 rnin
2.6.3
Chromatographic Behavior in Pressurized Flow-Driven Electrochromatography
Effect of Applied Voltage. Ppical separations obtained from applying a high voltage are shown in Figure 2.17 and Table 2.2 [2.3], [2.4]. The applied voltage was varied from 0 to 15 kV or 0 to -15 kV. The electric current was set at 0.2,4,7,10 and 17 pA at applied voltage 0, +2,5, +5, +7,5, 10, and 15 kV, respectively. A 7.5 cm x 0.5 mm tetrafluoroethylene microcolumn packed with 3 pm ODS-silica was used with methanoY1.5 x N phosphate buffer (pH 6.7) (88:12) as eluent. A high voltage was applied to the column (up to +2 kV/cm). The cis-N-methyl4-/?-styrylpyridium iodide solute was significantly retarded, although uracil was unaffected, owing to its lack of charge. Baseline separation of both solutes was obtained at
+
+
+
Table 2.2. Effect of voltage on retention time
Voltage
Current
Rentention time (x lo-’ rnin)
a
kV
PA
Uracil
CSI
R ,C S I ~ Rurnnl ~,
0 - 1.0
0 1 2 3 3 6 10 0 2 4 7 10 17
10.7 10.7 10.6 10.4 10.0 10.0
20.7 18.7 17.3 15.3 12.5 11.5 9.5 17.5 20.2 23.0 27.1 33.0 51.0
1.93 1.74 1.63 1.47 1.25 1.15 (1.0) 1.62 1.83 2.07 2.73 2.00 4.63
-2.5 -5.0 -7.5 - 10.0 - 15.0 0 f2.5 +5.0 +7.5 + 10.0 15.0
+
10.8 11.0 11.1 9.9 11.0 11.0
2.6 Pressurized Flow-Driven Electrochromatography on Microcolumns
33
M -
Y -
10-
L o -
0 -16
-I2
-I
-4
0
*4
I6
A P P L I E D VOLTAGE
+I2
I KVI
*la
Figure 2.17B. Retention times versus applied voltages [2.3]. Solute A) were uracil; solute B) CSI.
(ii)
3
(ii)
w 3
-+It 2 rnin
Figure 2.18. Chromatograms showing the effect of applied voltage on pressurized flow-driven electrochrornatography of PTH derivatives of (i) asparagine and (ii) 2naphthalenesulfonic acid, obtained (a) with no applied voltage and (b) with applied voltage [2.4]. Applied voltage for (i) and (ii) 7.4 and 11.8 kV,respectively.
34
2 Electrochromntogruphy in Analytical Chemistry
a positive voltage above 5 kV. Table 2.2 lists the retention times of the pyridium salt and uracil at different applied voltages, and its variation is shown in Figure 2.17B. The solute can be eluted over a shorter or longer period (compared with no applied voltage), if the applied voltages at the end of the column are positive or negative, respectively. Figure 2.18 shows separations of phenylthiohydantoin (PTH) derivatives of asparagine (A) and 2-naphthalenesulfonic acid (B) with and without applied voltage. In the chromatogram of PTH derivatives, two unknown peaks (1 and 2 in Fig. 2.18) are well separated with applied voltage, and in the chromatogram of 2-naphthalenesulfonic acid, peak 3 is retarded; its elution time is 3.5 times longer. However, peak 2 is not retarded with applied voltage and, therefore, may represent a neutral compound. This shows that electrochromatography is a good method for separating a group of charged components from a group of neutral compounds. Time Lag for Constant Elution Time. In electrochromatography, elution time and peak height of a solute depend on the time from the beginning of the voltage application. Variation of these values for methanol and adenosine-5'-monophosphate (AMP) are shown in Figure 2.19. A PTFE microcolumn and M phosphate buffer as eluent were used. Pure methanol was used as solute, which produces a UVdetector response. Elution times and peak heights for solutes were measured before, during, and after the application of voltage. Elution times of methanol and AMP stabilized 20 min after the application of the voltage, and when the voltage was released, the elution times became constant after 10 min. These time lags may be equal to the period necessary to alter the nature of column supports by the voltage.
10
0
______OFF
10 20 30 40 0 10 20 TIME (min) [m1 n) - - - - - - - - - - - ON OFF ELECTRO VOLTAGE
-
--
Figure 2.19. Variation of elution time and peak height with period of voltage application [2.4]. Peak height of methanol (squares); elution times, AMP (triangles) and methanol (crosses); applied voltage, 2.5 kV.
2.6 Pressurized Flow-Driven Electrochromatography on Microcolumns
35
Effect of Application of Voltage for Peak Height. With no applied voltage, the methanol peak in Figure 2.19 is low and broad, but, with applied voltage, it becomes high and sharp of twice the area obtained with no applied voltage. These phenomena, also observed in the case of AMP, may be due to the alteration of the column support; they were also observed in other experiments [2.33].
2.6.4
ChromatographicVariation due to the Application of High Voltage
Variations of elution times due to the applied voltage are shown in Figures 2.20 and 2.21 for benzoic acid, phenol, and trimesic acid at pH 6.3 and 8.0. The apparatus used for this experiment is shown in Figure 2.16B. The column used was a fused silica capillary (50 vm inner diameter, 7.5 cm long, with a frit, total capillary tubing 12.5 cm with a section for on-column detection) packed with ODs-silica (particle diameter 7 pm, Capcell 120 SG, Shiseido). The eluent was a mixture of methanol, a buffer of 10 mM malonic acid, and sodium hydroxide aqueous solution with a constant pressure operation mode, e.g., 50 atm. The chromatogram with applied voltage was obtained after the elution time of the solute had stabilized (usually ca. 15 min after applying the high voltage). In Figure 2.20, methanol and buffer (1:l) are used as eluent. At pH 6.3, benzoic acid is completely dissociated (pKa = 4.21) [2.38]. With application of a high voltage along the column, the elution time of benzoic acid increases in proportion to the voltage. The elution time of ethanol, shown as a negative peak, is also affected by the applied voltage; it is used as a marker of voltage-generated electroosmosis. The elution time of benzoic acid is delayed owing to its electrophoretic mobility at high applied voltage along the column, even though vosmincreases. Phenol does not dissociate at pH 8.0 (pKa = 10.0) [2.38], its elution time is affected by R , vpresrand vosm,and decreases when voltage is applied along the column. The mutual separations of benzoic acid and trimesic acid with and without applied voltage are shown in Figure 2.21. As the dissociation constants for trimesic acid are pK1 = 3.12, pK, = 4.10, and pK, = 5.18 [2.39], trimesic acid dissociates completely at pH 8.0. The elution order is reversed when voltage is applied. As the trimesic acid has three carboxylic groups, the electrophoretic mobility is larger than that of benzoic acid. Therefore trimesic acid can be drawn back and stays longer in the column at high applied voltage than benzoic acid.
2.6.5
Relation between Elution Time Ratio and pH
The elution time ratio e, is defined as: er = t e , benzlfe, ethanol
(14)
where te,ethanol is the elution time of ethanol in the same run, and the subscript benz means benzoic acid. The elution time ratio of benzoic acid with and without applied voltage is shown in Figure 2.22. Although e, is not directly related to applied voltage, this factor is expressed as follows,
lniii
lnin
_1
E
R
B
C
1
A
4-
1
Figure 2.21. Electrochromatograms for mixtures of benzoic (1) and trimesic acids (2) [2.7). Eluent: as Figure 2.20 except buffer pH 8.0. Applied voltage: 0 (A); 7.5 kV (B); 8.8 kV (C).
Figure 2.20. Electrochromatograms of benzoic acid and phenol [2.7]. Eluent: mixture (1:l) of methanol and the buffer (pH 6.3) of 10 mM malonic acid - sodium hydroxide aqueous solu-
tion; sample: ethanol solution of benzoic acid (A-D) or phenol (E, F). Applied voltage: 0 (A, E); 5 kV (B, F); 7.5 kV ( C ) ; 10 kV (D).
I
I
I
I
I
I
2.00
-
-
1.00
-
-
.: =
Y
\ Y
I 4.0
I
1
I
I
8.0
6.0
I
10.0
PH
Figure 2.22. Elution time ratio versus p H [2.7]. Eluent: mixture (1:1) of methanol and 10 mM malonic acid - sodium hydroxide buffer. Applied voltage: 0 (full circles); 5 kV (open circles); 7.5 kV (full squares); 10 kV (open squares).
The ratios are shown in Figure 2.22. The values of elution time ratio is larger at high applied voltage, and the value at applied voltages of 7.5 and 10 kvdecreases when pH of the eluent varies from 5 to 9.4. These tendencies derive partly from the increase of vosmin the alkaline region. The three curves shown in Figure 2.22 are focused at a same value, ca. pH 4, because benzoic acid does not form a dissociated ion in acidic conditions (pH < 4). The variation of the ratio with no applied voltage shown in Figure 2.22 corresponds well with the curve for the relation between the capacity factor of benzoic acid and the pH obtained with ODS columns [2.40].
2.6.6
Variation of Electrophoretic and Electroosmotic Flow Velocities with pH
The relations between the electroosmotic and electrophoretic velocities of benzoic acid versus pH are shown in Figures 2.23 and 2.24. To calculate v,,, from Equation (4), it is necessary to determine vpresand R for ethanol. We assume that R = 1 for trimesic acid at pH 9.4, because the three carboxylic acid groups of trimesic acid are completely dissociated. Thus, vpresis calculated by using the elution time of trimesic acid at pH 9. This valve of vpres= 0.103 cm/s at the constant
38
-
2 Electrochromatography in Analytical Chemistry L
i
I
i
I
I
I
I 4.0
I
I 6.0
I
I 8.0
I
6 . 0 0 ~
;. Y
0
* X
4.00:
h
E
-
u)
0 v
>
2.00
-
t
.o
PH Figure 2.23. Relation between electroosmotic velocity and pH [2.7]. Experimental conditions as Figure 2.22 except applied voltage: 5 kV (full circles); 7.5 kV (open circles); 10 kV (full squares).
inlet pressure of 50 atm, is assumed the same as in other experiments in which the composition of the eluent is the same, but the pH of the buffer varies. For example, R for ethanol is estimated as 0.82 from the chromatogram at pH 9.4. We calculate the values of R for ethanol and benzene at every pH. From these values, vOsmat different pH is estimated (Fig. 2.23). The curve first shows a steep increase from pH 3.5 to 4.8, and then changes to a gentle increase from pH 5 to 9. The value of vosmdepends on pH, owing to dissociation of unmodified silanol groups on the surface of the ODs-silica gel (the initial steep increase may correspond to that of the dissociation constant of silanol on the gel surface). The pK, may be around 4.3; it is estimated by using the relation between pH and electroosmotic flow velocity in capillary electrochromatography. The value of vmobis calculated from Equation ( 3 ) , in which vprea,v,,,, and the retardation factors of both ethanol and benzoic acid are included. The three former values are estimated in the same way as for calculating v,,,, and the last value is estimated as 0.85 at pH 9.4. The relation between vm,b and pH is shown in Figure 2.24. The value of vb , at pH 3.5 is almost zero, owing to the absence of the ionized form of benzoic acid; it increases very sharply according to the dissociation of the carboxylic acid group between pH 4 and 5. The value then becomes almost constant between pH 6 and 8. The value of v , , ~obtained is nearly proportional to the applied voltage.
2.6 Pressurized Flow-Driven Electrochromatography on Microcolumns
39
m
t 0.00 4.0
6.0
8.0
10.0
PH Figure 2.24. Relation between electrophoretic velocity of benzoic acid and pH [2.7]. Experimental condition and symbols as Figure 2.23.
2.6.7
Dependence of Electrophoretic and Electroosmotic Velocities on the Composition of Eluents Containing Methanol
The dependence of electrophoretic and electroosmotic velocities on the percentage of methanol in the mixture of methanol and buffer is shown in Figure 2.25. For these values, it is assumed that vPresis inversely proportional to the eluent viscosity, with vpresof the 1:l mixture taken as a standard. The values of v,,, and V,,b for benzoic acid are almost constant between 30 and 90 % methanol in the eluent. They increase markedly at more than 90 % methanol. Values at 99 % methanol content, were unstable, nearly one and a half times those at 95 % methanol. From Figure 2.25, the benzoic acid in the region 30-90 % methanol is completely ionized, and the value of v,,, and v,,~,benz are independent of the methanol content in this range. Increasing methanol content increases the dissociation constants of malonic acid and benzoic acid. The electrophoretic mobility of benzoic acid has a relatively high value at 95 % methanol, suggesting that the benzoic acid buffer is completely ionized. In general, v,,,,, and V,,,ob are expressed as [2.12], [2.42]: vnsm= A E & l ~ v,,,,b
=
d E c*DC q-'
(16) (17)
40
2 Electrochromatography in Analytical Chemistry
10.00 Q ,-I
Y
I
n 0
E
5.00
*
Y
40
60
vol.% of methanol
80
in
effluent
Figure 2.25. Dependence of electrophoretic velocity (full circles) and electroosmotic velocity (open circles) of benzoic acid on the percentage of methanol in the eluent [2.7]. Eluent: mixture of methanol and buffer (pH 4.92) composed at 10 mM malonic acid and sodium hydroxide aqueous solution.
where E = E~ D;D is the dimensionless dielectric constant (relative permittivity) and .c0 is the permittivity of free space; A E , I;, r ] , and C* are the potential gradient, zeta potential, viscosity, and a constant. The value of r] for a 40 % methanol-water solution at 20 "C is ca. 1.8 cP, and gradually decreases to ca. 0.6 CP at 100% methanol [2.43]. The dielectric constant of 40 % methanol-water at 20 "C is 61,and decreases gradually to 32 at 100 % methanol. These two parameters do not vary significantly between 80 and 90% methanol-water. Therefore, the marked increase of v,,,,, and vmObbeyond 90 % methanol may derive from the increase in zeta potential.
2.6.8
Ion-Exchange Chromatography in an Electric Field
Electrochromatography is also applied to ion-exchange chromatography [2.44]. The apparatus (Fig. 2.16 B) consists of a fused silica capillary (50 pm inner diameter, column length 18 cm, total capillary length 25 cm) packed with anion-exchange resins (TSKgel IC-Anion-sw, Toso) and a mixture (10: 90) of methanol and a buffer of 3 mM phthalic acid + 3 mM hexamethylenediamine + 0.15 mM 2-[4-(2-hydroxyethyl)-lpiperazinyl] ethanesulfonic acid (HEPES) aqueous solution as eluent. Indirect UV absorption (236 nm) was employed for detection of anions, except iodide.
2.6 Pressurized Flow-Driven Electrochromatographyon Microcolumns
I\ I
I 0
0.01
O.D.
41
f
I
I
I
I
5
10
15
18
mi
Figure 2.26. Electrochromatograms of inorganic anions [2.45). (a) C1-; (b) NO,; (c) I-; (d) C104; (e) SOP Eluent: mixture of methanol (10 %) and buffer (pH 6.4) composed of 3 mM phthalic acid - 3 mM hexamethylenediamine aqueous solution (90 %).
In Figure 2.26, the elution time of sulfonate double charged anion is reduced more than that of singly charged anions when the applied voltage along the column is increased from zero to + 2 kV (positive electrode was at the column outlet). The elution order of perchlorate and sulfonate anions is reversed at 2 kV. Figure 2.27 shows electrochromatograrns of five organic carboxylic acids. By applying voltage along the column, the separations between acetic and lactic acids and between tartaric and malonic acids are improved, and the retardation ratios, [(elution time with -3 kV)/(elution time without -1 kV)] 1.23, 1.59, 1.69 and 2.03 for lactic, acetic, tartaric, malonic and oxalic acid anions.
42
2 Electrochromatography in Analytical Chemistry
- 3 K V
vv
0
10
20
E
30
(min)
Figure 2.27. Electrochromatograms of carboxylic acids [2.45] (A) acetic acid, (B) lactic acid, (C) tartaric acid, (D) malonic acid, (E) oxalic acid Elucnt: mixture of methanol (10%) and buffer (pH 6.8) composed of 3 mM phthalic acid, 3 mM hexamethylcnediamine; applied voltage -3 kV (lower diagram); -1 kV (upper diagram).
Figure 2.28 shows the relationship between capacity factor and applied voltage. The capacity factor is [(elution time of anion)/(elution time of nonretained peak)]. The peak of the solvent in the sample solution was taken as a nonretained peak. The eluent was a mixture of 3 mM phthalic acid, 3 mM hexamethylenediamine, and 0.15% HEPES acid, aqueous solution. It is very interesting that the elution time of each of the monovalent anions has its own constant value when the applied voltage is increased to +7 kV. However, the elution times of these anions are increased when negative voltages are applied. The sulfonate anion value is increased by the application of positive voltages, and vice versa, like the ions in ODS-silica column chromatography. The effect of application of electrovoltage is clearly observed in the electrochromatography using ion exchange resins, as shown in Figure 2.27.
2.6.9
Voltage-Programmed Electrochromatography
Tjaden et al. [2.33] compared electrochromatography with constant and programmed applied voltages. They also tried to compare chromatograms obtained with isocratic and gradient modes in conventional chromatography and electrochromatography. Figure 2.29A shows the chromatogram of a test mixture of alkaloids run under isocratic conditions. Alkaloids are basic compounds that are neutral at high pH (pK, > 9). Although good efficiency was obtained for neutral compounds, the charged alkaloids showed severe tailing, resulting in a disappointing performance of the separation system. The use of an end-capped packing material improves the performance, but this study was not focused on optimum separation of. By applying a voltage over the separation column, an electrophoretic component is be superimposed on the chromato-
2.6 Pressurized Flow-Driven Electrochromatography on Microcolumns
43
. so,'-
c10,-
-
I
I
I
I
I
I
I
I
I
1
I
L - 3 - 2 -1 0 +1 + 2 + 3 +4 + 5 +6 +7
APPLIED VOLTAGE [KV] Figure 2.28. Variation of capacity factors of inorganic anions with applied voltage [2.45]. Eluent as Figure 2.27.
graphic migration. Figure 2.29B shows the chromatogram of the same test mixture, but with a applied voltage along the column. It is clear that a voltage influences the separation characteristics considerably. For the separation of nucleotides, triethylamine, an ion-pairing reagent, is added above 10 mM in order to achieve sufficient retention of nucleotides. For LC-MS, such a high concentration of triethylamine causes excessive ion-source contamination. Tjaden et al. [2.33] found the longer alkyl chains of dibutylamine (DBA) gave sufficient retention of nucleotides when used at concentrations as low as 1-2 mM. Figure 2.30A shows the chromatogram of three adenosine phosphates, separated under isocratic conditions. Retention was obtained, but ADP and ATP are eluted with poor performance. In Figure 2.30B the chromatogram of the same mixture is shown, with applied voltage + 5 kV on the electrode at the column end; anionic compounds are accelerated. Although the symmetry of the later eluting compounds is far from ideal, a real peak compression can be seen. The influence of the application of a voltage over the capillary column is even clearer in Figure 2.30C, in which a stepwise gradient of applied voltage is used. After the elution of AMP, the applied voltage was increased from 5 to 10 kV. The ATP peak is really compressed and becomes very sharp. The programmed applied voltage is very effective for improving chromatograms, like the solvent gradient elution used in conventional liquid chromatography.
+
+
44
2 Electrochromatography in Analytical Chemistry
(B)
(A)
moi
ine codeine
L
dimethyl-morphine thebaine
1
0
20
10
30
min O
-10 kV
T
Figure 2.29. Chromatograms of some morphine alkaloids [2.3.5]. Column, 1.50 mm X 220 pm inner diameter packed with 5 pm Nucleosil 100 CIS;mobile phase: mixture of acetonitrile and 2 mM ammonium acetate (2:3, voYvol); applied voltage: 10 kV starting at t = 9 min; UV detection at 220 nm, 0.01 a.u.f.s.; amounts injected, ca. 50 ng of each compound.
IADP
0
10 20 30 40
min
0
10 20 30 40
min
Figure 2.30. Separation of 17 ng of AMP, ADP and ATP [2.35]. Column: 200 mm x 220 pm inner diameter packed with 5 pm Nucleosil 100 C18;mobile phase: mixture of methanol and 2 mM dibutylamine at pH 5.0 (1:9); UV detection 260 nm, 0.0025 a.u.f.s. (A) Isocratic conditions with no applied voltage; (B) With applied voltage +S kV, (C) Starting voltage + 5 kV, increased to 10 kV at t = 9 min.
+
Voltage-programmed electrochromatography can be carried out in the isocratic mode. Therefore it is convenient to connect a microcolumn liquid chromatograph with a mass spectrometer, because the latter is sensitive to the change in solvent composition [2.33], (see also Chap. 6, Section 3).
2.7 References
2.7
45
References
[2.1] S. Otsuka, L. Listowsky, Anal. Biochem., 102, 419-422 (1980). [2.2] P. H. O’Farrell, Science, 227, 1586-1589 (1985). [2.3] T. Tsuda, Anal. Chem., 59, 521-523 (1987). [2.4] T. Tsuda, Anal. Chem., 60, 1677-1680 (1988). t2.51 T. Tsuda, Y. Muramatsu, J . Chromatogr., 515, 645-652 (1990). 12.61 T. Tsuda, 1. Chem. SOC. Jpn, Chem. Indu. Chem., 937-942 (1986) (CA 105: 126484r). [2.7] S. Kitagawa, T. Tsuda, J. Microcol. Sep., 6,91-96 (1994). [2.8] T. Tsuda, K. Nomura, G . Nakagawa, J . Chromatogr., 248,241-247 (1982). [2.9] J. H. Knox, I. H. Grant, Chromatographia, 24, 135 (1987). [2.10] T. Tsuda, LC-GC Zntl. 5 (9), 26-36 (1992). [2.11] T. Tsuda, J . Liquid Chromatogr., 12, 2501-2514 (1989). [2.12] T. Tsuda, “Control of electroosmotic flow in capillary electrophoresis”, Chap. 22, in Handbook of capillary electrophoresis, J. P. Landers (ed.), CRC, Ann Arbor, 1993. [2.13] J. H. Knox, I. H. Grant, Chromatographia, 32,317-328 (1991). [2.14] T. S . Stevens, H. J. Cortes,Anal. Chem., 55, 1356-1370 (1983). [2.15] J. C. Giddings, Dynamics of chromatography, Dekker, New York, 1965. [2.16] S. Kitagawa, T. Tsuda, 1994 unpublished. [2.17] C. E. Reese, R . P. W. Scott, J . Chromatogr., 18,479 (1980). [2.18] G. Taylor, Proc. R. SOC.London, Ser. A , 219, 186 (1953). [2.19] K. Ando, Thesis for bachelor degree, Nagoya Institute of Technology (Nagoya), 1984. [2.20] H. Wada, S. Hiraoka, A. Yuchi, G. Nakagawa, Anal. Chimica Acta, 179, 181-188 (1986). [2.21] V. Pretorius, B. J. Hopkins, J. D. Schieke, J . Chromatogr., 99, 23 (1974). [2.22] W. D. Pfeffer, E. S. Yeung, Anal. Chem., 62, 2178 (1990). [2.23] E E. P. Mikkers, E M. Everaerts, P. E. M. Verhaggen, J . Chromatogr., 169, 11 (1979). [2.24] J. W. Jorgenson, K. D. Lukacs, Anal. Chem., 53, 1298 (1981). [2.25] T. Tsuda, K. Nomura, G. Nakagawa, J. Chromatogr., 264,385 (1983). [2.26] W. D. Pfeffer, E. S. Yeung, J. Chromatogr., 557, 125-136 (1991). [2.27] M. Martin, G. Guiochon, Anal. Chem., 56, 614 (1984). [2.28] M. Martin, G. Guiochon, Y. Walbroehl, J. W. Jorgenson, Anal. Chem., 57, 559 (1985). [2.29] C. L. Rice, R. Whitehead, J . Phys. Chem., 69 (ll), 4017 (1965). [2.30] T. Tsuda et al., J . Chromatogr., 632, 201 (1993). [2.31] T. Tsuda, J. W. Sweedler, R. N. Zare, Anal. Chem., 62, 2149 (1990). [2.32] A. B. Littlewood, Gas Chromatography, Academic Press, New York, 1970. 12.331 E. R. Verheij, U. R. Tjaden, W. M. A. Niessen, 3. van der Greef, J . Chromatogr., 554, 339-349 (1991). [2.34] M. Nakashima, T. Tsuda, 1994 unpublished. [2.35] D. H. Shain et al., Anal. Biochem., 200, 47-51 (1992). [2.36] S. Kitagawa, M. Inagaki, T. Tsuda, Chromatography (Kuromatoguraphi), 14, 39R-43R (1993). [2.37] S. Hoffmann, L. Blomberg, Chromatographia, 24,416 (1987). [2.38] G . Kortun, W. Vogel, K. Andrussov (eds.), Dissociation Constants of Organic Acids Aqueous Solution, Buttenvorths, London, 1961. [2.39] N. Purdie, M. B. Thomson, N. Riemann, J. Solution Chem., 1, 465-476 (1972). [2.40] C. Horvath, W. Melander, I. Molr, Anal. Chem., 49, 142-152 (1974). [2.41] S. Kitagawa, T. Tsuda, J. Microcol. Sep. 7, 59-64 (1995). [2.42] R. J. Hunter, Zeta potential in colloid science, Academic Press, London, 1981. [2.43] S. Hjerten, Chromatogr. Rev., 9, 122-219 (1967). [2.44] International Critical Tables of Numerical Data Physics, Chemistry and Technology, McGram-Hill, Inc., New York, 1929, p. 22. [2.45] H. Watanabe, S. Kitagawa, T. Tsuda, 1995 unpublished.
This Page Intentionally Left Blank
3
Electroosmosis and Electrochromatography Takao Tsuda
3.1
Electroosmosis
In electrochromatography, an electroosmotic flow is generated if three essential elements are satisfied: a) there is an applied voltage along the column; b) there are charges on the surface of the packing support or the inner surface of a open-tubular capillary column; c) the eluent conducts electricity. Electroosmosis has ben studied by a number of workers [3.1]-[3.31]. It is affected by eluent pH, electrolyte concentration, temperature, applied voltage and current, column material, and surface adsorption.
3.1.1
Surface Charge of Silica Gel and Packing Support
In liquid chromatography, columns have been classified as: 1) open-tubular capillary columns [3.32]; 2) packed microcapillary columns [3.33], [3.34]; 3) slurry-packed capillary columns [3.35]; 4) microcolumns [3.36]; and 5) conventional columns. Microdimensional columns are preferable for electrochromatography, because of their abilitiy for effective heat dissipation. Electroosmosis has been studied by using open-tubular capillaries in capillary zone electrophoresis, in which electroosmosis is used as the essential solute driving power. Chromatography performed with electroosmosis as the only driving power has a great deal of similarity with capillary zone electrophoresis. Since the silica gel used as packing support has 3-10 silanol groups on an area of 10 A2 [3.37], it is easy to understand that the surface will be charged by SiO- groups resulting from dissociation of SiOH. The amount of SiO- groups depends on pH. However, the total charge on the surface cannot be explained solely by the dissociation of silanol groups [3.7], I3.301. The surface itself has a charge induced on it by surrounding electrostatic fields. For example, silica gels, glass beads, fused silica and tetrafluoroethylene tubing, have negative surface charges [3.7], while charcoal has a positive charge. Under applied voltage, electroosmotic flow will be generated provided the three essential elements set out above are met. Another way of inducing a surface charge is by means of a voltage along the crosssectional direction of the column, i. e., a radial field [3.21]-[3.29]. Even with no externally applied radial field, a certain radial voltage will be generated by applying a voltage along the column; at every site along the capillary column, a certain current will be released from the outer surface of the capillary to the medium, e.g., air or water, through the glass wall (resistance ca. 1015Q [7.27]). Experimentally we can observe, especially at high applied potential gradients along the column, e . g., 500-1000 Vkm, that a great deal of dust is forced onto the outer surface. As dust has
48
3 Electroosmosis and Electrochromatography
opposite charges, the current will be released every time a dust particle attaches to the surface. The applied potential along the column induces a potential gradient across the column, and hence some charge on its inner surface (i.e., a zeta potential).
3.1.2
Electrical Potential in the Vicinity of a Solid Surface
To understand the origin of electroosmotic flow, it is necessary to discuss briefly electrical potential and the physical environment of the interface. The discussion follows the approach of Hunter [3.38]. The electrostatic potential near an isolated charged object in vacuum is defined by Coulomb's law. If the object is a sphere of radius r the potential at a distance a from its surface is:
q = Q / [ e m o(r+a)]
(1)
where Q is the total charges on the surface and q,is the permittivity of free space [3.38]. The potential very close to the surface (less than 100 A) cannot be measured, because of interactions between the probe and the charge on the object. For r = 1 cm, the potential in vacuum is constant up to 100 pm from the surface of object and equal to:
Vo = Q / ( 4 n ~ )
(2)
where q0 is the potential at the surface of the object. Then the potential q then decreases with increase of the distance from the sphere [3.38]. The simplest model, attributed to Helmholtz, has two layers of charge at the interface between two phases (solid surface and liquid) fixed in parallel planes to form a molecular condenser, called an electrical double-layer (Fig. 3.1). Although the charge on a solid surface may be assumed to be located in a plane, the electrical forces on the ions in a liquid phase compete with the thermal diffusive forces, forming a diffuse double-layer [3.39], [3.40]. With this model, the fundamental electrostatic equation for the system is: V2 = -(4n%)-' [(4ne)/D]
(3)
where D is the relative permittivity and p is the volume charge density:
e = Cn,z,e
(4)
where z is the valency of type i ions and n, is the number of type i ions per unit volume:
n, = n,"exp (- z,e$dkT)
(5)
when q = 0, n, = n,". Substituting Equations (5) and (4) into Equation (3), we obtain the complete Poisson-Boltzman equation: V2q = d@/dx2 = - (4n~&'(4n/D) Cn,"z,e exp (-z,eqlkT)
(6)
This equation is simplified to one dimension because W varies only with x . If q!~is small everywhere in the double-layer (i.e., z,eq << kT), we approximate exp x by l-x and use the preservation of electroneutrality in the bulk electrolyte to obtain:
3.1 Electroosmosis
49
(7) where x = [(e' Xn~z?)/ ( E ~ T ) ] ' . ~
Equation (7) can be solved to give: dW/dx
=
-*
(9)
since dV/dx = 0 and qj = 0 in the bulk solution, far from the solid surface ( x -+ A second integration of Equation (9) using q~ = qjo at x = 0 gives:
1~ = vIIexp
co).
(10)
This shows the approximate form of the potential distribution near the wall. The general Equation (6) can be integrated, and treats most electrolytes as symmetric. It gives: dV/dx
=-
[(2xkT)/(ze)] sinh [zeq)/(%kl")]
(11.1)
The integrated form of Equation (11) can be expressed in many ways, the most compact of which [3.38] is: tanh(zq*/4) = tanh(zqo*/4)exp(-kx)
(11.2)
x
along cD1umn radial applierj
diffuse double layer
an electric double layer [an elertrrc or Lnner
Hs 1 mho I t L p l a n e ( IHP) 1
olid surface
B)
Electrostatic
Potential
Figure 3.1. A) Electrical double-layer. B) Schematic expression for the distribution of ions at, near, and within a solid surface, and for the variation of electrostatic potential from the surface of the solid to the liquid (x-direction).
50
3 Electroosmosis and Electrochromatography
where q~ = ely*/kTis a dimensionless potential parameter, which we will refer to as the reduced potential (at 25 "C, qI* = 1 when q~ = 2.57 mV). Figure 3.1 shows the electrical potential zq* in the double-layer versus xx according to Gouy and Chapman [3.41]. The distance l/x is referred to as the thickness of the double-layer (namely xx = l), although the varying potential extends to 2 % beyond its original value. The potential drop across the inner region of the double-layer (inner Helmholtz plane, IHP, Stern layer) is often around 0.1 V and since this occurs across a distance of less than 1 nm, the field strength is about 1O8V/rn.This is sufficiently high to polarize molecules, appreciably.
3.1.3
Origin of Electroosmotic Flow
The theory for electroosmosis was developed by von Smouluwski [3.38], [3.42], who considered the movement of a liquid adjacent to a flat, charged surface under the influence of an electric field applied parallel to the interface. The velocity of the liquid in the direction parallel to the wall v, rises from a value of zero in the inner Helmholtz plane to a maximum value v,, at some distance from the wall (ca. 314. The forces on unit volume of the liquid, which is generated by the friction of liquid, is equal to the force due to the attraction of ions under the applied voltage (Fig. 3.2):
E, Q = E,@Adw= VA (dv,/&), - VA(dv,/&),+~ or:
E,
@X =
-
-V(d2vZ/dx2)&
Electric
F i e l d
Area:A
m8 m8@-&+ +
Figure 3.2. Electroosmotic flow generated by movement of partial charge of molecules due to an electric field (2-direction).
3. I
Electroosmosis
51
where E,, Q, A, and v are the potential gradient applied parallel to the solid surface, number of charges in the liquid (ions), unit area which experiences the friction and local velocity of electroosmosis, respectively. The z-axis is parallel to the surface. Substituting for e gives: E , ( 4 n ~ )(D/4n) (d2q/dx2)dx = q(d2v,/d x2)&
(14)
This equation can be integrated from a point far from the solid surface where q = 0 and v, = vosmup to the inner Helmholtz plane where v, = 0 and = f. At first, both dq/& and dv,/dx are zero far from the solid surface. The result is: v0SmiE7
= pe = =-
- 4 n ~[ ( ~ ~ ) 4 4 ~ ~ ) 1
(&r)
(15)
(16)
where pe is electroosmotic mobility. In several reports, Equation (16) is expressed as follows [3.2], [3.6], [3.12], [3.431-[3.45] : vosm = [(DW(4nv)lf =
[(~fW(4n7~~~q)l
(17) (18)
According to Hunter (p. 357 of [3.38]),Equation (18) is not “rationalized”, and he recommends replacing D by 4 n D~ or 4 n ~Equation . (18) is still satisfactory, but it is better to use Equation (16). There are two equations for electroosmosis: vosm =
(DW(4nr)
vosm = ( 4 x 4 [ ( D M ~ =~ ( EI W V
(A.1) (A3
Hunter describes the reason (p. 357 of [3.34]), as follows. The factor 4 n appears because the original units were “unrationalized”. After rationalization by defining the force between two charged particles in a dielectric, the factor 4 n appears in formulae involving spherical symmetry and disappears for flat plates. Whenever D appears in the old formulae it should be replaced by 4nq, D or 4 n ( ~E = %D).They remark that D is a dimensionless quantity.
3.1.4
Thickness of the Double-Layer
The thickness of the double-layer 6 depends on the electrolyte concentration which enters through its effect on the parameter x (= 1/6), and for water at 25 “C [3.38] is: x = [(2000F2)/ (QDRT)]”.~ p 5 nm-’ = 3.288 p5
(19)
where Fand R are the Faraday (96485 C) and the molar gas constant, respectively. The ionic strength, I (mol/L), is: I
=
0.5 Fc,z/
(20)
52
3
Electroosmosis and Electrochrornatography
Table 3.1. Electrolyte concentration and double-layer thickness [3.38] ~
_
_
_
_
_
~
Electrolyte, moVL
Thickness*, nm
lo-‘
1.04 3.04 9.62 30.4
10-2 10-3
10
* Calculated from Equation (21) moYL, the value of l l x is 3.04 nm, and where ci is the concentration of ion i. At I = moYL (Table 3.1). These values are estimated for a flat chanit is 30.4 nm at I = nel. Another estimation of the thickness of the double-layer is given by Delahy [3.32]: S = (3 x 10-~1 zi 1 c , ’ ~ ) - cm ’
(21)
In slurry-packed capillary columns, the channels between packing supports are very narrow, estimated at one-tenth of the particle diameter [3.46], ca. 1-0.3 pm when the particle diameter of the packing supports is 10-3 pm. As the surface charge will affect up to ten times the thickness of the diffuse double-layer, its length will reach almost half the thickness of the channels in some cases. This will reduce the flow velocity of electroosmosis. The overlap of central flow with the diffuse double-layer may cause plug and laminar flow profiles. The cross-section of channels between packing supports may be square or rectangular, not flat or round. Rice and Whitehead [3.45] proposed that the equation for the planar model should be modified for application to a cylindrical model by the correction factor F ( x r ) :
F(xr)
=
1 - “ 2 z i ( x r ) ] / [ x rI,(xr)]j
(22)
The value of F ( x r ) , plotted as a function of log xr ( I = radius), is shown in Figure 3.3. Its value deviates from 1 when xr is less than 10. Thus, the electroosmotic flow velocity in a cylindrical capillary is given by:
0.0 I 0.0
J 0.2
0.4
0.8
0.8
1.0
Log
Kr
1.2
1.4
1.6
1.8
2.0
Figure 3.3. Correction factor for electroosmotic flow velocity; cylindrical geometry [3.45].
3.1 Electroosmosis
53
The parameter x is equal to the reciprocal thickness of the diffuse double-layer, b-', and is given by Equation (21). Correction factors for the cylindrical geometry are shown in Figure 3.3. Although the cross-section of the channels between packing supports in the column is not circular, the use of this correction factor gives a better approximation for the channels in silica gels. In open-tubular capillary columns, we usually use 100-25 pm inner diameter capillary. It is not necessary to use the geometric factor for the electroosmotic flow, because the thickness of the double-layer is less than 1 pm in general. However if we use a very narrow capillary tube (less than 5 pm inner diameter), the flat model geometry is no longer valid, and we should be very careful about the effect of the double-layer on flow profile, since the potential from the solid surface is generally effective up to 3 / x , i.e. ca. 1 pm at lo4 moVL (Table 3.1).
3.1.5
Charge Density on Silica Gel Surfaces
The estimation of charge density on the wall surface is the key to the calculation of q, capacitance, thickness of double-layer, and electroosmotic mobility. It is clear that the charge density can be controlled by a radially applied voltage. The charge density is also affected by its chemical environment, e. g., pH. The number of silanol groups per unit area of the surface, [SiOH],, depends on the dissociation.
c,
[SiO-] + [H']
[SiOH],
(24)
These ionized silanol groups generate a surface charge density a(SiO-), which is related to the solution pH by: a(SiO-)
=
[SiOH], ([SiO-],/{[SiOH],
+ [SiO-Is]) = f'/[l+ [H+]/K]
+
(25)
where f' = [SiOH], [SiO-1, and K is the dissociation constant. When there are 5 SiOH groups in an area of 10 A', the density of silanol groups, b(Si0H) b(SiO-) = 5 x 1014cm-', and a(SiO-) = 5 X lo7 cm-' at pH 7 ( K is estimated as The total charge density 4,is assumed to be given by the summation of the charges due to silanol groups and the radially applied voltage:
a, = a,,,, + a",+ a(SiO-)
+
(26)
where onatand a", are the charge density generated by the natural electric field (i. e., the electric field of the Earth or the radial electric field generated when a voltage is applied along the column) and the radially applied voltage, respectively. Equation (26) assumes that the electrostatic charges and the ionization of surface hydroxyl groups do not interact. The relation between a,and u,,, is derived [3.18] as follows: u,,,,, =
E,(~dq-)exp (-xx)(2kT/e) sinh-' [(a,,, + a,, + o(SiO-))] (500 ~ / E ~ c R T ) ~ . ~
(27)
where suffix b refers to the solution buffer. Although a,,, + u,, was originally defined as the density of the double-layer [3.19], it can be identified with the surface charge. From Equations (24) and (27) it is possible to estimate the pK, values for silanol groups on the surface of silica gels. Figure 2.23 of Chapter 2 gives pKa = 4.2 for silanol groups on the surface of silica gel modified with octadecylsilane [3.47].
54
3
3.1.6
Chemical Modification of the Inner Surface by Adsorption in Open-'Ibbular Capillary Columns
Electroosmosis and Electrochromatogruphy
Since E.- = ilk, where i and k denote current density (A/cm2) and specific conductance (Q-' cm-'), Equation (16) can be written in the form: vom
=
( i d ) (kq1-I
(28)
As R (resistance in Q) depends on the temperature in the medium, E (= RI) varies
with temperature, where I is the current. The zeta potential and diffuse double-layer thickness 6 , have the following relationship [3.48]:
c = (6 n,,,e)
(29)
($I
where n,,, is thc number of charges on the inner surface of the open-tubular column. Substituting Equations (29) and (21) into Equation (28) gives: v,
= (i n,,,e) I [3 x 10'
I z 1 c"* kq]
(30)
The product of k and q , called the Walden product, is independent of temperature 13.121. We obtain: v,,,
=AI
(i 4,,,e) 1 ( I z
I C''7
(31)
where A , is a constant. From Equation (30), the electroosmotic flow velocity depends on a,,, and its dircction depends on the sign of the charges. On the inner wall of the capillary there are two kinds of surface charge: I) surface charges which are induced by the Earth's field a,,d,; 2) surface charges due to the dissociation of SiOH groups, u(SiO-). These surface charges may be covered partially or completely by surface-active reagents, such as cetyltrimethylammonium bromide (CTAB) I3.101, [3.12] or tetrabutylammonium hydroxide (TBA) [3.20]. From the relation between the concentration of these surface-active reagents and electroosmotic flow velocity (Fig. 3.4), it is clear that the original surface has a certain amount of negative charge, a,,,, which decreases as increasing amounts of CTAB or TBA are adsorbed on the surface of the glass wall. The sign of the surface charge changcs from negative to positive at 3.5 x lo4 mol/L for CTAB. The number of positive charges increases, until the surface is completely covered. On the other hand, when we use a strong surface-active reagent with negative charges, such as sodium dodecylsulfonate, the amount of negative charge on the inner surface of capillary column may become more negative [3.12]. Therefore, we can control the amount of the surface charge and its sign by the addition of surface-active reagents. This is often done in capillary electrophoresis to obtain a short elution time of negative ions, since both solute mobility and electroosmotic flow operate in the same direction, to decrease the adsorption of protein on account of the electrostatic repulsion between the surface and the solute [3.24], [3.30], [3.31].
3.1
Electroosrnosis
55
=-N
I
E V u
+, 4 2
. 3
t
V .3
c,
0
E In
0
-10-
a
F
Figure 3.4. Variation of electroosmotic mobility v with concentration of cetylammonium bromide (CTAB). (Open circles) buffer for points 1-3 was 0.02 M-(N-morpholine) ethanesulfonic acid-L-histidine aqueous solution with 0.5 % ethylene glycol (pH 6.2); buffer for points 4-7 was 0.01 M phosphate buffer with 5 % ethylene glycol (pH 8.3). FEP (0.2 or 0.3 mm) and a fused silica capillary (50 pm inner diameter) were used for points 1-3 and 4-7, respectively L3.121; (Triangles) pH 3, 10 mM phosphate buffer containing 10% acetonitrile, 10 pm fused silica capillary [3.19]; (Squares) pH 7, 10 mM phosphate buffer, 10 pm fused silica capillary [3.20].
3.1.7
Effect of pH on Electroosmosis
The effect of pH, as described by Ewing et al. [3.28] is given by Equation (27) which includes the contribution of dissociated silanols on the glass inner surface. However, the estimate does not correspond to experimental values [3.27], [3.28]. It is possible that their estimation of other factors andor the additive law of charge densities are not valid. As shown in Figure 3.5, at pH > 8, most of the silanol groups on the inner surface of an uncoated capillary are dissociated, and the electroosmotic flow velocity is independent of pH. For pH < 4, v,,, is independent of pH because most of the silanols are indissociated. Therefore, v,,, varies at pH between 4 and 8. It is necessary to note that
56
3 Electroosmosis and Electrochrornatography 7.53 -
h
.--* .-
6.53
a
0
E
.V
-d
4.5.
Y
0
E v1
-
0
0
.l
w V Y
0.d
3
4
5
6
7
8
9
1
0
1
1
PH Figure 3.5. Dependence of electroosmotic mobility on pH for uncoated and coated capillary. (m) uncoated capillary; (A) Briji-35/0DS coated capillary [3.30]; ( 0 ) uncoated [3.7]; (*) uncoated, theoretical estimated value, and ( 0 ) experimental value [3.50].
the pK, of silanol is always dependent on the surface conditions and the buffer employed [3.7], [3.30]. Teflon tubing is also used for electroosmosis, and its pH dependence is similar to that of as fused silica capillary tubing [3.7], [3.49].
3.1.8
Electroosmotic Mobility in Open-Tubular Capillary Columns
The linear flow velocity in electroosmosis depends on the physical parameters of the solvent and also on the electrolyte concentration, from Equations (28)-(32). Electroosmotic mobility is listed in Tables 3.2A and 3.2B, in which data were obtained by using an uncoated Pyrex glass capillary column and coated fused silica capillary respectively. The order of the velocities of acetonitrile, water, and methanol is readily understood from the ratios of their dielectric constants to the viscosity of the solvent, 10, 7, and 6 , respectively. The effect of the concentration of disodium hydrogen orthophosphate in water on the electroosmotic mobility is also understandable from Equation (30). The electroosmotic mobility is shown in Figure 3.5.
3.1.9
Electroosmotic Flow Velocity in Packed Columns
The values of electroosmotic mobility kSosm are listed inTables 3.2A, 3.2B and 3.3 [3.7], [3.9], [3.47], [3.51], [3.52], in which both the references and our measured data are summarized. Cortes [3.53] found that the electroosmotic mobility depends on the size of the channels, because there is an effect from the diffuse double-layer, for extremely small channels, e.g. < 1 pm . However, Knox et al. [3.52] deny this effect. This contradiction may arise from the differences in experimental conditions. Coretes et al. used a neutral medium and Knox et al. used an alkaline buffer. Erni et al. [3.51] admit that
3.1 Electroosmosis
57
Table 3.2A. Linear flow velocity of electroosmosis at 100 V/cm Solvent
Linear velocity, cm/s
Experimental conditions*
Distilled water Methanol Acetonitrile 0.05 % Methanol-n-hexane 0.01 M NazHPO,-water 0.025 M Na2HP04-water 0.05 M Na2HP04-water 0.075 M Na,HPO.+-water 0.1 M NazHP04-water Methanol-benzene (1 :9)
0.061 0.034 0.14
1 1 1 1 2 2 2 2 2 Ref. [3.70]
0.0006
0.12 0.093 0.087 0.077 0.057 0.01
* 1) Capillary column, Pyrex, 57.6 cm x 132 pm; solute, benzene or pyridine; power supply, constant-voltage mode, 13 kV; 2) Capillary column, Pyrex, 60 cm X 132 pm; solute, as above; power supply, constant-currentmode, 200 pA (voltage gradient was varied from 302 to 96 Vlcm). Table 3.2B. Electroosmotic mobiliy KO, measured in various fused-silica capillaries Capillary
b,x
VPe
Q, pm
Acetonitrile-buffeP (2:3)
ODS ODS PSL-ODS~ ODS ODS Etched
5 10 10 25 50 50
2.40 2.38 2.18 2.49 2.43 2.37
a
cm2V-' s-' Methanol-buffer" (1:l) 1.16
1.13
0.1 M phosphate buffer (pH 7). PSL, capillary with a porous silica layer.
electroosmotic mobility depends on pH, increasing in the region pH 6-8 and then remaining constant for pH > 8. The same phenomenon is observed in the region pH 4-8 in open-tube capillaries by Regnier et al. [3.30]. In other words, Erni et al. [3.51] and Knox et al. [3.52] did their experiments with a high density of surface charge, due to the dissociation of the silanol groups on the silica packing materials. Although Erni et al. used a silica-modified packing material (silicaODs), they assumed that 50% of the silanol groups on the surface of the silica gel remain unreacted, and the groups may dissociate in alkaline medium following an increase in the electroosmotic mobility [3.28], [3.29]. The diffuse double-layer affects electroosmotic mobility, reducing the flow velocity by factors of 0.3 and 0.8 when 2 and 10 times the thickness of the diffuse double-layer are equal to the radius of the channels [3.29], [3.38], [3.45]. The estimate of the diffuse double-layer is 1 pm [3.14] to 3 nm [3.38] for 10-20 mM aqueous electrolyte solutions. The data in Table 3.3 can be explained as follows: in a pure medium (it does not contain electrolyte) the thickness of the diffuse layer is larger, and this may decrease the electroosmotic mobility in narrow channels. As in a buffer of pH > 4, the density of charge of the silica solid surface due to the dissociation of silanol groups is larger than that in an organic medium, and also in a buffer of pH < 4, it increases the electroosmotic mobility markedly, even though there is a small reduction from the effect of the
58
3 Electroosmosis and Electrochromatography
Table 3.3. Electroosmotic mobility in the packed microcapillary column
Packing
Supposed channel", Pm
Bio-Sil-A [3.53]
1 10 10 1 0.5
Patisil 10-Sil [3.53] Hypersil [3.52]
DeverosiK8 [3.53]
0.3 0.15 0.5
Capcell-ODS [3.54]
0.7
Develosil-ODS [3.9] Hypersil ODS [3.51]
0.3 0.3
Monospher ODS [3.51] 0.16
Electroosmotic mobility, X lo4 cm2V-' s-l 2.8 1.2 3.2 0.7 1.9 1.2 2.6 0.49 0.47 0.46 0.15 0.16 0.085 0.41 0.28 0.13 -0.088 0.48 0.16 4.1 2.9
Medium
methanol acetonitrile water methanol 2 mM NaH2P04+ H20 acetonitrile (3:7) as above as above ethanol ethano1:water (8:2) ethano1:water (95:s) with additivesb ethanol:water (80:20) with additivesb ethano1:water (70:30) with additivesb ethanol:water (80:20) with additives' methanol ethanol acetonitrile 20 mM CTAB ethanol (3:7) methanol 2 mM B4Na207(pH 8.7): acetonitrile (2:8) 4mM Na2B407(pH 9.2) H 2 0 4 mM Na2B407(pH 9.2): acetonitrile (4:6)
Channels estimated as one-tenth of particle diameter, d,. Ethylenediamine and polyoxyethylene lauryl ether (PELE) 5 mM and 0.1 YO,respectively. Ethylenediamine and PELE, 20 mM and 0.1 % , respectively.
a
diffuse double-layer. The coverage of silanol groups on the surface of silica gels by reaction with ODS decreases the electroosmotic mobility by a factor of half to one-sixth of the value for untreated silica materials, owing to the low charge density (Table 3.3). The relation between applied voltage and linear velocity of electroosmosis in a packed capillary column is shown in Figure 3.6 [3.51]. Electroosmotic flow velocity at a given potential gradient, v,,~,is given by kosm A E . Therefore, if we apply 1000 V/cm to a column packed with Capcell-ODS and methanol is used as eluent, v,,, = 0.4 mml s. This is a moderate flow rate for liquid chromatography. If more than 50 kVis applied to a 143 mm long capillary column, the system becomes unstable. This could be considered to be the result of heating [3.51]. In general the speed of electroosmosis is high enough to operate liquid chromatography with a 10-20 min analysis period, and application of a high potential gradient may yield high-speed analysis, in spite of the use of very small particles as column supports. Usually, the use of column supports of very fine particle diameter needs a high inlet pressure when operating liquid chromatography with pressurized flow, but not in the case of the use of electroosmosis. The another consideration for the estimation of electroosmotic mobility in packed columns is that the channels in a packed column are both tortuos and constricted [3.46]. Therefore it is necessary to estimate the structural parameter fst when we compare the value of electroosmotic mobility in a packed capillary with the value in an open-tubular capillary column.
3.2 Electroosmotically Driven Chromatography and Electrochromatography I
.
I
'
l
h
P
P
. '
59
I
6-
Y
-
h .rl c,
:: 4 -
r(
2 2
:2 -
.3
4
I
0
,
I
,
I
,
I
4
Potential Gradient (KV/cm)
Figure 3.6. Dependence of electroosmotic flow velocity on applied voltage. Capillary column, 50 pm internal diameter, 143 mm long; packing material, Hypersil ODS (3 pm); mobile phase, 2 mM sodium tetraborate (pH 8.7) 80% Acetonitrile; sampling 1.5 kV for 4 s; sample, thiourea [3.51].
Comparing the values listed Table 3.2 and Table 3.3, the electroosmotic mobility obtained for open-tubular capillary columns is generally higher compared with packed columns and it is reasonable to employ structural parameters for comparing the values.
3.2
Electroosmotically Driven Chromatography and Electrochromatography
In the following discussion, electrochromatography means that the solute is charged, and the electrophoretic mobility contributes to the separation. Chromatography means that the solute is neutral, and separation depends on partition under electroosmotic flow. Without pressurized flow, separation proceeds by electroosmosis, electrophoretic mobility, and partition with respect to the stationary phase. The separation efficiency is governed by the electroosmotic flow profile and by the velocity profile due to electrophoretic mobility. The plug-like flow profile of electroosmosis is advantageous for separation, especially if the capacity factor of the solutes is < 2. This effect on column efficiency decreases gradually to capacity factor > 2 [3.56], [3.57].
3.2.1
Electroosmotically Driven Electrochromatography
A typical example of electroosmotically driven electrochromatography is shown in Figure 3.7, in which a 50 cm X 10 pm open-tubular capillary column coated and crosslinked with 0.9 % (wthol) PS-264 (Petrarch Systems) was used with a laser-based fluorescence detector. Yeung et al. [3.20] have separated anions, using both their partition coefficients and electrophoretic mobilities under electroosmosis. They were able to separate anionic compounds with identical mobilities. The different separations obtained with capillary zone electrophoresis (CZE), electroosmotically driven electrochromatography, and open-tubular capillary liquid chro-
60
3 Electroosmosis and Electrochromatography
matography are also shown in Figure 3.7. CZE does not provide effective separation of naphthalensulfonic acid isomers, and open-tubular liquid chromatography has difficulty separating 2-amino-1-naphthalenesulfonicacid and 8-amino-2-naphthalenesul-
(4
4A1 N, 5A2N
-8A2N, 1H4N
-
-2A1N
c
.
1
2
0
4
6
10
8
1H4N
-
I
I
I
2
0
4
(4
1H4N
6
-I 2AiN
4AlN-
Y
0
2
k
.
4 Time (min)
-
iLI.
6
8
Figure 3.7. Comparison of elution profile obtained using A) CZE; B) Electroosmotically driven electrochromatography; C) Open-tube liquid chromatography 13.201. A) Column, 50 cm X 10 pm, uncoated, 40 cm separation distance; eluent, 10 mM phosphate buffer, pH 7.0, containing 1.25 mM tetrabutylammonium hydroxide; applied voltage, +21 kV; B) Column, 50 cm x 10 pm, coated with PS264; applied voltage, +21 kV; other condition as for (A); C) Column, as for (B); applied voltage, 0; Peaks, 4A1N = 4amino-1-naphthalenesulfonic acid, 5A2N = 5-amino-2naphthalenesulfonic acid, 8A2N = 8-amino-2naphthalenesulfonic acid, 1H4N = 1-naphthol-4-sulfonic acid.
3.2 Electroosmotically Driven Chromatography and Electrochrornatography
61
fonic acid (there is only a small difference in the partition coefficients and electrophoretic mobilities of these two peaks). Electrochromatography can solve this problem, providing complete separation.
3.2.2
Electroosmotically Driven Chromatography
Electroosmotic elution of neutral compounds has been used in open-tubular capillary columns 13.71, 13.191, 13.551, packed microcapillary (drawn packed capillary) columns [3.5],[3.62], and slurry-packed capillary columns [3.9], [3.51], [3.64] as an alternative method to pressurized flow elution.
3.2.2.1 Open-Thbular Capillary Columns Contribution of Flow Profile to Theoretical Plate Height. In an open-tubular column, the channels are completey straight and the separation mechanism is easy to treat theoretically. Pretorius et al. 13.21 used a column of 1mm inner diameter and 50 cm long, and found that the theoretical plate height H for the nonretained peak (benzene) in electroosmotic flow is half that obtained with pressurized flow. However, they did not show any chromatograms. Tsuda et al. [3.7] used an open-tubular capillary column, 42 cm long and 30 pm inner diameter, in which the inner surface was modified with ODS, to separate aromatic compounds (Fig. 3.8). The relation between H a n d vosmis shown in Figure 3.9. If we calculate the value of H for pyridine as: H = ?v124Dm
(32)
This equation is for open-tubular liquid chromatography; v = 0.09 c d s , column radius r = 61 pm, and the diffusion coefficient of pyridine in pure water D , = 9.2 X lo4 cm2s (estimated value), the calculated value of H = 152 pm [3.7]. If we assume that the flow profile in the capillary tube is plug-type and molecular diffusion alone is responsible for zone broadening, (i. e., axial diffusion), the value of H for a nonretained solute is obtained as follows:
d=2Dt
(33)
H = dlL
(34)
where t and L are the elution time and column length. The calculated value is 2 pm; the value obtained by experiment is 5.2 pm, about one-thirtieth the value in pressurized flow, and more than twice the calculated value. Therefore the assumption does not hold; the band broadening for a nonretained solute is due, not only to molecular diffusion, but also to the electroosmotic flow profile in the capillary tube. Although these experimental values were obtained in the early days of electroosmotically driven chromatography, it is clear that the electroosmotic flow profile is much flatter compared with pressurized flow and a little less flat than plug flow. This flow profile raises the question of the true nature of electroosmosis. Comparison of Plate Height for Pressure-Driven and Electroosmotically Driven Systems. Axial diffusion and slow equilibrium in the mobile and stationary phases are the main processes contributing to band broadening in open-tubular liquid chromatography. This is expressed in the theoretical plate height equations derived by Golay [3.59]
62
3 Electroosmosis and Electrochromatography
I
I
15
20
I
I
I
10
5
0
Elution
I
,
30
20
Time
( min)
I
I
0
10
Elution Time
b i n )
Figure 3.8. Separation of aromatic compounds on ODS capillary column [3.7]. A) Column, 42 cm X 30 pm inner diameter; eluent, acetonitrile-water (40:60); voltage, 13 kV, samples: (1) benzene, (2) naphthalene, (3) biphenyl, (4) fluorene + anthracene, (5) p-terphenyl, (6) chrysene, and (7) 1,3,5-triphenyIbenzene; B) Eluent: acetonitrile-water (30: 70); sample, as in (A) except ( 5 ) pyrene; other conditions as above.
for a pressurized flow system (PD), and by Martin and Guiochon [3.56] and Giddings [3.46] for an electroosmotic flow system (ED). The overall plate height equation for open-tubular capillary liquid chromatography is valid in both PD and ED systems: H
=
2 0 , (v)-’ + C,d;v ( D J 1
+ C, d:v
(DJ’
(35)
where D, and D,are the diffusion coefficients in the mobile and stationary phases, d, is the inner diameter of the column, and df is the thickness of the stationary phase layer. For the pressurized flow system: C,,,,
=
(1
+ 6k’ + Ilk’) [96 (1 + k’)]-’
(36)
3.2 Electroosmotically Driven Chromatography and Electrochromatography
0
0.1
v
0.2
(cm/sec)
63
Figure 3.9. Relationship between H and v [3.7]. Pyrex capillary tube, 60 cm x 132 pm;solvent: (circles) 0.025 M Na2HP04-H20,(triangles) 0.1 M Na2HP04-H20;solute, pyridine; applied voltage, (1) 193, (2) 136, (3) 96, and (4)96 V/crn.
and for the electroosmotic flow system, assuming a perfectly flat flow profile: C,,,,
=
k’2 [16 (1
+ k’)2]-1
(37)
The main difference between the ED and PD systems is the flow profile. This is expressed in different plate height curves, calculated by equations (35)-(37), and shown in Figure 3.10A, B for a 25 and a 10 pm inner diameter capillary and three k’ values. For a 25 pm inner diameter capillary the gain in efficiency by using E D instead of a PD system is almost threefold [3.55]. For a 10 pm inner diameter capillary, the same phenomenon occurs, but the slow mass transfer term is much less significant. In Figure 3.11, the term C,,, is plotted against k’. Both functions increase with increasing retention. It is also clear that C,,,, ED is always smaller than Cm, PD. The improvement is very large at low k’, but is still greater by a factor of 11/6 for k’ up to infinity. The lower C,,,, ED results in a lower plate height for the E D system when the flow profile of electroosmosis is plug-type [3.55]. Optimization of capillary columns based on the theory of capillary gas chromatographic operational conditions shows that a capillary column of inner diameter 2.5 to 10 pm is suitable in laminar flow when the mean linear flow velocity of open-tubular capillary liquid chromatography is the same or one-tenth of the valve for capillary gas chromatography. If a 25 pm inner diameter capillary E D system is compared with a 10 pm inner diameter PD system (Fig. 3.11), the increase in plate height is only a factor of three 13.551. This opens up the possibility of using larger diameters in electroosmotically driven open-tubular liquid chromatography, which has the advantage of fewer practical problems with regard to detection, loadability, and column preparation, again provided that the flow profile is plug-type [3.7], [3.55]. Relationship Between Reduced Plate Height and Linear Velocity. The real flow profile is supposed to be a little different from the plug profile [3.29], [3.69] [Chapter 2, Section 51, and the above supposition should be checked by experiment. Comparison of the reduction in plate height due the type of flow profile and column are examined in
64
3 Electroosmosis and Eiectrochromatography A
80
60
n
E5.
W
40
*aw
k' = 1 70
X
k' = 0
0
€3
n
E
3. W
n b
w
X
k' = 3
k' = 1 / k ' = O 0
08
0.4
v
1.2
1.6
(mm/s)
Figure 3.10. Theoretical HETP ( H ) curves for an electrosomotically driven system (ED) (solid lines) with A) 25 mm inner diameter capillary and B) 10 ym inner diameter capillary for three different k' values, f3.551.
65
3.2 Electroosmotically Driven Chromatography and Electrochromatography
a W
5
6
0.M
8 \
a
nd
4 0
0
k’ Figure 3.U. C, functions for a pressurized flow-driven system (dashed line) and for an electroosmotically driven system (solid line), plotted versus k’ [3.55]. Dot-dashed line, Cm, pdCm,ED.
Figure 3.12. Electroosmotic flow gives a slightly lower plate height. From the curves obtained by electroosmotic flow, we can see: 1) in the region below 1m d s , the effect of diffusion; 2) in the region above 1-3 m d s , the effect of mass transfer in both mobile phase and stationary phases, and between them. Clearly, there is some advantage in using electroosmotic flow to reduce plate height. An excellent chromatogram obtained by electroosmotically driven chromatography with a 10 pm open-tubular capillary column is shown in Figure 3.13 [3.55],for a capillary column with a porous silica layer and on-column laser-induced fluorescence detection [3.59]. Eight polycyclic aromatics (k’ = 0.0-0.24) are separated in less than 400 s, with baseline resolution in only 90 s. Town and Regnier [3.30] reported the separation of proteins using an octadecylsilane-modified capillary column with 0.01 M phosphate buffer (pH 7) containing 0.001 % surfactant (10-100 times less than the critical micelle condition; Fig. 3.14). Although they classify their report as capillary electrophoretic separation, it is really electroosmotically driven electrochromatography. Even though the inner diameter of capillary tubing is as large as 75 pm, the chromatograms show very fine separation. The differences in the chromatograms of Figure 3.14A and B may be due the different stationary phase conditions, as the TWEEN 20 and BRIJ are adsorbed on the ODS stationary phases. From their experiments, we can not estimate how much contribution the electrophoretic process and partition process each make during the solute travel in the column. At least it is certain that the partition process plays some role in the separation. We need to determine the optimum inner diameter for open-tubular capillary column chromatography with electroosmosis only. We must also keep in mind that if the capacity factor is greater than 2, the contribution of the mobile phase becomes relatively smaller compared with the contribution of the stationary phase, and
66
3 Electroosmosis and Electrochromatography
a
I
I
0
1
I
I
I
I
I
I
I
I
I
2 4 6 Linear V e l o c i t y (mm/s)
1I
Figure 3.12. Relation between reduced plate height and flow velocity. Slurry-packed capillary column 143 mm long, 50 pm inner diameter, packed with Hypersil ODS (3 pm); driving force, electroosmotic flow; mobile phase, 2 mM sodium tetraborate (pH 8.7) + 80 % acetonitrile; sampling, 1.5 kV for 4 s; sample, thiourea [3.51]; B) Slurry-packed capillary column 620 mm long, 50 pm inner diameter, packed with 1.6 pm Monospher ODS; driving force, electroosmotic flow; applied voltage, 5-35 kV; mobile phase, 4 mM sodium tetraborate + 60 % acetonitrile; sample, thiourea t3.511; C) Drawn packed capillary column 40 pm inner diameter; packing support, Hypersil 5 pm; driving force, pressurized flow; mobile phase, 70 % acetonitrile + 30% water containing sodium dihydrogen phosphate 2-6 mM (plate height independent of electrolyte concentration); sample, fluorene, non-retained under these conditions [3.52]. D) Driving force, electroosmotic flow; other experimental conditions as C t3.521; E) A slurry packed capillary column of 50 pm inner diameter, packed with 1.5 km Monospher; driving force, electroosmotic flow;other conditions as C t3.521.
the important factor to obtain good separation depends on the quality of the stationary phase, in which the diffusion coefficient between solute and molecules composed of stationary phase needs to be relatively high. The stationary phase must also be very thin for rapid equilibrium with the mobile phase (i. e., minimal mass transfer between stationary and mobile phases). 3.2.2.2 Packed Microcapillary (Drawn Packed Capillary) Columns
Characteristics of Packed Microcapillary Columns. Packed microcapillary columns (PMC) were proposed by Tsuda and Novotny [3.33], [3.34]. Their preparation is described in [3.34] and [3.52]. In principle, PMCs are made by first packing a thickwalled tube with a packing such as silica gel (the particle size can be 3-SO pm), and then drawing this tube down to the required final diameter. All packing procedures should be carried out very carefully to maintain the dryness of the packing materials and the glass tubing [3.33], [3.34], [3.52]. Electron micrographs of a cross-section of drawn packed capillary, ca. SO pm inner diameter and packed with 10 pm silica gel, are shown in Figure 15, and the plane of cross-section was inclined at 25". The packing state of silica gel in the PMC has been considered to be loose packed compared with
3.2 Electroosmotically Driven Chromatography and Electrochromatography
67
2
6
5
3
Figure 3.W. Chromatograms of eight polycyclic aromatics with 10 pm PSL (etched porous silica
.. I
I
0
5
I 6 ___ct
(rnin)
L_
I 7
layered)-ODS capillary. Applied voltage, 20 kV, column length 49 cm (from injection to detection 26.5 cm); current 0.7 FA; temperature, 25 “C; injection volume, 9 pL; mobile phase, 0.05 M phosphate buffer (pH 7) + methanol (1: 1). Peaks are (1) naphthoquinone (k’ = 0); ( 2 ) 9-anthracenemethanol ( k ” = 0.03); (3) 9-anthracenecarbonitrile; (4) anthracenecarbonitrile; (4) anthracene; (5) 7,8benzoflavone; (6) fluoranthene; ( 7 ) pyrene; (9) 9-vinylanthracene (k’ = 0.24) [3.55].
the packing state of conventional columns, but electron micrographs show it to be rather like that of a slurry-packed conventional column. However, flow resistance parameters Qi [3.60], [3.61] of the columns are about 100 [3.34], [3.52], very low compared with those of conventional columns (usually 500-1000). So the present column has a very desirable feature; it is well packed, but highly permeable. Some silica gel particles in Figure 3.15 are observed to have been inserted partly in the glass. For a more precise view of these conditions, an electron micrograph at higher magnification, ~ 7 5 0 0is, shown in Figure 3.15B. About one-tenth of the silica gel particles per cross-section were held in the glass wall, stabilizing the other silica gel particles; this is why the drawn capillary column was stable under a pressure of 200 atm per metre of column length. As silica gel is stabilized by the column materials, no frit was necessary at the ends of the PMC.
68
3
Electroosmosis and Electrochromatography
A
T O0IAU
z
8 c! W
V
T
,002AU
1
W
0
z
2
a
U
51 m
v,
m
m a
[L
0
m
a
a
.r.A
u I
I
1
I
I
4
8
12
16
20
Time (rnin)
Figure 3.14. Electrochromatograms obtained with an ODS capillary column doped with surfactants. Protein samples are: 1) Lysozyme; 2) Cytochrome c, 3) Ribonuclease A, 4) a-chromotrypsinogen; 5) Myoglobin. Columns preparation: inner surface modified with ODS, and doped with 0.5 % surfactant-aqueous solution for 2 h, followed by wash-out. A) Tween 20; B) BRIJI 35 [3.30].
Application of Drawn Packed Microcapillary Columns for Electroosmotically Driven Chromatography. The first application of this column was descried by Tsuda et al. (Fig. 3.16) [3.62]. Knox et al. have obtained good chromatograms by using an ODs-column t3.521, [3.63]. They used on-column fluorescence detection. In Figure 3.17, two chromatograms obtained by pressurized flow and electroosmotic flow are shown. Electroosmotic flow gives better peaks, with a slightly increased theoretical plate height. The effects of electrolyte concentration on electroosmotic velocity and plate height for a drawn capillary column packed with 5 pm Hypersil were examined. The electrolyte concentration in this case has little effect on the electroosmotic flow - 2 x lo-' M, but a significant improvement in plate velocity over the range 4 x efficiency occurs with increasing electrolyte concentration. Thus, with the 5 pm Hypersil, the reduced plate height is from 3-4 to about 1 as the electrolyte concentration is increased. The compromise which best maintains a high electroosmotic velocity and yet gives a minimum HETP is around 0.002 M. When they applied the voltage at electrolyte concentration > 0.006 M, they encountered the problem of bubble formation [3.52]. The electrolyte concentration depends on the efficiency of heat dissipation of the column. With a mixture of water and organic solvent as eluent, it is possible to use a concentration up to 20 mM [3.47].
3.2 Electroosmoticalfy Driven Chromatography and Electrochromatography
69
Figure 3.15. Electron micrographs of a cross-section of a packed microcapillary column. A) Magnification x 1500; B) detail of A x 7500 [3.34].
' I
I
2 0
1 0
'
Elution Time
(min)
Figure 3.16. Electrochromatogram from a packed microcapillary column 40 cm long, 42 pm inner diameter. Applied voltage, 10 kV; sample, aromatic compounds[3.62].
70
3 Electroosmosis and Electrochromatography
0
0
10
10
20
20 Elullon tlme
30
I
40
(mln)
Figure 3.17. Separation of an aromatic test mixture with pressurized (A) and electroosmotically driven chromatography (B). Column: drawn capillary packed with 3 pm Hypersil and subsequently derivatized by ODS, inner diameter 30 pm, 900 mm (A) and 800 mm (B) long. Solutes in order of elution: 1) Naphthalene; 2) 2-Methylnaphthalene; 3) Fluorene; 4) Phenanthrene; 5) Anthracene; 6) Pyrene; 7) 9-Methylanathracene [3.52].
3.2.2.3 Slurry-Packed Capillary Columns Electroosmotically driven chromatography using a slurry-packed column (its packing procedure was described in I3.351) was first reported by Pretorius et al. [3.2] for a packed column of 50 cm x 1mm inner diameter. Jorgenson et al. [3.15] used a narrow packed capillary column (68 cm x 0.17 mm inner diameter, packed with 10 pm ODSsilica), and a reduced plate height of 1.9 was obtained for a peak eluting at ca. 30 min. Tsuda et al. [3.9], [3.64] have demonstrated the possibility of separating aromatic compounds in a short analysis period by applying 12.4 kV (1.3 kV/cm) across a 9.1 cm x 400 prn capillary column, slurry-packed with 3 pm ODS (Fig. 3.18). Another group produced an excellent chromatogram obtained with a slurry-packed capillary column (Fig. 3.19) [3.65]. These columns and a system for electrochromatography, are commercially available. The fastest analysis by the present method has been demonstrated by Erni et al. [3.51] for a packed capillary column (143 mm long and 50 pm inner diameter, slurrypacked with Hypersil ODs, 3 pm particle diameter, Fig. 3.20). They examined the relation between applied voltage and linear velocity due to electroosmosis (Fig. 3.12). They found an almonst linear relationship, and remarked that it is possible to apply up to 2 kV/cm (total applied voltage 55 kVfor the packed column 143 mm long and 50 pm inner diameter) and obtain ca. 6 m d s electroosmotic flow. However they admitted that the electroosmotic flow became unstable above 50 kV applied voltage and higher than expected velocity (Fig. 3.12). In references [3.16] and [3.17], this curve is not linear in the region of high applied electrovoltage and is generally lower than expected.
3.2 Electroosmotically Driven Chromatography and Electrochromatography
71
A
1
I
Figure 3.18. Chromatogram showing separation of neutral compounds by electroosmotically driven chromatography. Columns: 9.1 cm X 0.4 mm, slurry-packed with 3 pm ODS (Develosil); eluent, 100 % methanol; applied voltage, 12.4 kV (current 2 FA). Peaks: A) Methyl benzoate; B) Anthracene; C) Pyrene (capacity factor 1.2) [3.9].
I
3 6 Time (min)
0
3 N = 176,000 plares/metet
J
I
I
1
I
I
I
0
3
6
9
12
15
Elution Time
= 178,000
Figure 3.19. Separation of aromatic mixture by electroosmotically driven chromatography. Mobile phase: acetonitrile + 4 mM borate buffer pH 8.0 (80:20); applied voltage, 20 kV, column: Electrochrom, 50 pm X 30 cm, packed with ODS 3 pM; ambient temperature; injection: electrokinetic at 20 kV, 0.08 min; detection: U V 254 nm, 50 pm on column. Samples: 1) Benzene; 2) Naphthalene; 3) Biphenyl; 4) Fluorene; 5) Anthracene; 6) Fluoranthene [3.65].
1 (
I
I
18
21
min)
72
3 Electroosmosisand Electrochromarography m
.-Ca
F
5
Figure 3.20. Rapid separation by electroosmotically
0
N
Q.!
driven chromatography.
a u
3E:
5!
,
!
Elution Time
3.2.3
(min)
Mixture of Isradipin, its byproducts, and thiourea. Column: 50 pm inner diameter, 143 mm long, packed with Hypersil ODS (3 prn); applied voltage, 30 kV;other conditions as in Figure 3.6. Flow velocity of electroosrnosis, 2.6 m d s (1.8 PA).
Advantages of Electroosmotic Flow for Liquid Chromatography
The use of the electroosmosis as the driving force has the following advantages: 1) Its flow profile is much flatter compared with the flow profile of pressurized flow (a high theoretical plate number is obtained especially for samples with capacity factor less than 2). 2) We can generate electroosmotic flow simply by applying high voltage along the column. This is a superior characteristic compared with pressurized flow. Therefore it is possible to use soft gels, such as agarose or polyacrylamide. These experimental conditions are used in capillary zone electrophoresis [3.66], [3.67]. However, in the field of liquid chromatography, we may have a chance to develop extremely useful columns by using soft gels or packing supports which could not be used for conventional liquid chromatography owing to their lack of hardness. Hjerten et al. [3.68] have used a solidified polyacryamide gel for electroosmotically driven chromatography. 3) Electroosmotic flow can pass through very fine micropores, unlike pressurized flow under ordinary pressure. Therefore it is possible to use crays or sludges [see also Chapter 7 and 81. 4) We can operate liquid chromatography without a mechanical pump. Therefore we may construct a very tiny liquid chromatography system. This might be helpful in a specialized field such as clean rooms for producing semiconductors. There are also disadvantages in using electroosmotic flow: 1) On applying a high voltage along a column, we always generate heat. Therefore it is necessary to dissipate it effectively. If we use a high potential gradient to obtain a high linear electroosmotic velocity, we need to use a capillary column. When we use a column of more than 1 mm inner diameter, we should be very careful to avoid bubble formation inside the column. It is essential to use a moderate or low potential gradient along the column. 2) Once a bubble is formed inside the column, we have to use pressurized flow to exclude the bubble from the column. The formation of bubbles means that we cannot continue operation because the electric current through the column will be unstable or stopped, and electroosmotic flow inside the column ceases. 3) We need to pay attention to the safety with the use of high voltages. Disadvantage (2) can be overcome by using a low pressurized flow during the run. For disadvantage (l),if we use an organic solvent as developing eluent or an aqueous
3.3 References
73
solvent with a low electrolyte concentration, generally the current falls to a low value, and we can avoid excessive heating inside the column. We still need to improve the stability of electroosmotically driven electrochromatography. The potential advantages of the technique encourage its development for use in specific fields.
3.3
References
[3.1] S. HjertCn, Chromatogr. Rev., 9, 122 (1967). [3.2] V. Pretorius, B. J. Hopkins, J. D. Schieke, J. Chromatogr., 99, 23 (1974). [3.3] E E. P. Mikkers, F. M. Everaerts, T. P. E. M. Verhaggen, J. Chromatogr., 169, 11 (1979). [3.4] J. W. Jorgenson, K. D. Luckacs, Anal. Chem., 53, 1298 (1981). [3.5] J. W. Jorgenson, K. D. Lukacs, J. Chrornatogr., 218, 209 (1981). [3.6] T. Tsuda, K. Nomura, G. Nakagawa, J. Chrornatogr., 264, 385 (1983). [3.7] T. Tsuda, K. Nomura, G. Nakagawa, J. Chromatogr., 248,241 (1982). [3.8] T. Tsuda, G. Nakagawa, M. Sato, K. Yagi, J. Appl. Biochem., 5, 330 (1983). [3.9] T. Tsuda, J . Chem. SOC.Jpn. Chem. Ind. Chem., 937 (1986). [3.10] T. Tsuda, J. High Resolut. Chromatogr. Chromatogr. Commun., 10, 622 (1987). [3.11] T. ’ISuda, Shimadzu Sci. Instrum. News, 28, 14 (1987). [3.12] T. Tsuda, J. Liq. Chromatogr., 12, 2501 (1989). [3.13] T. Tsuda, Chromatography, Maruzen, Tokyo (1989). [3.14] M. Martin, G. Guiochon, Anal. Chem., 56, 614 (1984). [3.15] M. Martin, G. Guiochon, Y. Walbroehl, J. W. Jorgenson, Anal. Chem., 57, 559 (1985). [3.16] C. Terabe, K. Otsuka, T. Ando, Anal. Chem., 57, 834 (1985). [3.17] X. Huang, W. F. Coleman, R. N. Zare, J . Chrornatogr., 48, 95 (1989). [3.18] X. Huang, M. J. Gordon, R. N. Zare, Anal. Chem., 60, 1837 (1988). [3.19] W. D. Pfeffer, E. S. Yeung, Anal. Chem., 62,2178 (1990). [3.20] W. D. Pfeffer, E. S . Yeung, J. Chromatogr., 557, 125 (1991). [3.21] C. S. Lee, W. C. Blanchard, C.-T. Wu, Anal. Chem., 62, 1550 (1990). [3.22] C. S. Lee, D. McManigill, C.-T. Wu, B. Patel, Anal. Chem., 63, 1519 (1991). [3.23] C. S. Lee, C.-T. Wu, T. Lopez, B. Patel, J. Chromatogr., 559, 133 (1991). [3.24] C.-T. Wu, T. Lopez, B. Patel, C. S. Lee, Anal. Chem., 64, 886 (1992). [3.25] C.-T. Wu, C. S . Lee, Anal. Chem., 64, 2310 (1992). [3.26] K. Ghowski, R. J. Gale, J . Chromatogr., 559, 95 (1991). [3.27] M. A. Hayes, A. G. Ewing, Anal. Chem., 84, 512 (1992). [3.28] M. A. Hayes, I. Kheterpal, A. G . Ewing, Anal. Chem., 65, 27 (1993). [3.29] T. Tsuda, “Control of electroosmotic flow in capillary electrophoresis”, Chap. 22, in Handbook of capillary electrophoresis, J. P. Landers (ed.), CRC, Ann Arbor, 1993. [3.30] J. K. Towns, F. Regnier, Anal. Chem., 63, 1126 (1991). [3.31] J. K. Towns, F. Regnier, Anal. Chem., 64, 2473 (1992). [3.32] T. Tsuda et al. J. Chromatogr., 158, 227 (1978). [3.33] T. Tsuda, M. Novotny, Anal. Chem., 50,271 (1978). [3.34] T. Duds, I. Tanaka, G. Nakagawa, Anal. Chem., 56, 1249 (1984). [3.35] Y. Hirata, K. Jinno, J. High Resolut. Chromatogr. Chromatogr. Commun., 6, 196 (1983). [3.36] D. Ishii et al. J. Chrornatogr., 151, 147 (1978). [3.37] K . K. Unger, “Porous silica”, J. Chromatogr. Library, 6, 1979; K. Nobuhara, private communications, 1993. [3.38] R. J. Hunter, Zeta potential in colloid science, Academic Press, New York, 1981. [3.39] G. Gouy, J. Phys. Chem., 9, 457 (1910). [3.40] D. L. Chapman, Phil. Mag., 25,475 (1913). [3.41] H. G. Th. Overbeek, in Colloid Science, vol. 1, H. R. Kruy (ed.), Elservier, Amsterdam, 1952. [3.42] M. Smoluchowski, in Handbuch der Elektrizitat und des Magnetismus (Graetz), vol. 11, Barth, Leipzig, 1921, p. 366.
74
3 Electroosmosis and Electrochromatography
[3.43] P. Delahay, Double layer and electrode kinetics, Wiley Interscience, New York, 1965. [3.44] R. A. Wallingford, A. G. Ewing, ...Capillary electrophoresis”, Adv. Chromatogr., 29, 1 (1989). [3.45] C. L. Rice, R. Whitehead, J. Phys. Chem., 69 (ll), 4017 (1965). [3.46] J. C. Giddings, Dynamics of Chromatography, Dekker, New York, 1965. [3.47] S. Kitagawa, T. Tsuda, J . Microcol. Sep. 6, 91 (1994). [3.48] P. Velaha, Double layer and electrode kinetics, Interscience, New York, 3rd ed., 1976. [3.49] T. Tsuda, 1983, unpublished work. [3.50] K. D. Lukas, J. W. Jorgenson, J. High Resolut. Chrornatrogr., 8,407-411 (1985). [3.51] H. Yamamoto, J. Baumann, F. Erni, J. Chromatogr., 593,313,319-328 (1991). [3.52] J. H. Knox, I. H.Grant, Chromatographia, 32, 317-328 (1991). [3.53] H. J. Cortes, C. D. Pfeffer, B. E. Richter, T. S. Stevens, J. High Resolut. Chromatogr. Chromatogr. Commun., 10,446-448 (1987). [3.54] T. Tsuda, M. Inagaki, 1993, unpublished work. [3.55] G. J. M. Bruin, P. P. H. Tock, J. C. Kraak, H . Poppe, J. Chromatogr., 517, 557-572 (1990). [3.56] M. Martin, G. Guiochon, Y. Walboel, J. W. Jorgenson, Anal. Chem., 57, 559-561 (1985). [3.57] M. Martin, G. Guiochon, Anal. Chem., 56, 614-620 (1984). [3.58] M. J. E. Golay, in D. H. Desty (ed.), Gas Chromatography 1958, Butterworths, London, 1958, p. 36-55. [3.59] P. P. H. Tock et al., Chromatographia, 24,617 (1987); J . Chromatogr., 477, 95 (1989). [3.60] J. H. Knox, M. T. Gilbert, J. Chrornatogr., 186, 405-418 (1979). [3.61] J. H. Knox, J. Chromatogr. Sci., 18, 453-461 (1980). [3.62] T. Tsuda, I. Isao, G. Nakagawa, 3rd Symp. Liquid Chromatography (Ekitai Kuronmatogurafi Toronkai), Abstracts p. 41-42, Oct. 20-21, 1982, Tokyo. [3.63] J. H. Knox, I. H. Grant, Chromatographia, 24, 135 (1987). [3.64] T. Tsuda, 2nd Symp. Liquid Chromatography (Ekitai Kuronmatogurafi Shotoronkai), Abstracs, p. 13-14, May 10, 1985, Osaka. [3.65] J. P. Chervet, brochure published by LC Packings, Switzerland, p. 16, 1992. [3.66] A. S. Cohen, A. Paulus, B. L. Karger, Chromatographia, 24, 15 (1987). [3.67] J. P. Landers (ed.), Handbook of capillary electrophoresis, CRC, Ann Arbor, 1993. [3.68] S. H j e r t h et al., 17th Intern. Symp. Column Liquid Chromatogr., Hamburg, May 9-14, 1993. [3.69] T. Tsuda et al., J. Chromatogr., 632, 201-207 (1993). [3.70] D. L. Mould, R. L. Synge, Biochem. J., 58, 571 (1954).
4
Electrochromatography with Radial Applied Voltage: Ion Separation by Electrochemical Approach Tsutomu Nagaoka
4.1
Introduction
This chapter describes electrochemical chromatography with a conductive stationary phase, which allows one to apply voltages in the direction normal to the stationary phase surface, not along the column as in standard electrochromatography [4.1]-[4.10]. The technique introduces the stationary phase potential as a parameter to control chromatographic separation, with several new features such as optimal retention (adjusted by the potential), concentration and elution of analyte on a single column, and potential gradient elution. This chapter focuses on the separation of electroinactive species that cannot be separated electrochemically. Electroinactive species are not reduced or oxidized at electrodes because their redox potentials are too low or too high. Figure 1 gives the basic principles of electrochemical chromatography, illustrating the conductive station-
layer Figure 4.1. Separation of ionic substances on conductive stationary phases. A) The direct electrostatic interaction; B) The indirect electrostatic interaction with the functional layer.
76
4
Electrochromatography with Radial Applied Voltage
ary phase particles. In this technique, the total charge at the surface can be controlled externally. Thus, as the potential of the stationary phase becomes more negative, the stationary phase accumulates negative charge (electrons). Similarly, the stationary phase accumulates positive charge as the potential increases. The voltage applied between a stationary phase and counter-electrode is usually less than a few volts thanks to a supporting electrolyte (an indifferent electrolyte) added to the mobile phase. Figure 4.1A illustrates a model for direct electrostatic interaction between an analyte cation and the charged stationary phase. As the potential of the stationary phase decreases, the interaction becomes strong, resulting in a larger elution volume. Both ionic species and neutral compounds can be separated on the conductive column; neutral molecules should adsorb most strongly at the zero charge potential (i. e., the potential at which no charge accumulates at the solution/electrode interface). The direct electrostatic model can be extended to the indirect model (Fig. 4.1B). The functional layer on the surface can be reduced or oxidized to take up ionic or neutral species, and enhanced selectivity is expected because of specific interaction with such species. The conductive stationary phases discussed here are confined mostly to carbonaceous materials, because their porous surfaces are easily modified with the various functional substances that have been studied extensively in electrochemistry [4.11].
4.2
Experimental Details
4.2.1
Design of the Electrode Column
The electrode columns usually contain carbon particles packed in a separator tube, with a counter-electrode and a reference electrode placed near the tube. The chromatographic system (Fig. 4.2) consists of standard HPLC components, the column electrode, and a potentiostat to control the potential of the stationary phase (the working electrode) versus the reference electrode. Figure 4.3 shows the electrode column used in our laboratory [4.2]-[4.6]. The column is made up of the conductive stationary
Drain Figure 4.2. Block diagram of an electrochemical chromatographic system. Conditions used in our experiments are given in the figure.
4.2 Experimental Details
77
Figure 4.3. Cross-sectional diagram of the electrode column. A) Counter-electrode (SUS316 stainless steel net); B) Vycor glass tube; C) Conductive stationary phase; D) Pt wire for stationary phase; E) PTFE tube; F) PTFE plug; G) Ag/AgCl wire; H) Counter-electrode compartment filled with 10 mM KCl, H20; I) 0-Ring; J) F'TFE porous filter.
phase, tightly packed into a Vycor glass tube separator (internal diameter 4.5 mm, effective length 4-16 cm), a counter-electrode (stainless steel net) encircling the separator, and reference electrodes. The counter-electrode compartment is filled with 10 mM tetraethylammonium perchlorate (TEAP) aqueous solution unless otherwise stated. The positions of the reference electrodes are important to obtain a uniform distribution of potential throughout the column. The reference electrodes keep to potential of the stationary phase constant; the potential of the counter-electrode varies during an experiment. The reference electrode used in our system is Ag AgCl I saturated KC1 I (SSE) placed every 4 cm along the column or an AgC1-coated Ag wire coiled around the Vycor glass. With the latter, open electrode, the counter-electrode compartment is filled with 1M KCI to keep the potential of the wire constant. The capacity factors of halide ions on a polyaniline-coated stationary phase, which is discussed later, are almost independent of the column length (k' = 8.6 k 0.2) [4.6]. The independence suggests that the potential is fairly even. The steady state of current the column is about 100 FA. The mobile phase used in this technique should contain a supporting electrolyte, which increases the magnitude of the electric field at the mobile phase/stationary phase interface. In addition, the separator tube must be porous to establish the ionic conductivity between the counter-electrode and stationary phase. A tube of Nafion, Vycor glass, or porous stainless steel has been used for this purpose [4.2]-[4.10].
I
4.2.2
Preparation of Stationary Phases
Table 4.1 summarizes the characteristics and preparation procedures of the stationary phases that have been reported in the literature. To achieve high selectivity, the stationary phases are modified with functional substances such as oxidized carbon, crown ethers, or conductive polymers. The substrate is usually carbon powder and can be modified electrochemically after being packed (columns 4, 6, 7, 9 in Table 4.1), or either chemically or electrochemically before being packed (2, 3, 5 , 8).
a
ion-exchange with polymer
Vinylferrocene/ maleic anhydride copolymer Crown ether on carbon
Polypyrrole on carbon
n-x interactions with
Polypyrrole on carbon
+
anion insertion into the oxidized polymer cathodic stripping
the polymer
anion insertion into the oxidized polymer
Polypyrrole on carbon
Polyaniline on carbon
carbon particlese (- 40 pm) coated by percolating a polymer solution through the column activated carbon' powder (- 45 pm) covered with a DCEgsolution containing 50 mM dibenzo-18-crown-6 50 mM TBA . TPhBh polymer deposited at +0.95 V on GC' (- 75 pm) from aqueous 0.5 M aniline and 1 M HCI for 5-20 min polymer deposited at + 1.0 V on G C powder (- 75 pm) from aqueous 0.1 M pyrrole and 0.1 M NaCl for 10 min polymer electrodeposited on RVC' (< 40 pm) from aqueous 0.2 M pyrrole and 0.1 M sodium dodecylsulfate
direct electrostatic adsorption
Oxidized carbon
electrostaic ion transfer induced by formation of a crown ether complex anion insertion into the oxidized polymer
carbon powderb modified with trimethyl chlorosilane after oxidation with r. f. plasma GC' powder (- 45 pm) oxidized in air at 400 "C for 4 h
electrosorption on hydrophobic surface
polymer deposited on GCa (70-230 pm) from 0.1 M pyrrole and 0.1 M sodium p-toluenesulfonate
+
GC" (60-100 mesh), Ag (30-40 mesh) grain
Silated carbon
+
electrodeposition anodic stripping
Glassy carbon
Preparation
Separation
Stationary phase
Mobile phase
anthracene, naphthalene, m-toluic acid, benzoic acid adenosine triphosphate, monophosphate
halides, SCN-, carboxylate,
halides, SCN-, [Fe(CN)6]4"-
alkali metal ions
10 mM TEAPd in H,O
H', alkali metal ions, alkaline earth ions methylviologen
+
-
10 mM LiC104 (pH 5.0)
CH30H/H20 CH3CNm20 NaOAc
H20
10 mMTEAP 10 mM HCIOl in H 2 0 (pH 2) 10 mM TEAP in
+
0.005 M NaCIO, (70:30 HzO :CH,CN) 10 mM TEAP + saturated DCE in H20
0.1 M NaCIO, in CH3CH
toluene, phenol, pyridine
Pb2+,Cu2+,CdZ+ 0.1 N HCI + 1Yo N&HCI
Analyte
[4.10]
[4.25]
Reference
'
Glassy carbon, grade unknown, Tokai Carbon. Ambersorb XE-347,270-325 mesh, Rohm and Haas. Grade GC-20 glassy carbon, Tokai Carbon. Tetraethylammonium perchlorate. Carbopack C, Supelco. Katayama Chemical. 1,2-Dichloroethane. Tetrabutylammonium tetraphenylborate. Vitreous carbon, Energy Research Generation.
No.
Table 4.1. Characteristics and preparation procedures of the conductive stationary phases and the compositions of the mobile phases
4 00
4.3 Redox Separation of Electroactive Metals on the Conductive Stationary Phase
79
Redox Separation of Electroactive Metals on the Conductive Stationary Phase
4.3
The idea of separation of electroactive metal ions with a conductive column is relatively old [4.1], [4.12], [4.13]. It is based on the electrodeposition of analyte metal ions on a conductive stationary phase that consists of carbon or noble metal particles. When the stationary phase potential is more negative than the standard redox potential (E") of the metal ion, the ion is reduced and deposits on the column as a metal film; it does not deposit when the potential of the stationary phase is more positive than E". The deposited metal can be eluted (oxidized) by stepping the potential to a value more positive than E". The partition coefficient (K,) in this technique can be related to the Nernst equation if the redox process is reversible. In this case, for electron transfer equilibrium between metal (M) and its ion (M"') is
{
nF K D = Cs/CM= [M]/[M"'] = exp -117'(E - Z?') where R is the gas constant, Tis the temperature, Cs is the concentration of the metal in the stationary phase, C, is its concentration in the mobile phase, E is the stationary phase potential, and E"' is the formal potential of the M/M"' couple. The formal potential, which is a practical version of E", involves the effect of the ionic strength and/or complex formation in a given medium [4.14]. It is important to point out here that K , is extremely sensitive to a small change in potential. For example, when n = 1and T = 298 K, Equation (2) predicts that a decrease in E by only 59 mV brings about a ten-fold increase in KD. Since the E"' values of metal ions span more than 3 V, this technique would often result in either too long or too short elution times, even if the stationary phase potential were carefully selected.
El
+ \ \ \ \ \ \ \ \ \ \ \ \
W
! [ / \ A A E N
L
M,
M2 Distance from inlet
M3
-
Figure 4.4. Redox separation for metal ions. A) Porous tube; B) Spiral counter-electrode. C) Carbon powder. Potential E is referred to the counter-electrode, and bias voltage, El, is applied across the column. Tis the surface concentration of a deposited metal, and the Ep' of metals is assumed to be in the sequence M 3 < M2 < M,. After deposition, E l is removed, and the ions then elute in the sequence of M3, Mz, MI.
80
4 Electrochromatography with Radial Applied Voltage
To overcome this difficulty, a variety of stripping techniques have been examined. First, the potential is set to a value sufficiently negative for any ion in a sample to deposit quantitatively. After deposition, each metal is stripped by a potential scan in the positive direction, eluting the metal ion with the most negative F"' first. Fujinaga et al. took a different approach (Fig. 4.4). They used a column with a potential gradient (column 1 in Table 4.1) [4.1], [4.15], [4.16]; analyte ions deposit at different positions according to their E"', and the deposited metals elute after the removal of the bias potential. Separation of metal ions such as CdZ+,Pb2+, and Cu2+ have been reported by this method. These techniques are extensions of stripping analysis, and the separation mechanism is quite different from what we expect for chromatography.
4.4
Direct Electrostatic Interactions for Potential-Dependent Separation of Electroinactive Species
All the studies outlined in the previous section have been based on redox reactions, so that analytes are limited to electroactive species. However, electroinactive species can also be separated by the electrostatic field. Such direct electrostatic interactions can occur on charged stationary phases, irrespective of whether the species have a net charge or not.
4.4.1
Pretreated Carbon for the Separation of Metal Ions
Figure 4.1A illustrates the interaction in this category. Studies on glassy carbon have revealed that oxidative treatment of the carbon creates a polar, porous layer on the surface, which can adsorb ionic species when the surface is oppositely charged [4.2], [4.17]-[4.21]. The amout of adsorbed anion increases exponentially with an increase in potential, and the amount of the cation increases exponentially with a decrease in potential. Therefore, this potential-dependent adsorption can be applied to chromatographic separation (column 3 in Table 4.1) [4.2], [4.3]. Indeed, the chromatographic peaks display a potential dependence for Li+ and K', and K + is retained on the column to a higher degreee than Li'. The k' values for Li' and K + are 0.4 and 1.0 at f0.25 V versus SSE, and 1.8 and 4.5 at -0.25 V [4.22]. The longer retention of K+ is attributed to the smaller hydration sheath of K+, leading to a stronger interaction with the negatively charged carbon surface.
4.4.2
Stationary Phase Coated with Crown Ether for the Separation of Alkali Metal Ions
A stationary phase coated with a crown ether layer (column 5 in Table 4.1) can separate metal ions when the ions are transferred to the layer at negative potentials [4.3]. Here, the carbon particle is covered with a 1,Zdichloroethane (DCE) solution containing dibenzo-18-crownd (Fig. 4.1B). Alkali metal ions can be separated at negative potentials (Fig. 4.5) and the elution time of each ion depends on the potential (Fig. 4.6). The
4.4 Direct Electrostatic Interactions for Potential-Dependent Separation
2oo
6oo
' 2oo
I\ 400
Time s
Time s
6oo
81
Figure 4.5. Separations of alkali metal ions with dibenzo-18-crown-6 at stationary phase potentials of A) +0.35V and B) -0.1 V for 20-pL injections of 10 mM (solid line) Li', (dashed line) Na+, (dashed-dotted) K+.
DCE layer plays an important role here, because there is a much smaller potential dependence if DCE is removed in vacuum before the particles are packed in the column. Results are similar if the DCE phase without the crown ether is used. Since K+ fits the dibenzo-18-crown-6best of all the alkali metal ions [4.23], the greatest elution time for K+ is explained in terms of a potential-induced extraction. The equilibrium of K+ with the crown ether at the interface of the aqueous/organic phases can be written:
KZ,
+ crownorg= {K+crown}org
(3)
where the suffixes aq and org stand for the aqueous and organic phases, respectively. The complex ion formed in the organic phase has a positive charge, so that the complex interacts with the negative charge of the carbon particle. In this case, the crown ether adds selectivity for the transfer of K'. Samec and Papoff have studied the ion transfer voltametry of alkali metal ions at a water I DCE interface in the presence of dibenzo-18-crown-6, reporting that the halfwave potentials (El,*)for the transfer of K+, Na', and Li+ from water to DCE are 0.43, 0.54, and 0.61 Vversus Ag I AgCl 1 0.01 M C1- [ , [4.24]. Our results cannot be compared quantitatively to their data because measurement of the potential difference between the aqueous and thin DCE layers is difficult. However, the sequence of the potentials at which elution time rises is consistent with that of the Ela values.
Potential
/ V vs. AglAgCl
Figure 4.6. Potential dependence of elution time for injections of: K+ (open squares); Na' (circles); and Li+ (full squares). The experimental conditions are the same as in Figure 4.5.
82
Electrochromutography with Radial Applied Voltage
4
As seen in Figure 4.6, the elution time of all the ions is independent of potential in the positive region, and K+ is retained there much longer than Na’ and Lit. The otential-independent retention of K+ suggests that a neutral ion pair such as K+crown)TPhB-is extracted to the organic phase, irrespective of potential (TPhB- = tetraphenylborate). It has been shown that Li+, Na’, and K+ ions are extracted by 0 YO, 1.6 YO, and 25.2 % to DCE in an H,O/CDE system with dibenzo-18-crown-6 and picrate, [4.23], which explains the retention time at positive potentials.
P
4.4.3
Electrosorption for the Separation of Neutral Organic Compounds
The principle of electrosorption coupled with chromatographic processes has been applied to the separation of electroneutral organic molecules. Antrim and Yacynych attempted separation of such compounds (toluene, phenol, and pyridine) and found that their capacity factors increase with an increase in the potential (column 2 in Table 4.1) [4.8]. Usually, organic compounds adsorb on electrodes most strongly at potcntials close to the zero charge potential and desorb at both positive and negative potentials that are remote from the zero charge potential, because of competitive adsorption of polar water molecules. It is not certain if their results follow this rule because the zero charge potential is unknown for their system. However, the high dependence of k’ on potential suggests that potential-controlled separation is feasible. The k’ values reported for toluene and phenol are 1.3 and 16.6 at +0.8 V versus an Ag quasireference electrode and 0.6 and 2.0 at -0.8 V, while the k’ of pyridine does not change significantly with potential. Ge and Wallace have discussed the potential effects for a polypyrrole-coated stationary phase, which gives a favorable z-zinteraction with organic molecules (Fig. 4.7, column 8 inTable 4.1) [4.25]. The k’ of caffeine increases with a decrease in the applied potential, while k’ values for more acidic compounds decrease. Beyond + 1.0V k’ for all compounds decreases markedly, which may be due to oxidation of polypyrrole.
1
-2 0
1
1
1
1
1
1
1
1
1
-1 0
1
1
1
1
1
1
1
+20
E (V)
Figure 4.7. Effects of potential on the capacitance factor of 1) Caffeine; 2) Theophylline; 3) Benzoic acid; 4) mToluic acid. Elucnt: 50:50 CH3CN:0.2 M CHICOONa buffer (pH 4.5); flow rate, 1 mWmin [4.25].
83
4.5 Indirect Electrostatic Interactions for Potential-Dependent Separation
4.5
Indirect Electrostatic Interactions for Potential-Dependent Separation of Electroinactive Species
Applied voltages can induce chemical changes of functional materials on stationary phases (Fig. 4.1B), and, indeed, materials such as conducting polymers create an ionexchange site as a result of redox reactions. If the site formation is reversible, the number of the sites can be varied with the potential, controlling the mean velocity of analyte in the column.
Redox Polymer with Ion Exchange. The modification of the carbon surface with a functional substance such as a redox polymer is an attractive way to achieve controlled separation. Ghatak-Roy and Martin have studied ion-exchange chromatography using a stationary phase coated with a film of vinylferrocene/maleic anhydride copolymer, which is conductive after swelling in solution [4.7]. H
H
H
Fc
I
H
H
I
H
H
I I I fc-c-j(-c I I I H Fc+ L o - 0 C +o
H
o//c'o-j
,,
\ \
\ \
Red
H
'M"2'
'
ox
When the ferrocene unit in the polymer is in the reduced state (Red), methylviologen (MV2+)as analyte is eluted in the eighth through thirteenth volume fractions (8-12 mL). In contrast, when the polymer is in the oxidized state (Ox), MV" is not retained on the column, eluting in the first fraction ( 5 1 mL).
4.5.1
Conducting Polymers for Separation of Anions
Ion-Exchange Mechanisms of Polyaniline. Conductive polymers such as polyaniline and polypyrrole are insulators in their reduced forms, while conductive in the oxidized forms via doping anions. Thus, the polymers are expected to exchange anion in the oxidized states and the number of ion-exchange sites can be controlled by the potential applied to the polymers. Insertion of anions from solution to polyaniline films has been studied extensively [4.26]-[4.28]:
84
4 Electrochrornatography with Radial Applied Voltage
(B)
0 0
4.0u
6.0
2.0
0.00,
20
10
'
Deposition time
30
I
min
Figure 4.8. Cyclic voltammograms for a column electrode coatedwith (solid line) and without (dashed line) polyaniline films obtained at a flow rate of 0.5 mL min-' and at a sweep rate of 5 mV/s. A) Polyaniline was deposited from an aqueous solution containing 0.5 M aniline 1 M HCI at +0.95 V vs. SSE for 5 min, and the voltammogram was recorded in the same aniline solution at 0.5 mL m i d . The base line voltammogram (dashed line) wasrecordedin 1 M HCI. B)The dependenceof loading Q on deposition time. The hatched area of the voltammetric peak defines Q (C cm-').
+
1
-0.2
1
.
I
0.2
I
I
0.6
.
I
-
I
Potential / V vs. AglAgCl
Figure 8 shows the cyclic voltammograms of the polyaniline film recorded at the electrode column. The first (less positive) anodic and cathodic peaks have been assigned to the insertion and release of anion as a result of film oxidation and reduction, respectively (Eq. 4). The second peaks have been ascribed to the degradation of the film, which involves release of anion from the polymer [4.26].
Potential Dependence of Anion Separation. The oxidized polyaniline (PAN04*+)film exchanges analyte anion A- in the mobile phase according to the equation: PAN04X'(c10q)4X
+ 4~ A-M
+ 4~ ClO,
= PAN04' +(A-).lX
M
(5)
where the suffix M denotes an ion in the mobile phase. We have shown that the potential-dependent separation can be done on a stationary phase coated with polyaniline (Fig. 4.9, column 6 in Table 4.1). Since the mobile phase contains TEAP in our experiments, the film initially contains C104- as dopant. The elution time of I- and SCN- depends on the potential because the number of the cationic sites (PAN04X ') increases with increase in potential. Table 4.2 lists the potential dependence of elution time for various organic and inorganic ions. Figure 4.10 shows that the larger the ionic size of the inorganic ion, the greater the elution time. However, organic acid ions such as CF,COO-, which are much larger than the inorganic ions, show virtually no retention on the column, even at positive potentials (Table 4.2), and the potential dependence of retention is quite similar to that of C1-. Since polyaniline film displays a dramatic decrease in the diffusion coefficient for ions with ionic radii greater than 0.37 nm [4.29], the short elution time for the organic acid anions would result from the exclusion of such ions from the film. The decreases in elution time at potentials above 0.3 V in Figure 4.10 are probably due to a release of anion via the second oxidation step for the polymer (Fig. 4.8).
4.5 Indirect Electrostatic Interactions for Potential-Dependent Separation
-1500
0 500
2500
0 500
Time 1 s
1500
2500
Time s
85
Figure 4.9. Potential dependence of chromatographic peaks for 20-pL injections of: 10 mM CF3COO- (solid line); 1 mM I- (dashed line); 10 mM SCN(dashed-dotted line). Potential: A) -0.3 V, B) 0.3 V; mobile phase, 10 mM TEAP 1.0 mM HC104; Q = 0.17 C cm-'. CF3COO-, I-, and SCN- were detected at 200,220, and 240 nm, and absorbance values (arrowed) are given in each figure.
+
Table 4.2. Elution of anionic species on polyaniline-coated and polypyrrole-coated stationary phases as a function of potential"
PoIyaniIineb
Polypyrroie"
ATRJAE", Elution time, s sV-' -0.3V +0.3V
Anion*
Elution time, s -0.3V +0.3V
Fumaric acid Malonic acid Succinic acid Tartaric acid Citric acid Benzenesulfonic acid 1,3-Benzenedisulfonic acidg Acetic acid' Trifluoroacetic acid CIBrI- k SCN[Fe(CN),l"
142 142 140 138
148 148 145 140
172 364
(186)' N. D.h
(47)
156 141 148 156 162 192
188 146 216 304 438 (336)'
53 8.3 113 247 460 (262)
10 10 8.3 3.3
A TRIAF , sV-'
130
168
125 124 135 154
162 172 166 (406)'
63 80 51 (630)
130 133
191 151
102 30
152 152 166 132
203 246 412 (161)'
85.7 156 411 (73.8)
65
a Flow rate 0.5 mL m i d ; UV detection 200-250 nm; potential vs. SSE. Deposition at +0.85 V for 10 min (Q = 0.12 C cm-'); mobile phase 10 mMTEAP + 10 mM HCIO4aqueous solution (pH 2). Deposition at + 1.0 V for 10 min; mobile phase 10 mM TEAP aqueous solution (pH 5.5 at +0.3 V). Injection 20 pL of 10 mM analyte ions + mobile phase solution. Organic acids exist mainly as monoanions in the mobile phase used with the polyaniline column. Average potential dependence of elution time A TR/AE = (TR, - TR,x2) I (x, - xz), where TR,I is elution time at potential x; Elution time at +O.O V, no peak detected at +0.3 V. Disodium salt. No peak detected at -0.1 V, Retention time at +0.1 V, no peak detected at +0.3 V. Sodium salt. kInjection 20 pL of 1.0 mM I- + mobile phase solution. Retention time at +0.25 V, no peak detected at +0.3 V.
'
'
86
4 Electrochromatography with Radial Applied Voltage 2000
v)
1500
.-E
.I-
c
.-0
I000
4-
C
a)
4-
$
Figure 4.10. Potential dependence of the elution time. Analyte: SCN- (lozenges); 1- (squares); Br- (triangles); CI- and CF3COO- (circles, both showed 0 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 virtually the same behavior). Q = 0.22 C cm-'; other experimental conditions are the same as in Potential " ~Ag,AgC, . Figure 4.9.
500
,
1.2
, l . l . l . l . l . l . l .
0.8
3c
0.4
D l
-0
0
-0.4
0
-0.8 -18
-14
-10
-6
log (C, / mol g-carbon-')
-2
Figure 4.11. Dependence of the capacitance factor, k', on the site concentration CR. ?I = 0.240 V; n = 1; T = 298 K. CRis calculated for: oxidatively treated carbon (full circles); and untreated carbon (lozenges, squares). CRwas also obtained experimentally (open circles). Q = 0.22C cm-' (lozenges); Q = 11 C cnid othenviese. Other experimental conditions are the same as in Figure 4.9.
Figure 4.11 shows the dependence of the capacitance factor on the concentration of the cationic site, = N'H. The dependence is linear over wide ranges of concentration [4.5]. When polyaniline is deposited on oxidatively treated carbon particles, the slope becomes much gentler, suggesting that the film structure created on such particles is very different from that for untreated particles. In contrast, the loading of polyaniline Q (Fig. 4.8) does not greatly influence the slope, so that the film structure is not very sensitive to the loading. The linear dependence can be analyzed by the equilibrium: A-
K + ( = N + H ) . C104 - + (-N+H). A- + ClO,
(6)
4.5 Indirect Electrostatic Interactionsfor Potential-Dependent Separation
87
-
where (= N’H) . C10,- and (= N+H) A- show Clod- and analyte bound to the stationary phase. The capacity factor can be related to the equilibrium constant K as follows [4.30]: log k’ z lOg(V,/V,)
+ log K + lOg(CR/CL)
(7)
where V,and V,,, are the volumes of the stationary and mobile phases, CRis the concentration of the cationic sites (= N’H), and C, is the concentration of C10,- in the mobile phase. CR can be evaluated both theoretically and experimentally. Theoretically, CRcan be calculated by the following equation, if the redox reaction of the polymer is reversible:
where C, is the total concentration of the oxidized (cationic) and reduced (neutral) sites in the film and should be equal to Q/nE Since Equation (4) suggests an n value of unity for the electron transfer at each site, the relationship between log k’ and log CR can be plotted from an experimental elution time and potential via Equations (7) and (8). CRat potential E can also be obtained experimentally by integration of the voltammetric peak from Eo to E (Fig. 4.8). Equation (7) predicts that the log k’-log C, plots should have unit slope, but slopes of 0.1-0.2 were obtained experimentally. Since an n value smaller than unity reduces potential dependence of C,, the deviation from theory may be attributed to the formation of triple ions such as ( 3 N0.’+H)&, which have been reported [4.26].
Irreversible Uptake of Highly Charged Large Anions into the Film. Large and/or highly charged ions are taken up irreversibly into the oxidized film at positive potentials [4.5]. Since such ions can be eluted by a potential perturbation in the negative direction, the uptake allows both concentration and separation on a single column. The mechanism is quite similar to that described in Section 4.3, and the procedures explained there can be applied. Deinhammer et al. have separated adenosine monophosphate (AMP) and adenosine triphosphated (ATP) on a polypyrrole-coated stationary phase (column 9 in Table 4.1), using potential-step stripping techniques [4.10]. Both AMP and ATP are totally retained at -0.3 Vversus SSE, a potential which corresponds to the moderate oxidation of polypyrrole film. Since ATP interacts more strongly with the polymer, AMP elutes at a smaller elution volume than ATP, when the potential becomes more negative in the stripping stage. The irreversible uptake is predominant for polypyrrole (column 7 in Table 4.1) than for polyaniline (column 6 in Table 4.1) and most ions studies in Table 4.2 are taken up in the polypyrrole film, at leat to a certain degreee, which would make it difficult to use this polymer in quantitative analysis. For these irreversible uptake, the application of the continuous pulse technique is more attractive [4.31].
88
4
4.6
Summary
Electrochromatography with Radial Applied Voltage
Although chromatography has been developed as one of the most commonly used separation techniques, the virtually unalterable properties of separation columns allow only a limited number of experimental parameters to be varied. In this connection, the idea of the stationary phase potential has attracted many electrochemists to the introduction of an additional parameter for controlled elution. To see if this idea is feasible, columns comprising conductive stationary phases have been constructed. The discussions in this chapter mainly focus on the separation of electroinactive species which cannot be separated by redox reactions at an electrode. The separation mechanisms have also been discussed from the point of view of direct and indirect electrostatic interactions between analyte and stationary phase. The direct interaction occurs between analyte ion and charged stationary phase, and changes in the field strength at the stationary phase alter the retention of analyte. Alkali metal ions have been separated on stationary phases coated with oxidized carbon and with crown ethers, by varying the potential. The separations of neutral organic compounds by electrosorption have also been discussed. Separations with indirect interactions are based on changes in the number of ion exchange sites, which is controlled by the potential of the stationary phase. The separation of ionic species by redox polymer and conducting polymer stationary phases has been discussed. Acknowledgements I express my sincere thanks to Dr. J. Yano, Professor S. Yamamoto, and Professor K. Ogura for many helpful comments and discussions. Many students have also contributed their talent to our work: Y. Uchida, K. Kakuno, H. Nakao, and, especially, M. Fujimoto. Grant support was obtained from the Ministry of Education.
4.7 [4.1] (4.21 [4.3] [4.4] [4..5] [4.6] [4.7] [4.8] [4.9] [4.10] [4.11] [4.12] [4.13] [4.14] [4.15] [4.16] [4.17]
References T. Fujinaga, T. Nagai, S . Okazaki, C. Takagi, Nippon Kugaku Zasshi, 84,941-942 (1963). T. Nagaoka, M. Fujimoto, H. Nakao, K. Ogura, Runseki Kagaku, 40,785787 (1991). T. Nagaoka, M. Fujimoto, Y. Uchida, K. Ogura, J. Electroanal. Chem., 336,4555 (1992). T. Nagaoka et al., J. Electroanal. Chern., 350, 337-344 (1993). T. Nagaoka et al., J. Electroanal. Chern., 364, 179-188 (1994). T. Nagaoka et al., J. Electrounal. Chem., 368, 31.5-317 (1994). A.R. Ghatak-Roy, C. R. Martin, Analytical Chemistry, 58, 1574-1575 (1986). R. F. Antrim, R. A. Scherrer, A. M. Yacynych, Anal. Chim. Acta, 164, 283-286 (1984). H. Ge, P. R. Teasdale, G. G. Wallace, J. Chrornatogr., 544, 305-316 (1991). R. S. Deinhammer, K. Shimazu, M. D. Porter, Anal. Chern., 63, 1889-1894 (1991). M. Fujihira, in Topics in Organic Electrochemistry, A. J. Fry, W. E. Britton (eds.), Plenum Press, New York, 1986, pp. 25.5-294. W. J. Blaedel, J. H. Strohl, Anal. Chem., 36, 1245 (1964). D. K. Roe, Anal. Chem., 36,2371-2372 (1964). A. J. Bard, L. A. Faulkner, Electrochemical Methods, Wiley, New York, 1980, pp. 51-52. T. Fujinaga, C. Takagi, S. Okazaki, Kogyo Kagaku Zasshi, 67, 1798-1801 (1964). T. Fujinaga, S. Kihara, CRC Crit. Rev. Anal. Chem., 6, 223-254 (1977). J. Schreurs, J. van den Berg, A. Wonders, E. Barendrecht, Recl. Trav. Chim. Pays-Bas,
103,251-259 (1984). [4.18] G. E. Cabaniss et al., J. Am. Chem. Soc., 107, 1845-1853 (19x5).
4.7 References
[4.19] [4.20] [4.21] [4.22] [4.23] [4.24] 14.251 [4.26j
89
E. Hollax, D. Sh. Cheng, Carbon, 23, 655-664 (1985). T. Nagaoka,Y. Uchida, K. Ogura, J . Chem. Soc., Furaday Trans. I , 85,3757-3765 (1989). T. Nagaoka et al., Anal. Chem., 60,2766-2769 (1988). T. Nagaoka, H. Nakao, unpublished work. C. J. Pedersen, H. K. Fresndorff, Angew. Chem. Internat. Edit., 11, 16-25 (1972). Z. Samec, €? Papoff, Anal. Chem., 62, 1010-1015 (1990). H. Ge, G. G. Wallace, Anal. Chern.. 61. 2391-2394 f1986). W . 4 . Huang, B. D. Humphrey, A. G. MacDiarmid,'J. &ern. Soc., Faraday Trans. I, 82, 2385-2400 (1986). I4.271 D. Orata, D. A.'Buttry, J. Am. Chem. Soc., 109, 3574-3581 (1987). [4.28] C. Barbero, M. C. Miras, 0. Haas, R. Kotz, J . Electrochem. Soc., 138, 669-672 (1991). [4.29] K. Tsubaki, A. Kitani, K. Sasaki, Ann. Meet. Chem. Soc. Jpn., Chugoku-Shikoku and Kyushu Branch, Tottori, Oct. 1991, Paper 2L07. [4.30] S. Yamamoto, K. Nakanishi, R. Matsuno, Zon Exchange Chromatography of Proteins, Marcel Dekker, New York, 1988. [4.31] T. Nagaoka et al., J. Electroanal. Chem., 371, 283-286 (1994).
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5
Electrochromatography in Biomolecular Analysis Daniel H. Shain
5.1
Introduction
A fundamental problem in the biological sciences is the identification and purification of molecules which appear to be interesting. In most cases, this is accomplished by employing some form of chromatography or electrophoresis. Both of these techniques have become highly developed and diversified in recent years and are each capable of remarkable resolutions. As the details of biochemical pathways are explored further, however, it has become necessary to analyze complex mixtures and to distinguish between closely related molecules which cannot always be resolved by conventional procedures. In these instances, it is often effective to combine two separative dimensions into a single technique. For example, two-dimension (2-D) electrophoresis separates proteins according to their isoelectric point and also by their molecular weight. The number of proteins which can be resolved by coupling these two dimensions far exceeds the number resolved when using each dimension alone E5.11. When electrophoresis and chromatography are combined into a single system, the resulting technique is called electrochromatography (EC). In EC, a substance migrates according to its electrophoretic mobility as well as its interaction with a supportive matrix [5.2]. Recently, EC has been expanded to encompass a myriad of related techniques [5.3], all of which utilize some aspect of chromatography and electrophoresis. In this chapter, EC methodologies which pertain to the analysis of biomolecules will be examined. These will include a number of earlier experiments which have provided the foundation for more modern EC technologies. The goal of this chapter is to demonstrate the practical aspects of EC in the biosciences and its potential advantages over conventional techniques.
5.2
Significance
Perhaps one of mankind’s most challenging problems is to understand the processes which constitute life. In its simplest sense, life may be considered as a network of biochemical interactions that result in a replicating unit, a process which is poorly understood. When considering higher eukaryotic organisms, such as ourselves, the problem becomes quite complex. Highly organized systems, including the circulatory, immune, respiratory and nervous systems, all have specialized roles yet they are dependent upon each other for survival. The different systems are comprised of tissue specific cells, each of which contains numerous compartments and organelles. These cellular structures can be broken down further and further until one arrives at the individual biomolecules which provide the foundation for cellular activity. It is at this level that
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5 Electrochromatography in Biomolecular Analysis
scientists are beginning to unravel what took billions of years of evolution to achieve. Nevertheless, it is astounding that processes such as ontogeny or consciousness can arise from the interaction and spatial organization of a finite number of molecules. Understanding these processes represents a formidable obstacle at the present time; however, much progress can be made in this direction by identifying the individual components of each system and then determining their function. In this case, it is the properties of individual biomolecules which need to be studied. Thus, it becomes necessary to obtain, in pure form, biomolecules which are suspected to play important roles in higher biological processes. In molecular evolution, ancestral molecules are modified by random mutations and those mutations which are favorable are maintained by natural selection. Since evolution proceeds in small steps, the similarities between two functionally distinct molecules may far outweight their differences. For example, a single amino acid substitution in the active site of a protein can completely abolish its activity. If the mutant and wild type proteins are compared at the structural level, however, they are essentially identical. While this represents an extreme example, within the cell there are numerous molecular families which have all evolved from recent common ancestors. As a result, closely related biomolecules abound in living organisms, yet many of these have distinct roles within the cell (e.g., transcription factors, olfactory receptors, etc.). As the intricacies of cellular mechanisms are gradually explored, it is of paramount importance that interesting biological molecules can be identified and purified to homogeneity so that the system may be understood in its entirety. The applications of such knowledge will be invaluable to basic research, biotechnology, medical science and many other disciplines.
5.3
Characteristics of Biomolecules
An emphasis will be placed on the analysis of macromolecules such as nucleic acids (deoxyribonucleic acid - DNA and ribonucleic acid - RNA) and proteins since they constitute the majority of the cell’s dry weight [5.4]and are representative of types of separations which can be achieved by electrochromatography. Smaller biomolecules, which are often precursors to macromolecules, will be considered when appropriate. The structure of double standed DNA (dsDNA) is shown in Figure 5.1. DNA is a polymer of deoxyribonucleotides, each of which contains a phosphate, a deoxyribose sugar and a nitrogenous base (adenine, cytosine, guanine or thymidine). In the cell,
Figure 5.1. Structure of dsDNA. DNA is a polymer of deoxyribonucleotides linked together by phosphodiester bonds. In most cases, DNA has a continuous double helical structure containing a major and a minor groove.
5.3 Characteristics of Biomolecules
93
Figure 5.2. Representation of mRNA. RNA is a polymer of ribonucleotides linked together by phosphodiester bonds. mRNA is often found as a linear molecule while transfer RNA (tRNA), ribosomal RNA (rRNA) and ribozymes have highly folded structures. RNA has numerous functions within the cell including its most recently discovered role in enzyme catalysis.
DNA is organized into chromosomes, each of which contains hundreds to thousands of individual genes. Chromosomal DNA is often digested by restriction endonucleases into fragments which are relatively easy to manipulate. These DNA fragments provide the foundation for molecular biology and are used, for example, in the construction of libraries which facilitate the cloning and characterization of individual genes. Almost all recombinant DNA techniques require size fractionating DNA. Two inherent features make DNA suitable for such analysis. First, DNA is negatively charged due to its phosphodiester backbone. Second, since dsDNA has a continuous double helical structure (with some exceptions), its charge-to-mass ratio is constant regardless of its length. Thus, dsDNA fragments are readily separated by size exclusion matrices in both electrophoresis and chromatography. DNA contains the archives of information necessary to build a living organism. By itself, however, DNA does very little in the cell and must first be transcribed into messenger RNA (mRNA) before its information is translated into proteins. RNAis chemically very similar to DNA with a few exceptions. Most notably, RNA contains a ribose sugar group in place of the deoxyribose found in DNA. In addition, the base thymidine (present in DNA) is replaced by uracil in RNA. RNA is a single stranded molecule (Fig. 5.2) that can acquire unique structures upon folding (e.g., ribozymes, ribosomal RNA). RNA is typically fractionated by size ecxlusion matrices under denaturing conditions (to remove 2' and 3' structures) using electrophoresis, chromatography or density gradients. Proteins represent the most diverse group of biomolecules and are associated with almost every structural and functional aspect of life. In contrast with DNA and mRNA, they fold into complex three-dimensional structures and often interact among themselves. A molecular model of hemoglobin is shown in Figure 5.3 to demonstrate the complexity of a typical protein. Each protein folds into a particular conformation depending upon its linear sequence of amino acids, of which only twenty are used by living organisms on Earth. Once folded, each protein assumes a specific role whether it is, for example, in the recognition of antigens (antibodies), capturing photons of light (rhodopsins) or in the transport of ions across the plasma membrane (i.e., Na+/K+pump). Proteins migrate in an electric field due to the presence of charged amino acid residues within their polypeptide chain. The net charge of a protein is largely dependent upon which types of amino acids are most abundant.Proteins rich in lysine, arginine
94
5 Electrochromatography in Biomolecular Analysis
Figure 5.3. Structure of hemoglobin. Hemoglobin and other proteins are polymers of amino acids linked together by peptide bonds.
or histidine tend to carry a positive charge while proteins abundant in aspartic acid or glutamic acid are generally electronegative. Thus, the amino acid composition of a protein usually determines which direction it migrates in an electric field. The exceedingly large number of polypeptide conformations and compositions found in nature presents the greatest challenge in the development of macromolecular separative techniques. Nonetheless, many proteins can be separated, to some extent, by conventional chromatographic and electrophoretic procedures.
5.4
Development of Electrochromatographic Techniques
The origins of chromatography and electrophoresis are controversial [5.5]-[5.8]and, as might be expected, the time at which the two techniques were first used in combination is also questioned [5-21, [5.7], [5.8].The term electrochromatography appears to have been used first by Berraz in 1943 in describing a form of paper electrophoresis [5.9]. Four years prior to this, however, Strain performed the technique by inserting electrodes into an alumina column and separating dyestuffs using an applied voltage [5.10]. Also in 1939, Coolidge conducted electrophoresis of proteins in a tube packed with glass wool [5.11]. Numerous experiments followed which used alumina [5.12],
5.4 Development of Elecfrochromafographic Techniques
95
[5.13], asbestos fiber 15.141, glass [5.15], [5.16], silica [5.17]-[5.21], filter paper [5.22]-[5.24], cellulose [5.25]-[5.28], pevikon C-870 [5.29], [5.30] or sephadex [5.31] as a supporting material to which an electric field was applied. Although many of these matrices introduced a sorptive component into the separation, it should be noted that some were used in an effort to control the problem of convection which plagued free solution electrophoresis [5.11], [5.15]. The supportive matrix, however, directly influenced all of these separations by interacting with the sample, even though these interactions were not always favorable. In fact, some materials
-
+
Sickle ccll Luemoglobln
e 5
Figure 5.4. “Finger prints” of human normal and sickle-cell hemoglobins. Electrophoresis at pH 6.4, chromatography with n-butyl alcohoUacetic acidwater (3: 1:1). The shaded and the stippled spots are those belonging to the peptide showing the difference. (Reproduced with permission from Macmillan Magazines Limited)
96
5 Electrochromatogrriphy in Biomolecular Analysis
were chemically modified to suppress electro-osmosis and adsorption [5.25], two properties which have been exploited in modern EC techniques. Nevertheless, it is clear that the benefits of combining electrophoresis and chromatography have been recognized for almost half a century. This is demonstrated by the experiments of Hauguord and Kroner who, in 1948, performed a two dimensional separation of amino acids by simultaneously performing chromatography and electrophoresis at right angles to each other [5.22]. Of the supportive matrices mentioned above, paper and thin-layer electrochromatography became the most popular since they were simple to use, inexpensive and they separated materials into distinct bands which could be identified and isolated. Consequently, a variety of conditions were developed for the analysis of proteins [5.30], [5.32]-[5.34], peptides [5.19]-[5.21], [5.24], [5.28], [5.30], [5.35]-[5.37], amino acids [5.7], [5.37], nucleic acids [5.7], [5.30] and other biological compounds [5.7]. Of particular interest is the two dimensional separation of hemoglobin peptide fragments by Ingram in 1957 [5.24]. Using a combination of electrophoresis and chromatography on Whatman No. 3 MM paper, trypsinized hemoglobin was resolved into approximately 30 different peptide fragments as shown in Figure 5.4. When the finger print of normal hemoglobin was compared to that of a sickle cell anemia patient, it was found that one peptide fragment was consistently displaced. This change was later found to be the result of a single amino acid substitution,thus demonstrating the sensitivity of this technique and its applications as a diagnostic tool. The technology of combining electrophoresis with paper and thin-layer chromatography underwent a period of rapid development and a wide range of experimental conditions were established. Although these techniques are still in use [5.38]-[5.44] and sophisticated systems are available today (see Chapter 1) the development of this field was overtaken by revolutionary advances in gel electrophoresis.
5.5
Gel Electrophoresis
The idea of electromigration through a gelatinous material dates back more than a century I5.451. The field received limited attention, however, until 1955 when Smithies introduced starch gels as a method for separating serum proteins L5.461. Since starch provided a molecular sieving effect, a protein migrated according to its size and shape in addition to its electrophoretic mobility. In 1959, the field was revolutionized with the development of polyacrylamide gels [5.47]. In contrast with starch gels, polyacrylamide gel electrophoresis (PAGE) enabled a wide range of pore sizes to be selected, each of which yielded high resolution [5.37]. As a result, PAGE became the method of choice for separating biomolecules ranging in size from small peptides to viruses. Subsequent modifications to PAGE greatly expanded its versatility. For example, sodium dodecycl sulfate-PAGE (SDS-PAGE) denatures and swamps a protein with negative charges, causing it to migrate according to its approximate molecular weight [5.48]. Isoelectric focusing separates proteins according to their isoelectric point [5.49], [SS O ) , while 2-D electrophoresis combines isoelectric focusing and SDS-PAGE (perpendicular to each other) and is one of the most powerful techniques currently available for protein separation [5.1], [5.4]. Gel electrophoresis has had such an enormous impact on biochemical analyses that it is generally placed into a category of its own. However, gels cast of polyacrylamide or agarose provide a molecular sieving effect (and in some cases a sorptive component [SSl]) which constitutes a major separative factor. Consequently, gel electrophoresis is
5.6 Electrochromatography in Preparative Columns
97
classified as a form of electrochromatography [5.2]. It may be more appropriate, however, to consider gel electrophoresis as an extreme form of EC called EC with infinitely slow pressurized flow since the system operates in the absence of a solvent flow [5.52]. No attempt will be made to cover the plethora of techniques which have been developed in this field; however, some particularly relevant applications in the analysis of macromolecules will be considered in later sections.
5.6
Electrochromatography in Preparative Columns
The successful application of electrophoresis performed on filter paper, thin layers of sorptive material and in gels prevented its extensive exploration in packed columns [5.31], [5.53], [5.54]. Nonetheless Neremberg and Pogojeff used a Sephadex G-100 preparative column with a specialized adapter which permitted chromatography to proceed by gravity, while an electric field was applied in either direction. Significantly, higher resolution of serum proteins was achieved when chromatography and electrophoresis were performed simultaneously as compared with each technique used alone [5.31]. In 1980, a preparative column which performed electrophoresis and chromatography in sequential steps was described by Otsuka and Listowsky [5.55]. The resolving power obtained by this apparatus sparked a new interest in EC which has been influential in the rapid development of EC over the past decade.
5.6.1
Sequential Electrochromatography
In electrochromatography, electrophoresis and chromatography can be performed simultaneously or in sequential steps, both of which have been utilized in paper and thin-layer electrochromatography (see Section 5.4). One of the first examples of sequential EC performed in a column is that described by Otsuka and Listowsky [5.55]. In their apparatus, a polyacrylamide gel is deposited on top of a column packed with a chromatographic support, as shown in Figure5.5. A sample is electrophoresed through the polyacrylamide layer and into the chromatographic support. At this point the applied voltage and the upper, polyacrylamide layer are removed. The sample now begins the chromatographic phase of the separation in which the flow rate is controlled by gravity. In the experiment done by Otsuka and Listowsky, the H and L subunits of human liver ferritin were separated using a 7.5 % polyacrylamide gel (in 9 M urea, 0.125 M Tris buffer, pH 6.8) and Bio-Gel P-300 (Bio-Rad Laboratories, Gaithersburg, MD) as the chromatographic support. Since these subunits have similar molecular weights (H subunit = 21,000; L subunit = 19,000), similar isoelectric points and are structurally related, they provide an example of closely related macromolecules which are difficult to separate by conventional methods, including ion-exchange and gel-filtration chromatography. Although separation is achieved using 2-D electrophoresis, this technique cannot be used effectively on a preparative scale. Using the conditions described above, approximately 7mg of dissociated ferritin was resolved in a single run as demonstrated by Figure 5.6. Recoveries of each subunit ranged from 70-90 % of the total protein applied. The main drawback to this system is the duration of the run which took more than 60 h (18 h electrophoresis and >40 h chromatography) to complete. When considering the amount of pure protein that is recovered, however, the time factor becomes somewhat less significant.
98
5 Electrochromatogruphy in Biomoleculur Analysis
Figure 5.5. Apparatus used for electrochromatography procedures. The column was packed with Bio-Gel P-300, and the layer of polyacrylamide above the resin is indicated by (A). The graduate cylinder and the funnel above the column contained the electrophoresis and eluting buffer 9 M urea, 0.125 MTris, pH6.8. Platinum wires were immersed in the electrode buffers and connected to the terminal leads. This photograph was taken 14 h after the sample was applied for electrophoresis. (D) indicates the position of the tracking dye. (Reproduced with permission from Academic Press)
ELUTION VOLUME (ml)
Figure 5.6. Elution pattern for ferritin subunits after electrochromatography. Human liver ferritin samples were treated with 9 M urea, pH2.5, for dissociation into subunits. Electrophoresis was carried out until most of the tracking dye migrated into the cathode buffer chamber. The column was then fitted with a stopcock and the elution pattern from the Bio-Gel P-300 chromatographic step was monitored using 0.5 mL fractions. Sodium dodecyl sulfatepolyacrylamide gel electrophoretic analysis of the protein containing fractions (within the dotted lines) are shown above the elution pattern. Each channel in the gel represented one 0.5 mL fraction and lanes were numbered to correspond to the volume in the elution pattern shown below the gel. (Reproduced with permission from Academic Press)
5.6 Electrochromatography in Preparative Columns
99
By using different combinations of electrophoretic and chromatographic conditions, it is likely that this form of EC could separate a variety of closely related biomolecules on a preparative scale. The key feature in this technique is its smooth transition from polyacrylamide gel electrophoresis to gel filtration chromatography. Without efficient coupling a significant loss in resolution is inevitable. This was accomplished by electromigrating the ferritin subunits from the polyacrylamide gel directly into the gel filtration column. It may also be possible to employ a system whereby solutes are transported from one separative dimension to the next via a peristaltic pump or an alternative pressure source, as examined below.
5.6.2
Electrochromatographywith Modem Adaptations to Liquid Chromatography
Numerous attempts have been described which combine electrophoresis with some aspect of liquid chromatography. This group of techniques typically employs a pressurized flow which follows electrophoresis and thus enables an eluted solute to be transported for detection and/or recovery. A commercially available apparatus which utilizes this idea is called high-performance electrophoresis chromatography (HPECTM)
Figure 5.7. Schematic of the HPEC apparatus. A sample is applied to a gel electrophoresis column mounted between two specially designed blocks housing the electrodes and the upper and lower buffers. A continuous flow of elution buffer, under helium pressure, carries the separated bands to the detection and fraction collection systems. A specialized elution trap fitted with dialysis membrane enables macromolecules to be recovered quantitatively. (Reproduced with permission from Applied Biosystems)
100
5
Electrochromatography in Biomolecular Analysis
Figure 5.8. Purification of synthetic oligonucleotides. HPEC successfully resolves and allows recovery of a 90-mer from 2.0 O D of crude synthetic product. Recovery of the fractions shown is 0.18OD, compared to a theoretical yield of 0.32 OD. The fractions are further analyzed on a 12 % polyacrylamide gel and their purity is confirmcd by the autoradiograph of the gel. Gel: 2.5 x 100mm. 20 ‘Xu polyacrylamide. Power: 0.8 mA, constant current. Detector: 260nm. (Rcproduced with permission from Applied Bi osyst e ms)
75-517
Figure 5.9. Separation of double-ctanded DNA fragments. Excellent peak resolution is achieved when separating 1 pg of a 1-kilobase ladder, from yeast 2 pm circle, on the Model 230A (HPEC). lZun conditions can be further optimized for the collection of specific peaks. Since fractions from the enlire \ample can be collected, additional peaks are available without the need to run additional gels or manually cut bands. Gel: 3.5 X 100mm, 0.4 Yo agarose. Power: 0.5 mA, constant current. Detector: 260nm. (Reproduced with permission from Applied Biosystems)
1
RNA-re 8 8
Figure 5.10. Analysis of the low molecular weight polypeptides of a bovine pancreatic ribonuclease R preparation. Using the Model 2 3 0 h , ribonuclease B is resolved from ribonuclease B”. The two compounds differ only by the extent of glycosylation. The mixture is analyzed by HPEC and on a 15 % SDS-PAGE slab gel. Gel: 2.5 x SO m m , 7.5 % SDS gel. Power: lSOV, constant voltage. Detector: 280 nm. (Reproduced with permission from Applied Biosystems)
5.6 Electrochromatography in Preparative Columns
n
101
Crude
I\
340 nm
J L
GBPDH activity
I
Figure 5.U. On-line activity assay. For on-line detection of enzyme activities, substrates and enzyme cofactors can be added to the elution buffer system of the Model 230A (HPEC). In this example, G3PDH is monitored by adding sodium phosphate, glyceraldehyde 3-phosphate and NAD to the elution buffer. NADH is monitored at its extinction coefficient of 340nm. Gel: 2.5 x 50mm, 5 % alkaline gel. Power: 300V, constant voltage. Detector: 340nm. (Reproduced with permission from Applied Biosystems)
(Model 230A, Applied Biosystems, Foster City, CA). The HPEC apparatus is shown schematically in Figure 7. HPEC is similar to high-performance liquid chromatography (HPLC) except that an electrophoretic column is used in place of a conventional HPLC column. Although the HPEC apparatus is somewhat complex in design, requiring three buffer chambers under helium pressure and a specialized elution trap which is fitted with dialysis membrane, it is also quite functional [5.56]-[5.58]. For example, more than ten proteins have been purified to apparent homogeneity from rat cerebrospinal fluid using HPEC as a final purification step [5.58]. Following a buffer exchange, these proteins are suitable substrates for microsequencing without further manipulations. Electropherograms demonstrating the capabilities of HPEC in resolving oligonucleotides, double-stranded DNA fragments and polypeptides are shown in Figures5.8, 5.9, and 5.10, respectively. HPEC has also be used for on-line activity assays by adding enzyme cofactors and substrates to the elution buffer (Fig. 5.11). A number of systems similar in function to high-performance electrophoresis chromatography have been developed, many of which can be assembled from standard laboratory equipment [5.59]-[5.65]. The system that will be described, called electrofractionation (EF), represents an efficient and versatile example of such an apparatus [5.65]. A schematic of the EF system is shown in Figure 5.12. Construction and operation of the apparatus were performed as follows. Teflon tubing (0.d. = 1.52mm7 i.d. = 0.86mm) was fitted into the bottom of a 3mL syringe (Becton-Dickinson, Rutherford, NJ). A nylon mesh filter with a pore size of about 1mm was cut so that it fit exactly inside the syringe. One end of a glass column (i.d. = 4 or 6mm, od. = 8 mm, length = 120mm) was sealed with parafilm and a polyacrylamide or agarose gel was cast inside. After polymerization the parafilm was removed and the glass column was placed into the column holder (syringe). Four symmetrically oriented holes ( l m m in diameter) were made with a heated needle 1cm from the bottom of the column holder to permit current and buffer flow. (A potential problem of EF appears to be the restriction in current flow between the column and column holder. This has been circumvented by increasing the distance between these surfaces [5.66], or by eliminating the column holder altogether [5.67].) Due to the design of the elution trap no dialysis membrane was required. An Eldex (San Carlos, CA) Model A-30-S pump was used to draw buffer from the lower reservoir into the column holder, across the bottom of the
102
5 Electrochromatogruphy in Biomoleculur Analysis
Power
supply
1
I
>
Buffer
Glass Column
el
Figure 5.12. Schematic of the electrofractionation (EF) apparatus. The EF system is similar to a conventional column gel system except that the column gel is enclosed by a column holder. Buffer is pulled through the intake holes and across the bottom of the gel which allows the electrical current to be maintained. As a sample elutes off the column it is carried by the buffer to the detection and fractionation systems. (Reproduced with permission from Academic Press)
gel, and into the outlet tube. As a substance eluted from the gel it entered the continuous buffer flow which resembles that found in HPLC. Buffer containing the eluent then passed through a detection system (Hitachi (Tokyo, Japan)) 655 A variable wavelength monitor and a Fisher (Pittsburg, PA) Recordall Series 5000 recorder) which was coupled to an automated fraction collector (Isco Retriever 11, Lincoln, NE). Samples were loaded onto the column using a capillary tube and run at ambient temperature. The flow rate of elution buffer was between 25-100pL/min and the applied electric field ranged from 1-20 V/cm depending upon the sample. Electrophoretic columns were used repetitively (as in HPLC) and recoveries were generally greater than 80 % . Typical electropherograms of oligonucleotide, DNA fragments and protein separations using the E F apparatus are shown in Figures5.13, 5.14 and 5.15, respectively. A modified EF apparatus was also used successfully for separating biologically active proteins in a non-denaturing separative gel [5.66]. Interestingly, this analysis was not successful using HPLC due to protein adsorption in the dialysis membrane. Electrofractionation has also been used successfully to size fractionate DNA for the construction of lambda bacteriophage libraries (Fig. 16) which are used widely for the cloning of genes (see Section 5.3). Subsequent ligations and packaging of lambda, two sensitive steps associated with library construction, are uninhibited by the EF procedure [5.66]. Since almost all libraries, whether they are genomic, sub-genomic or cDNA libraries, require size fractionation of nucleic acids, E F seems particularly well suited for this application in molecular biology. High-performance electrophoresis chromatography and electrofractionation are versatile techniques in that they may be scaled up or down by adjusting the diameter of the column. For strictly analytical purposes, however, these techniques do not compare with capillary electrophoresis (CE) (see Section 5.7). Nevertheless, both techniques are capable of detecting nucleic acids in the nanogram range which is comparable
5.6 Electrochromatography in Preparative Columns
103
2
1
A264
n
io
30 80 Minutes (A)
(B)
Sb !A
1;o
Minutes (C)
Figure 5.W. EF analysis of oligonucleotides. A) Electropherogram of an unpurified 42 base pair oligonucleotide (peak 2) with a significant failure sequence at 15base pairs (peak 1). The small middle peak represents failure sequences between 16-41 base pairs in length; 25 pg of sample was loaded onto a 30 X 4mm, 20 % acrylamide/7M urea column gel (TBE buffer) and electrophoresed at 20V/cm. The flow rate was 50 pWmin. B) Autoradiogram of the unpurified oligonucleotide sample (lane 1) and the radioactivity corresponding to the 42base pair species recovered from peak2 (lane2). Electrophoresis was performed on a 0.75 mm, 20 % polyacrylamide/7 M urea slab gel. C) Electropherogram of oligonucleotides 18 (peak2), 20 (peak3), 25 (peak4) and 33 (peaks) base pairs in length. Peakl, bromophenol blue. The sample conditions used were the same as described in (a) except the gel length was 75 mm. (Reproduced with permission from Academic Press)
to that of conventional methods, e.g., ethidium bromide stained agarose gels. Thus, HPEC and EF are suited for routine analytical procedures such as determining concentration or purity. Sensitivity could be increased significantly if on-column detection units were used, as demonstrated by automated sequencing [5.69] and CE procedures. The most applicable feature of high-performance electrophoresis chromatography and electrofractionation lies in their ability to separate macromolecules on a micropreparative or a preparative scale. Approximately 300 pg of protein have been purified by HPEC in a single run [5.56] and up to 1mg of partially purified proteins analyzed using modified EF [5.66]. These amounts may be increased if the diameter of the column is enlarged with the limiting factor being the dissipation of heat from the center of the column. Since electrophoretic mobility increases approximately 2 % per "C [5.2], differential heating within the column results in a parabolic flow profile and a subsequent loss of resolution. This problem has been circumvented to some extent in the Model 491 Prep Cell (Bio-Rad, Gaithersburg, MD) which circulates buffer through a tube within the gel. Although horizontal gel systems provide an attractive alternative to columns with regard to heat dissipation [5.70], [5.71], these apparatuses have other limitations (i.e., lack of operation ease). Recently, several analytical techniques have coupled HPLC with capillary electrophoresis [5.72], [5.73] or SDS-PAGE [5.74]. Others have coupled gel electrophoresis
104
5 Electrochromutogruphy in Biomoleculur Analysis
3 2 1
1
3
2
4
A26( I
\
L
Ir (B)
Figure 5.14. EF analysis of DNA fragments. A) Electropherogram of DNA fragments generated from a Pstl digestion of pcDNAllneo. Peak 1, bromophenol blue; peak2, 922 base pairs (130ng); peak3, 2054 base pairs (290ng); and peak 4, 4147 base pairs (580ng); 1pg of sample was loaded onto a 30 X 6mm 1% agarose (TAE buffer) column gel and electrophoresed at 7 Vlcm. The flow rate was 50 pL/min. B) Collected fractions were ethanol precipitated and electrophoresed on a 1% horizontal agarose gel. Lane 1, pcDNAll neo digeted with Pstl; lane 2, DNA recovered from peak 2; lane 3, peak 3; and lane 4, peak 4. (Reproduced with permission from Academic Press)
with liquid chromatography [5.75], and size-exclusion chromatography with capillary zone electrophoresis. These systems have resolved a number of complex protein mixtures which could not be separated when using each technique alone. An appropriate step for the improvement of high-performance electrophoresis chromatography or electrofractionation would be adaptations to include additional separative dimensions. In this way, it is likely that the components of complex mixtures could not only be detected but they could also be purified to homogeneity at a micropreparative level. To date, separations of biological materials using high-performance electrophoresis chromatography and electrofractionation have employed the inert matrices of polyacrylamide or agarose [5.56]-[5.68]. There are no restrictions, however, with regard to its supportive matrix and it is likely that conventional chromatographic matrices could enhance the quality of some separations (see Sections 5.7 and 5.8).
5.6.3
Focusing in Columns
The development of carrier ampholytes [5.77] provided the foundation for isoelectric focusing, the conventional method which has been used to focus proteins according to their isoelectric point. Despite the elegance of this technique [5.49], [55.0], isoelectric
5.6 Electrochromatography in Preparative Columns
105
2
1
2
3
Ovalbumin-
A28
!
.
I
.
Lysozyme-
.
30 60 90 120 Minutes (A)
(B)
Figure 5.15. EF analysis of protein. A) Electropherogram of lysozyme (14,400 Da, peak 2) and ovalbumin (45,00ODa, peak3). Peak 1, bromophenol Blue; 8pg of lysozyme and 14mg of ovalbumin were loaded onto an 18 (15 mm resolving gel and 3 mm stacking gel) X 6 mm 12 % SDS-PAGE column gel and electrophoresed at 7.5 V/cm. the flow rate was 50 FL/min. B) Collected fractions were dialyzed against water and aliquots were electrophoresed on a 12 % SDS-PAGE slab gel and stained with Coomassie brilliant blue. Lane 1, lysozyme and ovalbumin standards; lane2, lysozyme (peak2); and lane 3, ovalbumin (peak 3). (Reproduced with permission from Academic Press)
focusing is not readily adapted to separate proteins on a preparative scale. Consequently, a novel approach has been developed which can potentially concentrate milligrams of material at unique positions on a chromatographic column. In this method, called counteracting chromatographic electrophoresis (CACE) [5.74], chromatography and electrophoresis oppose each other in a manner similar to what has been described by Neremberg and Pogojeff [5.31]. With an appropriate combination of chromatographic matrices, electrophoresis dominates in one part of the cotumn while the solvent flow dominates in another. As a result, a substance becomes concentrated at the point where these two opposing forces equal each other. The mechanism by which this process occurs has been described by O’Farrell in referring to Figure 5.17 [S.74]. “A column (Fig. 1)was packed with a restrictive matrix, BioGel P-10 (Bio-Rad), on top of a bed of a much more porous matrix, BioGel A-50m. The colored protein ferritin is found to chromatograph rapidly downward through the upper part of this column (where it is excluded from the matrix beads) but more slowly through the lower part of the column (where it is included in the matrix beads). As illustrated in Figure 1, at the appropriate applied voltage, the upward electrophoresis rate, RE, will exceed the downward rate of movement with solvent flow, RF, in the bottom matrix but will be
106
5
Electrochrornatography in Biornolecular Analysis
L8.W 1 2 3 4 5 6 7 8 9101112
Row A
Row B
Row c
Figure 5.16. Size fractionation of genomic DNA using EF. Rat genomic DNA (10 pg) was digested with EcoR1, applied to a 15 X 6mm 1% agarose (TAE buffer) column gel and electrophoresed at 0.8 V/cm. The flow rate was 40 pWmin. Fractions (2.50~1)were collected, ethanol precipitated and electrophoresed on the horizontal agarose gel shown. The size marker UHind 111 appears in lane 1 in all rows. Row A,lane 2 is an aliquot of rat genomic DNA digested with EcoR1. Row A, lane 3-12 correspond to aliquots from fractions ranging in size from about 0.3 kb-1.5 kb; rowB, lanes2-12 correspond to fractions from 1.5 kb-3.0 kb; and row C, lanes 2-12 correspond to fractions from 3.0 kb-4.5 kb. Following electrophoresis, the DNA was blotted onto nylon and probed with a radiolabeled oligonucleotide. DNA fractions which showed a positive signal were used for constructing a sub-genomic library in the vector hGem 4 (Promega).
insufficient to counteract the more rapid downward RF in the top matrix. At this voltage, the net movement (R,)of ferritin is downward in the top part of the column and upward in the bottom part of the column. The ferritin is thereby highly concentrated at an equilibrium position adjacent to the interface of the P-10 and A-50m gel beds.” Reproduced with permission from the AAAS. Copyright 1985 by the AAAS. Once concentrated, a sample can be recovered from a port located at the equilibrium zone or by removing the electric field and eluting as in liquid chromatography. CACE also has the advantage of a continuous buffer flow which effectively removes heat from the column, a problem which has plagued other preparative methods. The column described above demonstrates the fundamental principle of CACE, but this column could not resolve a complex protein mixture since almost all proteins would focus at the same interface. Resolution is achieved in CACE by increasing the number of chromatographic layers within the column. Under the appropriate conditions, a protein will concentrate at a particular interface while closely related proteins will concentrate at another interface or will be removed from the column either by electrophoresis (RE) or by the pressurized flow (RF).A relatively simple column containing six different layers (different compositions of BioGel A-5m and P-300) is shown in Figure 5 -18. Four different proteins (hemoglobin, myoglobin, ferritin and cytochrome C) were readily focused on this column and, in fact, no two proteins could be forced to reside on the column at the same time [5.78]. The sensitivity of the column
5.6 Electrochrornatography in Preparative Columns
107
t e of movement w l t h - R afluld flow RE - R a t e o f movement b y electrophoresle
RF
RM
- Net
rate: balance o f R f and R g
voltage drlvee 0 Applled components u p w a r d
1 1 11
Fluld f l o w c a r r l e e c o m p o n e n t s down t h e column
Equ I ilb r l u m z o n e
0
Figure 5.17. Schematic representation of the CACE apparatus. A 50-cm glass column (0.7 cm internal diameter) was packed to a height of 25 cm with BioGel A-50m (Bio-Rad), and the remainder of the column was filled with BioGel P-10. The column was connected to electrode reservoirs by short sections (1cm) of large-bore (1.2cm internal diameter) tubing plugged with 15 % polyacrylamide gel. Additional ports allowed the column to be connected to a peristaltic pump to give a regulatable flow of the carrier solvent (10mM tris acetate, pH7.4) through the column. Ferritin loaded on this column was brought to equilibrium with the flow rate set at about 0.17mUmin and the applied voltage adjusted to 600 V. The flow of the carrier solution is indicated by the group of arrows at the top. Vectors to the right indicate the magnitudes and directions of the electrophoretic rate (RE), the rate of solvent flow (RF), and net (RN) solute mobilities. In the equilibrium zone, RE and RF are equalized by concentrationdependent effects. (Reproduced with permission from the AAAS. Copyright 1985 by the AAAS)
was estimated by the percent change in voltage required to shift a protein from one interface to another. Using this criteria, it was determined that a 4 % difference in RE,which corresponds to a difference of a few charged amino acids, was sufficient to focus myoglobin at an adjacent interface. When considering the wide variety of chromatographic matrices available and the potential of using continuous gradients, CACE could have applications in a wide range of preparative fractionations (see Sections 1.6 and 6.1). At present, the main drawback to CACE in the biosciences appears to be in its lack of establishment. Although the idea is relatively simple, the development of the optimal conditions for focusing a particular protein represents a formidable task. Thus, it seems that some fundamental parameters of CACE need to be established before its potential can be realized.
108
5 Electrochrornatography in Biomoleculur Analysis
@
c lnterface
A- . 6 m l P - 3 0 0 1100
A
20100 B 40/80
C
D
60140 80120
E
1001
t
0 Figure 5.18. Use of a multilayered column to measure resolution. A column (20cm by 0.7cm internal diameter) was packed with BioGel A-5m, BioGel P-300, and mixtures of these matrices to produce a series of layered beds as indicated. This layering gives rise to subtle but discontinuous changes in properties so that the rate of movement of myoglobin with solvent (50mM tris acetate, pH7.4) flow (RF) differs in each layer. A protein will be focused at the interface between any of these pairs of layers if REis between the RFvalues of the two bounding layers. RE is determined by the product of the electrophoretic mobility (M) of the solute and the voltage gradient [dVldD), where D is the distance]. The fractional change in dVldD required to change RE to a value, RE', that will cause myoglobin to move from one interface to another provides a direct measure of the resolving power of the method. That is, as shown by the formulas at the right, REand RE' can be related, respectively, to M and M ' , the mobilities of two proteins that differ in electrophoretic mobility by the smallest amount that will permit complete resolution. (Reproduced with permission from the AAAS. Copyright 1985 by the AAAS)
5.7
Electrochromatography in Microcolumns
Most macromolecular separations are currently performed by slab gel electrophoresis or some form of column chromatography (HPLC, anion exchange, size exclusion etc.). Although these methods are quite functional, they all have drawbacks of being either labor intensive, expensive, semiquantitative or a combination of all the above. These problems have been reduced substantially in recent years with the development of capillary electrophoresis (CE). CE is typically performed in fused silica capillary tubes having an i d . <200 pm. Due to their large surface area to volume ratio, capillaries dissipate heat very efficiently and thereby allow high voltages (15-30 kV) to be applied without overheating. As a result, both high resolution and rapid analysis times can be obtained with essentially any biological material. In addition, CE is readily automated and on-column detection (e.g., laser-induced fluorescence) can provide sensitivity in the attomole/zeptomole (10-'8/10~*o) range using injection volumes of only a few nanoliters [5.79]-[5.82].
5.7 Electrochromatography in Microcolumns
109
Not surprisingly, the use of fused silica capillaries in the biological sciences is becoming quite popular. Many have utilized the technique of capillary zone electrophoresis (CZE) which separates molecules according to their electrophoretic mobility in blank capillary tubes [5.83]-[5.85]. CZE has produced many exceptional separations of biological materials, but it is mentioned here only for completeness and the reader is referred to several sources which describe this technique in detail [5.80]-[5.82]. It should be noted that a problem which has emerged in CZE is the adsorption of molecules to the inner wall of the capillary. To counteract this problem the interior wall has been coated with a variety of substances [5.86]-[5.91], some of which have sorptive properties and therefore constitute a form of electrochromatography. These separations will be considered in the following section. While CZE can be tuned by modifying the conditions of electrophoresis I5.791-[5.82], [5.92], its resolving power is limited to a single, electrophoretic dimension. As the need to separate complex biological mixtures becomes increasingly important, the idea of modifying existing CE techniques by introducing additional separative dimensions is receiving considerable attention. Since many of these methods introduce a sorptive component into the system, they have been classified as capillary E C (CEC). Within this group there are a number of subcategories [5.3] which have been described in detail elsewhere in this book (see Chapters2 and 3). Relevant techniques will be mentioned briefly here to demonstrate their potential in the analysis of biomolecules.
5.7.1 Electro-Osmotically-Driven Electrochromatography Electroosmosis is the flow of liquid which ensues following the application of an electric field to a solution containing stationary, charged particles; for example, the walls of a capillary tube or packing material which has been immobilized in a column. This phenomenon has been described in detail elsewhere [5.89]-[5.91]. The fundamental principle of electro-osmosis involves the formation of an electrical double layer between oppositely charged ions at all surfaces. If the surface bears a negative charge, then an excess of positive ions accumulate and form a sheath which surrounds a core of uncharged liquid. When an electric field is applied, the positive ions within the sheath migrate and propel the core along with it towards the negative electrode. In contrast with the parabolic flow profile observed in pressure driven systems, electroosmotic flow exhibits a flat flow profile similar to that of plug flow [5.93]. As a result, electro-osmotically driven systems have sharper bands and are less sensitive to matrix irregularities since flow is uniform throughout the system [5.84], [5.93]. In addition, oppositely charged and neutral molecules can be analyzed in a single run [5.84], [5.93]. The potential of using electro-osmosis in the analysis of macromolecules was first demonstrated by Mould and Synge in the early 1950s [5.96], [5.97]. Polysaccharides of various lengths were carried by electro-osmotic flow and separated according to their differential adsorption to collodion membranes [3.97]. This method of generating electro-osmotic flow was later applied to both thin-layer and column chromatography using microparticulate silica 15.931 and, in 1981, Jorgenson and Lukas described it in conjunction with their paper on capillary zone electrophoresis [5.84]. In their report, an electro-osmotically driven separation of peptides from a tryptic digest of chicken ovalbumin was demonstrated (Fig. 5.19). While many peptides are resolved by this analysis, it is likely that others have been masked under larger peaks. To obtain a complete separation of this peptide mixture the conditions of the separation could be modified (i.e., pH, salt concentration, etc.) or an additional separative component could be introduced. In many cases, the latter approach has been more effective. For exam-
110
b
5 Electrochromatography in Biomolecular Analysis
1
I
1 0
20
Time (min)
Figure 5.19. Electropherogram of fluorescamine labeled peptides obtained from a tryptic digest of chicken ovalbumin. (Reproduced with permission from Elsevier Science Publishers B.V.)
ple, closely related naphthalensulfonic acids have been resolved by capillary electrochromatography using capillaries coated with cross-linked PS-264 (a sorptive reagent) [5.98], while CZE and open tubular liquid chromatography have been unable to separate these compounds. Matriccs such as Chirasil-Dex [5.89], silica [5.99] and octadecylsilane (ODS) [5.100]-[5.103] have also demonstrated superior resolving power (see Chapter 3). Despite their potential in analytical procedures, electroosmotically driven systems may make their greatest contribution in the purification of macromolecules on a micropreparative or preparative scale. Since electroosmotic flow can be generated in packed columns of large diameter [5.84], [5.85], conditions which have been optimized analytically could be scaled up for preparative applications. In addition, the problem of heat dissipation is substantially reduced in electroosmosis due to the continuous flow of buffer through the column. Taken together, these features make electroosmotically driven EC an attractive alternative to the conventional preparative methods currently used to separate macromolecules (e.g. , column chromatography, HPLC, column electrophoresis, etc.). Despite its great potential, electroosmotically driven EC has undergone limited experimentation in the biosciences. This is due in part to the rapid developed and wide success of CZE and its related techniques [5.79]-[82], [5.85], [5.90], [5.91]. Since E C introduces a chromatographic component into the system, however, it is quite possible that electroosmotically driven EC techniques will some day outperform strictly electrophoretic procedures in a variety of biochemical applications.
5.7 Electrochromatographyin Microcolumns
5.7.2
111
Pressurized Flow Driven Electrochromatography
Solvent flow in pressurized flow driven EC is generated either by gravity or by an LC pump. Early experiments in this area utilized large columns (i.d. >2cm) in which the flow rate was determined by gravity [5.31], [5.55]. Recently, Tsuda has introduced an HPLC-like apparatus which contains additional hardware to accommodate an electric field [5.104], [5.105]. In this system, much smaller columns are used (i.d. <0.5mm) and pressure is generated by an LC pump. Columns which have been packed with chemically bonded ODS and subjected to a high voltage (up to 15 kV) have produced high quality separations in a fraction of the time it has taken previous systems (see Chapter 2). If mobility is equal to or greater than the pressurized flow velocity, then a sample may be injected continuously and concentrated on the column [5.105], [5.106] in a manner similar to that of CACE (see Section5.6.3). Pressure driven E C is also suited for applications on a preparative scale and, at the present time, may have advantages over electroosmotically driven EC since the solvent flow is more readily controlled. A variation of this technique, called electrochromatographic solid phase extraction (SPE), has been used to concentrate and partially purify cimetidine from serum by employing a C-18 cartridge with voltage assisted eluteion [5.107]. This technique may have general applications as a pretreatment and preconcentration step in the analysis of crude biological samples.
5.7.3
Micellar Electrokinetic Capillary Chromatography
Micellar electrokinetic capillary chromatography (MECC) may be considered an extreme form of EC, although it has already been placed into a category of its own [5.3], [5.81]. Since its introduction in 1984 [5.108], MECC has developed into a powerful tool for separating both charged and uncharged compounds [5.109]-[5.111]. MECC is unique in that it employs an electroosmotically pumped mobile phase and a moving stationary micellar phase [5.111]. Micelles are generated in the system by the addition of detergents such as sodium dodecyl sulfate (SDS) or cetyltrimethylammonium bromide (CTAB) [5.111], [5.112]. Once the critical concentration is reached, individual detergent molecules aggregate to form micelles containing 20-100 or more monomers [5.111]. A solute migrates in free solution due to both electrophoresis and electroosmosis (as in CZE) while, at the same time, partitions between the free solution and the pseudo-stationary micellar phase (as in micellar liquid chromatography). Exceptional separations have been obtained using MECC in the analysis of oligonucleotides [5.113], vitamins [5.110], drugs [5.114], glucosinolates [5.112] and peptides I5.1151. It can be expected that other biological molecules will be equally amenable to this technique.
5.7.4
Capillary Gel Electrophoresis
As with conventional gel electrophoresis, capillary gel electrophoresis (CGE) has been placed into a category of its own [5.81], [5.82], although it may be considered as a form of electrochromatography. CGE is essentially a scaled down version of tube gel electrophoresis and all the benefits of conventional gel electrophoresis apply to CGE. With the added feature of efficient heat dissipation, CGE has produced among the highest separation efficiencies of any analytical method to date [5.81]. Despite its attractive features, CGE has several practical limitations including the sensitivity of gels (particularly agarose) to high temperatures and the difficulties currently associated with cast-
112
5 Electrochromatography in Biomolecular Analysis
ing gels inside capillary tubes. In addition, CGE can not separate oppositely charged or neutral compounds since electroosmosis is suppressed. This drawback is likely to hamper CGE considerably in the analysis of complex biological mixtures. Nevertheless, for the analysis of linear biomolecules such as double stranded DNA and denatured proteins, it is doubtful that any technique will surpass the efficiency of CGE in the near future. It is possible that CGE will soon replace conventional procedures used for DNA sequencing, restriction fragment length polymorphisms (RFLPs), SDSPAGE and Southern blot analysis.
5.8
Conclusions and Future Directions
We are currently in an exciting period of growth and development in the biological sciences with discoveries being made almost daily. As our knowledge advances, however, we are constantly reminded of the complexity which underlies the fundamental processes of life. From viral replication to consciousness, evolution has engineered highly regulated pathways by selecting biomolecules having unique properties. It has been the goal of modern researchers to identify and isolate these biomolecules so that their roles in higher biological processes may be determined. It can be expected that progress in this direction will contribute immensely to basic research, biomedicine, and industrial sciences. Nested deeply in these efforts are the fundamental techniques of chromatography and electrophoresis. In most cases these methods are used independently of each other. Due to the complexity of natural samples, however, the idea of combining electrophoresis and chromatography into a continuous system (EC) is receiving considerable attention. The primary advantage of EC, in comparison with each technique used alone, is relatively simple. If two different molecules have identical electrophoretic mobilities, there is a good chance that their partition coefficients will differ. Likewise, two molecules having identical partition coefficients are likely to have different electrophoretic mobilities. Thus, in the analysis of complex mixtures or closely related molecules, the chances of obtaining a complete separation are enhanced when two separative components are employed instead of only one. The pioneering experiments of electrochromatography were performed almost half a century ago using filter paper [5.22]-[5.24] and thin layers of sorptive material I5.181, [5.97]. In the past decade, EC has experienced rapid growth which has produced a variety of new methodologies [5.3].These range from preparative columns [5.55], [5.76] to electro-osmotic E C separations performed in microcolumns [5.89], [5.98]-[5.107]. As the field continues to grow, it can be expected that new innovations will be introduced. For example, two dimensional separations within capillary tubes are being explored [5.114], [5.116] and additional separative dimensions, such as mass spectroscopy, have already been coupled with EC [5.117]. Despite the early success of electrochromatography in analytical chemistry, most EC procedures are still in their infancy in the biosciences. This is due to the dominance of capillary zone electrophoresis and high-performance liquid chromatography which have experienced tremendous success in the separation of biological molecules. It suffices to say, however, that EC may be used in parallel with CZE and HPLC, and with potentially greater resolving power since two separative components are employed instead of one. In addition, EC is capable of separating oppositely charged and neutral molecules which is a major limitation of CZE. While CE techniques proved numerous advantages in analytical applications, capillary tubes can not be used practically on a preparative scale. Due to this limitation,
5.9 References
113
electrochromatographic techniques may be among the most efficient procedures for obtaining biomolecules in preparative quantities. In contrast with CZE, several E C techniques which have been utilized in capillary tubes have the potential to be scaled up. These include electro-osmotically driven EC, pressure driven EC and capillary gel electrophoresis. In addition, the unique separation method used by CACE is also well suited for preparative applications. Of these methods, capillary gel electrophoresis is the most developed in the biosciences due largely to its similarities with conventional gel electrophoresis. Scaled up versions of CGE have been used effectively for macromolecular separations in high-performance electrophoresis chromatography, electrofractionation and other preparative apparatuses (e.g., Bio-Rad Model 491 Prep Cell). While these techniques have been developed for the inert matrices of polyacrylamide and agarose, they also provide excellent systems for exploring the potential of other EC methods on a preparative scale, such as electro-osmotically driven EC. Modern electrochromatography is in its early stages of development and is only beginning to make its way into the biological sciences. As the resolving power of conventional techniques become inadequate for the analysis of complex biological mixtures, it is likely that the benefits of combining electrophoresis and chromatography will gain popularity. Progress in this direction could revolutionize the methodologies for capturing biomolecules in their pure form, but this should be expected only after a period of considerable progress in the field. It has taken billions of years of evolution to put the molecules of life together and we are only beginning to develop methods to separate them.
Acknowledgements I thank Randy DeBey for assistance with molecular structures, and Dr. Jakyoung Yo0 for helpful comments on the manuscript.
5.9
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[5.72] M. M. Bushey, J. W. Jorgenson, Anal. Chem., 62, 978-984 (1990). [5.73] H. Yamamoto, T. Manabe, T. Okuyama, J . Chromatogr., 515,659-666 (1990). [5.74] W. G. Burton et al., J . Chrornatogr., 443, 363-379 (1988) [5.75] D. J. Rose, G. J. Opiteck, Anal. Chem., 66, 2529-2536 (1994) [5.76] A . V. Lemmo, J. W. Jorgenson, J. Chromatogr., 633, 213-220 (1993) [5.77] H. Svensson, Acta Chem. Scand. 16, 456-466 (1962). [5.78] P. H. O’Farrell, Science 227, 1586-1589 (1985). [5.79] C. Schwer, E. Kenndler, Chromatographia, 30(9/10), 546-553 (1990). [5.80] Z. Deyl, R. Struzinsky, J. Chromatogr., 569, 63-122 (1991) [5.81] S. E Y. Li, Capillary Electrophoresis: Principles, Practice and Applications, Amsterdam, Elsevier Science Publishers, 1992. [5.82] J. P. Landers et al., Biotechniques, 14(1), 98-11 (1993). [5.83] J. W. Jorgenson, K. D. Lukas, Anal. Chem. 53, 1298-1302 (1981). [5.84] J. W. Jorgenson, K. D . Lukas, J . Chromatogr. (1981) 218,209-216. [5.85] J. W. Jorgenson, K. D. Lukas, Science, 222,266-272 (1983) [5.86] Y. Tanaka, W. Thormann, Electrophoresis, 11, 760-764 (1990). [5.87] L. R. Gurley, J. E. London, J. G . Valdez, J. Chromatogr., 559,431-433 (1991). [5.88] W. Nashabeh, Z. E. Rasi, J. Chrornatogr., 559, 367-383 (1991). [5.89] S. Mayer, V. Schurig, J. High Resolut. Chromatogr., 14, 129-131 (1992). [5.90] J. T. Smith, Z. E. Rassi, J. High Resolut. Chrornatogr., 15, 573-578 (1992). [5.91] J. K. Towns, J. Bao, F. E. Regnier, J . Chromatogr., 599, 227-237 (1992). [5.92] W. D. Pfeffer, E. S. Yeung, Anal. Chem, 62, 2178-2182 (1990). [5.93] V. Pretorius, B. J. Hopkins, J. D . Schieke, J. Chrornatogr., 99,23-30. (1974) [5.94] A. W. Adamson, Physical Chemistry of Surfaces, New York, Wiley-Interscience, 1976,3rd ed., chap. 4. [5.95] J. H. Knox, I. H. Grant, Chromatographia, 24, 135-143 (1987). [5.96] D. L. Mould, R. L. M. Synge, Analyst (London), 77, 964 (1952). [5.97] D. L. Mould, R. L. M., Biochem. J., 58,571-585 (1954). [5.98] W. D . Pfeffer, E. S. Yeung, J . Chromatogr., 557, 125-136 (1991). [5.99] T. S. Stevens, H. J. Cortes, Anal. Chem. 55, 1365-1370 (1983). [5.100] T. Tsuda, K. Nomura, G . Nakagawa, J . Chromatogr. 248,241-247 (1982). [5.101] N. Tanka, H. Kinishita, M. Araki, T, Tsuda, J. Chromatogr., 332, 57-69 (1985). [5.102] J. H. Knox, I. H. Grant, Chromatographia, 32, 317-328 (1991). [5.103] H. Yamamoto, J. Baumann, F. Erni, J. Chromatogr., 593,313-319 (1992). [5.104] T. Tsuda, Anal. Chem., 59, 521-523 (1987). [5.105] T. Tsuda, Anal. Chem., 60, 1677-1680 (1988). [5.106] T. Tsuda, Y. Muramatsu, J. Chromatogr., 515, 645-652 (1990). [5.107] H. Soini, T. Tsuda, V. Novotny, J. Chromatogr., 559, 547-558 (1991). [5.108] S. Terabe et al., Anal. Chem., 56, 111-113 (1984). [5.109] S. Terabe, K. Otsuka, T. Ando, Anal. Chem., 57, 834-841 (1985). [5.110] H. Nishi, N. Tsumagari, T. Kakinoto, S. Terabe, J . Chromatogr., 465, 331-343. [5.111] K. Kleparnik, F? Bocek, J. Chromatogr., 569, 3-42 (1991). [5.112] S. Michaelsen, P. Moller, H. Sorensen, J. Chromatogr., 608, 363-374 (1992). [5.113] A. S. Cohen, S. Terabe, J. A. Smith, B. L., Karger, Anal. Chem. 59, 1021-1027 (1987). [5.114] R. Weinberg, I. S . Lurie, Anal. Chem., 63, 823-827 (1991). I5.1151 S. H. Swedberg, J. Chromatogr., 503, 449-452 (1990). [5.116] W. Nashabeh, Z . E. Rassi, J . High Resolut. Chromatogr., 15, 289-292 (1992). [5.117] E. R. Verheij, U. R. W. M. A . Niessen, J . Chromatogr., 554, 339-349 (1991).
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6
Electromatography for Focusing, Counter-Current , Mass Spectrometry, and Two-Dimensional Separation Takao Tsuda
6.1
Continuous In-Column Sample Focusing
Electrochromatography uses two processes in the development of a sample in the column. If we can adequately control these two processes, we can achieve in-column focusing. A solute will remain in the column when the velocity due to mobility, V,,,&, is larger than the flow velocity due to pressurized flow, vpres, and the two flows are in opposite directions. Figure2.4 in Chapter2 illustrates one condition in which a solute will be retained in the column. In this instance, the diffusion coefficient of the solute in the mobile phase is assumed zero for simplicity, X,, is the point of injection and the region X > 0 corresponds to the column; for X < 0 is necessary to work out the retarded solute at the column inlet. At time to the solute is introduced as a plug. The sample remaines in the column under the condition 2 vpresC - V,b because the highest laminar flow velocity is twice the mean linear-flow velocity. For any chromatographic process, there is a diffusion coefficient, and a narrow zone is always formed. Here, the center of the zone occurs equal to vpres.Therefore, most of the sample remains under the condition vpres< - y,,,&, if vpresis slow enough to produce a sharp distribution of the sample zone.
6.1.1
Multiple Injection for Accumulating a Charged Component in the Column
A typical example is shown in Figure6.1, where N 1 , N,, and N3 are a series of experiments [6.1]. Experiment N , involved an applied voltage; the chromatogram shows three minor peaks. After several injections with the voltage applied, the voltage was switched off (N, at time to) and a chromatogram was immediately obtained. After a large peak was eluted, the baseline became straight and chromatogram N3 was obtained for injection with no applied voltage. The N-methylphenylpyridinium perchlorate peak was eluted only when no voltage was applied. Under the applied voltage, only minor components in the sample of N-methylphenylpyridinium perchlorate were eluted. During injections with the applied voltage, the main peak of Nmethylphenylpyridinium perchlorate remained in the column. After the voltage was removed, it emerged, it emerged as a large peak, N z . The reverse phenomenon was also observed: after application of a voltage to the column, several peaks were eluted without any injection. These peaks may be from components in the sample solution or from the eluent itself.
118
6 Electromatogruphy for Focusing, Counter-Current, Muss Spectrometry
A N3
I
0
20
10 Rt
30
(mini
Figure 6.1. Retained solute in column under the influence of an applied voltage. N , with applied voltage; N2 and N3 with no voltage. N2 obtained just after release of the applied voltage at time zero. Solute, N-methylphenylpyridium perchlorate (A) and its impurities (other peaks).
6.1.2
Continuous Sample Introduction for In-Column Sample Focusing
Although the chromatograms in Figure 6.1 were obtained with individual injections, it is possible to introduce the sample continuously into the column head. The sample can then be concentrated by the applied voltage, and collected once the applied voltage is switched off (A$).The instrumentation for continuous injection is shown in Figure 6.2.
% 14
Figure 6.2. Schematic diagram of the apparatus for continuous injection. 1) Column (5 cm x 4 mm) of Pyrex, packed with ODS-silica; 2) Injector; 3) Silicone rubber septum; 4) Frit; 5 ) Connector; 6) Platinum tubing (inner diameter 0.5 mm) for terminal; 7) Three-way connector; 8), 8 ) , 9), 9' Fused silica capillary tubing; 10) Connector; 11) Stainless steel tubing (outer diamter 0.16cm); 12) Eluent pump; 13) Sample pump; 14) UV detector (254 nm); 15) d. c. high-voltage supply.
6.1 Continuous In-Column Sample Focusing
119
a
0
6
C
31
I+-
5 rnin C
Figure 6.3. Chromatograms with continuous injection of a negatively charged solute. Sample, 10mM aqueous 1, 2, 6-naphthalenesulfonic acid; sample injection rate, 10 pL/min; eluent, 0.1 mM phosphate buffer (pH7) - methanol (40:60); applied voltage, + 5 kV (75 PA). Peaks (a) and (b) belong to the sample, and peak (c) is the solvent peak. A) Voltage on; B) Voltage on, no injection; C) Voltage on, 5min injection; D) Chromatogram obtained just after stopping the applied voltage at time zero.
The sample is injected via a fused silica capillary tube, the extremity of which is placed at or just inside the head of the column packing material. The amount of sample injected was proportional to the duration of injection. (The injection was performed by a pump used exclusively for this purpose.) The rate of sample injection was adjusted by setting the flow rate of the pump. This device can be a very good system for electrochromatography. Since the fused silica capillary works as an electrical insulator, the process of injection is safe. This system was tested with 1% benzene - methanol as a sample, and there was no tailing due to injection, i.e., the electroosmotic flow in the fused silica capillary due to the applied voltage is not sufficient to cause continuous leakage of sample stored in the pump. The chromatographic behavior with continuous injection of sample solution (1,2,3naphthalenetrisulfonic acid aqueous solution, lo-*M) into the column inlet is demonstrated in Figure6.3. Continuous injection for 5 min (total sample 50 pL) is performed. During this period a positive electric field was applied in order to retain negatively charged sample components. In chromatogram C (of Figure 6.3) there is a large solvent peak due to the relatively long period of sample injection. After switching off the applied voltage, sulfonic acid that had been retained in the column was eluted. As the total length between the two terminals was ca. 12cm, the real voltage applied to the column was ca. 40% of the actual applied voltage. We used samples of relatively high concentration, and the sample injection rates were 4-10 pL/min. Although neither the sample injection rate nor the total amount injected in 5min are large, this is the first experiment that shows the possibility of liquid chromatography with continuous injection [6.2]. Even if a solute has a high mobility, e.g., - vmOb> 2 vpres, it is possible to retain it in the column. Hence it is possible to concentrate a very minor component in the sample solution by passing an amount of sample through the column under a high applied voltage. Electrochromatography can be a very effective method for concentration of some types of charged compounds.
120
6 Electromatography for Focusing, Counter-Current, Mass Spectrometry
O’Farrell [6.3] also proposed in-column focusing using a column packed with two kinds of porous resin, BioGel P-10 (used for size exlusion in the mass range 15 000-20 000) as the upper bed, and BioGel A-50m (used for size exclusion in the mass range ca. 100000). He suggested that vpresdepends on the inner pore volume of the gels, and a solute has capacity factors kfupand kfdown for upper and lower beds in the column. Therefore, a solute in the upper bed moves at(Rupvpres),where R is a retardation factor (1 k’)-’. He also considered that the potential gradient along the column was the same at any location along the column. Therefore a solute at the upper bed is carried with velocity (Rupvpres-vmob), and at the lower bed (Rdown vpres-vmob). When the flow velocity of solute at the upper bed is positive and the value at the lower bed is negative, the solute will be focused at the interface of the two beds. O’Farrell made one mistake. When vpres slows, the total cross-sectional area of the channels, in which the mobile phase flows, increases. So the potential gradient decreases, corresponding to the ratio of the cross-sectional area of the beds. The retardation factor R‘ is always effective for both vpresand Vm&. Therefore, the flow velocity of the solute should be expressed as [R’ (vpres- v,&)]. The value of (vpres- v,,,) is the same at both the upper and lower beds, and it is impossible to concentrate the sample at the interface of the two beds. This mistake may hamper the development of the method for in-column focusing (see also the discussion sections 1.6 and 5.6).
+
in-Column Focusing with l h o Potential Gradients. We could use one more factor for concentrating a solute in column, namely the potential gradient. When we apply a lower potential gradient at the upper portion of a column compared with that at the lower portion of the column, we can concentrate the sample in the middle of the column. For this it is necessary to place an electrode in the middle of the column, and apply different potential gradients at the upper and lower portions.
6.2
Rotation Locular Counter-Current Electrochromatography
In counter-current chromatography, there are two liquid phases, a lighter and a heavier liquid phase, which are not mixed with each other and correspond to the stationary and mobile phases, respectively [6.4]-[6.6]. Counter-current chromatography has a high sample loading capacity, and is often used for sample purification. The application of an electric field along the column gives two separation processes, liquid -liquid partition and electromigration of solutes in the heavier layer (mobile phase). Selective purification becomes possible.
6.2.1
Instrumentation
Kabasawa [6.7] applied a voltage along a column in counter-current chromatography. Kabasawa’s home-made instrumentation is shown in Figure 6.4. The column consisted of a Pyrex tube (12 mm inner diameter, 100cm long), 100 loculi (Teflon disks inserted into the glass tube, 1cm apart each with a 2mm diameter hole at its center), an injection port, and two salt bridges to electrode reservoirs via rotating seals. Heavier liquid is introduced via the injection port to the column at 0.5 mumin. Additional makeup flow of heavier liquid, 0.2 mumin, is fed at the other end of the column for fractionation of the eluent, which exists from the right of the column in Figure 6.4. The column
6.2 Rotation Locular Counter-Current Electrochromatography Sample injection port
+Salt
121
Rotating seal
bridge
P t electrode
1M KCI Figure 6.4. Rotation locular counter-current electrochromatography. Separation column made by placing 99 disks in a glass tube (12 mm inner diameter, 100cm long) at regular intervals. Solvent, n-butanol - acetic acid, water (4: 15).
itself is rotated during the separation at 70rpm. The total volume of lighter liquid placed in the upper port of each loculi is 15 mL, with 55 mL of the heavier liquid in the column. The solvent system used was n-butanol, acetic acid, and water (4: 1:5). This solvent was split into two liquid sections, namely lighter and heavier layers after equilibration. Figure6.5 shows the device for passage of the eluent. One end of a capillary tube, 1mm inner diameter, is inserted in the final loculus, which the eluent is forced to pass through for fractionation. Pump
Oil seal packing Lighter phase
+ Fractiona t ion
t bridge
Heavier phase Figure 6.5. Device for transferring eluent to a fraction collector.
122
6 Electromatography for Focusing, Counter-Current, Mass Spectrometry
O.D. xmpuritic.
10
20
1:: I 30
40
50
100
in c
(620nn)
.I
110
120
130
140
0 0.7
0.s
0.3
0.1
Fraction, 5ghube
Figure 6.6. Elution pattern of Ponceau SX (P), NaphtholYellow S (N) and Guinea Green B (G). A) Separation by rotation locular counter-current chromatography with no applied voltage (rotation 100rpm, heavy phase 0.5mLhin); B) Separation with 2000V applied voltage (rotation 70 rpm),operation period 54 h.
6.2.2
Preparative-Scale Electrochromatography with a Counter-Current Chromatographic Column
A typical example of electrochromatography with rotation locular counter-current chromatography, is shown in Figure 6.3. A mixture of Naphthol Yellow S (8-hydroxyl-5,7dinitro-2-naphthalenesulfonic acid disodium salt), Ponceau SX (4-hydroxyl-3-(5-sulfo2,4-xylylazo-)-l-naphthalenesulfonicacid disodium salt), and Guinea Green B (sodium salt of N-ethyl-N-[4-[a-[4-[N-ethyl-(3-sulfobenzyl)amino]phenyl]benzyliden]-2,5cyclohexadien-l-ylidene]3-sulfobenzylaminiumhydroxide) was injected as sample (0.5 mL containing 0.35% of each component). The upper chromatogram is without applied voltage, and the lower with an applied voltage (2000V, 1.5-2.0mA). Each fraction contained 5g of eluent which corresponds to collection every 7-10min. Kabasawa estimated the partition coefficients of Naphthol Yellow S, Ponceau SX, and Guinea Green B as 0.42,1.03, and 0.23, with ratios of migration distance due to applied voltage 6, 5 , and 2.6, respectively, from paper chromatography. As these values are obtained under different experimental conditions, we cannot adopt these values directly, but it is worth referring to them to check the results of the chromatograms in Figure 6.6. Separation is attained after 54 h. The partition coefficients are up to 1.The impurities in Guinea Green B eluted at same fraction number both with and without applied voltage. All the components in the mixture, except impurities of Guinea Green B, are eluted earlier when the applied voltage is on. Therefore, the three components have to travel to the outlet by their electrophoretic mobilities, especially Naphthol Yellow S and Poneau SX. Preparative-scale electrochromatography can provide effective separation.
6.3 ElectrochromatographyIMass Spectrometry
6.3
123
Electrochromatography/Mass Spectrometry
Continuous-flow fast-atom bombardment (CF-FAB) has become a valuable technique for interfacing different separation techniques with mass spectrometry. This is due to the good performance of this type of interface, permitting FAB ionization in mass spectrometry combined with liquid chromatography (LC/MS) [6.8]. With growing interest in the analysis of compounds such as peptides, (glyco)proteins, and (o1igo)nucleotides, separation techniques that permit the analysis of these polar and often charged compounds are needed. Although the efficiencies obtained in CE are better than those in liquid chromatography, the use of narrow-bore fused silica capillaries in CE results in peak volumes that are of the order of only tens of nanoliters. Even though mass concentration of the peak may be relatively high, the absolute masses are very low. Thus in some cases it is not appropriate to use the combination of CElMS [6.9]. Since electrochromatography can produce a compressed peak, i.e., a high mass concentration in peak volumes of the order of pl or sub pl, the combination of electrochromatography/mass spectrometry may be worth exploring [6.10].
6.3.1
Instrumentation
The instrumentation used for electrochromatophy - mass spectrometry is shown in Figure 2.16C and D of Chapter 2 for microliquid chromatography continuous-flow fast-atom bombardment (Fig. 6.7) [6.10]. Micro-LC - CF - FAB Interface. Verheij et al. [lo] used a liquid junction interface [6.11]. The 75pm fused silica capillary is polished and inserted 10mm into a 100 mm x 350 pm inner diameter fused silica capillary, and fixed with epoxy. The 350 pm capillary is connected to a T-piece with a Vespel ferrule. The 50 pm capillary coming from the micro-LC system is passed through the T-piece and inserted in the 350 pm capillary until it reaches the 75 pm capillary. The makeup flow containing 15% glycerol was applied at flow rates of 5-10 pL/min. The total flow rate to the mass spectrometer was 7-14 pL/min. The mass spectrometry used had a saddle field gun, which generated 7-kV xenon atoms. A gold-plated target was used in combination of pressed paper at the bottom of the ion volume to ensure stable ionization conditions [6.10]. 0 50 Pm n o 3 5 0 prn,
p-LC 0-5 pl/min
:
JCI
-
I 075pm
b CF-FAB 5-10 ul/min
FAB Matrix 5-10 pl/min
Figure 6.7. Liquid junction micro-LC-FAB interface for post-column FAB matrix addition.
124
6 Electromatography for Focusing, Counter-Current, Mass Spectrometry
1
6.3.2
Combination of Pressurized Flow-Driven Electrochromatography and Mass Spectrometry
Figure 6.8 shows mass chromatograms of ionsine-5’-triphosphate (ITP) under isocratic and electrochromatographic conditions. The mass chromatogram obtained under isocratic conditions shows a broad peak, but it becomes a single sharp peak under electrochromatographic conditions l6.101. Figure 6.9 gives another example of the potential of electrochromatography. Figure 6.9a shows the results obtained in solvent gradient mode and Figure 6.9b shows
0
0
5
10
10
15
20
20
30 Time (rnin)
25
40
30
50
Figure 6.9. Comparison of summed mass chromatograms of 250 ng suramin. A) LC - MS with gradient elution (from 5 % to 60% methanol in 30 min); B) Pressurized flow-driven electrochromatography - MS with isocratic elution.
6.3 Electrochrornatography/Mass Spectrometry
125
[M-H? mh 403*483
177 [H,P20,1-
0
10
i
-
20
' .IL.
- m/z
30
min
(c)
-
UTP
483 [M-HI'
159 [HP20al-
+ . '
100
0
200
-
300
,I.
400
500
E 10
m/z Figure 6.10. Mass chromatograms of the [MH]+ions from long of UTP and UTP; B) Corresponding negative-ion FAB mass spectra obtained by electrochromatography.
the results of electrochromatography. Suramin is used as sample, in which there are six sulfonate groups. The theoretical plate numbers of the peaks are 5000 and 34000, respectively. This example shows that electrochromatography gives a better chromatogram than the solvent gradient mode [6.10]. Figure 6.10 shows a chromatogram and spectra obtained by electrochromatography/ mass spectrometry. The negative-ion FAB mass spectra were obtained after injection of 10ng of two nucleotides into the eIectrochromatography system [6.10]. Electrochromatography gives high mass concentration and enough total mass for an adequate measurement of mass spectra. Electrochromatography has definite advantages over the solvent gradient mode of conventional microcolumn liquid chromatography. However, Verheij et al. [6.10] pointed out the following disadvantage. Dilute buffers are required in pressurized electrochromatography with 220 pm inner diameter columns, in order to avoid excessive heat generation within the capillary, and there is a greater risk of air bubble formation. Buffer concentration exceeding 5 mmol/L results in unacceptable high electric currents and lowers the performance of the system considerably. To overcome this disadvantage, we need to ensure effective heat dissipation, cooling the column by liquid media or adopting a narrower capillary column. Alternatively, we can use an organic solvent as eluent to decrease the current. Under these conditions, we can use an eluent containing a moderate concentration of electrolyte.
126
6 Electromatography for Focusing, Counter-Current, Mass Spectrometry
6.4
Tho-Dimensional Preparative Chromatography with Continuous Injection
It is possible to perform two-dimensional chromatography. For example, the mixture is separated by paper chromatography, followed by paper electrophoresis [6.12]. Simultaneous two-dimentional separation was performed with a tapered paper in which pressurized flow (z-direction) and applied voltage (x-direction) were applied [6.13]. Martin proposed a two-dimensional method for separation with continuous sample introduction. The apparatus was a continuous chromatograph that included an annular bed of adsorbent moving with respect to a feed stream and eluate collection points [6.14]. This concept was considered theoretically by Giddings [6.15]. Practical instrumentation employs a rotating cylinder containing an active annulus of adsorbent, with stationary feed and eluent streams as well as stationary eluate collection ports [6.14]-[6.18]. There are also several reports of continuous electrophoresis in a free zone [19]-[21]. -INNER ELECTRODE
POWER SUPPLY
r
COOLANT ANNULUS
ELUENT
ELUENT
I
FE
COOL ANT PRODUCT RECOVERY POSITIONS
EFFLUENT
L
EFFLUENT COOLANT
Figure 6.11. Continuous electrochromatography using a rotating annular system [6.22]. A) Flow through the system; B) Detail of the inner electrode; C) Rotation geometry.
6.4 Two-Dimensional Preparative Chromatogruphy with Continuous Injection B
127
FEED INTO STATIONARY NOZZLE 0 . 5 c m BELOW SURFACE OF SORPTION -ELUENT INTO STATIONARY NOZZLE JUST ABOVE SORPTION BED
NOMINAL- 0 - 6 4 C LUCl TE W A L L ROTATING ANN EL EC TROCHRO GRAPH (10.2 c 30 c m I---
SORPTION MEDIA
OUTER WIRE MESH ELECTRODE
1.25 c m - D I A M I GRAPHITE E L E
u ELUATE E X I T S
Figure 6 . l l B FEED INTRODUCTION
INNER ELECTRODE
OUTER ELECTRODE
e=o
Figure 6.llC
6.4.1
Continuous Preparative Electrochromatography: a Large Cylindrical Column with Rotation
Both continuous chromatography and continuous electrophoresis with a rotating cylinder are combined in continuous electrochromatography using a rotating annular system [6.22] (Fig. 6.11).
128
6 Electromatography for Focusing, Counter-Current, Mass Spectrometry
The system is composed of an annular column of adsorbent that is slowly rotated past a stationary feed point and the eluate takeoff points. The annulus is relatively wide with respect to the feed stream, introduced through a nozzle approximately at the center of the annulus. Typically, two electrodes are placed within the annular column, close to the inner walls. An inert sweep gas is introduced into the top headspace to remove potentially explosive electrode gases, and to provide an over-pressure. All entrance and exit streams and electrode connectors at the top of the column progress through a central stationary shaft with a pressure seal, thus allowing the annulus to be rotated under pressure. Exits ports are arranged in radial rows around the bottom of the annulus. Each component is collected by placing a stationary channel at the angular and radial position expected for elution. Continuous annular electrochromatography provides two-dimensional separation. Solute is transferred under pressurized flow (z-direction), and at the same time the bed is rotated with angular velocity o(rad/s). The solute is also forced to migrate via its electrophoretic mobility to one of the inside walls by the radial electric field. Therefore the elution position of the exit port is characterized by the angle, O(rad), and radius, r [6.22]. O = ( L w / u ~[)E
+ (I - E) K ]
where L, uf, E, K , r,, v,,,,~,and to are the vertical distance from feed point to exit (cm), eluent velocity through the column ( c d s ) , void fraction in the chromatographic column bed (fluid phase)/(total volume), distribution coefficient, radial position of feed point (cm), velocity due to electrophoretic mobility ( c d s ) , and elution time of nonretained solute, respectively. This type of electrochromatography has an unique character: the radius depends only on the electrophoretic mobility of the solute, and the angle 6 depends only on the distribution coefficient of the solute. Therefore, the position of the exit port for a given solute is defined by these two parameters.
6.4.2
Separation of a Test Mixture
The separation of the mixture of Blue Dextran and hemoglobin has been demonstrated for a cylindrical column packed with size-exclusion gel, Bio-Gel P-150, molecular exclusion limit 15 x lo4.As the molecular mass of Blue Dextran is 2 x lo6, its elution time indicated that the void fraction of the packed bed was 0.40. The flow velocity of the eluent was 8.OmL/min, and the feed rate of the mixture (each component 0.01 %) was 0.25mllmin. As hemoglobin is retained much longer (elution time 100 min), the stream of hemoglobin showed in a somewhat greater dispersion under an electric field (1OV). It begins to separate as two extremes (hemoglobin itself is the mixture of several components). Scott [6.22] also pointed out that, in the absence of an electric field, 77% of the original material still exited at a single nozzle. Continuous preparative electrochromatography is very attractive, because the method is very selective and has the potential to separate complex mixtures on a semiindustrial scale. To realize its full potential, we need to take care of the temperature rise due to Joule heating, to remove the gas evolving at the surface of the electrodes, and to avoid electrochemical reactions on the surface of electrodes.
6.5 References
6.5
129
References
[6.1] T. Tsuda, Anal. Chem., 60, 1677-1680 (1988). [6.2] T. Tsuda, Y. Muramatsu, J. Chromatogr., 515, 645-652 (1990). [6.3] P.H. O’Farrell, Science, 227, 1586-1589 (1985). [6.4] Y. Ito, R. L. Bowman, J. Chromatogr., 8, 315 (1970). [6.5] Y. Kabasawa, H. Nakazawa, M. Yamamoto, T. Tanimura, Bunseki Kagaku, 38, 16-20 (1989). [6.6] Y.Kabasawa, S. Kimura, T. Tanimura, Yakugaku Zasshi, 109 (3), 168-171 (1989). [6.7] Y. Kabasawa, J. Chem. SOC.Jpn, Chem. Znd. Chem., 1355-1359 (1990). [6.8] Y. Ito, T. Takeuchi, D. Ishii, M. Gotto, J. Chromatogr., 346, 161 (1985). [6.9] M. Caprioli, T. Fan, J. S. Cottrell, Anal. Chem., 58, 2949 (1986). [6.10] E. R. Herheij, U. R. Tjaden, W. A. Niessen, J. van der Greef, J. Chrornatogr., 554, 339-349 (1991). [6.11] R. D. Minard, D. Chin-Fatt, P. Curry, Jr., A. G . Ewing, 36thAnnual Conference on Mass Spectrometry and Allied Topics, ASMS, San Francisco, CA, 1988, p. 4950. [6.12] J. Rybarska, K. Bobrzeka, L. Konieczny, Haematologia, 18 (2), 13-139 (1985). [6.13] H. H. Strain, Anal. Chern., 30, 228-231 (1958). [6.14] A. V. P. Martin, Disc. Farady SOC.,7, 332 (1949). [6.15] J. C. Giddings, Anal. Chem., 34, 37 (1962). [6.16] C. D. Scott, R. D. Spence, W. G. Sisson, J. Chromatogr., 126, 381 (1976). [6.17] R. M. Canon, J. M. Begovich, W. G. Sisson, Sep. Sci. Technol., 15, 655 (1980). [6.18] J. M. Begovich, C. H. Byers, W. G. Sisson, Sep. Sci. Technol., 18, 1167 (1983). 16.191 K. Hanning, Z. Anal. Chem., 181, 244 (1961). [6.20] A. Kolin, J. Chromatogr., 26, 164 (1967); ibid. 26, 180 (1967). [6.21] T. Vermeulen et al. Ind. Eng. Chem., Process Des. Dev., 10, 91 (1971). [6.22] C. D. Scott, Sep. Sci. Technol., 21,905-917 (1986).
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Part 2 Applications of Electric Fields in Industrial Processes
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7
Electroosmotic Dewatering Masashi Iwata
7.1
Introduction
The term electroosmosis refers to the motion of liquid induced by an applied electric field. Such motion occurs when an electric field is applied across a porous material. Flow results from the presence of an electric double layer along a solid - liquid interface. The application of the electric field causes the ions in the double layer to move toward one electrode or the other (Fig. 7.1). Since the ions in the double layer are predominantly of one sign, their motion gives rise to a body force on the liquid in the double layer, which sets the liquid in motion. Electroosmosis was first described in 1809 by Reuss [7.1]. He observed that, on passing an electric current between two electrodes contained in glass tubes and separated by clay, the water was found to rise in the cathode compartment while it fell in the anodic compartment. Electroosmosis has been used to treat a wide variety of materials. The purpose of treatment has predominantly been the dewatering of compressible solid - liquid mixtures. It is especially effective in removing liquid from sludges of colloidal particles, for which conventional mechanical dewatering is not very successful. Colloidal sludge is usually highly compressible and mechanical dewatering is impeded by hydrodynamic resistance. On the other hand, the high surface charge density of colloidal particles leads to high electroosmotic flow under an applied electric field.
Figure 7.1. An electric double layer along a solid - liquid interface. The broken line shows the slip plane.
134
7 Electroosrnotic Dewatering
In spite of substantial development of industrial equipment for electroosmotic dewatering in the past decade, the theory of dewatering is far from complete. This chapter aims to discuss the mechanism of electroosmotic dewatering. We will first discuss the electroosmotic flow through a capillary with a circular cross-section. Then, we will extend the theory to the flow through compressible porous media, and finally develop an analytical method for practical electroosmotic dewatering. We will make clear that electroosmotic dewatering is a kind of consolidation process which accompanies an increase in solid compressive pressure in the material. Industrial applications of electroosmosis are also described.
7.2
Electroosmotic Flow through a Capillary
The electroosmotic flow induced in a capillary tube has been modeled in a variety of ways. Kobayashi et al. [7.2] solved the Navier - Stokes equation, taking into account the applied electric field strength Eo and the electric field strength in the double layer Eicaused by the contact potential difference qj. The equation of motion is
Dv
p-
DO
=
-vpL
+ p v 2 v + pg + p,(Eo + El)
(1)
where p is the density of the liquid, v the velocity vector, 8 the time, DlD8, the substantial time derivative, p,. the liquid pressure, p the viscosity of the liquid, g the gravitational acceleration, and pe the volumetric charge density of the liquid. Gauss's theorem for the electric flux density is div[D(Eo + Ej)] = pr
(2)
where D denotes the dielectric constant of liquid. Based on Equations (1) and (2), the electroosmotic flow through a capillary is analzyed in cylindrical coordinates (Fig. 7.2). The working conditions and assumptions are as follows: 1) The fluid velocity and applied electric field are in the z-direction. 2) Gravity is ignored.
Figure 1.2. Potential distribution in a capillary.
7.2 Electroosmotic Flow through a Capillary
135
3) The electric field strength Eicaused by contact electric potential acts in the radial direction of a capillary. 4) The fluid flow and electric current are steady, and the applied electric field in the zdirection, E,,', is constant in the capillary. 5) D is constant. 6 ) Interfacial electric conduction owing to electric charges adsorbed on the capillary surface is ignored. 7 ) The solid material is an insulator. 8) Capillary end effects are ignored. Substituting these conditions into Equations (1) and (2), the following equations are obtained: d r dv2 - _ + p -1dz rdr dr) d
~
~
(
d
+ p e ~ O=z o
(3)
where uz is z-component of the velocity. The value of pe varies radially; to simplify the derivation, an average value is used here. Substituting the boundary conditions: dv,ldr
=
0 and dvldr = 0 at r = 0
(5)
+ = 5 at r = d!2 - 6
(6)
vz = 0 and
into Equations (3) and (4), the following equations are obtained:
@ =
+ *{(d 160
- 2 ~ 3)~
c
where is the potential at the slip plane, 6 the distance from the capillary wall to the slip plane; and d the diameter of the straight capillary tube. The average velocity uz.au in the capillary is expressed as follows:
Kobayashi et al. arrived at different solutions of Equations (1) and (2),considering the radial distribution of pe [7.2].It should be noted that the average velocity uz.avdepends on the capillary diameter, d. In contrast, if the curvature of the solid - liquid interface is ignored and a zero-pressure gradient condition is introduced, the conventional Helmholtz - Smoluchowski equation is derived:
This does not depend on the size of the capillary.
136
7 EEectroosmoticDewatering
14 7.3
4 4 1 h 4'
Figure 7.3. Model of a flow path in a solid-liquid mixture.
Electroosmosis in Porous Media
Kobayashi et al. 17.31 extended Equation (9) to describe electroosmotic flow in porous media in much the same way as the derivation of the Kozeny - Carman equation [7.4]. A flow path of the porous media is approximated by a winding tube (Fig. 7.3). Referring to Equation (9), the average velocity of fluid uc along the local axis of the winding tube is:
where D, is the effective diameter of the tube, K2 the coefficient which expresses the complexity of the tube shape, x, the coordinate in the direction of the local axis of the tube, and E, the electric field strength along the x,-axis; u,, E,, dxe can be transformed into variables measured in the direction of the material thickness; v,.,,, E, and dx, by means of the tortuosity T of the flow path.
E,
=
EIT
(13)
dx,
=
Tdx
(14)
The effective diameter of the path D, is defined by D,
4rtf (wetted cross - sectional area of the flow path) = 4 (wetted perimeter of the flow path) (wetted cross - sectional area)(thickness of the material) = 4 (wetted perimeter)(thickness of the material) (void volume of the material) = 4 (total solid surface area in the material)
=
(15)
where rH is the mean hydraulic depth of the flow path. Thus D, can he represented in terms of the porosity E and the volumetric specific surface So of the material:
7.4 Electroosmotic Dewatering of Compressible Solid - Liquid Mixtures
137
The apparent liquid velocity q through the porous material is defined by the flow rate per unit cross-sectional area of the material (Fig. 7.3) and represented by: 4 = EVx.au
(17)
Combining Equations ( l l ) , (12), (13), (14), and (16), one obtaines:
where KO= K2/16, and k ( = T2&) is the Kozeny constant. The first term of the righthand side of Equation(18) represents an electroosmotic flow in a porous material, while the second term shows a pressure flow in the material, both including the tortuousity and size of a flow path. If E = 0, Equation (18) is reduced to the Kozeny - Carman equation [7.4] which represents the apparent flow rate through a porous material under a liquid pressure gradient. Kobayashi et al. examined Equation(18) by the use of incompressible sintered glass beads [7.3]. They found that a pressure flow is induced by electroosmosis through a nonuniform porous material. This pressure flow cancels out the electroosmotic flow when q = 0.
7.4
Electroosmotic Dewatering of Compressible Solid Liquid Mixtures
7.4.1
Experimental
Electroosmotic dewatering is the separation of liquid from a solid - liquid mixture between two electrodes. The apparatus used to study electroosmotic dewatering is shown in Figure7.4 [7.5]. It consists essentially of a piston and a cylinder, both made of acrylic resin. As the experimental material, bentonite clay (Hohjun Kogyo, Annaka, Japan) was used. A main constituent of bentonite is sodium montmorillonite, a clay mineral consisting of silica, alumina, and magnesia. Bentonite absorbs large quantities of water, so it swells into a jelly-like mass. A bentonite-deionized water slurry was preconsolidated in the apparatus under a pressure psl of 98.1-490 kPa, resulting in a semisolid material with a uniform void ratio el. (“Semisolid” refers to a nonfluid solid-liquid mixture in which the solid particles contact each other.) A d.c. voltage was applied between the electrodes. Since bentonite is negatively charged, the piston surface is used as anode, with the cylinder bottom as cathode. The preconsolidation pressure was maintained in the apparatus during an electroosmotic dewatering test to ensure that the material was in good contact with the electrodes. The volume of liquid removed per unit drainage area ZI was measured over time 13.
7.4.2
Theory
As shown in Figure7.5, liquid passes through the lower electrode, and the material near the upper electrode dries out. As dewatering progresses, the thickness of the material decreases. In the analysis of electroosmotic dewatering, it is more convenient and accurate to use the moving material coordinate w [7.6], [7.7] instead of the fixed
138
7 Electroosmotic Dewatering PRESSURE
1
Figure 7.4. Experimental apparatus for electroosmotic dewatering [7.5].
LlOUlD
PSI
0- ....................
I .............- w =wo Qky::::.;'):.:
.....................
....,..... ... ,.. .,.... .:. . ..... ...... . . . . . . .. . .. . . . . .. . . . . . .. . . . . . . . . . . . . . . . .
. . . . . .
q=o
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
WET
.
.
W=O
LIQUID
ps =psi PL=O
Figure 7.5. Schematic diagram of experimental conditions [7.5].
spatial coordinate x, because both the liquid and the solid particles move during dewatering. Here w denotes the net solid volume per unit cross-sectional area extending from the drainage surface up to an arbitrary position in the material. Rearrangement of Equation (18) by substitution of w yields
where ps denotes the true density of solids, the effective charge on the solid surface per unit volume of solids, i the electric current density, and p~ the specific electrical resistance of the material. a is the specific hydrodynamic resistance defined by
7.4 Electroosmotic Dewatering of Compressible Solid - Liquid Mixtures
Figure 7.6. Forces acting over the element dx of the material [7.5].
‘-MEDIUM
a=
139
k$(1
- E)
Ps e3
The negative sign before the pressure flow term of Equation (18) becomes positive in Equation (19), since the flow toward the drainage surface is hereafter defined as positive (Fig. 7.5). To discuss the internal mechanism of electroosmotic dewatering of compressible materials, the relation between liquid pressure p L and solid compressive pressure p s is essential. In Figure 7.6, forces acting over the element dx of the material are shown schematically. FD and FE are the viscous drag and the electrokinetic force acting on unit surface area of solid particles, and Fs is the compressive force on the particles. The balance of forces acting on the solids and the liquid over dx can be represented by the following equations:
where A is the cross-sectional area of the material. Combining Equations (21) and (22), and defining the solid compressive pressure by p s = FJA yields:
-dPL + - = odPs dx dx or, on the material coordinate:
These equations imply that the change in the liquid pressure in the material is offset by the change in the solid compressive pressure. In other words, if a negative liquid pressure distribution appears in the material, a positive solid compressive pressure distribution also appears. This is essential for understanding the mechanism of electroosmotic dewatering. Using Equation (24), one can rewrite Equation (19) as:
140
7 Electroosmotic Dewatering
The continuity equation relating the change in q to the change in void ratio e can be obtained from a mass balance of liquid with respect to a volume element dw in the material:
Substituting Equation (25) into Equation (26) gives the basic differential equation which controls the progress of electroosmotic dewatering:
As described above, a preconsolidation pressure p S lis applied to the material during electroosmotic dewatering; integration of Equation (24) with respect to w yields: PL
+ ps = Psl
(28)
Therefore (Fig. 7.5), the initial and boundary conditions for Equation (27) can be described as: ps = pSl at 0
=
0
(29)
ps = pSl at w = 0 (drainage surface) aP9
ao
e
- GiPE at w = E
wg
(30)
(upper electrode)
Iem
(32)
Here q, denotes the total volume of solid per unit cross-sectional area. Equation (31) is the impermeable surface condition, i.e., q = 0. In other words, based on Kobayashi's results [7.3], we suppose that the pressure flow and the electroosmotic flow cancel each other out at the impermeable wall. In Equation(32), e, represents the critical void ratio at which electroosmotic flow ceases. To solve Equation (27), the right-hand side is represented by finite differences, in place of derivatives. Numerical calculations based on the Runge - Kutta - Gill method are then made to obtain the change of e over time 0 and position w . The liquid volume removed per unit drainage area, v is calculated by: u = l u " ( e l - e)dw
where el is the initial void ratio of the material.
(33)
7.4 Electroosmotic Dewatering of Compressible Solid - Liquid Mixtures 6
I
I
I
I
I
1
I
I
I
I
141
P
4
'
e 7.4.3
Figure 7.7. Specific electrical resistance pEof bentonite clay [7.5].
Physical Properties of the Material
-
Compression Permeability Characteristics. Relations among a, e, and p s can be determined by use of the conventional compression - permeability (C - P) cell [7.8], 17.91, which is essentially the same as the apparatus shown in Figure7.4. The solidliquid mixture is first compressed in the C - P cell under constant pressure p s until equilibrium compression is attained. The equilibrium void ratio e is calculated from the equilibrium thickness of the material. On measuring the pressure flow resistance of liquid through the material, the specific flow resistance a is determined. These values for bentonite clay are: a
=
4.68 x lO'exp((22.9 - e)/1.28} 2 < e
e
=
22.9 - 1.61lnp,
45 < p, < 500 kPa
< 6.5
(34) (35)
Specific Electrical Resistance. The electrical resistance of bentonite in the equilibrium compression state was measured by a resistance meter and correlated with the e-value as shown in Figure 7.7 [7.5]; pEincreases as e decreases, because bentonite acts as an insulator. Effectice Electric Charge Density on the Solid Surface. The Electroosmotic dewatering rate dvfd0 decreases over time as shown in Figure7.8. The initial dewatering rate (dv/d8)o=ois equivalent to the first term of the right-hand side of Equation (25), i.e.,
because the pressure flow term of Equation (25) is negligible at the beginning, if the material is uniform. uscan be determined from Equation (36) and the intercept shown in the figure; it increases as the void ratio e decreases (Fig. 7.9) [7.5]. Critical Void Ratio. We assume that Equation (25) does not hold in the region e <em. In the electroosmotic dewatering of bentonite semisolid, we use the average void ratio of the material near the upper electrode as the critical void ratio e,, although a porosity distribution exists in that layer. Since the average void ratio ranged from 0.64 to 1.44 [7.5], the arithmetic mean of 1.12 is used as the value of e,.
142
7 Electroosrnotic Dewatering I
I
lo-'
I
I
I
0.219 cm PSI = 98.1 kPa
WO =
-
VT'4V
-
-
0 0
I
I
-
I 2
I
I
e
I
0
-
I 'Io
CSl
Figure 7.8. Change of electroosmotic dewatering rate d d d 0 over time (constant applied voltage) [7.5].
0
I
7.4.4
2
3
4
e
5
6
7
Figure 7.9. Effective electric charge density usof bentonite clay [7.5].
Comparison of Theory and Experiment
Figure 7.10 shows the results of electroosmotic dewatering under constant electric current [7.5]. Since the total electrical resistance of the material increases over time, the applied voltage V Tshould be increased to maintain a constant electric current. The broken line in the figure represents the theoretical result calculated from Equation (27). The close agreement between observed and calculated values supports the validity of the theory described above. Figure 7.11 shows the result for constant applied voltage. The current density i decreases over time because of the increase in the electrical resistance of the material. This is also due to the increase in the contact resistance between the upper electrode and the material. The solid line in the figure is the result from Equation (27), assuming that the effective voltage applied to the material does not change over time, while the broken line represents the result using the empirical electric current density i in Equa-
7.4 Electroosmotic Dewatering of Compressible Solid - Liquid Mixtures 10-3
I
1
1
I
I
I
143
I 14
w0=0.222cm, Ps1=98.IkPa
i = 12.9A/m2
0
I
0
I
I0000
1
1
e
I
I
Figure 7.10. Comparison of theory and experiment; constant electric current [7.5].
0 30000
[SI
XIO-3
W o = 0 . 2 2 2 cm,
I
20000
I
I
I
PSI = 98. I kPa
N t-7
Y
.-
0
10000
20000
e
CSI
30000
Figure 7.U. Comparison of theory and experiment; constant applied voltage [7.5]
tion (27). That is, good agreement between theory and experiment is obtained if we use the empirical value of i. This also supports the validity of the theory. The enormous difference between the solid line and the observed values may originate from the increase of contact resistance at the upper electrode. That is, as dewatering proceeds, gas generation and drying occur near the electrode so that contact between the material and the upper electrode becomes insufficient. Figure 7.12 illustrates the change in effective voltage over time, which is a substantial driving force of electroosmosis and is calculable from Equation (27), using the empirical current density shown in Figure 7.11. If the applied voltage VTis4 V, 75% of VTisconsumed in the electrode reaction and only 1V is used as the effective voltage that results in electroosmotic flow through the material. The effective voltage decreases drastically over time (Fig. 7.12). In designing an electroosmotic dewatering operation at constant voltage, we should estimate the decrease in the effective voltage in advance.
144
7 Electroosmotic Dewatering
EFFECTIVE VOLTAGE
I
00
I I0000
I 20000
I
e
I
J
Figure 7.12. Change of effective volt-
30000 age V over time; constant applied volt-
age [7.5].
CSI
Figure 7.13 illustrates the changes of e andp, distributions over time at constant electric current, calculated numerically from Equation (27). In the derivation of Equation (27), we supposed that the local solid compressive pressure p s is related to the local void ratio e by Equation (35). This is acceptable; if the solid particles do not move, the p L distribution results in a pressure flow to compensate the electroosmotic flow. A p s distribution also arises (Eqs24, 28). The solid layer, with void ratio e, begins to be compressed when the local compressive pressure p Sreaches the values given by Equation ( 3 9 , because that value represents the strength of the layer against compressive action. To simplify the calculation, the value of p s at the upper wall was fixed when it reached a value corresponding to em. In the figure, w/w, represents an arbitrary position in the material (dq, = 0 at the lower electrode, and fo/@ = 1 at the upper elec0 ) , e and p s values are constant throughout the trode). At the start of operation (@= material, because it is uniform as a result of preconsolidation. The decrease of void
WO
0.222 crn , Psi = 98. I kPa
D = 490 kPa. i = 12.9A/m2
1
-I
)THEORETICAL
7 6
E
&E
Y
> 4
2
0 0
2
I
8
CSl
3 XI04
Figure 7.W. Changes of e and p s distributions over time; constant electric current [7.5].
7.4 Electroosmotic Dewatering of Compressible Solid - Liquid Mixtures
1 wo = 0.222 cm ,
Psi = 98.1 kPo
I
w0 = 0.222 cm,
0.8
-
0 0
0.5 WWO
I
0
PSI
145
= 98.1 kPa
i = 12.9A/rn2
I
I
0.5 w/wo
I
Figure 7.14. Results of combined electroosmotic and mechanical dewatering [7.12].
ratio e starts from the layer near the upper electrode, as shown in the figure, i.e., dewatering proceeds from the layer near the upper electrode. The positive value of dp,ldo means that there is a pressure flow toward the upper electrode; the pressure flow propagates from the upper to the lower electrode. The impermeable upper electrode plays an important role in the progress of dewatering. Since q = 0 at the upper electrode, the liquid pressure gradient d p L / d oshould be negative (= -qip$&). The negative dpLldw is offset by the positive dp,ldw arising from the force balance (Eq 24), resulting in the increase of solid compressive pressure p s near the upper electrode. This increase of p s leads to a reduction of local void ratio e. Thus, electroosmotic dewatering is a kind of consolidation, since it accompanies the increase of solid compressive pressure in the material. At equilibrium, deld0 in Equation (27) should be zero, i.e., the electroosmotic and pressure flows cancel each other out in all layers of the material. Thus, e and p s distributions are not uniform at 0 = ~0 (Fig. 7.13). This coincides with the experimental observations of Yoshida et al. [7.10] and Yamaguchi et al. [7.11]. P
. . . . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . . . . . . .
.
.
.
.
.
,
.
LIQUID
.
.
Figure 7.15. Schematic diagram of electroosmotic and mechanical dewatering.
146
7 Electroosmotic Dewatering
5
w0=0.222cm, Ps1=98.1kPa
wo =0.222 cm, PSI = 98.1kPa
p = 490 kpa , i = 12.9 A/m2
p = 490 kPa
. i = 12.9 A/m2 I
-ELECTRO. +MECH. _ _ ~ ELECTROOSMOTIC _
W
[-
;
I'
ELECTRO. MECH. ELECTROOSMOTIC
em> I
OO
0.5
0.5 w/wo
Figure 7.16. Changes of internal conditions of the material over time for combined electroosmotic and mechanical dewatering [12].
Figure 7.14 compares the combined operation of electroosmotic and mechanical dewatering with each operation alone [7.12]. The semisolid material, which was preconsolidated under a pressure of psl,was expressed under a pressure p and concurrently subjected to a constant electric current i (Fig. 7.15). (The boundary condition of the combined operation is p s = p at w = 0 in place of Equation 30.) In electroosmotic dewatering alone, the decrease of moisture starts from the upper electrode (Fig. 7.13), while the mechanical consolidation proceeds from the lower electrode, if the upper one acts as an impermeable wall. The dewatering in the combined operation proceeds from both upper and lower electrodes, thus leading to a higher dewatering rate than in each operation alone (Fig. 7.14). Figure 7.16 shows changes in the internal conditions of the material for the combined operation. It can be seen that the dewatering proceeds from both upper and lower electrodes. The broken lines represent the e and p s distributions of electroosmotic dewatering alone. The difference between the solid and broken lines accordingly represents the contribution of the mechanical consolidation. Figure 7.17 compares the electrical energy consumption J for the combined operation with that of electro-osmotic dewatering alone; both were calculated by:
pE.av in the upper half o f the figure is the average specific electrical resistance calculated by: = V./
(i
x
(cake
(38)
It consists of the cake resistance and the contact resistance between the electrode and the material. Since the theoretical average cake resistance p E . & of bentonite is a unique function of v and does not depend on the mode of dewatering (broken line),
7.5 Industrial Applications
0 0
I
2
I
4
I
I
6
147
Figure 7.17. Energy consumption for electroosmotic and combined electroosmotic - mechan-
the difference between b.av in the two operations depends on the difference in contact resistances. The enormous improvement in energy consumption under the combined operation is consequently due both to the contribution of the mechanical dewatering and to the decrease of contact resistance.
7.5
Industrial Applications
The applications of electroosmosis can be divided into two general categories: those involving the use of large fixed electrodes for the dewatering of mine residues and for decontamination or stabilization of soils; and those using machinery based on moving feedstock for the dewatering of clay, sludge, food products, and so on. Danisch et al. reported a state-of-the-art study for the Canadian Electrical Association, mainly on the application of electroosmosis for dewatering [7.13]. They focused on the energy consumption of electroosmotic dewatering in comparison with that of competing mechanical and thermal techniques. Quantitative data to estimate relative energy consumption were taken from over 100 studies. They concluded on the basis of the energy data that electroosmosis can be reasonable, or preferable in some cases, and is at least competitive with conventional mechanical methods of dewatering. In all cases it is considerably better than thermal technology. Electroosmosis is especially effective in removing liquid from sludges for which conventional mechanical dewatering is not very successful. The use of electroosmotic phenomena in industrial dewatering has just begun; drum or filter-press equipment is used
148
7 Electroosmotic Dewatering
Figure 7.18. Filter-press electroosmotic dehydrator. 1) Oil pressure cylinder, 2) Electrical supply bar; 3) Filter cloth holder; 4) Sludge feed; 5) Filter plate; 6) Dewatered cake; 7) Water pan. (Courtesy of Shinko Pantech, Kobe, Japan)
~
-FILTER P L A T E -MEMBRANE -ELECTRODE
$z
F I L T E R CLOTH
Figure 7.19. Filter chamber of filter-press electroosmoticdehydrator. (Courtesy of Shinko Pantech, Kobe, Japan)
for dewatering of waterworks sludge and sewage sludge, and for the conditioning of clay sludge in the ceramics industry. Voltages are usually 20-100 V. Figures 7.18 and 7.19 illustrate filter-press equipment (Shinko Pantech) [7.14]. The filter chamber consists of filter cloths, two electrodes, the membrane for expression, and the filter plates. After the chamber is filled with sludge, it is filtered under a pressure of ca. 200 kPa, and then expressed by inflation of the membrane under a pressure of ca. 200-400 kPa. A d.c. electric field (ca. 40 V between the electrodes) is applied at a predetermined time after the filtration is started. If the electric field is applied before the chamber is filled with filter cake, both electrophoresis and electroosmosis occur in the chamber, accelerating cake formation. The polarity of the electrodes is periodically reversed to remove scale. Kondoh et al. reported that, in electroosmotic dewatering of excess activated sludges, the moisture content of the sludges reaches 50-60 wt% after dewatering [7.14]. Figure7.20 shows a mechanism based on a revolving drum and moving belt (Fuji Electric) [7.15], [7.16]. The surface of the rotary drum and the caterpillar belt are used as electrodes. The applied voltage and the pressure between the electrodes are 40-120 V and 100-3OO kPa. The scale deposited on the electrodes is continuously
7.6 Conclusions
149
D.C. POWER SUPPLY ROTARY DRUM ANODE
CATERPILLAR BELT CATHODE FILTER CLOTH
Figure 7.20. Electroosmotic dewatering 1
PRESSURE APPARATUS APPARATUS DRAINED WATER
machine based on a revolving drum and moving belt [7.16].
removed. The zone of compression created by a decreasing belt-to-drum distance, combined with electroosmosis, produces a product with about 50 wt% solids. The most important factor in the commercialization of electroosmotic dewatering is the life of the electrodes [7.14]. Many of the recent patents for electroosmotic dewatering machinery include the specification of materials, as well as the configuration of the electrodes.
7.6
Conclusions
Electroosmotic dewatering is very effective in removing liquid from difficult sludges which contain fine colloidal particles; such sludge is usually highly compressible, and conventional mechanical dewatering is impeded by hydrodynamic resistance. In summary: 1) Considering the curvature of a solid - liquid interface, the electroosmotic flow rate through a capillary depends on its diamter. It follows that the electroosmotic flow rate through a porous material depends on the structure of flow channels in the material, as expressed by the following equations. E3
= k g ( 1 - &)*y(
s>
~-3
These are very different from the conventional Helmholtz - Smoluchowski equation. 2) The basic differential equation which controls the progress of electroosmotic dewatering is:
3) Electroosmotic dewatering is a kind of consolidation which accompanies the increase of solid compressive pressure in the material. The impermeable side of the
150
7 Electroosmotic Dewatering
material plays an important role in the progress of dewatering. The liquid pressure becomes negative near the impermeable wall to cancel out the electroosmotic flow. This negative liquid pressure is offset by the positive solid compressive pressure arising from the force balance:
resulting in a decrease of local porosity of the material. This propagates from the impermeable electrode to the drainage surface until the pressure flow cancels the electroosmotic flow in all layers of the material.
Nomenclature
7.7
m2 Dielectric constant of liquid, F/m D, = effective diameter of the flow path in the solid-liquid mixture, m d = diameter of a capillary, m E = electric field strength in the direction of material thickness, V/m E, = electric field strength along the local axis of a flow path, V/m Ei = electric field strength by contact electric potential, V/m Eo = electric field strength by applied voltage, V/m e = void ratio defined by d(1--E) e, = void ratio of pre-consolidated material em = critical void ratio; if e < emelectroosmosis does not occur FD = viscous drag force acting on unit surface area of solid, N/m2 FE = electrokinetic force acting on unit surface area of solid, N/m2 Fs = compressive force propagated by solid structure, N g = gravitational acceleration, m/s2 i = electric current density, A/m2 J = electric energy consumption per unit sectional area, J/m2 k = the Kozeny constant p = expression pressure, Pa p,. = local liquid pressure, Pa = local solid compressive pressure, Pa = pre-consolidation pressure, Pa = apparent velocity of liquid, m/s = mean hydraulic depth of a flow path, m = volumetric specific surface, m2/m3 = tortuosity of a flow path defined by dxJdx = liquid volume removed per unit drainage area, m = velocity, m/s = voltage, V = applied voltage, V = average velocity of liquid along the local axis of a flow path, m/s = average velocity in the direction of the material thickness, rnls v, = local velocity in z-direction, m/s v , . ~= ~average velocity in z-direction, m/s = coordinate in the radial direction of a capillary, m r
A D
= cross-sectional area of a solid-liquid mixture, =
7.8 References x x,
z
a 6 E
I; 0 p p
pE pe ps a, t/j
w
wo
7.8
151
spatial coordinate in the direction of material thickness, m coordinate in the direction of the local axis of a flow path, m coordinate along a capillary axis, m specific hydrodynamic resistance, d k g distance from capillary wall to slipping plane, m local porosity I;-potential, V time, s liquid viscosity, Pa s = liquid density, kg/m3 = specific electric resistance, Qm2/m = volume density of true electric charge of fluid, Urn3 = true density of solid, kg/m3 = effective charge on solid surface per unit volume of solids, C/m3 = contact electric potential, V = net solid volume per unit cross-sectional area extending from the drainage surface up to an arbitrary position in the mixture, m; dw = (1-e)dx = total volume of solid per unit cross-sectional area, m = = = = = = = = =
References
~7.11F. F. Reuss, Memoires de la Societt! Irnperiale des Naturalistes de Moscou, 1809, vol. 2, 327-337. 17.21 K . Kobayashi, M. Iwata, Y. Hosoda, H. Yukawa, J . Chem. Eng. Japan, 12, 466-471 (1979). L7.31 K. Kobayashi et al., J. Chem. Eng. Japan, 12,492-494 (1979). "7.41 P. C. Carman, Trans. Inst. Chem. Eng., 15, 150-166 (1937). 17.51 M. Iwata, H. Igami, T. Murase, H. Yoshida, J. Chem. Eng. Japan, 24, 45-50 (1991). [7.61 M. Shirato, T. Murase, H. Kato, S. Fukaya, Kagaku Kogaku, 31, 1125-1131 (1967). 17.71 M. Shirato,T. Murase, A . Tokunaga, 0.Yamada, J. Chem. Eng. Japan, 7,229-231 (1974). ~7.81H. P. Grace, Chem. Eng. Prog. 49, 303- 318 (1953). P.91 M. Shirato, Kagaku Kogaku Binran, 4th ed., Maruzen, 1140 (1978). [7.10] H. Yoshida, T. Shinkawa, H. Yukawa, J. Chem. Eng. Japan 18,337-342 (1985). [7.11] S. Yamaguchi et al., Kagakukogaku Ronbunshu, 18,959-961 (1992). [7.12] M. Iwata, H. Igami, T. Murase, H. Yoshida, J. Chem. Eng. Japan, 24, 399-401 (1991). 17.131 L. A . Danisch, Report CEA 716 U 629, Canadian Electrical Association, 1989. [7.14] S. Kondoh, M. Hiraoka, Wit. Sci. Echn., 22, 259-268 (1990). [7.15] M. Yamagchi,T. Arai, H. Matsushita, Water and Waste, 28, 36-41 (1986). [7.16] H. Yoshida, Drying Technology, 11, 787-814 (1993).
This Page Intentionally Left Blank
8
Electrophoretic Forming of Ceramics Takao Tsuda and Emile H. Ishida
There are two representative methods for producing ceramics: pressure forming and slip casting using capillary attraction. In the former method dynamic pressure is applied directly to the raw materials. The third method is electrophoretic forming (electrophoretic and electroosmotic forming), in which electrophoretic mobility due to surface charges of the raw materials and the phenomenon of dewatering in an electric field are used [8.1]-[8.16]. In this chapter we will discuss the process of forming by the third method, its potential and advantages. During the past five years, one of the authors has been developing a new continuous forming method by using both electrophoretic and electroosmotic mechanisms, applied to the continuous production of an endless ceramic sheet [8.1]-[8.3]. We review briefly the method of electrophoretic forming and introduce our new work on forming of an endless ceramic sheet. Raw materials are usually natural clay and nonplastic fine particles, e.g., quartz andor feldspar. When clay and for these fine particles are mixed with water, a very viscous solution (slip) is obtained. The slip is
0 0
i/ 8 Me"'
0 0 I I I
-I-
Cations
I I I
Anionic particles
Figure 8.1. Electrodeposition of slip under an applied electric field.
154
8 Efecrrophoretic Forming of Ceramics
used for forming under an electric field, and then forming is continued by glazing and firing. The slip is a very viscous solution, e.g., 10% solids. The mechanism of electrodeposition of the slip onto the electrode under an electric field is shown in Figure 8.1.
8.1
Electrophoretic Behavior of Inorganic Particles in Aqueous Solution
The electrophoretic mobility of particles is expressed as: vnwb
= f ( x , a)
(1)
&
c,
where q, a, E , and x are the viscosity of medium (liquid), particle diameter, dielectric constant, zeta potential, and reciprocal thickness of the electric diffuse doublelayer, respectively. The factor f ( x , a) depends on the relative values of particle diameter and double-layer thickness. When the particle diameter is large enough compared with the thickness of the double-layer, its value becomes 1 1 4 ~When . xu becomes zero, the factor becomes 1/6n. When inorganic oxides are dispersed in water, their surfaces become solvated, and the solvated water shows the functional behavior of hydroxyl groups. The apparent surface charges of the solvated particles may be expressed as:
M-OH*- +OHM -OH +H++OHM -O-+H++H20
(acidic regions) (neutral regions) (alkaline regions)
Each particle has a characteristic isoelectric point at which the particle becomes electrically neutral. The isoelectric points for oxides are listed in Table8.1 [8.4]. They depend on the processes of synthesis, e.g. the history of heat treatment. The mobility of the particle is proportional to the value of the zeta potential (Equation l). The zeta potential depends partly on the manufacturing processes,e.g., the relationships between zeta potential and pH of the medium for commercial alumina powders obtained by different conditions of calcination in lo” M KN03 solution [8.5] Table 8.1. lsoelectric points of oxides [8.4]
Material
lsoelectric point
a-Al,03 CUO FeO a-Fe203 y-FezO3 MgO SiO, SnO, TiO, ZnO ZrO,
5-9.2 9.5f0.4 6.5f0.2 5.2-8.6 6.7k0.2 12.4k0.3 2.2 7.3 4.7-6.2 8.7-9.3 4-11
8.2 Selection of Organic Solvents for Electrodeposition of Inorganic Particles
75
155
A
50 h
>
5 -
--s
25
.-a
o
0 Cl
5a, -25
N
-5c
-75
*
4
6
PH
8
10
Figure 8.2. Zeta-potential of commercial alumina powders (Baikowski International) obtained by different manufacturing pro12 cesses in terms of calcination in 10- M KN03solution [8.5].
(Fig. 8.2). Although the curves in Figure 8.2 are of similar form, they are widely scattered. Clay particles are generally complex, with two kinds of charges: permanent charge and the charge induced by the pH of the medium. The permanent charges are located on the surface of the clay. They come from the replacement of Si4+(silicon bonded to four oxygen atoms in tetrahedral sheets) by A13+,especially in montmorillonite, or the replacement of A13+in octahedral sheets by Mg2+or Fez+.After the displacement, the net surface charge is -1, owing to the loss of one positive charge per displacement. On the other hand, the induced charge of the clay appears on the edge of clay. Oxygen a n d o r hydroxyl groups located at the edge are generally unstable, and they are affected by hydrogen and hydroxyl ions in water.
8.2
Selection of Organic Solvents for Electrodeposition of Inorganic Particles Dispersed in Organic Solution
@Alumina is the most difficult substrate for applying conventional forming methods. Powers [8.6] examined the conditions for its electrodeposition dispersed in a range of organic solvents. His experimental results are shown in Figure 8.3. “Electrodeposition” here refers to the layer solidified around the electrode. Solvents marked with a full circle give positive results. Powers concluded that the solvent should have dielectric constant in the range 12-25 [8.6]. Pyridine acts as a reagent to make the surface charge on palumina positive, so that it is deposited at the negative electrode.
156
8 Electrophoretic Forming of Ceramics
ELECTROPllORETlC DEPOSIT
0 METIIAHOL
ETHYLENE
O H 0 DEPOSIT
ETHANOL
t
-
ETHYL
L
GLyCoL
1
I - PROPA N OL
I- HEXANOL
2
$ 1(~-~k PyR!"E'
.3-PENTANONE I - DECANOL
0 V
u
2 W R
lo?-
v,
-
OETllYL ACETATE
IO-~ OMETIIYL ACETATE dDENZENE 10 - , y P - D I O X A N E
--
8.3
Electrophoretic and Electroosmotic Forming
8.3.1
History
There are few reports on electrophoretic forming. The electrophoretic forming process has been applied to the formation of vessels by a dipping technique [8.7]. Inorganic particles in the solution were deposited electrophoretically on the electrode and the thickness of the deposition layer was controlled by the period of dipping. Controlling the speed of withdrawing the electrode, and the applied voltage, determines the shape of the vessel. In forming with molding, the material and geometry of the electrode are important for product quality. Porous electrodes made from a mixture of carbon and gypsum [8.8],carbon and clay [8.9], or carbon and cement [8.9] are recommended. Ryan et al. [8.9] used the last mixture and obtained good results: a deposited layer 10mm thick in only 3-4min. Bentley [8.10] suggested that a mixture of carbon or silicon carbide and clay is the best electrode, and the problem in this process is how to control or exclude gas generated at the electrode. Forming of Alumina Ogives. Although there are several reports of electrophoretic forming, the work of Andrew et al. [8.11]is the only one to include a photograph of the products. Andrews et al. used the apparatus shown in Figure8.4 for electrophoretic
8.3 Electrophoretic and Electroosmotic Forming
u
Pump
u
157
Figure 8.4A. Pumping arrangement forl5-cm ogives. Aluminum ogives and the slip in the tank are the positive and negative terminals [8.7].
Figure 8.4B. Forming of alumina ogives; former with metallized plastic sheath, deposition electrode, and whole and sectioned deposits [8.7].
forming of alumina ogives. They used a slip composed of 50% solid in ethanol, recycled by a centrifugal pump. A voltage of 100-200V (0.3 mA/cm2) was applied. Beyond this voltage range it was difficult to achieve electrodeposition. The flow of the slip around the electrode is very important for operational stability; the flow carries the slip around the electrode, avoiding local variation of pH induced by the applied voltage. As the density of the product depends on the temperature of the slip (e.g., 1.6-1.9 g/cm' at 20 "C and 2.1 g/cm3at 40 "C), higher temperatures giving a product of higher density. The electrode is covered with conductive plastic resin to ease the detachment of product from the electrode. The period of operation for forming was 15min. The products obtained by electrophoretic and electroosmotic forming are then fired [8.11]. The rocket ogives, shown in Figure8.4B, are 15cm in height and 5mm thick. Powers [8.6] suggested for the case of P-A1203that higher density products are obtained at higher concentration of solid in the slip, under the following conditions: medium, amyl alcohol; applied voltage, 280-1400 V/cm; electrodes, closed tube of 36-Invar; counter-electrode, 18-8 stainless steel.
158
8 Electrophoretic Forming of Ceramics
Small additions of aluminum tristearate in the slip give stable operation [S.l2]. The products of electrophoretic forming have the same strength as products of the cold isostatic pressing, and their strength is increased by the addition of Zr02. [8.12.]
8.3.2
Industrial Applications of Electrophoretic and Electroosmotic Forming
Clay has both permanent negative charges and induced charges due to the equilibrium of the medium and its edges. Although the net charge depends on the pH of the medium, it has significant electrophoretic mobility. If the electrophoretic mobility is 5 x 104cm2V ' s ? in an aqueous solution (viscosity ca. 1cP), its value in the slip (viscosity ca. 500cP) by Equation (1) is 1 X 104cm2V-' 8'.The distance traveled due to its electrophoretic mobility under an electric field of 100V/cm is lo-' cm in 10 s, very short compared with that in aqueous solution. It may be necessary to introduce local viscosity in a very viscous matrix. The local viscosity is not dependent on the viscosity of the matrix. For example, we can prepare the slip with a mixture of kaolin, feldspar, quartz, and water (solids content 70%). Micro-spaces surrounded by nonplastic material in the slip is filled with the mixture of water and very fine particles of kaolin and feldspar. The local viscosity of the mixture in a micro-space may be very different from the apparent viscosity. If the viscosity of the solution in the micro-space is 5 CP (100 times less than the apparent viscosity of the slip), the distance traveled under the applied electric field in the above experimental conditions may be 1mm in 10 s. For industrial production, the water content in the layer after forming is an important factor; it should be ca. 20% for easy handling. At the same time as the electrophoretic movement, dewatering by electroosmotic mobility also occurs in the opposite direction. Thus the water molecules in the slip located near the electrode are forced to transfer with the flow velocity, owing to their electroosmotic mobility. Neutral water, partially ionized water molecules and solvated hydronium ions experience the applied electric field and the effect of the surface charges of the particles of kaolin and/or feldspar. These surface charges act as an electric capacitor. Water molecules are forced to move by induced dipoles, as if they were positively charged. This phenomena is called electroosmosis (see also Chaps. 2 and 3). Therefore the dewatering contributes to the decrease in water content of an electrically deposited layer around the electrode (see also Chap. 7). The speed of dewatering depends on the electroosmotic mobility of water molecules. Its value is generally (3-0.2) x 104cm2V-'s-' toward the negative electrode in aqueous solution. The distance traveled by water molecules in a slip might be 0.6-0.04 mm in 10 s when the potential gradient and the local viscosity of the medium are 100V/cm and 5 cP. Electrophoretic forming is based on both electrophoretic and electroosmotic phenomena. Owing to the flow of electricity, metal ions are released from a metal electrode, and are forced to migrate in the slip. After electrical forming, the process of firing is carried out, converting the metal ions to metal oxides, sometimes coloring the product, which may be undesirable for many products. Handle [8.13] has proposed the following equation for the amount of electrodeposition m. m = kTCE' 6s 6t
(2)
where k, 'i; C, E ' , ds and dt are an empirical operational constant, the temperature of the slip, concentration of the solid in the slip, potential gradient, surface area of the electrode, and period of operation, respectively.
8.3 Electrophoretic and Electroosrnotic Forming
159
The behavior of m with respect to electric field, slip concentration and temperature is shown in Figure 8.5. Increases in the latter three factors give high values for m.The use of a high potential gradient has the advantage for giving a high value of m and at the same time accelerating the dewatering process. From an industrial standpoint, it is favorable to decrease the water content in the deposition very rapidly period. However, when we use a high potential gradient, the variation water content perpendicular to the surface of the electrode increases, and so does the amount of gas generated at the electrode. These two problems may lower quality. The factor k depends on the dielectric constant of the medium, the viscosity, the potential, the anode material, and the solid particle diameter. We can control the last four factors. The particle diameter relates to the c-potential and electrophoretic mobil-
c-
A
0.35
f
0.30 .
0
E .8 L
0.25
P
U c
g
0.20
2
.-=
I: n
0.15
In
0.10
, / , 30
Figure M A .
Slip concentration (wt%)
Figure SSB.
160
8 Electrophoretic Forming of Ceramics
ity of the particles, and the particle size distribution of clay and feldspar relates also to the electroosmotic mobility. Therefore, a key factor is control of the particle diameter of the clay and feldspar. Another factor is the selction of high-efficiency electrode. C
0.30
'?
0.25
5
-.0)
L
8n
:
0.20
m 9
E
.-=u g
fn
0.15
0.10
I
20
30
40
Slip temperature ('C)
2-20pm
5ornass %
Figure 8.5. Influence of control factors on deposition. (A) Electric field strength; (B) Slip con, centration; (C) Temperature. o (Reproduced with permission from INAX Corp.)
2201.4~1
Figure 8.6. Relation between particle size distribution and packing rate after forming. Solid lines are contours of packing rate, which increases with increasing amount of coarse particles. The maximum is indicated as HPA - high packing area. (Reproduced with permission from INAX Corp.)
8.4 Control of Particle Diameter for High Productivity and a Low Water Content
161
c2um
2-20pm
50rnass%
a20pm
Figure 8.7. Influence of particle size distribution on moisture content of deposition. (Reproduced with permission from INAX Corp.)
8.4
Control of Particle Diameter for High Productivity and a Low Water Content in Electrical Forming
The relations between the size distribution of particle diameter in the mixture of the clay and nonplastic raw materials (feldspar and quartz) and the density of the product are shown in Figure 8.6. The high-packing area (HPA), in which high-density products are obtained with low water content, is composed mainly of a high content of particles of diameter more than 20 ym and less than 2 ym. We examined the relation between moisture content and particle size distribution (Fig. 8.7). The numbers inside the triangle are the percentages of water after the forming. When the particle size distribution is close to the HPA, the water content decreases after forming. Therefore, it is clear that formings in the HPA contain a low percentage of water. We need to find raw materials that have a particle size distribution in the HPA. The size distributions of natural clays (high and low grades) supplied by Set0 region (Japan) are shown in Figure 8.8. As the size distribution of low-grade clay varies with each lot, we cannot use it as a main raw material for industrial production. It is better to use high-grade clay which has a constant size distribution. We prefer a high-grade plastic clay and nonplastic materials feldspar and quartz, with coarse particle sizes more than 20 Vm. The nonplastic materials are ground in a ball mill. The period of grinding corresponds directly with the size distribution of feldspar and quartz (Fig. 8.9). Powders milled for 3-5 h give the best composition. A mixture of powdered feldspar and quartz processed by ball milling and high-grade clay A in Figure 8.8, is the ideal raw material. The size distribution of the mixture corresponds to the HPA in Figure 8.6. However, the grinding period of 3-5 h is rather short for obtaining reproducible results since grinding with a ball mill is affected greatly by the initial size distribution of raw materials, water temperature, etc. Therefore we prefer jet milling (Fig. 8.10) instead of ball milling as the grinding method.
162
8 Electrophoretic Forming of Ceramics
c2um
Figure 8.8. Stability of particle size distribution of clay. A and B indicate high- and low-naturally-elutriated clays, respectively. These clays are from Set0 (Japan), the major source for Kibushi (plastic clay). (Reproduced with permission from INAX Corp.)
50mass%
- --r..-
Figure 8.9. Variation of particle size distribution with milling period (wet). (Reproduced with permission from lNAX Corp.)
In jet milling, the particles are accelerated to high speeds by compressed air (7-8 kg/cm2), and collide into each other at position 3 in Figure8.10. The photographs shown in Figure 8.11A and B are feldspars obtained by conventional ball and jet milling. Jet milling gives a rougher surface, with twice the surface area, and with a higher zeta-potential. Jet milling also gives reproducible size distributions and improved physical properties for industrial production.
8.4 Control of Particle Diameter for High Productivity and a Low Water Content
163
Figure 8.10. Air jet milling system. 1) Raw material; 2) Air compressor; 3) Crushing and milling zone; 4) Transportation pipe; 5) Separation zone; 6) Separator; 7) Storage. (Reproduced with permission from INAX Corp.)
Figure 8.U. Particles crushed by ball milling (A) and air jet milling (B). Numbers indicates milling period in hours. (Reproduced with permission from INAX Corp.)
164
8 Electrophoretic Forming of Ceramics
Table 8.2. Effects of ion exchange on zeta-potential of kaolinite Exchanged ion
PH
Zeta-potential, mV
Viscosity, cp
Nontreated Ca2+ Mg2+
5.30 5.60 5.60 6.00 6.30
-27 -29 -34 -40 -45
4660 3730 3740 2240 1660
K+ Na'
8.5
Zeta-Potential of Raw Materials
The relationship between zeta-potential and pH of media are shown in Figure8.12. The zeta-potential was estimated in M KC1. Talc from Shantung (China), kibushi clay from Set0 (Japan), and illitic clay from Westerwald (Germany) each have characteristic relationships. The zeta-potentials can be modified by other ions; some of the surface ions of the clay are exchanged with other ions in the medium or washing solution. The characteristics of samples treated with special ions are listed in Table 8.2. The shift of zeta-potential to a high value is one advantage, as we may obtain a large k value in Equation (2). At the same time, the clay can be a very unstable raw material, and it is necessary to handle it carfully to maintain the constant behavior. It is better to use raw materials with a high zeta-potential.
8.6
Electrodes
The electrode itself affects the k value given by Equation (2) in the following ways: its geometrical design, oxygen gas generated at the surface of electrode, and coloring by metal ions liberated from the surface. The smoothness of the electrode surface is also important for detaching process electrophoretic formings. The variation of electric current with deposition period for different electrode materials is shown in Figure 8.13. Although all of the electrodes have a tendency degrade
-80'
'
2
"
4
"
6
PH
"
8
"
10
'
12
Figure 8.12. Variation of zetapotential for different materials. Talc from Shantung (China) (tnangle); Kibushi clay from Seto (Japan) (square); Illitic clay from Westerwald (Germany)(circle); Zeta-potential measured in M KC1 aqueous solution. (Reproduced with permission from INAX Corp.)
8.6 Electrodes
P: deposition '0
3 4 Time (rnin)
5
6
165
Figure 8.W. Variation of current with deposition period for different electrodes under constant voltage. (Reproduced with permission from INAX Corp.)
with time, Fe and Zn give a higher amount of deposition compared with the others. In the case of aluminum electrodes, aluminum oxide may form on the surface. Metal ions liberated from the electrode surface penetrate into the layer of the forming in the direction of the counter-electrode. The penetration distance is almost onethird of the thickness of the formings. This curious result has been observed at each different layer formed by electrophoretic deposition with current density 5-10 mA/cm2. Poorten [8.15] has tried to explain this phenomenon. After leaving the surface, the metal ions may attain equilibrium with the edge portion of clay on which there are sev-
1.2
Thickness of deposition (mm)
Figure 8.14. Variation of metal ion concentration with deposition thickness. (Reproduced with permission from INAX corp.)
166
8 Electrophoretic Forming of Ceramics
c
c
Figure 8.15. Zones of iron migration from the dram surface (anode). Arrows show migration fronts. (Reproduced with permission from INAX Corp.)
era1 functional groups. Metal ions may displace hydrogen ions from the surface of the clay, changing the local pH. Due to this local high pH, coagulation is induced, and the following layer becomes thicker. These schemes are illustrated in Figure 8.1. The migration distances of metal ions in the electrophoretic deposition layer are generally one-third of the thickness of the deposition. We have confirmed this result [8.2] (Fig. 8.14, 8.15). The zinc electrode is stable up to 15mA/cm2 and reacts instantly with any oxygen generated. This has the great advantage of excluding oxygen from the vicinity of the electrode.
8.7
Electrophoretic Forming Continuous Ceramic Sheet
A continuous electrophoretic forming machine was patented in 1977 by Chronberg [8.16]. This patent is of revolutionary importance in the development of electrophoretic forming. With this machine, called the elephant, it is possible to produce an endless sheet. However the machine has never been used on an industrial scale, per-
8.7 Electrophoretic Forming Continuous Ceramic Sheet
167
4
A
0
W
I
Figure 8.16. The “elephant” forming process. (A) 1) Zinc anode; 2) Counterelectrode (cathode), 3) Slip inlet; 4) Excess slip, outlet; 5) Double-layer band of ceramic body; (B) Detailed view, ( C ) External appearance. (Reproduced with permission from INAX Corp.)
168
8 Electrophoretic Forming of Ceramics
haps because at that time there was little information about controlling the characteristics of the slip, and a lack of detailed studies of operational conditions. In 1988 test operation of a modified elephant machine, with several unique characteristics was reported [8.1]-[8.3]. One advantage is that production is highly economical in terms of energy. The arrangement of the elephant is shown in Figure 8.16. It consists of a pair of zinc drams (anode) and blades (cathode). The drams are c cled at constant speed, and pressed toward each other with a force of several kg/cmY. The layer formed by electrophoretic movement on the surface of the dram is dewatered by electroosmosis. The portion of the layer near the dram surface is subjected to more dewatering compared with the remote portion of the layer. The layers formed at both drams are then pressed into each other by cycling. The period from the start of forming to the facing operation is about 10s. The upper portions of the forming surfaces contain high amounts of water, and these portions are pressed together by both drams. During pressing, they are dewatered and a uniform layer is formed ( 5 in Figure 8.16(A)). The slip used contains 70% solids, and its characteristics are: density 1.7 gkm', viscosity 500 cP, electrical conductivity 1000pS/cm, zeta-potential 20mV. The slip is supplied from position 3 in Figures8.16(A), and excess is removed at position 4.The surface of the cathode is kept clean by the flow of the slip. The drams shown in Figures 8.16 are 1500mm in diameter and 700 mm wide, and the constant voltage and electric current used are 20V and 90A [8.2]. The surface area used for forming is around 0.6m2per dram, and the thickness of slip formed is 5-6mm. The continuous layer is produced at a speed of over 100cdmin. The water content in the layer emerging from the elephant is about 20%, so it is easy to handle. The total energy used for 1000 kg of product is only 10-12 kW.Forming of layer is followed by firing, to give the final product (Fig. 8.17). It is easy to produce a continuous plate 1-6 mm thick. Plate of this thickness is difficult to produce by doctor blade, dry pressing, or extruding methods. During electrophoretic and electroosmotic forming, the anode (zinc drams) is oxidized. It is necessary to remove the zinc oxide layer before the next cycle, by polishing the surface of the dram (Fig. 8.16(C)). A trapezoidal probe is used to supply a reagent for smoothing the surface of the dram, making layer detachment easy. The layer formed is subjected to conventional ceramics production processes, e.g., firing at 1200-1250 "C for 30-60min. By controlling the mixture of raw materials, it is possible to obtain different products; one the example has an elasticity of half to twothirds of that of conventional ceramics.
Figure 8.17. Double-layer band of ceramic body. (Reproduced with permiss#ion from INAX Corp.)
8.8 References
8.8
169
References
[8.1] H. Ishida, Annual report of the Ceramic Research Lab, Nagoya Institute of Technology, 1, 3-19 (1991). [8.2] H. Ishida, J. Mineralogical SOC.Japan, 22, (2), 79 (1993). [8.3] H. Ishida, “Forming and Sintering of Fine ceramics concerning Large Scale Products”, in Committee of The Society of Chemical Engineering, Japan, Report no. 1025, 27-141 (1989). [8.4] G. A. Parks, Chem. Rev., 65, 177 (1965). [8.5] A. S. Rao, Adv. Ceram., 21, 517 (1987). [8.6] R. W. Powers, J. Electrochem. SOC., 122, (4), 490 (1975). [8.7] Demande De European, Office Europeen des Brevets, 1980 AL-0022113. [8.8] H. Kawado, Japan Patent 52-28450 (1977). [8.9] W. Ryan, E. Massoud, Interceram, 28, (2), 117 (1979). [8.10] S. N. Bentley, Br. Ceram, Trans. J . , 87, (2), 46 (1988). [8.11] J. M. Andrew et al. “Proceeding of the British Ceramic SOC.”,Fabrication Science, 2, (12) 211 (1969) [8.12] G. H. Heavens, BE Ceram. Proc., 38, 119 (1986). [8.13] F. Handle, Keram. Z., 32, (4), 185 (1980). [8.14] H. Ishida, 0.Watanabe, Seramikkusu Ronbumshi, 98, (12), 1356 (1990). [8.15] H. V. Poorten, Silic. Ind., 46, (9), 156 (1981). (8.161 M. S. Chronberg, E Handle, Intercerurn., 27, (I), 33 (1978).
This Page Intentionally Left Blank
9
Control of Viscosity by Electric Fields: Novel Materials for Electro-Rheological Fluids and their Applications Yuichi Ishino, Tasuku Saito, Norio Goshima, and Kazuya Takano
9.1
Introduction
The viscoelectric effect is the phenomenon where the viscosity of a liquid is increased when an electric voltage is applied. At the end of the nineteenth century, Duff [9.1] and Quinke [9.2] reported that uniform polar liquids showed increased viscosity when a voltage was applied, but this increase was relatively small. In the 1940s, Winslow, an electrical engineer in the United States, worked on the experiment of Johnson - Rahbeck effect in which the electrostatic attractive force is increased when a high voltage is applied to a solid dielectric material. In this experiment he found that a suspension of dielectric powder in oil showed a very large viscosity increase when a high voltage was applied. He patented this finding as the electro-fluid power-transforming system [9.3]. The term electro-rheological fluid (ERF) is commonly used to describe a suspension of a dielectric powder in insulating oil, showing a large viscosity change when a high voltage is applied. With silica gel as the dispersed phase, applications of ERFs such as car clutches were extensively studied, but no practical industrial product resulted. In the 1970s, the British Ministry of Defence funded a number of university and industrial groups to study ERFs and their applications. As the result, many patents of ERF devices were applied for in Britain. Among these, Stangroom’s ERF [9.4], with lithium polymethacrylate as dispersed phase, attracted attention because, unlike silica gel ERFs, it was not abrasive. In the 1980s, small, simple ERF devices were developed. Car and car part manufacturers became interested in ERF devices because of developments in computer technology and small high-voltage supplies [9.5]. Many Scientists and engineers in universities and industry are now working on ERFs and their applications [9.6]-[9.14]. We have been involved in this development for several years.
9.2
Fundamental Characteristics of ERF
Winslow’s ERF consists of a dielectric powder dispersed in an electrically insulating oil. Figure 9.1 demonstrates the viscosity increase, of ERF, which is called electrorheological (ER) effect. When a voltage is applied, the ERF is maintained between the electrodes, but when it is turned off, it flows downward . The mechanism of the ER effect has been extensively studied [9.10], [9.11], [9.14] and has been established well by microscopy [9.15]. Figure9.2 shows the structural change when the electric field is turned on and off. When the electric field is applied, the particles form chains between the electrodes. This is explained by the electrostatic forces between particles (Fig. 9.3).
172
Control of Viscosity by Electric Fields: Novel Materials for Electro-Rheological Fluids
9
ON
OFF
OFF
ON
OFF
Elsct rods
ON
L Figure 9.3. Principle of the ER effect. (Reproduced with permission from Bridgestone
Corporation) When the electric field is turned off, the electric charges in the particles are randomly distributed and the particles become electrically neutral. When a high voltage is applied, negative charges in the particles are attracted to the positive electrode and vice versa. The electrically polarized particles attract each other, and chains of particles are formed between the electrodes. Although the viscosity increase is normally called the E R effect, this is not strictly accurate. Figure 9.4 shows ERF data for sodium polyacrylate in silicone oil evaluated by a rotational viscometer in which high voltages can be applied between the inner and outer cylinder. In Figure9.4, the horizontal axis is the number of revolutions of the viscometer and the vertical axis is the torque detected. If the fluid is Newtonian, the
9.2 Fundamental Characteristicsof ERF
173
BOO
17.5KV/cr
600
n
E
0
cn
W
(II
J 0L
400
0
I-
200
.
0
0
I
I
200
400
Number
of
600
Revo 1ut i on (RPM)
Figure 9.4. Shear rate dependence of the ER effect. Torque is measured when voltage is increased at different rotation speeds. (Reproduced with permission from Bridgestone Corporation)
torque is proportional to the number of revolutions and the slope represents the viscosity of the fluid. When the voltage is turned off, the ERF is Newtonian, but when the electric field is increased, the slope does not increase; instead the intercept on the vertical axis increases. This means that the fluid starts to flow after a certain shear stress is applied to the fluid. This phenomenon is called Bingham flow. The intercept on the vertical axis is the “yield stress”. In other words, the ER effect is the increase in yield stress of the fluid when the voltage is applied. This can be expressed as follows. When the electric field is off: 00 =
qP
(1)
where oois shear stress at zero voltage, q is the viscosity of the fluid at zero voltage (initial viscosity), and P is the shear rate. When the electric field is applied: aE=
qy+r(E)
(2)
where a, is shear stress when a voltage is applied, z (E) is the yield stress, and E is the electric field.
174
9 Control of Viscosity by Electric Fields: Novel Materials f o r Electro-Rheological Fluids
1000
BOO
m
E
o 600 m U al J 0L
400
200
0
I
I
I
100
200
Electric F i e l d
300 Squared(KV 2/crn2>
400
Figure 9.5. Electric field dependence of the ER effect. Volume fraction of sodium polyacrylate powder is changed from 16.9% to 37.6%. (Reproduced with permission from Bridgestone Corporation)
Since the viscosity q of a Newtonian fluid is expressed as aoly,when the electric field is applied, the apparent viscosity qappis:
qaPp= o E / f
=q
+ t (E)/f
(3)
This equation shows that the increase of apparent viscosity due to the electric field is simply t ( E ) / f ,i.e., inversely proportional to the shear rate. Thus, when the shear rate is zero, the increase of the apparent viscosity becomes infinite and vice versa. At a given shear rate, the electric field dependence of the apparent viscosity is shown in Figure 9.5, where the torque change for different volume fractions of sodium polyacrylate is plotted against the square of the electric field. The plots are nearly linear. Thus, when the electric field is applied, the shear stress produced is: = qf+ K E ~
(4)
where K is a coefficient which depends on the type of material and composition of the ERE Obtaining larger values of K at lower values of E is the focus of ERF material development. For some applications, especially automotive applications, where ERFs
9.3 Development of the Dispersed Phase
175
have to be used at high temperatures, the temperature dependence of the E R effect and the durability of the fluid at high temperature become important. Low electric power consumption is desirable in all applications in order to reduce the cost of electric power supply. However, conventional aqueous ERFs, e.g., with sodium polyacrylate as the dispersed phase show very high electric currents over 60 "C. In order to overcome this drawback this drawback ERF, we have tried to develop new materials for the dispersed phase.
9.3
Development of the Dispersed Phase
9.3.1
Chelate Resin
Since ERFs containing sodium polyacrylate show very high current over 60 "C, we thought that this might be due to the presence of strongly ionic functional groups. Accordingly we tested a chelate resin whose functional group is a salt of iminodipropionic acid as the dispersed phase. The salt has a metal chelate structure:
-N
/CH?CH.COo-
\CH2CHzCOO We used an epoxy resin modified by iminodipropionic acid groups. Sodium and calcium salts were tested. Chelate resin powder was dispersed in silicone oil. The ERF containing the sodium salt showed the E R effect, but high current at room temperature. On the other hand, as shown in Figure9.6, ERFs containing the original calcium salt did not show any ER effect, but when a small amount of water was added to the dispersed phase, an ER effect was observed at low current. In addition, as shown in Figure 9.7, fluids containing the original calcium salt showed no E R effect, but as temperature was increased, the ER effect was observed. Even at 94 "C this ER effect occurred at low current. The water content has a strong influence on the E R effect. ERFs containing a relatively large amount of water in the dispersed phase show a large ER effect at room temperature; those containing a relatively small amount of water show a small E R effect at room temperature, but a large effect at high temperature. The chelate resin was supplied as pulverized commercial resin, with average particle size 50 p and prone to sedimentation.
176
9 Control of Viscosity by Electric Fields: Novel Materials for Electro-Rheological Fluids 2000
Water Added 1500
n
E
0
07
U
; 1000 0L
0
I-
500
Not Added
1 0 0
I
I
I
5
10
15
20
Electric F i e 1d (KV/cm> Figure 9.6. Effect of water in a chelate resin ERE (Reproduced with permission from Bridgestone Corporation)
9.3 Development of the Dispersed Phase 2000
1500
A
E
u
m 2J!
v
I-
iooo
0L 0
500
2 6 0 0
I
I
I
5
10
15
'C
20
Electric Field (KV/crn> Figure 9.7. Temperature dependence of the ER effect. Dispersed phase is chelate resin. (Reproduced with permission from Bridgestone Corporation)
177
178
9 Control of Viscosity by Electric Fields: Novel Materials for Electro-Rheological Fluids
9.3.2
Porous Silica Microspheres
Since inorganic particles are durable at high temperature and are smaller than polymer particles, they were tested as the dispersed phase. The inorganic powder we obtained was porous silica microspheres made from silica sol. When this powder is used as the dispersed phase, even at relatively large volume fractions, the initial viscosity is lower than that of the ERF containing conventional silica particles (white carbon) which is normally used in the rubber industry. As shown in Figure 9.8 the ERF containing silica microspheres show the same level of ER effect from room temperature to 85 "C. Since silica microspheres absorb water, the ERF is essentially aqueous. However, the ER effect shows a relatively smaller temperature dependence compared with polymer dispersed phases. Figure 9.9 shows the response characteristics of the ER effect when the electric field is pulsed. The vertical axis represents the viscosity change and the horizontal axis represents time. The ERF containing silica microspheres shows better characteristics than the ERF containing white carbon. These results can be explained by the difference in their microstructure. The silica is spherical, but white carbon is a complicated agglomerate of submicron particles. Thus, with porous silica microspheres as dispersed phase, temperature dependence, electric power consumption, and response characteristics are all improved. However, after this ERF is heated at 150 "C for 6 h, the E R effect disappears, owing to evaporation of water. This poor durability at high temperature is a very serious problem.
0 0
5
10
15
Electric Field (KV/crn>
20
Figure 9.8. Temperature dependence of the ER effect. Dispersed phase is silica microsphere. (Reproduced with permission from Bridgestone Corporation)
9.3 Development of the Dispersed Phase
Porous Silica
179
White C a r b o n
Microbeads /T J
7
7
To
To I
I
I
Time (mid
9.3.3
I
I
Figure 9.9. Response characteristics of the ER effect. T torque with electric field on; Totorque with electric field off. (Reproduced with permission from Bridgestone Corporation)
Carbonaceous Particulate Phases
Since aqueous ERFs show poor temperature durability, after many trials and errors, a carbonaceous particulate was developed as a dispersed phase. Figure 9.10 shows the temperature dependence of the E R effect of an ERF with a carbonaceous particulate dispersed phase. The vertical axis is the E R effect and the horizontal axis is temperature. A relatively constant ER effect is observed from room temperature to 100 “C. Moreover, the durability at high temperature is greatly improved. As shown in Figure9.11, after heating at 150 “C for 6h, the E R effect of this ERF is essentially unchanged. Therefore, this “anhydrous ERF” is suitable for devices used as car parts.
9 Control of Viscosity by Electric Fields: Novel Materials for Electro-Rheological Fluids
180 800
15KV/ce
600
n
E
u 07 w 3 0-
h
ao
L
0 I-
200
BKV/cfl X
”
X
n
OKV/ca
0 U
0
I
0
X
A
U
I
Y
I
0
V
I
20
Temperature ( O C) Figure 9.10. Temperature dependence of the ER effect. Dispersed phase is carbonaceous particulate (Reproduced with permission from Bridgestone Corporation)
I
9.4 Applications
o
: :
Before Heating After Heating at 1 5 0 t
I
0
5
181
I
/
I
I
10
15
20
Electric Field (KV/cm) Figure 9.11. Temperature stability of an ERF with carbonaceous particulate as dispersed phase. (Reproduced with permission from Bridgestone Corporation)
9.4
Applications
Utilizing ERFs intelligent ERF devices whose properties are instantly adjustable can be designed. They are simple in design, with no moving parts. Although an E R F cannot generate a force by itself, it can generate a resistance against an external force. Figure9.12 shows several ways to control the resistive force. In many applications, the shear stress is controlled by the ERF either with a fixed electrode or with a moving electrode. Some applications, the vertical force, such as compression or tension stress, can be controlled by ERFs. Since the distance between the electrodes has to be changed in these cases, the electric field is varied as the distance changes. Historically, the clutch and the damper have been considered as suitable applications [9.3], [9.6], [9.7]. However, the most practical ERF application is in anti-vibration mountings [9.16]. The mounting is used to prevent the transmission of vibration from a vibrating source such as the engine to the interior of the car. Figure 9.13 shows the structure of an anti-vibration mounting utilizing an ERE The mounting has two fluid chambers connected through an orifice. The rheological properties of the fluid can be controlled by the voltage applied by electrodes built into the orifice, enabling the mechanical properties of the mounting to be controlled. Figure 9.14 shows the dynamic character-
182
9 Control of Viscosity by Electric Fields: Novel Materialsfor Electro-Rheological Fluids
Figure 9.12. Working mechanism of an ERE (Reproduced with permission from Bridgestone Corporation)
Rubber Membrane
Figure 9.13. Structure of ERF-filled anti-vibrationmounting. (Reproduced with permission from Bridgestone Corporation)
9.4 Applications
4 00 tan 6 (E = 0 )
-------- tan 6 (E = 2 KV/mm) --- -Kd IE = 0)
---
300 -
Kd
- 3.0
(E = 2 KV/mm)
-
tan 6
5#
-
\
r" 2 0 0 -
- 2.0
s
100 -
-
-/ ---
_ _ _ - - #
0
1
I
1
I
10
20
30
40
6 -
---
r 5-
.-
50
E=O E = 2 KVIrnm
U
2
4 -
.-UIul
E 3-
t
:
21-
0.
I
1
183
184
9 Control of Viscosity by Electric Fields: Novel Materials for Electro-Rheological Fluids
istics of the mounting. The vertical axes are the dynamic spring constant and tan 6 of the mounting, and the horizontal axis is the vibration frequency. When the voltage is turned on, the dynamic spring constant is increased over a wide range of frequency and tan S is also increased, especially at lower frequency. This means that both the resonance frequency and damping of the vibration system including the mounting are increased, especially at lower frequency when the voltage is turned on. Figure9.15 shows the vibration characteristics of the system consisting of the mounting and the attached mass. The vertical axis is the transmissibility of vibration and the horizontal axis is the vibration frequency. When voltage is turned off, the system has a resonance frequency around 17 Hz; when voltage is applied, the resonance frequency moves to higher values and the transmissibility of vibration is remarkably reduced, especially at lower frequency. Thus ERFs can control the characteristics of the vibration system and minimize vibration over a wide frequency range.
9.5
Conclusion
The history of ERFs is extensive, but so far no industrial product utilizing them has appeared. However, the development of new dispersed phases such as carbonaceous particulate has overcome many of the drawbacks of aqueous ERFs, and offers the possibility of widespread applicatons in the near future.
9.6
References
[9.1] A. W. Duff, Physical Review, 4, 23 (1896). [9.2] G. Quinke, Ann. Phys., 62, 1 (1897). [9.3] W. M. Winslow, US Patent 2417850 (1947). [9.4] J. E. Stangroom, US Patent 4129513 (1978). [9.5] D. Scott, J. Yamaguchi, Automotive Engineering, 93, Nov. 75 (1985). [9.6] D. A. Brooks, Design Eng., Nov., 41 (1982). [9.7] R. Stanway, J. L. Sproston, N. G. Stevens, J. Electrostatics 20, 167 (1987). [9.8] N. Sugimoto, J. Jpn. SOC.Lubr. Eng., 30, 859 (1985). 19.91 Z. P. Shulman et al., J . Eng. Phys., 52, 175 (1985). [9.10] A. F. Sprecher, J. D. Carlson, H. Conrad, Muter. Sci. Eng., 95, 187 (1987). [9.11] H. Block, J. P. Kelly, J. Phys. D.Appl., Phys., 21, 1661 (1988). [9.12] T. C. Jordan, M. T. Shaw, ZEEE Trans. Electr. Insul., 24, 849 (1989). [9.13] R. Pool, Science, 247, March, 1990. [9.14] D. J. Klingenberg et al., J . Chem. Phys., 94, 6160 (1991). [9.15] T. G. Duclos et al., Machine Design, 42, Jan., 21 (1988). [9.16] T. Ushijima, K. Takano, T. Noguch, Society of Automotive Engineer. Technical Paper No. 880073 (1988).
10 Application of Electric Fields to Solvent Extraction Manabu Yamaguchi
10.1
Introduction
With the growing needs of separation engineering and the development of new extractants, solvent extraction technology has developed rapidly and has played an increasingly important role in hydrometallurgy, chemical and oil industries. Consequently, many types of contactors have been developed for achieving effective mass transfer, each with its particular advantages. They may be roughly divided into two classes, the stagewise contactor and the differential contactor (Fig. 10.1). In the stagewise contactor, two liquid phases are fed to a stage, brought to equilibrium, separated, and passed countercurrently to the next stage, e.g., mixer - settler type contactors (Fig. 10.2A). In the differential contactor the two phases are kept in continuous contact as they pass through the column in opposing directions. Mass transfer occurs during their movement, and the phases are separated at the top and bottom ends of the column, e.g., spray contactors (Fig. 10.2B). in separation processes dealing with liquid drops, various attempts have been made to enhance mass transfer. Generally, the enhancement can be obtained by producing a larger interfacial area for diffusion and a higher degree of turbulence outside and inside the drops for eddy diffusion. The combination of tur-
E x t r a c t i o n Equipment
I
I
I
S t agew i se
Differential
I
Mixer - S e t t l e r
T---1
Co I umns
r--l
Non-Agitated
SP r ay
I
Packed Perforated Plate
Centrifuges
Agitated
i
Vertical
Horizontal
I
1
RDC Graesser ARDC Kuhni Scheibel Oldshue Karr Pulsed Packed Sieve-P I a t e Pu I s e
Figure 10.1. Classification of liquid liquid extraction equipment. (Reproduced with permission from Principles and Practices of Solvent Extraction, p. 467, Marcel Dekker, New York)
186
10 Application of Electric Fields to Solvent Extraction agitator
A
1 i q u i d-1 i q u i d
mixer t a n k
l i g h t phase
heavy phase
continuous phase phase inter face
Figure 10.2. Liquid - liquid extraction equipment. A) Mixer - settler; B) Spray column. (Reproduced with permission from Principles and Practices of Solvent Extraction, p. 467, Marcel Dekker, New York)
bulence and increased interfacial area is difficult to achieve because small drops do not have high relative velocities, nor do they exhibit marked internal circulation patterns. Generation of droplets also makes the phase separation difficult. The application of an electric field to overcome these problems was proposed by Thornton [lO.l] and co-workers [10.2], [10.3]. This technique has some advantages in that small charged drops can be produced easily by using an electrostatic force and the charged drops can move through a continuous phase with higher velocity, owing to the Coulomb force, which induces a high degree of fluid turbulence outside and inside the drops. Thus, mass transfer operations can produce larger interfacial areas coupled with enhanced transfer coefficients. Furthermore, the direct utilization of electrical energy is potentially extremely efficient because the electric field acts only at the interface between the drops and the continuous phase, rather than throughout the bulk of both phases.
10.2 Theory The performance of conventional liquid - liquid extractors depends on their hydrodynamics which is controlled by three mechanisms: motion, break-up, and coalescence of the drops. Electric fields influence all three mechanisms by:
10.2 Theory
187
1) Increasing or decreasing drop velocity, owing to the electrical forces (hybrid flow, enhanced mass transfer). 2) Disintegrating drops by electrostatic dispersion at high field strength (interfacial renewal, increased interfacial area). 3) Promoting drop - drop coalescence under favorable conditions in the electric field (phase separation, enhanced liquid mixing inside the drops).
10.2.1 Hybrid Flow and Enhanced Mass 'Ikansfer Around Drops The flow outside and inside a liquid drop moving under the influence of gravity through a viscous medium was solved analytically by Hadamard [10.4] and Rybczynski [10.5], independently. Figure 10.3 shows the flow outside and inside the drop. The circulation inside the drop has been shown, both theoretically and experimentally, to play an important part in enhancing the rate of mass transfer between the drop and the continuous liquid phase. When a drop is placed in another liquid phase in the presence of an electric field, how does the drop behave? Taylor [10.6] studied theoretically on the behavior of a stationary drop of a leaky dielectric liquid in another such liquid phase in a uniform electric field. The potentials & and q&, outside and inside the drop in the electric field ar given by:
$d
=
3Eor2
1
+ R cOs*
where R = hJ& with h, and & being the resistivity of the continuous and drop phases, and Eo the strength of the uniform electric field. The electric stress acting on the surface of the drop has radial and tangential components, (z,,)~ and ( ~ ~ 0 ) ~ :
Figure 10.3. Hadamard - Rybczynski flow pattern outside and inside the drop.
188
10 Application of Electric Fie1d.s to Solvent Extruction
where S = E J E ~ ,with E~ and &d being the permittivity of the continuous and drop phases, and En and E, the normal and tangential components of the electric field strength on the surface of the drop. The tangential stress can only be balanced by stresses associated with hydrodynamic flow outside and inside the drop. The approximate stream functions qcand qdfor both flows, corresponding to the appropriate angular distributions of stress are as follows:
(;:+ )
qc = A -
q d =
Ba2 sin26cos6
(: + .;) C-
D-
(5)
sin20cos0
The four unknown coefficients in Equations ( 5 ) and ( 6 ) can be determined from the continuity conditions for velocity components on the drop surface, r = a. Consequently, A = -B = C = -D. The hydrodynamic stress components at r = u can be obtained in terms of the coefficient A as follows.
The equilibrium equations for the drop shape are:
(Zr@)E
+ ( t r @ ) c - (xr8)d
=
0
(10)
where C' determines the difference between the internal pressure and the normal component of stress due to surface tension acting over the spherical surface of the drop. By substituting Equations(3), (4), (7), and (8) into Equations (9) and (lo), a function for the drop shape in the electric field can be derived as follows:
where X = qdlqcwith qd and qc the viscosity of the drop and continuous phases. If 4 ( S , K , X ) is positive, the drop will be prolate in shape; if it is negative the drop will be oblate. When Cp = 0, the drop is spherical. From Equation (TO), coefficient A can be obtained as follows:
10.2 Theory
189
Figure 10.4. Taylor flow pattern outside and inside the drop in a uniform electric field (SR > 1.0). Flow pattern reverses when SR > 1.0. (Reproduced with permission from Proc. Roy SOC., [10.12])
The surface velocity of a spherical drop is derived from Equations (5) or (6) as follows:
In Equation (13), the sign of UJE)changes, depending on the electrical properties of the liquids used. That is, the direction of fluid flow around the drop can be either from the equator toward the pole (for RS> 1) or from the pole toward the equator (for RS < 1). Figure 10.4 gives an example of the flow pattern outside and inside the drop for the case of RS<1 in which the direction of flow is indicated by arrows [10.6]. McEwan and de Jong [10.7] confirmed Taylor's theory experimentally by using a silicone oil drop in a mixture of castor oil and corn oil in a d.c. field (Fig. 10.5). The patterns are similar to those in Figure 10.4. When the drop moves through another liquid phase under gravity in the presence of a uniform electric field (parallel to the gravitational field), what are the flow patterns around the drop? Chang et al. have solved the problem analytically for the low Reynolds number, Re
">
+ (": + ++B2 r2 B3 r3 1.4 -
vd
= (El?
sin 8 cos6
+ E3r4)sin28 + (FI? + F3r4)sin% cose
(16)
where both qjc and qd,are dimensionless stream function. The twelve unknown coefficients in Equations (15) and (16) can be determined by the appropriate boundary conditions and the auxiliary Galerkin orthogonality conditions [ 10.91. Consequently, A2-A4; El and E3 were expressed in terms of A,, and B2- B4. Fl and F3were expressed in terms of B1 with three parameters Reynolds number, Re; viscosity ratio X , and a dimensionless parameter, W
190
10 Application of Electric Fields to Solvent Extraction
A
Figure 10.5A. Flow pattern inside a silicone oil drop in a mixture of castor oil and corn oil in a d.c. field (Eo= 64.9kV/m (Reproduced with permission from Proc. Roy SOC., [10.12])
Figure 10.5B. Flow pattern outside a silicone oil drop in a mixture of castor oil and corn oil in a d.c. field (Eo= 162.3 kV/m (Reproduced with permission from Proc. Roy SOC., [10.12))
where U,,dE)is the maximum surface velocity of the drop at 6 = n/4 in Equation (13). U, is the constant velocity of the drop falling or rising in the continuous phase. As W depends on U,,dE)(Eq. 13), if Wis positive, the electrically induced surface flow of the drop occurs from the pole toward the equator; for negative Wit is reversed. Thus, Wis a new dimensionless parameter that characterizes the relative importance of the electric field and the translation of the drop due to gravity. For instance, even if the drop is moving slowly, (Re <1), separation of fluid flow around the drop occurs in the flow field of I W 1 > 1 [10.8]. For intermediate Reynolds numbers, examples of the flow patterns calculated by Equations (15)-(17) are shown in Figure 10.6. They are quite different for positive or negative values of W [10.9]. The flow patterns are more complex than those shown in Figures 10.3 and 10.4. Chang et al. investigated in detail the effect of such hybrid circulations on the rate of mass transfer between the drop and the continuous phase at high Peclet number Pe, where the boundary layer of momentum transfer is thicker than that of diffusive mass
10.2 Theory
( a ) Re
=37, W=6
( b ) Re
191
Figure 10.6. Hybrid flow pattern outside and inside the drop moving in both electric and gravitational fields at intermediate Reynolds number for I W I = 6 and X = 2. A) Re = 37, W = 6; B) Re = 110, W = -6. (Reproduced with permission from Elsevier Science)
=110, W=-6
transfer, for low and intermediate Reynolds numbers. They obtained an analytical solution for the mass transfer rate in the continuous phase at intermediate Reynolds number; assuming a thin diffusional boundary layer on the outside of the interface, the steady-state convective - diffusion equation was solved by similarity transformation and numerical techniques, their expression for the Sherwood number of the continuous phase, Sh, is:
where: (sin3@~ ~ c o s-t 1l)
m
and css(for positive F )
=
F -
4
dtl
sin48 + case -
1 3
-C
+ 23
O S ~ ~-
where F is FlIE3, with Fl and E3 being coefficients expressing flow intensity inside the drop in Equation (16). In Equations (19) and (20), is a similarity variable in terms of polar angle (surface position) and penetration thickness of diffusive mass around the drop translating in the uniform electric field. Figure 10.7 shows the relation between the calculated dimensionless mass transfer rate Sh,c"/Sh,") and dimensionless electric field strength, for various Re nolds numbers, together with the creep flow at a viscosity ratio of X = 2 [ 10.91, Sh,(ElSh,"' is the ratio of the continuous-phase Shenvood number for the drop in both electric and gravitational fields to the corresponding value for the drop translating by gravity alone (the Sherwood number is the dimensionless mass transfer rate; i.e., the convective mass transfer rate divided by the diffusive mass transfer rate). It is seen that the rate of mass transfer increases roughly in step with the field strength. In the region of creeping flow, the enhancement is significant.
cSs
Y
192
10 Application of Electric Fields to Solvent Extraction
E
Figure 10.7. Relation between the ratio of the Sherwood number in the continuous phase for a drop moving in both electric and gravitational fields to that in the gravitational field, only and dimensionless electric field strength for various Reynolds numbers.
10.2.2 Electrostatic Liquid Dispersion As stated in the preceding section, a stationary spherical drop or a moving spherical drop in a liquid phase in the presence of a uniform electric field is subject to electrical stresses, which are produced by the interaction of the electric field with the charge accumulated on the drop surface. It has been shown that the hybrid flow around the drop is induced electrically and when 4 (Eq. 11) has negative or positive values the drop will be oblate or prolate. With increasing field strength, the drop deforms prolately in the direction of the electric field, becomes unstable, and finally disintegrates into a large number of smaller droplets (Fig. 10.8) [10.11,10.12]. Such behavior may be useful in creating large interfacial areas for mass transfer (interfacial renewal, increased specific surface area). It is very important in the design of extractors to predict the volume and electric charge of the drops, coupled with their motion in the electric field. The volume of the drop formed from an electrified nozzle has been widely studied. Takamatsu et al. 0
0 Eo
0
0
I-*
Eo
Figure 10.8. Disruption of a water drop in hydrocarbon oil by formation of sharp points of after instability in a uniform electric field. (Reproduced with permission from Proc. Roy Soc., [10.11])
10.2 Theory
193
,ode
L --- --------_-----
lower e l e c t r o d e
Figure 10.9. Schematic diagram of charged drop formation on a nozzle extended into a uniform electric field Eo. DF drawing force; RF restraining force.
[10.13] have proposed an equation for predicting the volume of the drop, modeled as two-stage drop formation; the original model was proposed for uncharged drop formation by Scheele and Meister [10.14]. In the first stage, a pendant charged drop expands at the nozzle tip until the force balance acting on the drop breaks down. The force balance is composed of gravitational, inertial, and electric forces (drawing force) and interfacial tension forces (restraining force). In the second stage, when the drawing force exceeds the restraining force, the drop begins to break away from the nozzle, but continues to grow during the necking process (Fig. 10.9). The force balance is given by [10.131: 20vcQnDn(32"v-2'3
+ xD,o + K(Qz Di,inp,pApg)'"
restraining force
-
drawing force The electric force F, was derived by the method of images as follows:
=
{ kY0.34
for a = 1 for a > 1
where p is a force coefficient depending on a = (1 + a)/a, which expresses the effect of a protruding nozzle in the uniform electric field. They confirmed the model experimentally by using a system of water drops in six kinds of organic phases. Figure 10.10 shows the pendant drops in air just before detachment at three different electric field strengths. Figure 10.11 shows the relation between calculated and experimental volumes of charged drops formed at a low flow rate of the dispersed phase, as a function of the protrusion length of the electrified nozzle t10.131.
194
I0 Application of Electric Fields to Solvent Extraction
Figure 10.10. Water drop formation in air in a uniform electric field (D, = 0.1 cm, i = 1.Ocm. A) Eo = 0; B) E = 2 X lo5V/m; C) E" = 3 x 10sV/m. (Reproduced with permission from J. Chem. Eng. Japan, 14, 178 (1981)
Figure 1O.ll. Dependence of charged drop volume on electric field strength and nozzle protrusion length for water drops in cyclohexane. D. 0.1 cm, Q,= 0.0032cm3/s. open circles I = 0.1cm; full circles 1= 0.4 cm. (Reproduced with permission from J. Chem. Eng. Japan, [10.13])
E , , X ~ O - ~V/cm
10.2.3 Electrostatic Drop - Drop Coalescence The action of high electric fields on dispersed water drops suspended in a dielectric liquid causes the drops to attract each other and to form an extended chain in the direction of the field. These phenomena result from polarization of the drops in the electric field. Consider the dipoles induced in two uncharged drops (radii ai,aj) placed in a uniform electric field Eo. The dipole moment pj, in the drop a, is the sum of the moment p,o induced by the external field and that due, to the additional polarization induced by the other dipole moment pi;it is derived by the method of images as follows:
p.. = I'
a3
r
r?. 11
wherefis a constant depending on the conductivity of the drop phase.
The attractive force F,; between the two drops in the liquid phase is given in polar coordinate as follows [10.15]:
10.3 Electrostatic Liquid - Liquid Extractor
195
where pi and piare the dipole moments of drops aiand aj and rijis the distance between the drops. The drops align in the direction of the electric field under the influence of F,i;further details are given in Chapter 11. Larger drops are then formed by coalescence between the drops within the chain, but as trapping in the local higher field, they are redispersed into smaller drop (see Fig. 10.8) (phase separation, internal mixing of drops, interfacial renewal).
10.3
Electrostatic Liquid - Liquid Extractor
10.3.1 Electrostatic Spray Column In the spray column, one of two liquid phases is dispersed into the other phase at one end of the column and they are allowed to fall (or rise) freely against (counterflow) the continuous phase. The column does not yield good mass transfer coefficients because it is subject to severe axial mixing which reduces the mean transfer driving force. Yamaguchi et al. [10.16]-[10.18] proposed that mass transfer coefficients could be improved by means of the electric fields. They examined experimentally the hydrodynamic and mass transfer characteristics of an electrostatic spray column, in which fourrod electrodes or a pair of parallel-plate electrodes were installed vertically along the inner wall of the column. Figure 10.12 shows a schematic diagram of the laboratoryscale column used. The dispersed phase, introduced into the continuous phase between the electrodes across which a d.c. voltage is applied, is allowed to fall, uniting and redispersing the drops in zigzag and spiral motion. Experimental results for electrostatically enhanced mass transfer have been reported for binary 110.21 and ternary liquid - liquid systems [10.17-10.211. Figure 10.13 shows an example of extraction performance as a function of the dispersed phase flow rate at different applied voltages, in which acetic acid in a continuous xylene phase was extracted by a dispersed water phase [10.20]. The degree of extraction FExincreases with increasing applied voltage Epfor a constant flow rate wd. This may be due to drop - drop coalescence (liquid mixing inside drops) and redispersion of the drops (interfacial renewal) caused by the electric force. The characteristics of this type column have been summarized as follows [10.16-10.181: 1) The coalescence and redispersion behavior of charged drops in an electric field significantly improve the performance of the column. Their behavior is easily controlled by means of the applied voltage. 2) The axial mixing coefficient decreases markedly with increase in the voltage because the forward axial mixing of the continuous phase can be reduced and radial mixing of the phase promoted by the complex motion of the drops.
dispersed phase
plate electrode liquid-liquid interface
Figure 10.12. Schematic diagram of electrostatic spray column.
continuous phase
196
10 Application of Electric Fields to Solvent Extraction
XW
tr
Figure 10.W. Relation between degree
0
0.1 0.2
0.3 0.4 0.5 0.6 0.7 of extraction and dispersed phase flow rate at various applied voltages for of xylene - acetic acid and water.
w*(cm/s)
Table 10.1. Preliminary mass transfer results: water tinuous phase)"
-
acetic acid (dispersed phase)/MIBK (con-
Device
Theoretical stagekm of column
Rel. performance
YSC (600 + rpm)h PCC (5000 + rprn)',' EPC
0.1 0.17 1.71
1.0 1.7 17.1
a Aqueous feed contained 16% acetic acid by weight. Perry and Chilton t10.371. column height approximated using the radius of the contactor. YSC York - Scheibel column. PCC Podbielniak centrifugal contactor. (Reproduced with permission from Ind. Eng. Chem. Res., [10.22])
3) The extraction efficiency and overall volumetric mass transfer coefficient of the column are somewhat better than those of the rotating disk column (RDC). Scott and Wham [10.22] developed a novel electroextractor which is essentially the type shown in Figure 10.12. The electric field between the plate electrodes was provided by an a.c. voltage or by a pulsed d.c. voltage. It is called the emulsion phas contactor (EPC). The EPC system is a continuous, countercurrent, multistage extraction device that uses electric field interactions to control droplet formation, translation, and droplet - droplet coalescence. The contactor performance was examined by using water - acetic acid as dispersed phase and methylisobutyl ketone as continuous phase; the contactor had 1.7 stageskm, outperformed laboratory-scale versions of the YorkScheibel column (YSC) by a factor of 17, and the Podbielniak centrifugal contactor (PCC) by factor of 10 (Table 10.1) [10.22].
10.3.2 Electrically Assisted Mixer - Settler Extractor Without Agitator. In the electrostatic spray column, the liquid phase is dispersed using electrified multiple nozzles or electrified liquid distributors. If one of the liquid phases can be dispersed directly into the other without any liquid distributor, development of a novel liquid extractor becomes feasible. Yoshida et al. [10.23,10.24] have developed a novel extractor with a pair of inclined parallel-plate electrodes (IPEE). Figure 10.14 is a schematic diagram of a laborator-
10.3 Electrostatic Liquid - Liquid Extractor
197
scale extractor, in which a water film flowing onto an inclined plate electrode is dispersed directly into a dielectric liquid phase by means of a d.c. voltage applied perpendicular to the film. Figure 10.15 shows the electrodispersion of the water phase. Some favorable characteristics of the flow (Figs. 10.14 and 10.15) are as follows: a) The drops were produced from the waves formed by field-induced stress on the water film. They had an induced charge and rose against gravity toward the upper electrode through the continuous phase because of the Coulomb force as the interaction of the charged drop with the electric field;
Figure 10.15. Side view of dispersion from a water film in cyclohexane.
198
10 Application of Electric Fields to Solvent Extraction
b) They collided with the upper electrode and their polarity was reversed;
c) They fell down through the continuous phase toward the lower electrode under gravity and the Coulomb force; d) They coalesced instantaneously with the film phase. Yoshida et al. [10.25] applied the IPEE successfully to the resolution of oil-in-water emulsions. The IPEE has some advantages [10.23, 10.241: 1) Liquid dispersion technology in the extractor can be utilized as a mixing tool for liquid - liquid systems because the dispersion produces marked interfacial turbulence between the two liquid phases as well as a bulk mixing in the continuous phase phase. 2) The liquid dispersion and drop - film coalescence between two “weirs” on the electrode are equivalent to more than one stage in conventional plate columns. 3) Phase separation between the dispersed and continuous phases is superior because charged drops coalesce instantaneously with the water film. 4) As the column does not have obstacles such as plates, agitators, etc. the pressure drop for liquid transportation is much lower than that in conventional columns. The Electro-Dynamic extractor has plate electrodes set vertically face-to-face in the contactor (Fig. 10.16B) [10.2b]. Figure 10.16A shows the shape of the pulsed d.c. voltage between the electrodes and the corresponding behavior of the drops supplied by a liquid distributor [10.2b]. The field strength and its variation with time are controlled locally. The extractor retains the organic phase within the grid section for 188s and provides 9 extraction cycles of mixing/coalescing/settling at a rate of 21 s per cycle. The extractor was successful in the extraction of soluble salts from petroleum to an aqueous phase. An example of the extractor performance is shown in Figure 10.17, in which the average inlet and outlet salt concentrations were 56.1 und 2.2ppm [10.27].
With Agitator. The electrostatic spray column and the IPEE have advantages for liquid dispersion, mixing of the liquid phases, and phase separation without moving parts in the column, but the dispersed phase holdup was low.
MIXING
COALESCING
SETTLING
VOLTS 45,000
0 DILUTION WATER
7
40,000
35,000
30,000
25,000
li EMULSION FLOW
TIME (SECONDS)
Figure 10.16A. Schematic diagram of the electrodynamic extractor and the shape of the applied voltage corresponding to drop behavior.
10.3 Electrostatic Liquid - Liquid Extractor
199
Figure 10.16B. Internal view of the electrodynamic extractor.
Several investigators [ 10.28-10.301 proposed application of an electric field to the phase separation zone rather than to the liquid dispersion. The dispersed phase holdup in the column was kept high by rotary agitators, and the phase separation in the settling zone was enhanced by electrically assisted drop - drop coalescence without inhibition of drop formation at the agitators. Agaiev and Abdullaev [10.31] used an electric field in a RDC for the purification of petroleum products by means of furfurol and, at a field intensity of 5 kV/cm, obtained a reduction in the height-equivalent theoretical plate (HETP) value by two-thirds in comparison with a spray column and by one-half in comparison with a packed column with Raschig rings. Bailes and Stitt [10.28] developed a novel liquid extraction column with high-shear mixing impellers and electrostatic settlers. They obtained a reduction in the height of transfer unit (HTU) value from 0.424 (1000rpm, 2kV) to 0.134 (1400rpm, 2kV), for extraction of cumene in isoparaffinic hydrocarbon (continuous phase) by a mixture of N-methyl-2pyrrolidinone and etylene glycol (dispersed phase). Nakashio et al. [10.32] developed a new process, using a mixer - settler extractor, in which extraction was performed in a water-in-oil emulsion with a hydrophobic complexing agent; after extraction the emulsion phase was resolved into the aqueous and oil phases by an electrocoalescer. They successfully applied this process to the separation of rare earth metals.
ok--h-’
10
20
30 t day
40
50
60
Figure 10.17. Salt extraction performance of the electrodynamic extractor. Average inlet salt concentration 56.1 ppm; average outlet salt concentration 2.2ppm (Reproduced with permission from Solvent Extraction, [10.27])
200
10 Application of Electric Fields to Solvent Extraction c .- .ca .o+ ;:.?2 0
high voltage generator
,
1
,
0
2 2 t;"
baffle electrode
Figure 10.18. Schematic diagram of the electrostaticpseudo-liquid membrane raffinate phase
concentrated phase
extractor. (Reproducedwith permission from J. Membrane Sci., [10.33])
10.3.3 Electrostatic Pseudo-Liquid Membrane Extractor Gu and co-workers [10.33-10.361 developed a new type of separation method called electrostatic pseudo-liquid membrane (ESPLIM) extraction. They applied it in metal extraction and waste water treatment. A schematic diagram of the ESPLIM is shown in Figure 10.18 [10.33, 10.361. A reaction tank is divided into an extraction compartment and a stripping compartment by an integrated baffle electrode. The organic phase only can pass freely through baffle electrodes while the feed and stripping aqueous solutions are completely separated. When a high voltage is applied simultaneously across the extraction and stripping compartments, the aqueous solutions are dispersed into numerous droplets in the continuous phase. The droplets in the compartments fall in a complicated zigzag motion between the electrodes. In the extraction compartment, metal ions in the aqueous droplets react selectively with the extractant in the continuous phase, and are extracted as metal complexes into the continuous phase. The complex, driven by its own concentration gradient, diffuses through the buffle plate into the stripping compartment and there the complex is stripped off and the extractant is regenerated. The extractant diffuses back to the extraction compartment through the buffle plate. The complicated motion of the drops and the circulation inside the drops in the electrostatic field assist renewal of the interface, and mass transfer is thus accelerated. Based on the principle of liquid transportation, the extraction and stripping processes are performed simultaneously in the column. This process has been applied to the separation of rare earth metals. The feed aqueous solution contained l.Og/L, Y3+or La3+at initial pH5.0; in the extraction compartment, the metal in the feed was
10.4 Nomenclature
Z 1501
0'6
P
t hr
201
'A Figure 10.19. Relation between concentration of rare earth metals in the raffinate phase and operating time for recirculation of stripping solution. Open triangles, D2EHPA-La; full triangles, D2EHPA-Y, open circles, HMHPA-Y; full circles, D2EPHA- all rare earth metals. (Reproduced with permission from Proc. Second Sino - Jap. Symp. Liquid Membranes [10.351)
extracted by D2EHPA or HMHPA in a kerosene solution. In the stripping compartment, the metal - extractant complex in the kerosene was stripped by an aqueous hydrogen chloride solution. During the experiment, the feed solution was passed through the extraction compartment only once, while the stripping solution was recirculated. Figure 10.19 shows extraction performance for the system of rare earth metals and D2EHPA or HMHPA in kerosene [10.35]. The metal concentrations in the raffinate phase were plotted against, the operating time for recirculation of the stripping solution. Optimum extraction performance was maintained for 40 h, after which the metal concentration in the raffinate phase increased with time. This is because hydrogen ions in the stripping solution were consumed and consequently the metal complex was accumulated and free extractant in the oil phase decreased. A multicompartment device specially designed for the ESPLIM process with parallel cascades has been developed [10.34].
10.4
Nomenclature
A, Ai, A29 A39 A4 coefficient, a drop radius, m B, Bi, B2, B3, B4 coefficient, C coefficient, -, concentration, moYL coefficient, -, diffusion coefficient, m2/s D D, nozzle outer diameter, m D,,in nozzle inner diameter, m strength of electric field, N/C E strength of uniform electric field, N/C Eo EP applied voltage, J/C El, E3 coefficient, d, drop diameter, m e, unit vector, -
202
10 Application of Electric Fields to Solvent Extraction
unit vector, electric force, N FEx extraction degree, F' Harkins correction factor, F,, F3 coefficient, f constant, I,, integral defined in equation (19), k mass transfer coefficient, m/s nozzler length extended from electrode, m 1 Pe Peclet number, U,'O)d,/D,Q,] flow rate of dispersed phase through nozzler, m'/s R resistivity ratio, A,/&, Re Reynolds number, pcU$"d,lqc,r distance in r-direction, m distance between drop(i) and drop(j), rij S permittivity ratio, &,/Ed, Sherwood number in continuous phase, kcd,/D, Sh t time, s terminal velocity of drop, m/s U, U,'E' interfacial velocity of drop in the presence of electric field, m/s dispersed-phase linear velocity through nozzle, m/s u, v drop volume, m3 dimensionless velocity parameter defined in Equation (17), W superficial velocity of liquid phase, m/s w X viscosity ratio, qd/qc, ee F,
dimensionless nozzle length parameter, force coefficient, permittivity x8.854 lo-'*, F/m similarity variable, viscosity, Pa.s electrical conductivity, S/m electrical resistance, l/S electric dipole moment, Cm dimensionless electric field strength defined in d r n / \ I C U t I WC I Wc= f I),-density, kg/m3 density difference, kglm3 interfacial tension, N/m shear stress, N/m2 electric potential, V stream function, m3/s Subscript c continuous phase d dipersed phase E electrical n normal component r radial component t tangential component 8 polar angle component
10.5 References
10.5
203
References
[10.1] J. D. Thornton, Rev. Pure Appl. 18, 197-218 (1968). [10.2] P. J. Bailes, J. D. Thornton, Proc. ZSEC,1011-1027 (1974) [10.3] J. D. Thornton, J. E. Porter, Symp. Electrochem. Eng. 3, 3-38 (1971). [10.4] J. Hadamard, Compt. Rend. 152, 1735 (1911). [10.5] W. Rybczynski, Bull. Acad. Sci., 1,40 (1911). [10.6] G . Taylor, Proc. Roy. Soc., A280, 383-397 (1964). [10.7] A. D. McEwan, L. N. J. De Jong, Proc. Roy, Soc., A291, 166 (1966). [10.8] L. S. Chang, T. E. Carleson, J. C. Berg, Znt. J. Heat Mass Transfer, 25,1023-1030 (1982). [10.9] L. S. Chang, J. C. Berg, Znt. J. Heat Mass Transfer, 26,823-832 (1983). [10.10] A. E. Hamielec, A. I. Jonnson, Can. i. Chem. Eng. 40,41-45 (1962). [10.11] C. G. Garton, Z. Krauski, Proc. Roy. Soc., A280, 211-116 (1964). [10.12] G. Taylor, Proc. Roy. Soc., A291, 159-166 (1966). [10.13] T. Takamatsu, M. Yamaguchi, T. Katayama, J. Chem. Eng. Japan, 15, 349-355 (1982). [10.14] G. F. Scheele, B. J. Meister,AZChEJ., 14, 9-19 (1968) [10.15] T. Ise, M. Ymaguchi,T. Katayama, Kagak u Kogak u Ronbunsy u, 15,1087-1094 (1989). [10.16] M. Yamaguchi, H. Sugaya, T. Katayama, J. Chem. Eng. Japan, 21, 179-183 (1988). [10.17] M. Yamaguchi, H. Sugaya, T. Katayama, J. Chem. Eng. Japan, 22,25-29 (1989). [10.18] M. Yamaguchi, Solvent Extraction 1990, B, T. Sekine (ed.). Elsevier, 1992,pp. 1357-1380. [10.19] P. Bailes, Proc. ZSEC., 1, 233 (1977). [10.20] W. Kowalski, Z. Ziolkowski, Znt. Chem. Erg., 21, 323-327 (1981). [10.21] L. Martin, P. Vignet, C. Fombarlet, F. Lancelot, Sep. Sci. Echn., 18, 1455-1471 (1983). [10.22] T. C. Scott, R. M. Wham, Znd. Eng. Chem. Res., 28, 94-97 (1989). [10.23] F. Yoshida, M. Yamaguchi,T. Katayama, J. Chem. Eng. Japan, 19, 1-7 (1986). [10.24] F. Yoshida, M. Yamaguchi,T. Katayama, J. Chem. Eng. Japan, 21, 123-128 (1988). [10.25] F. Yoshida, M. Yamaguchi, T. Katayama, J . Chem. Eng. Japan, 21, 428-430 (1988). [10.26] NATCO commercial materials of process and environmental systems. [10.27] K. W. Warren, J. J. Byeseda, Solvent Extraction 1990, B, T. Sekine (ed.). Elsevier, 1992, pp. 1417-1422. [10.28] P.J. Bailes, E. H. Stitt, Chem. Eng. Res. Dev., 65, 514-523 (1987). [10.29] M. Dilley, M. T. Errington, D. Naden, Proc. ZSEC, 1, 140-147 (1993). [10.30] A . Kumar, F. Ansermet, S. Hartland, Can. J. Chem. Eng. 339-341 (1987). [10.31] A . A. Agaiev, P. C. Abdullaev, Zzv. VUZ. Neft i Gaz, 3, 4135-4143 (1969). [10.32] Y. Miyake, M. Goto, F. Nakashio, 24th Autumn Meeting of the Society of Chemical Engineers, Japan, Nagoya, Oct. 1991, 247. [10.33] Z. M. Gu, J. Membrane Sci. 52, 77-88 (1990). [10.34] Z. M. Gu, Q. F. Wu, L. R. Jin, Proc. Second Sino-Jap. Symp. Liquid Membranes 68-69 (1991). [10.351 Q. E Wu, Z. M. Gu, L. R. Jin, D. X. Wang, Proc. Second Sino-Jap. Symp. Liquid Membranes 70-72 (1991). [10.36] Q . Zhou, Z. M. Gu, Water Treatment, 3, 127-135 (1988). [10.37] R. H. Perry, C. H. Chitton, Chemical Engineering Handbook, 5th ed.; McGraw-Hill, New York. 1973.
This Page Intentionally Left Blank
11 Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions Manabu Yamaguchi
ll.1
Introduction
Water-in-oil (W/O) or oil-in-water (Om)emulsions are utilized in many industries, e.g., the food, chemical, and pharmaceutical industries, but in the petroleum industry, demulsification is a major activity accounting for hundreds of millions of dollars each year. A number of different methods for breaking down petroleum emulsions have been developed: centrifuges, filters, chemical additives, and electrical treatments, etc. [ 11.11. Electrical treatment has been successfully employed for crude oil refining by Cottrell precipitators. The technology offers in lower operating and equipment costs and its adaptability to large throughputs. How and where are petroleum emulsions formed? Some are formed during production of crude oil (well fluids) in the oil fields, and some are formed during desalting processes in the petroleum refinery. They may also be formed during transportation of curde oil through pipelines and pumps 111.21. In oil wells there are many subterranean formations which contain vast quantities of highly viscous crude oil that cannot be recovered by primary production mechanisms. In such situations, various recovery operations are employed to stimulate the flow of the well fluids, e.g., hot aqueous fluids may be injected into the formations [11.3]. The well fluids recovered by the injection method are usually a mixture of highly viscous crude oil, large volumes of water, other impurities, and solids. The amount of water in the fluids is highly variable and depends on the order of the recovery operation: W/O, Om,or water-in-oil-in water (W/O/W) emulsions. The fluids are roughly separated in a gravity or mechanical separators into an oil-free aqueous phase and a wet crude oil phase. The wet crude oil must be dehydrated to a water content of less than 3 ~ 0 1 % . The dehydrated crude oil is shipped by tankers or transported by pipelines to the refinery. In the refinery, residual impurities in the crude oil are removed completely, because they would cause corrosion and fouling of the refinery distillation equipment. For this purpose, fresh water, ca. 5 vol% , are mixed with the crude oil, and the impurities are dissolved in the dispersed fresh water. The resulting mixture is then subjected to an electric field to separate the crude oil and the water phase, which is a brine carrying the extracted impurities and salts to give desalted crude oil. The desalted crude oil can be refined without further treatment. The electrostatic technology for dehydrating crude oil, often in conjunction with desalting, is widely employed in large-scale petroleum plants, and this knowledge has been applied to other industries.
206
11 Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions
11.2 Fundamental Relationships Separation of W/O emulsions is performed by their aggregation and subsequent repeated coalescence between the droplets. Presumably, in the bulk phase the coalescence proceeds to the point at which sedimentation is effective. The application of highvoltage electric fields to W/O dispersions promotes separation. A number of different mechanisms have been proposed for this electrical resolution t11.41. The mechanisms involve: 1) formation of droplet chains and coalescence; and 2) droplet - droplet coalescence by random collisions. They relate to dielectrophoretic and electrophoretic forces resulting from motion of the droplets; dielectrophoretic forces between droplets resulting from induced dipoles in an a x . or a d.c. field, and electrophoretic forces from the interactions between a unidirectional applied field and the charges on the droplets.
ll.2.1
Dielectrophoresis
Consider water droplets in an electric field; the droplets are polarized owing to the dipolar nature of the water molecules and acquire induced charges. They approximate electric dipoles, a number of droplets tending to line up progressively into an extended chain in the direction of the field [11.5]-[11.7]. Ise et al. [11.8] obtained analytical solutions for the attractive force and the formation of the droplet chains. Consider two uncharged droplets (radius ai, a,) of permittivity E ~ in a continuous oil phase of permittivity E ~ in, an electric field Eo (Fig. 11.1).The dipole moment, p,, of droplet a, is given by the sum of the moment P,~,induced by Eo,and that due to the additional polarization induced by the dipole moment pL,of droplet a,:
' ii
EO='
O0 kV'm
ai=ai'5k'm
U
20 pm
Figure U.1. Lines of force between two water droplets with induced dipole moments in a dielectric liquid phase in a uniform electric field. (Reproduced with permission from Kagaku Kogaku Ronbunshu, [11.8])
,
11.2 Fundamental Relationships
207
where f is a parametric constant depending on the conductivity of the droplet. The electric force F , , acting on droplet aj, due to droplet ai,is as follows [11.8]:
where rj, is the distance between the centers of two droplets in polar coordinates and the droplets are assumed to move in a two-dimensional plane (I$= 0). The line of electrical force through any position P(r,, 0,) in the field is as follows [11.9]: sin0 r=rjsinOj
J
cos0 cost),
-
(4)
The lines of force around a dipolar ai,in the electric field can be calculated from Equation (4) and are shown in Figure 11.1. The arrows represent attractive and repulsive directions. Each droplet moves through the lines of force and, finally they make contact with each other. Such behavior is the fundamental scheme of formation of a droplet chain. If it is assumed that droplet motion is governed by Equation (3) and resistance by Stokes’s law, i.e., if inertial forces are negligibly small, the force balance on the droplets can be given as:
where we assume, F, = -F, and r represents a relative position vector (r,-ri).The time during which droplet a, moves from position (r,, 0,) to position (0,O) by the pathway shown in Figure 11.1 can be obtained by integrating Equation (5) after substituting by Equations (3) and (4). In the case of 0, # 0:
=
A?
v(5/2,5/4) - 1(3/2,1/4;4!2)] [(l - p ) 2 . 5 p ]
In the case of 0, = 0:
tc =
2 -A2 5
208
11 Applications ofthe Electric Fields to the Resoltrtion of Water-+Oil Emulsions S
n
Figure U.2. Interaction of a lengthwise dipole chain and a droplet in a uniform electric field. (Reproduced with permission from Kagaku Kogaku Ronbunshu, [11.8])
1
in which t, is the time during which the droplet moves from position (rc, 0) to position (0, 0), where r, is the distance between the centers of two droplets in contact with each other. The value of A in Equations (6)-(8) is as follows:
The lapse time, during which two uncharged droplets in Figure 11.1move through the lines of force and finally make contact with each other can be calculated as follows:
t = tr
-
tc
(10)
Consider a chain formed by n droplets and an uncharged isolated droplet a, (Fig. 11.2) [11.8]. The electrical force F,, acting on droplet a, due to the chain is as follows [11.9]:
where r,yirepresents a position vector at distance rSi,from a droplet ai within the chain. Using F, in place of Fj in Equation ( 5 ) , the lapse time during which an isolated droplet is drawn toward the chain and comes in contact with its end can be obtained.
11.2.2 Computational Simulation of Chain Formation Rearrangement of the droplets in a uniform electric field can be simulated computationally by using Equations (5)-(11), and the results are shown in Figure 11.3 for the system of water droplets in a kerosene phase [11.8].The open and dashed circles in the
3A
38
3c
Figure 11.3. Computational simulation of dielectrophoretic movement of water droplets in kerosene under a uniform electric field. (Reproduced with permission from Kagaku Kogaku Ronbunshu, [11.8])
11.2 Fundamental Relationships
209
Figure ll.4. Contours of lapse time during which an isolated droplet moves through the lines of force field shown in Figure 1 to makes contact with a chain of two droplets.
figure represent the initial and final positions of the water droplets, and the direction of droplet movement is indicated by arrows. Figure 11.3A shows the paths of movement of two droplets, of different size and placed at the initial positions shown by open circles. A small droplet is attracted faster toward a large droplet compared with a large one toward the small one, because of the large viscous resistance of the continous phase against a large moving droplet. When two droplets of equal size are placed side by side with a slight deviation in their dipole axes, they are initially repulsive toward each other, and move through the paths shown in Figure11.3B, eventually making contact with each other. Figure 11.3C shows the behavior of three droplets of equal size situated at the initial positions indicated. They finally line up into a lengthwise chain in the direction of the field. Figure 11.4 [11.10] shows some contour lines of the time during which an isolated droplet placed in any position away from a two-droplet chain is drawn into contact with it. The most remote droplets which the chain can attract are on the t9 = 0 axis.
U.2.3 Observation of Chain Formation The validity of the theory for the formation of droplet chains has been confirmed experimentally by optical microscopy. An example is shown with applied voltage and droplet radius as parameters (Fig. 11.5) for two droplets situated on the 13= 0 axis [11.8]. The value of r is the relative distance between the centers of the two droplets and the value o f t is the lapse time during which the droplets travel the distance r. The results represent the migration behavior of the droplets, i.e., each of the lowest values of r corresponds closely to the distance between the centers of the droplets in contact, and the droplets become more distant with time. The figure shows both experimental and calculated values. Figure 11.6 shows the relation between experimental and calculated lapse time during which an isolated droplet approaches a droplet chain and comes into contact with it [11.8]. Chain formation and coalescence of small water droplets in mixed organic phases in an electric field were examined by optical microscopy.
210
I1 Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions I k e y EP
a,
a*
[am1
[VI
[/Iml
80
9 8 . 7 71.0
0
A I0 27.7 29.2 0 50 16.0 15.9
- c.31. L=1.12 m m
a =o
-
Figure 11.5. Relation of lapse time during which two droplets in contact migrate along the 8 = 0 and the relative distance between centers of the droplets (system 1). r
[mml
10
I L = l . 12 m m
:
1
I
8
0.1
/g
I
0.0 1 0.0 1
0.1
n
EP
1
d r o p l e t radius
[-I [ V I
[Urn]
a.
key system
a,
a,
a.
ad
as
a,
0
I
3
50
16.9 24.1
12.3 28.7
0
1
3
7U
31.0 10.6
6.6
2
1 4 5
10
2 6 . 7 3 6 . 5 26.0 3 1 3
60
16,450.527.1 24.730.~
40
39 I 29 3 3n 4 3 5 . 2 19.5 19.5
5 6
50
2 2 . 8 3 9 . 1 4 2 . 3 2 3 . 4 3 3 . 9 22 8
50
2 3 . 1 20.8 11.7 4 2 . 7
o
2
A
z
A
I
v
I
40.3 9.8
3 3 . 6 36
a
28.8
system 1 : 5 2 . 3 w t X K e r o s e o r t 3 8 . 5 w t % C C L t 8 . l w t % L i q p a r a f f i n+U. 5wtSLSpan 80 system 2 : 59.6ut%Keror~net39.Sut%CCI,tO S w l % S p a n 80
Figure 11.6. Relation bctween experimental and calculated lapse time during which an isolated droplet makes contact with a droplet chain. (Reproduced with permission from Kagaku Kogaku Ronbunshu, [11.81)
Mixtures of organic phases were prepared, of equal density to the water droplet phase to obtain different viscosity values. Figure 11.7 shows photomicrographs of chain formation and droplet - droplet coalescence in a uniform electric field [11.10].
11.2 Fundamental Relationships
11.2.4
211
Electrophoresis
When water droplets in an oil phase in an electric field have intrinsic charges, the droplets are subjected to electrophoretic force and move unidirectionally. The electric force F, is a product of the droplet charge and the electric field strength [11.11, 11.121:
in which Q is the charge on a droplet, Eois the mean strength of the external electric field, E~ is the permittivity of a continuous oil phase, a is the droplet radius, and yis the ratio of the net charge of the droplet to its saturation charge. In fact, y varies from 1 to 0, depending on the relaxation times of the various dielectrics that prevent direct contact between the droplets and the electrified electrodes [11.4]. As the droplets subjected to the force given by Equation (12) move in the electric field, they will collide with one another. Bailes and Larkai proposed a model based on random collisions of the drops for electrical coalescence of water drops dispersed in an oil phase [11.13, 11.41. According to their model, the work done per collision of the drops leads to the following expression, assuming y = 1 :
in which S is the mean distance separating each drop from its neighbor under the assumption of a rhombohedra1 pacing of the drops, and c$w is the fractional volumetric holdup of the drop phase in the continuous oil phase. It is assumed that any special field effects are unaffected by a finite holdup of the drops, and each collision involves two drops. The total work done per second W,can be obtained as follows:
where m is the toal number of the drops in the continuous phase and N , is the mean collision frequency, defined as the mean number of collisions that each drop undergoes in one second. The relation between the work and drop - drop coalescence is derived from Equations (13) and (14):
Wt
=
[(!$y
4 Epl, = 4.95-na3~,mN,E~ 3
- I]
in which EPis the mean applied voltage and I , is the mean conducting current. It is assumed that the work done per collision is equal to the electrical power consumption EPIm.The term 4 na3m/3 in the equation is the total volume of the drops in the continuous oil phase and is equal to the volumetric holdup of the drop phase, GWVwhere Vis the volume of a coalescer. They derived a general expression for calculating the mean collision frequency, including measurable variables, as follows:
212
11 Applications of the Electric Fie1d.v to the Resolution of Water-in-Oil Emulsions
(-0.37 s e c )
(t2.36 s e c )
(t0.61 s e c )
(t2.66 sec)
Figure 11.7.
11.2.5 Effect of Electric Fields on Resolution of Water-in-Oil Dispersions Bailes and Larkai [11.13, 11.41 devised a box-type electrostatic coalescer and experimentally tested the effect of various electric fields on resolution performance of waterin-oil dispersions using a system of aqueous H,SO, solution and kerosene containing 20% LIX 64N (Henkel). The applied voltage was varied between 0.2 and 10kV and the frequency was variable over the range 0.5-60Hz for square and half-wave pulse forms. Figure11.8 shows performance plotted as a function of mean collision frequency in which all the data are successfully brought together on a single curve, irrespective of the frequency and the form of the applied electric field [11.4]. Equation (17) was applicable for holdup fractions of the dispersed phase up to 0.5.
11.3 Electrostatic Resolution of Emulsion in the Oil Industry
(t3.20 s e c )
(t4.03 sec)
213
(t3.63 sec)
(+4.23 sec)
Figure 11.7. Sequence of microscopic behavior of aqueous droplets in a uniform electric field EP= 70 Vd.c. electrode gap 1mm, aqueous solution droplets (CNa= 2wt%) in kerosene containing Span 80 (Cs= 4wt%). Negative and positive values in parentheses represent the time before and after the voltage is applied to the emulsion.
11.3
Electrostatic Resolution of Emulsion in the Oil Industry
11.3.1 Dehydration The connate crude oil in the wells that cannot be pumped up by primary production mechanisms is recovered by various methods, called enhanced oil recovery (water injection or gas injection methods, miscible drive, chemical processes, steam injection, etc.) [11.3]. Figure 11.9 shows a schematic diagram of oil recovery by hot water injection, where the injected hot water raises the temperature of the connate crude oil, whereby its viscosity is reduced, and sweeps away the oil to production wells [11.3]. The well fluids pumped up from production wells are usually a mixture of the highly viscous, connate crude oil and large volumes of water. In an extreme case, the fluids
214
11 Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions
Nc
Is-']
Figure 11.8. Relation between phase separation performance and calculated mean collision frequency.
-v
3 C
0 .c U
0 -7
.*E: Figure ll.9. Oil recovery by water injection and fluid distribution.
may consist primarily of water with some interspersed crude oil, i.e., the volumetric ratio of crude oil to water is below 1to 10.This mixture is roughly separated by gravity or mechanical separators into a wet crude oil phase and an oil-free aqueous phase, together with silt and sand. The water content of the wet crude oil, after heating, is reduced by electrostatic coalescers to marketable values (dehydrated crude oil) [11.7]. Recently, a compact hybrid technology for a dehydration of well fluids has been developed. Figure 11.10 shows a flow sheet of the pilot plant tested at the British Petroleum onshore oil field at Wareham f11.141.Three different technologies are employed: a preseparator hydrocyclone, a pulsed-d.c. electrostatic coalescer, and a rotating-body hydrocyclone. According to the report, the preseparator hydrocyclone in the first stage
11.3 Electrostatic Resolution of Emulsion in the Oil Industry
-
I
gas+
215
gas
electrostatic rotary
oil+ water
pre-separation hydrocyclone r
2-3% o i l
20% O i l
25% o i l
&--
40ppm o i l
water discharge
multistage deoiling hydrocyc lone
Figure 11.10. The hybrid dehydration process.
can reduce the crude’s water cut from 75% to 10%. The electropulse inductive coalescer (EPIC) in the second stage can reduce the water content in crude oil from 22% to 6%. When the EPIC was switched off, the gravity separator had no effect. The rotating-body hydrocyclone in the third stage can reduce the water and sediment in Wareham crude oil from 25% to 3%. It is reported that the hybrid process will be able to cut residence time from minutes to seconds, with a correspondig reduction in size and weight.
11.3.2 Desalting The wet crude oil obtained from the well fluids is best dehydrated to marketable values by an electrical treatment. Residual impurities such as asphaltic material, waxes and organic acids, inorganic salts, etc., must be removed completely because they are detrimental to the ultimate use in chemical products. Further, such impurities cause corrosion and fouling of the equipment in refinery distillation process. For this purpose, fresh water, ca. 3-5 vol% is deliberately emulsified in the dehydrated crude oil to aid the removal of impurities. The resulting mixture forms highly stable water-incrude oil emulsions in which the impurities accumulate on the surface of the water droplets to produce a structural barrier which effectively retards droplet - droplet coalescence. In order to promote breakdown of such emulsions, the barrier has to be weakened or removed completely. After heating and pressurizing the mixture to suitable levels and subjecting it to subsequent electric fields, breakdown is facilitated. The former plays a part in reducing in the mechanical strength of the interfacial films and the viscosity of the oil phase, and the latter promotes droplet movement and coalescence between droplets. The mixture is separated by an electrostatic coalescer into a desalted crude oil phase and a water phase (brine plus extracted impurities). Recently, Taylor observed the electrical coalescence behavior of water-in-crude oil emulsions by microscopy, and showed that the behavior differed depending on the oilproducing district (the nature of the oil) even in a specified electric field [11.2]. He classified the behavior into two types on the basis of the conducting current during coalescence related to the interfacial rheological properties on the surface of water
216
11 Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions
Figure 11.11. Microscopic behavior of water-in-crude oil emulsions in a uniform electric field. A) 5% water-in-Kuwait crude oil emulsion; B) 5% water-in-Kuwait crude oil emulsion; containing 300pprn Span 80;C) 5% water-in-Romashkino crude oil emulsion. (Reproduced with permission from Colloids and Surface, [ 11.21)
drops in the crude oil: for incompressible films droplet - droplet coalescence is prevented, and chains of water droplets (type 1) are formed: for compressible films efficient coalescence (type 2) occurs. Figure 11.11 shows the microscopic behavior of water-in-crude oil emulsions in an electric field [11.2]. Figure 11.11A shows the behavior of a 5% water-in-Kuwait crude oil emulsion: a change from the original emulsion dispersed uniformly (micrograph 1) to the formation of droplet chains (micrographs 4 and 5; type 1). This behavior can be altered dramatically by addition of a surfactant. By adding 300 ppm of Span 80 (sorbitan monooleate, Koch-Light) to the water-inKuwait crude oil emulsion, the formation of chains is prevented and droplet - droplet
11.3 Electrostatic Resolution of Emulsion in the Oil Industry
217
O I L OUT
V E L O C I T Y A D JUSTOR
Figure 11.12. The Petreco cylectric desalter: flow behavior of WIO emulsion and droplet - droplet coalescence. (Reproduced with permission from Chem. Eng. Progress, [11.6])
coalescence can be effectively promoted as shown in Figure 11.11 3 (type 2). On the other hand, in the water-in-Romashkino crude oil system, both types of behavior can be observed in Figure 11.11C: type 2 behavior (micrographs 1-3) occurs in the initial stage and as coalescence proceeds, but the behavior reverts to type 1, and formation of droplet chain can be observed (micrographs 4 and 5). Various types of electrical treatment in dehydration and desalting processes have been developed for achieving high efficiency. Generally these can be classified as filter, liquid extractor, and emulsion breaker types. The emulsion breaker and liquid extractor types are widely used in large-scale plants commercial. Figure 11.12 shows a Petreco cylectric desalter [11.6,11.7] a typical emulsion breaker, in which a water-in-crude oil emulsion produced by mixing fresh water and crude oil is discharged directly into an electric field through a parabolic poppet valve of high-
218
I1 Applications of the Electric Fields to the Resolution of Water-in-Oil Emulsions
velocity distributor type. The electric field is generated in the space between two plate electrodes which are arranged for the emulsion to flow horizontally. Droplet - droplet coalescence in the electric field is promoted by various effects: fragility by droplet deformation, random collision between the droplets, etc. Repeated coalescence of the droplets causes small droplets to grow. The mixture supplied from the valve sweeps the emulsion liquid phase, including the enlarged drops, outside the field. The rate of settling of the drops is aided by forced ciculation toward the lower water phase within the vessel. Unsettled drops are recycled into the field under the jet pump effect of the distributor. By proper balance between the hydraulics of the emulsion phase and the electric force, an optimum desalting performance and maximum rate of throughput may be achieved commercially of ca. 15000 kL/day per unit. In the dehydrating capacity of a commercial plant which had throughput of 3600 kWday per unit in 1965, the dehydrating efficiencies were up to 98% for 15 vol% fresh water added to the crude oil, while at a desalting capacity of 15 000 kWday per unit, removal efficiencies were up to 99% for 100-200 ppm of salt in the inlet crude oil [11.61. In this type of equipment, the breakdown behavior of the emulsions can be interpreted by the random collision model discussed in Section 11.2.4. The removal of the impurities in petroleum is only a special case of liquid - liquid extraction: the feed crude oil is a heterogeneous fluid containing fine water droplets
aqueous
phase
phase
organic
_--------
phase
Figure ll.W. Idealized electrostatic liquid - liquid contactor.
11.4 Electrostutic Phase Separation of Rich Wuter-in-OilEmulsions
219
(water-in-oil emulsion) rather than a homogeneous fluid, i.e., the corresponding solute has fine droplets of micrometer size rather than molecular solutes. The problem is how the fresh water effectively scavenges the fine droplets in the crude oil, i.e., how the dispersed fresh water makes contact with the droplets and unites with them. Reduction of the water content in the feed crude oil depends on effective mixing between the fresh water and feed oil phases, the rate of settling of enlarged drops, the continuous phase viscosity, the dispersed phase holdup, the filed strength, etc. Figure 11.13 shows a schematic diagram of an idealized electrostatic contactor for achieving these compromises, in which declining field strength in a large space can enhance drop growth and increasing field strength in a narrow space can produce drop shatter and electrostatic mixing of the liquid [11.15]. An Electro-Dynamic contactor, devised as a commercial plant using an idealized electrical treater and typical of the liquid - liquid extractor type is shown in Figure 11.13. The external appearance of a commercial plant is shown in Figure 11.14A, and the shape of the applied pulsed d.c. voltage between the electrodes and the corresponding behavior of the drops is shown in Figure 11.14B [11.16] (see also Chapter 10). The electric field is generated in each space by composite electrodes which are installed vertically at equal intervals, and the fresh water and the crude oil containing fine droplets flow counter-currently through the space. The throughput of this contactor can be ca. 182000kL/(m2-day) with the crude oil phase linear velocity 0 . 0 0 2 d s C11.161. The shape of the applied voltage between the electrodes and the corresponding mixingkoalescinghettling behavior of drops per cycle is also shown in the figure. The mixing phase shown is at 35 000 V and settling occurs at 16000 V. The coalescence phase is shown by the electric field responding to the voltage reduction. Electrostatic mixing in which the voltage is periodically modulated between the dispersion voltage and the coalescing voltage has been applied to the extraction of soluble salts from the crude oil. Performance in this contactor is evaluated by removal of salts and water from the crude oil. (Figure11.17 in chapter 10 shows the desalting performance of soluble salts from the crude oil to the fresh water phase.) Dehydrating performance is shown in Figure 11.15 [11.7] where the solid and dashed lines represent residual water in the crude oil for contactor 1 and contactor 2, respectively. For supplying 5~01% fresh water, the average effluent water content of contactors 1 and 2 was 0.06% and 0.11%, respectively. Dehydrating efficiencies were 98.8% for contactor 1 and 97.8% for contactor 2, where coalescence between droplets may be controlled by the formation of droplet chains as illustrated in Figures 11.4 and 11.11.
11.4
Electrostatic Phase Separation of Rich Water-in-Oil Emulsions in Liquid Membrane Separation
U.4.1 Membrane Recovery in Liquid Membrane Separation As an industrial application of a rich water-in-oil (W/O) emulsion, a novel emulsion liquid membrane technology was developed in 1968 by Li [11.18]. Its application to the extraction and concentration of very dilute solutes has received a great deal of attention in waste water treatment, hydrometallurgy, and fermentation etc. The process consists of: 1) an emulsification step; 2) a permeation step; 3) a settling step; and 4) a breakdown step of the emulsion. The flow sheet of the process is shown in Figure 11.16. In hydrometallurgy, for instance, a W/O emulsion is dispersed by stirring in a bulk water phase containing metal ions (step 2). Under certain conditions, these ions will
220
11 Applications of the Electric Fields to the Resolution of Wuier-in-Oil Emulsions
MIXING
VOLTS 45,000
40,000
1
35,000
-
30,000
-
25,000
-
20,000
-
I
-
COALESCING
SETTLING
(
0
0
DILUTION WATER
-
. _ ) _ _~~ _
15"
~
EMULSION
___________
FLOW
B)
TIME (SECONDS)
Figure 11.14. The Electro-Dynamic extractor. A) External appearance of commercial plant; B) Shape of applied voltage and corresponding
drop behavior.
11.4 Electrostatic Phase Separation of Rich Water-in-Oil Emulsions
1.0
0.8 -7
1
I
1
1
-c o n t a c t o r
1
--___ c o nt a c t or
2
20
1
2
-
I
30
40
221
50
60
t [day]
Figure ll.15. Dehydration performance of the Electro-Dynamic extractor. (Reproduced with permission from Solvent Extraction, [11.17]) W/O e m u l s i o n d r o p e m u l s i f i e d
0.1-0.3 mm
droplet
phase 3
raffinate phase 3 extract
-
I
1
A
e m u l s i f i c a t i o n permeation
settling
breaking step
step
step
o f emulsion
step
phase 1
Figure 11.16. Flow sheet of the emulsion liquid membrane process.
permeate through the oil phase of the emulsion drops into the inner water phase emulsified in the drops, where the oil phase contains extractants to extract the metal ions and surfactants to emulsify the inner water phase (step2). After permeation, the emulsion drops are separated from the bulk water phase (step 3) and have to be broken (step4). An efficient operation for breaking the emulsion is a key step in the entire process. In this step, breakdown of inner water phase (droplets) containing the metal ions enriched in the extraction step is necessary, as is full recovery of the oil phase containing surfactants and extractants without loss and deterioration, because they are used to remake the emulsion and the entire process is operated continuously. Since the pioneering work on electrically aided phase separation in liquid - liquid dispersons by Bailes and Larkai [11.13, 11.41, much useful information and equipment has been published [11.19-11.301. Continuous-flow electrostatic coalescers [11.19,
222
I1 Applications of the Electric Fields to the Resolution of Water-in-OilEmulsions DC g e n e r a t o r
. rome te r
demul s i f i e d water
Figure ll.17. The W/O emulsion coalescer with perforated clectrodes. (Reproduced with permission from Kagaku Kogaku Ronbunshu, [11.28])
11.21, 11.22, 11.24-11.291, with agitator [11.23], and with a centrifugal field [11.31]. The characteristics and performance of coalescers without agitator, and with an electrode and rotating body will be discussed here. Figures 11.17 shows a schematic diagram of a coalescer without agitators, used for continuous electrical resolution of a rich W/O emulsion [11.29]. Two bare, perforated plate electrodes are installed in the middle section of the column, parallel to one another; the upper electrode is connected to a high-voltage d.c. generator and the lower one is grounded. The feed emulsion is demulsified during flow between the electrodes: the demulsified clear water phase is removed through the lower perforated electrode to the column, and the emulsified oil phase through the upper perforated electrode to the column. The W/O emulsion used is a system of aqueous NaCl solution and kerosene containing Span 80 (Wako), and the demulsification performance is evaluated by the residual water content in the demulsified oil phase GW.Figure 11.18 shows the relation between the performance QW, and the apparent residence time t,, of the emulsion phase through the electric field, where an emulsion with initial water content Qtwi = 0.3 is fed continuously under applied voltages EP= 500 V and 1000V with L = 5 cm between the electrodes. For a long residence time, i.e., for a low superficial velocity of the feed emulsion, high performance is obtained irrespective of the applied voltage. If residence time is short, GWvalue becomes high and the performance is poor,
11.4 Electrostatic Phase Separation of Rich Water-in-Oil Emulsions
0.1 :
--
"05_ as
0.01 I --
\
I
I
I
I
a
I
I
I
d ~ ~ 2 [gml. . 5 6 , , = 0 . 3 [-I. L=5 [cml
4 '&\
key
EP [VI
A 0 A
a
0
500
rectangular column cylindrical column
1000
--
-8 \ .
0.005 --
\!.------
I
I
I
0-
:--:
0.002
--
-
1000
500
223
I
I
I
a I
obviously because the emulsion passes between the electrodes without being sufficiently demulsified. Deterioration of the surfactant is tested qualitatively by the relation between utilization frequency of the demulsified oil N , the corresponding @ ,,, and droplet size in each reemulsified liquid phase. An example of the results is illustrated in Figure 19 [11.29] up to N = 3, increases slightly and is almost constant for N = 4-7. This indicates that the demulsified oil phase can be reused repeatedly without appreciable deterioration in the efficacy of the surfactant in the oil phase. The figure also shows the current density i between the electrodes during demulsification, which is seen to decrease when N > 5 . The size distribution of water droplets in all reemulsified liquids was almost the same. Effects of electric fields, constant d.c., pulsed d.c., and a.c. voltages on the behavior and the resolution performance of the emulsion were investigated experimentally using the system mentioned above. As an example of the results obtained, Figure 20 shows the relation between the rate of demulsification and the electrical energy consumption, calculated as the product of applied voltage and conducting current [11.27]. The behavior of droplet coalescence is the same in the constant d.c. and pulsed d.c. fields, but the behavior in the a.c. field is different from those in the d.c. fields. Bailes and Watson have pointed out the following problem for phase separation of a rich W/O emulsion [11.31]. The water drops which have grown to a large size during emulsion breakdown fall to the bulk interface under the influence of gravity, but do not coalesce with it immediately. This is because the rate at which drops coalesce with the bulk interface is governed by the rate at which the continuous phase between the drop and the interface drains. As emulsion breakdown proceeds, a condensed layer of surfactant in the vicinity of the bulk interface is formed due to the discharge of surfactant from the drops. Consequently, with growth of the condensed layer the rate at
eW
224
11 Applications ofthe Electric Fields to the Resolution of Water-in-Oil Emulsions
0.02-
I
I
0.2
I
I
I
L1-~--A--A---&--
0.01 -
lO:.\
-
-
\ E
-~-0-o0--o-~lpa5 '
4
8
k\
-
0.005 -
Y
.-
-
a
EP= 1000 CVI
6 wr=O. 3 [-I
-
N
a
-
ds~2.5
Figure ll.19. Relation between demulsification performance and utilization frequency of demulsified oil. (Reproduced with permission
P O . 0077 tcm/sl
L=5 [cml
0.001
I
I
I
I
I
I
I
I
05
1
I
I
I
I
5
10
20
P [J/(ma.s)l
Figure 11.20. Relation between demulsification rate and electrical energy consumption. Aqueous solution droplets (CNa= 2wt%) in kerosene containing Span80 (Cs = 2wtY0).
which the enlarged drops coalesce with the bulk interface becomes gradually less than the rate at which the drops arrive at the interface. This leads to buildup of the drops at the interface and a decrease in breakdown rate of the emulsions. To overcome this problem, they proposed superimposing a centrifugal field on the electric field: i.e., the electric field causes water droplets to grow to a size sufficient for their removal by a centrifugal force which makes the drops coalesce effectively with the bulk interface.
11.4 Electrostatic Phase Separation of Rich Water-in-OilEmulsions
225
W/O e m u l s i o n
organic phase
Figure ll.21. The continuous spinning electrostatic coalescer.
They developed a novel continuous spinning electrostatic coalescer (CSEC) shown in Figure 11.21 [11.31]. The effects of applied voltage, pulsation frequency of the d.c. field, and rotation speed of the coalescer on demulsification performance were tested by using a W/O emulsion which was a mixture containing 1~01%Span80 and equal parts by volume of distilled water, light liquid paraffin, BPC, and Isopar M (kerosene). An example of the results obtained is shown in Figure 11.22, in which the best performance is obtained at 25 Hz pulsation frequency and the water content (&i = 0.33), of the feed emulsion is reduced to less than 0.02 at a rotation speed of 1500min-' and a feed emulsion phase of 1.25mWs.
' h
0.98.-0C
-a 2 L
VI
a 0
-_ - -
0.96 -
4
,a/---
0.94-
n = 1500 min-1
0.92 -
key
0
a
w 4
0
5
10
2 Hz
0
25Hz 50Hz
15
-
frequency
0
A
0.86
-13
20
25
nominal v o l t a g e [ k V I
Figure 11.22. Relation between aqueous resolution and applied voltage. Water droplets in a mixture of kerosene and liquid paraffin (BPC) containing Span80 (C, = lvol%). (Reproduced with permission from solvent Extraction in the Process Industries, [ 11.311)
226
I1 Applications of the Electric Fields to the Resolution of Water-in-OilEmulsions
11.5
Conclusions
Electrostatic technology in dehydrating and desalting processes has been successfully applied to many commercial plants in the oil industry, and no major problems are to be expected, even in the operation of large-scale plants. For electrical demulsification of rich W/O emulsions in liquid membrane process, however, there are still some important problems to be solved prior to industrial application, e.g., establishment of a rate equation for demulsification, and development of suitable technologies for recovery of the solutes, surfactants, and extractants without their physical deterioration.
11.6
Nomenclature
concentration of NaCl, wt% concentration of surfactant, wt% Sauter-mean diameter of droplet, m unit vector, electric force between droplet (a,) and droplet (al), N electric force acting on droplet (aJ) by droplet chain, N flow rate of demulsified water phase, m3/(m2s) conducting current density, A/m2 I-function, mean conducting current, A distance between electrodes, m total number of drop, rotation speed, min-' utilization frequency of demulsified oil, collision frequency between drops, electrical energy consumption, J/(m2s) dimensionless distance parameter, true charge, C distance, position in polar coordinate, m position vector, N distance between centers of two droplet, m distance between droplet (a,) and droplet (al),m distance between droplet (rs) and droplet (a,), mean distance separating each drop, m lapse time for which drop contacts with another drop or drop chain, s apparent residence time of the emulsion phase in the coalescer, s lapse time for which drop moves radius of another drop, s lapse time for which drop moves a given distance, s superficial velocity of emulsion phase in the coalescer, m/s coalescer volume, m' rate at which work is being done in the coalescer, J/s /$function, ratio of true charge of droplet to saturation charge of droplet, permittivity, x8.854.lo-'', F/m viscosity, Pa.s
11.7 References
227
polar angle, rad dipole moment, Nm 6 cos8 & volumetric fraction of aqueous phase in oil phase, qWiinitial volumetric fraction of aqueous phase in oil phase, 8
p
Subscripts c continuous cal. calculated d dispersed exp. experimental i i-th droplet j j-th droplet r r-component 8 8-component
ll.7 References [11.1] K. L. Lissant, Demulsification, Surfactant Science Series 13, New York and Basel, Marcel Dekker, Inc., 1983. [11.2] S . E. Taylor, Collids and Surfaces 29, 29-51 (1988). [11.3] Sekiyu Gijutsu Kyokai (ed.), Sekiyu Kogyo Benran, Chap. 10.2, pp. 568-591 (1983). [11.4] P. J. Bailes, S. K. L. Larkai, Pans. Inst. Chem. Eng. 60, 115-121 (1982). [11.5] C. A. R. Pearce, Brit. J. Appl. Phys., 5 , 136-143 (1954). [11.6] L. C. Waterman, Chem. Eng. Progress, 61, 51-57 (1965). [11.7] L. C. Waterman, J. D . Winslow, IEEE Convention Record, 9-22 (1966). [11.8] T. Ise, M. Yamaguchi, T. Katayama, Kagaku Kogaku Ronbunshu. 15, 1087-1094 (1989). [11.9] G . Zebel, Stuub, 23, 263-268 (1963). [11.10] M. Yamaguchi, unpublished data. [ll.ll] A. Y. H. Cho, J. Appl. Phys., 35,2561-2564 (1964). [11.12] S. E. Sadek, C. D. Hendricks, Znd. Eng. Chem. Fundum., 13, 139-142 (1974). [11.13] P. J. Bailes, S. K. L. Larkai, Pans. Inst. Chem. Eng., 59, 229-237 (1981). [11.14] Chem. Eng., 11, s31 (1993). [11.15] K. W. Warren, F. L. Prestridge, Apparatus for Application of Electrostatic Fields to Mixing and Separating Fluid, US Patent 4161439, 1979. [11.16] NATCO commercial materials of process and environmental systems. [11.17] K. W. Warren, J. J. Byeseda, Solvent Extraction 1990, B, T. Sekine (ed.), Elsevier, pp. 1417-1422 (1992). [11.18] N. N. Li, Membrane Separation Process, US Patent 3410794, 1968. [11.19] M. Goto, J. Irie, K. Kondo, F. Nakashio, J. Chem. Eng. Japan, 22,401-406 (1989). [11.20] T. Hano, T. Ohtake, T. Takagi, J. Chem. Eng. Japan, 21,345-351 (1988). [11.21] H. B. Hauertmann, W. Degener, K. Schugerl, Sep. Sci. Tech., 24, 253-273 (1989). [11.22] E. C. Hsu, N. N. Li, Sep. Sci. Tech., 20, 115-130 (1985). [11.23] H. Ino, N. Imaishi, M. Hozawa, K. Fujinawa, Kagaku Kogaku Ronbunshu, 9, 263-268 (1983). [11.24] T. Kataoka, T. Nishiki, Kugaku Kogaku Ronbunshu, 12, 16-22 (1986). [11.25] A. Kriechbaumer, R. Marr, ACS Sympo. Series, 381-398 (1985). [11.26] Y. Mori, W. Eguchi, Proc. World Cong. Chem., 3, 235-238 (1986). [11.27] M. Yamaguchi, K. Ito, Solvent Extraction in the Process Industries, D. H. Logsdail, M. J. Slater (eds), Elsevier Applied Science, 2 pp. 809-816 (1993). [11.28] M. Yamaguchi, A. Kobayashi, T Katayama, Kaguku Kugaku Robunshu, 11, 599-602, (1985). Continuous Demulsification Method and Their Equipments, Japan Patent 16 66727, 1992.
228
I1
Applicutions of the Electric Fields to the Resolution of Water-in-Oil Emulsions
[11.29] M. Yamaguchi, A. Kobayashi, K. Ohbori, T.Katayama, Kaguku Koguku Robunshu, 11, 729-734 (1985). [11.30] Z . Yan, S. Li, Y. Yu, X. Zheng, Desalination, 62, 323-328 (1987). 111.311 P. J. Bailes, M. Watson, Solvent Extruction in the Process Industries, D. H. Logsdail, M. J. Slater (eds). Elsevier Applied Science, 1, pp. 126-133 (1993).
Part 3 Applications of Electric Fields for Concentration, Immunoassay, and Molecular Orientation
This Page Intentionally Left Blank
12 Dynamic Electroconcentration Processes in Analytical Chemistry Takao Tsuda
Concentration in analytical chemistry is important for the detection of very dilutes solute. The role of electric fields in concentration processes has a unique character. The solute can be concentrated by simple and selective mechanisms based on the physical nature of the solute and matrix (e.g., oxidation - reduction potential or electrophoretic mobility) or by means of flow and ion-exchange membranes. By using electroconcentration techniques it is possible to vary the composition of the concentrate by changing the conditions under which electrochemical processes proceed. The use of flow makes it possible to concentrate the solute with a high ratio or high selectivity from dilute sample solutions of a relatively small volume. The techniques do not in general require large amounts of reagents and are accessible to practically any laboratory. In this chapter we describe several methods for concentration andor preconcentration by electric fields. All these methods include dynamic processes.
l2.1
Collection of Metal Ions from Acid Solutions by Hydrodynamic Recycling
A solution containing metal ions can be recycled by a pump for continous electrodeposition onto a carbon electrode. An electrolysis cell for this purpose is shown in Figure 12.1.The solution is passed continuously through the carbon electrode, in which there is a hole of 2.8 mm inner diameter and 9 mm long, via a Teflon pump (20-30 mL/ min). As an electric voltage is applied between the carbon electrode and a platinum
Figure 12.1. Dynamic recycling electrolysis cell [12.11. 1)Teflon reservoir (40mL); 2) Electrolysis cell, graphite tube cathode, 2.8 mm inner diameter, 9 mm long, 5 mm outer diameter; 3) Housing for cathode and anode; 4) Pt/Ir anode; 5) Teflon pump; 6) Voltage supply; 7) Teflon tubing; 8) Holder at end of anode; 9) Cathode contact.
232
12 Concentration Processes in Analytical Chemistry
Table 12.1. Yields of metal ions under various experimental conditions [12.1]* Element
Electrolyte
Period of Operation, h
Fe (100 ng)
NH4F NH4F (NH4)2S04 (NH4)2S04 NH4NO3 NH4F NH4F (NH4)2S04 (NH4)2S04 NH4N0, NHdN03 NH4F NH4F (NH4hS04 (NHdzS04 NH,NOq NH4NO3 NH4F NH4F
0.3 1.3 0.3 7 22 0.3 22 0.3 5 0.3
c o (6 ng)
Zn (150 ng)
Bi (0.5 ng)
8 0.3 3.3 0.3 2 0.3 5.3 0.3 2
Yield, % 45.7 2 98
8.7 3 98
60 12.8 3 98 25.8 3 98 20.4 2 98 31.4 3 98 38.2 3 98 24.6 98 29.8 3 98
* Current, 400mA; applied voltage; Fe5.5V; Co5.7V; Zn4.9 V, Bi 5.0V, solution pH 5.0; electrolyte volume 40 mL; electrolyte concentration 5%. wire positioned at the center of the inner hole, metal ions in the electrolyte are deposited continuously [12.l]. The percentage collection-(yield) of Fe, Co, and Zn from solutions of different pH are shown in Figure 12.2. The optimum pH ranges for Fe, Zn, and Co are 5-8,5-8 and 5-6, respectively [12.1]. The yield of ''Co2+, measured by a gamma-spectrometry, increases for longer operational periods (Fig. 12.3). The relationship between yield and operational period is independent the electrolyte composition, i.e., the yields
PH
Figure 12.2. Relation of pH of electrolyte solution and yield of metal ions [ 12.11. Current, 400 mA; applied voltage, Fe 5.0V, Co 5.7V, Zn 4.9 V; electrolyte solution used 40mL, amount of metal added to the solution: Fe 100ng, Co6ng, Zn 150ng; period of operation, 20min; the electrolyte solution, 5% HF and NH4E
12.2 Electrodialysis for Desalting or Concentration of Metal Ions
233
n @
%
I
W
a I
Q) . I
b 20
0
2
0
Operation
_____, 20 period (h)
Figure 12.3. Relation of yield of 6 ng %o in 40 mL solution to operational period [12.1]. pH 5; current, 400 mA; applied voltage,5.7V.
from solutions of 5% NH4F, 10% NH4F, and 10% NH4F containing 200mg Be as BeS04 all follow the relationship shown in Figure 12.3. Table 12.1 summarizes yield under different experimental conditions [12.1]. Volland et al. [12.1] conducted their experiments in strongly acidic media. In such media, most metal ions are stable, and their movement depends only on their electrophoretic mobility. Volland et al. demonstrated the selective electric coilection of metal ions when a large amount of Be is present in the solution. The graphite cathode can be used directly for a sensitive determination of the deposited elements, e.g., by flameless atomic adsorption [12.1].
12.2
Electrodialysis for Desalting or Concentration of Metal Ions by Multiple Ion-Exchange Chambers
Sodium chloride is common in natural and clinical samples. It is sometimes necessary to exclude salt because of interference in the determination of the minor components in a sample. For desalting, ion-exchange membranes are useful (Figure 12.4, 12.5). Cations and Anions in the sample solution are forced to migrate through cation- and anion-exchange membranes by an applied voltage, and both are kept in the solution chamber collection; Once a cation has passed through a cation membrane and reaches the collection chamber (2 in Fig. 12.5), it cannot migrate to the next chamber, because it cannot pass through the anion membrane, by the reverse process (Fig. 12.4). Therefore, these ions are led to the collection reservoir. The membrane also acts as a selective filter; certain molecules of relatively high molecular mass cannot pass through it. These characteristics are useful for desalting or excluding unfavorable inorganic ions from the sample solution. A typical example is as follows. The number of chambers for sample and taking-up solutions are 20 and 19, respectively. Each chamber is separated by both anion- and cation-exchange membranes (total effective membrane area 50 cm’). The electrolyte
234
12 Concentration Processes in Analytical Chemistry
Figure 12.4. Ion migration through the pores in the cation membrane. As the anion-exchange membrane has positive charges around its pores, anions are able to pass through, but cations cannot. (Reproduced with permission from Asahikasei KWYO)
solution used in the electrode chamber is 50 g/L Na2S04aqueous solution (pH 7). The sample solution was 1000ml aqueous solution containing 1N NaCl, 10-8 g/L alcohols and/or glycols. The collecting solution was 0.1 NNaC1. Both the sample solution and the solution for collecting salts were circulated by pumps. The aqueous solution containing a high amount of NaCl and a minor amount of several kinds of ethylene glycols was desalted for 50min under an applied voltage of 0.4-0.55 V for a pair of chambers at a constant current of 3 A/dm2 (Fig. 12.6). NaCl in the sample solution is completely excluded by this process and the most of ethylene glycols remain in the sample solution (only a few are observed at chromatogram C , Fig. 12.6). Therefore membranes can present ethylene glycols from passing through, especially polyethylene glycols. The Asahikasei Kogyo sells this instrument under the name Acilyzer. There are several classes of Acilyzer, depending on the volume of sample solution: 50-100 pL (type GO), 2-20mL (typeGl), industrial use (type G3), etc. The apparatus is also used for the recovery of metal ions from dilute sample solutions. When, for example, we use 10 L of 0.01 N sample solution and 0.15 L of collecting the concentration of the collecting solution for the metal ions is enhanced 40 times to 0.4N with. During operation of the apparatus the sample solution is not diluted, because the ions in the sample solution always migrate, accompanied by water molecules and together then pass through the membrane. Therefore, the volume of the sample solution decreases during operation. This can give selective solute concentration in the sample solution during desalting process. The pH of the sample solution is one of the most important factors for desalting (Fig. 12.7) A solution containing phenylalanine and 200mM of phosphate buffer is desalted with anion-exchange membranes (AC-110, Asahikasei Kogyo). The desalting period for H,PO?-, is much shorter at p H 3 than at pH7 or 9, suggesting that its through the membranes is faster at low pH. The present method is applicable to the following cases: 1) Desalting from solutions including oligosaccarides, amino acids, peptides, nucleotides, and proteins 2) Recovering of heavy metal ions, e.g., Cu2* 3) Collection of alkali from dilute sample solutions, as its concentration is possible 4) Counter-ions exchange, e.g. from sodium to potassium salts
12.2 Electrodialysis for Desalting or Concentration of Metal Ions -I+
424
235
236
12 Concentration Processes in Analytical Chemistry
3EGthaCI
c
A
PECXi03
f EC: 1000
\ 502-
I
I
10
15 (min)
Figure 12.6. Chromatograms of sample solutions obtained before (A) and after (B) desalting, and chromatograms of the enriched solution after desalting (C). The aqueous sample solution before desalting was 1 N NaCI, 10gL of mono-, di- and triethyleneglycols, and 10 g/L of polyethylene glycols molecular mass of 1000-3000. Desaltings conditions: 25 "C, 3 A/dm2; 50min. Analysis by liquid chromatography: Asahipak GS-220 (7.6min X 50cm); mobile phase, 0.1 N NaCl (pH 7) or distilled water; flow rate, 1.0mUmin; temperature, 30 or 25 "C; detector, R1. (Reproduced with permission from Asahikasei Kogyo)
12.3
Counter-Current Electroconcentration
Pressurized flow and counter-migration, due to electrophoretic mobility of solute, can be used for sample concentration [12.3], [12.4] (Fig. 12.8).
12.3.1 Principles The velocity of the pressurized flow and migration due to electrophoretic mobility can be controlled by a pump (4 in Fig. 12.9) and the applied voltage. These two flows are usually in opposite directions. The sample solution in chamber A is aspirated into a n open tube. The solute, which is assumed to have a negative charge, is also aspirated into the open tube. With no applied voltage at either end of the tube, the solute zone front adopts the profile suggested by Taylor [12.5]. Each solute has a local velocity, v ~ ~where ~ ~ n ,represents ~ , the cross-sectional direction (perpendicular to the column
12.3 Counter-Current Electroconcentration n
E
E
W
237
200
I\ \\
t\
cn
* m
I
a
Ccl
0
C 0 . I
Y
rn h C a4
u
C 0
u "0
120
240
Period of desalting
360
(min)
Figure l2.7. Dependence o f desalting time on pH. The sample solution was phenylalanine and 200 mM of phosphate buffer. The recovery of phenylalanine at p H 3 , 7 , and 9 are 82,91, and 91%, respectively, after desalting. Phosphate buffer concentrations at pH 3, 7, and 9 are 0.5, 98, and 185 mM, respectively. (Reproduced with permission from Asahikasei Kogyo)
Figure 12.8. Principle counter-current electroconcentration [12.4]. The direction of pressurized flow is toward the negative electrode.
+
axis z ) . Most of the local velocity is in the region of vprra 26, where vprcaand 6 are the mean pressurized flow velocity and its standard deviation. With applied voltage along the tube, the negatively charged solute moves due to its electrophoretic mobility vmObtoward the positive electrode. We assume a plug flow profile of the zone front due to mobility. When the absolute velocity of the solute due to electrophoretic mobility is larger than the absolute value of the flow velocity due to pressurized flow, the solute will be concentrated in chamber A (Fig. 12.9), because the velocity of migration due to elec-
238
12 Concentrutinn Processes in Analytical Chemistry
Figure 12.9. Schematic diagram of counter-current electroconcentration [12.3]. Chambers A and B are polyethylene vials; I) n b e ; 2), 3) Platinum wire electrodes; 4) Pump for aspiration of solution.
+
B
C
A
.
L
Figure 12.10. Concentration of 2,6naphthalenedisulfonic acid, 47-fold [12.3]. Sample and final volumes were 10 and 0.2 mL, respectively. Analysis by capillary zonc electrophoresis. Capillary column, 100 pm inner diameter, 30cm long; applied voltage, 11kV, detection, UV (254 nm); medium, 8 mM phosphate buffer (pH8) containing 0.5% ethylene glycol.
trophoretic mobility is fast enough to emerge from the tube after the solute has been aspirated into the tube. The flow profile of pressurized flow is parabolic, and the profile of migration of solute due to electrophoretic mobility is assumed to be plug type. The flow velocity due to mobility vmob,depends on the potential gradient. The apparent local flow velocity of the solute vaPpis given by: vpres,x = vpres
+Avx
(1)
12.3 Counter- Current Electroconcentration
239
The value of Avx is given by v ~ ~ ~ and ~ depends , ~ - von ~the~ mean ~ ~ velocity of the pressurized flow, diffusion coefficient, and tube radius [12.5]. The maximum and minimum apparent flow velocities of a solute are as follows: Vapp,max = v p r e s Vapp,min
= vpres
+ 2s-
+ Zs
vmob
(4)
- vmob
(5)
If v , ~ has ~ a, negative ~ ~ ~value, most of the solute can be drawn back into the reservoir of chamber A, where the solute can be drawn back into the reservoir of chamber A, where the solute is concentrated. Therefore, if:
most of the solute is forced back to the chamber containing the positive electrode. When vpreris too rapid and the contribution of the diffusion coefficient to the zone front profile of a solute is minor, vpres,xat the central position of the tube is twice the mean velocity of pressurized flow. Equation (6) becomes:
For concentration, the solute must have sufficient electrophoretic mobility against the pressurized flow. When the solute has enough mobility and Equation (6) or (7) applies, most of the solute can be concentrated in chamber A. As a consequence of Equation (7) Vmob must be at least twice vPres.
12.3.2
Apparatus
A schematic diagram of counter-current electroconcentration is shown in (Fig. 12.9). The apparatus consists of two chambers (A and B), one or two small glass tubes which connecting them, a pump for generating a constant pressurized suction flow in a small glass tube (1.0-1.7mm inner diameter, 10-17mm long), an electrode in each chamber, and a high-voltage d.c. power supply. The electrode for chamber B is inserted in a small hole at the side of the polyethylene vial and sealed with epoxy glue. The small tubes are connected by two vials, also with epoxy glue. The solution containing solute for concentration is kept in chamber A during operation, it is added using a drop-wise syringe method if necessary. The operational procedure is as follows. The system is filled with the sample solution containing the solute for concentration in 2 mM ammonium acetate, 20 mM arginine, or 20 mM asparagine buffer. A voltage (300-1100 V) is applied under suction pressurized flow (100-400 pL/min). The pressurized flow is directed from chamber A to chamber B, and the flow of the solute due to electrophoretic mobility is toward chamber A. The concentrate remaining in chamber A (finalsolution) is subjected to analysis [12.3], [12.4], [12.6].
240
12
Concentration Processes in Analytical Chemistry
12.3.3 Examples The predicted enhancement V(ratio): V(used)/V(final), where V(used) is the volume of the solution used for concentration and V(fina1) is the volume of the solution in chamber A after the run. When V(used) = 10 mL, V(fina1) = 1.0 or 0.2mL means that their predicted enhancements are 10- and 50-folds, respectively. The concentration enhancement E = (concentration of solute in the final solution)/ (concentration of solute in the original solution). The value of E is usually calculated from the peak areas of solutes in the electropherogram. In Figure 12.10 the predicted enhancement for 2,6-naphthalenedisulfonic acid is 50, and the concentration enhancement 47. The mobility of 2,6-naphthalenedisulfonic acid is estimated as 7.6 X 104cm2V-'s-' from its elution time (obtained by capillary zone electrophoresis). The applied voltage is 600V along the tube (1.7 mm inner diameter, 17mm long), Vmoh for this solute is -18.2cm/s, and vpresis set at 5.5cmlmin to satisfy Equation (6). Therefore, vaPpfor the solute is -12.7 cm/s. A negative linear flow velocity means that the flow is from reservoir B to A in Figure 12.9 [12.3]. Herring DNA lo-'& is concentrated 9.6 times using two tubes (1.5 mm inner diarneter, 15mm long) when applied voltage, vpres,and V(ratio) are 600V, 7.6cm/min, and 10, respectively (Fig. 12.11). As the mobility of DNA is estimated to be 9.8 x ~ ~ ~ c m * from ~ - ' its s ~elution ' time in capillary zone electrophoresis, Vmob and vaPp are 23.5 and -15.9cds 112.31.
a
H lmln
A
B
a
Figure 12.11. Concentration of herring DNA [12.3]. A and B are original and concentrated solutions, respectively.
12.4 Electroosmosis and Electrophoretic Mobility
l2.3.4
241
Operational Stability
To obtain a constant pressurized flow in the tube, the suction pump should be operated under very stable conditions. As the inside of the tube has the highest electrical resistance in the apparatus, most of the Joule heat is generated in the tube, owing to the large potential gradient (300-800 Vkm). The heat must be dissipated from the outer surface of the tube. For this purpose, part of the tube is cooled by immersion in a water bath, with current and applied voltage 1mA and 600 V. With ampholite buffer and electric current less than 0.4mA, we do not encounter bubble formation in the tubes, even though the apparatus is operated without cooling. If a bubble is formed in the tube, the electric current becomes unstable or clases; it is essential to avoid bubble formation. There is some possibility that solute and buffer will be chemically changed due to electron transfer andlor reaction at the electrodes. Water is electrolyzed to hydrogen and oxygen at the negative and positive electrodes, respectively. Therefore, the area around electrodes is highly oxidative or reductive. If we concentrate 0.1mM 2,6naphthalenedisulfonic acid (aqueous solution) 10-fold to obtain a 1mM solution, we note the formation of byproducts. However, byproducts are not observed when we concentrate the 0.01 mM original solution 10-fold (final solution 0.1 mM 2,6naphthalenedisulfonic acid), i.e., we can avoid the formation of byproducts by keeping the concentration of the solute low. The pH of the medium varies from the beginning to the end of the operation. The degree of variation of pH depends on the nature of the sample to be concentrated. The use of a buffer gives less pH variation and more stable electric current. It is better to use a buffer composed of a weak base and a weak acid. For this purpose, ampholite buffer is best [12.6]. The current increases at the end of the operation, owing to the concentration enhancement of the solute in reservoir A. It is better to keep the pH variation within 1 unit during operation [12.4], [12.6]. Counter-current electroconcentration is based on the simple principle of countercurrent migration and the operation is also easy to implement.
12.4
Electroosmosis and Electrophoretic Mobility for Counter-Current Electroconcentration
Counter-current electroconcentration has been proposed by Tsuda et al. [12.3] using pressurized flow. However, the use of electroosmosis is also possible. The electroosmosis is generally generated inside porous filters or capillary tubes under applied voltage. In concentration processes under applied voltage, the medium is forced to move by electroosmosis, and the ions themselves migrate due to their electrophoretic mobilities. If we can handle both flow velocities, we can achieve counter-current electroconcentration by electroosmosis. Electroconcentration has the unique feature that we can take advantage of the flow profile of electroosmosis (nearly plug-type [12.7], see Section 2.5 (Chap. 2)). Therefore, the local flow velocity in the small channels which exist in capillary tubes, frits, or membranes, is nearly constant at any point on their cross-section, as is the flow velocity due to electrophoretic mobility. Therefore we can concentrate ions if the flow velocity of the solute due to electrophoretic mobility is larger than the electroosmotic flow, if the flows are in opposite directions.
242
12 Concentration Processes in Analytical Chemistry
Figure 12.12. Apparatus for
Apparatus for couii te rcurren t e 1 ec t roconcen t ra t I on
counter-current electroconcentration by electroosmosis (12.61. l), 2) Reservoirs 1ml polyethylene vials; 3) Frit; 4), 5) Electrodes (platinum wire).
The apparatus used for countercurrent electroconcentration by electroosmosis is shown in Figure 12.12A glass frit (class G3) or porous silica frit consisting of a mesoporous silica skeleton with ca. 1pm through-pores is used. When voltage is applied along the frit, electroosmosis is generated. The flow velocity is fast enough for electroconcentration of a solute. As the volume of the original solution in the reservoir is decreased by electroosmosis, the original sample solution is repeatedly added. The total sample solution used was 15 mL. The concentration of the small volume of final residue in reservoir 1 is 237 times greater than that in the original solution (Fig. 12.13). For maintaining the pH of the reservoir, amino acid is used as ampholite buffer [12.6]. The pH in the experiment of Figure 12.13 varied in the final stage of the concentration, owing to the accumulation of solute in the reservoir. The pH of the medium is very important because the flow rate of electroosmosis depends on [12.8]. About 0.16mL/ min of the solution is carried by electroosmosis at pH 11 when the potential gradient along the frit is ca. 1.7 kV/cm t12.61. This counter-current electroconcentration method is a very effective for the concentration of sample solutions of milliliter or submilliliter volumes.
12.5
Electroosmosis for Sample Transfer
Electroosmosis is also generated inside the channels of the partly fused particles of ion-exchange resins under applied voltage. We can use this electroosmotic flow to force the medium toward the electrode at same time as the ions themselves are forced toward the electrode by their electrophoretic mobilities. The electrode is positioned behind the thin layer of ion-exchange particles. This technique, has the unique feature of avoiding electrochemical reactions at or near the electrode; the ions to be collected can be trapped by the ion-exchange resin before reaching the electrode. Electroosmosis here is used purely as the driving force of the solute. The cell used is shown in Figure 12.14 [12.9], [12.10]. Positively charged ions are carried by electroosmosis into the ion-exchange bed (H-form), followed by the charged ions to be trapped by the ion-exchange resin. Positively charged porous membranes of thickness 2.5-3.0 mm were prepared by fusing a mixture (3 : 2) of chromatographic aluminum oxide and Teflon powder at 380 "C. At least 99% of trace elements (Co, Cu,
12.5 Electroosmosis for Sample Transfer
243
\
c
A U
lmin
Figure 1 2 3 . Counter-current electroconcentration of naphthalene-1-3, 6-trisulfonic acid trisodium salt [12.6]. Filter, G3 glass filter, 3 mm thick, 5 mm diameter; original solution, 2.3 pM; concentration period, 94 min; medium, 20 mM L-arginine aqueous solution; applied voltage, 520 V, pH of original solution, after concentration in reservoir 1, and after concentration in 2 were 11,7, and > 11,respectively. Chromatograms A , B and C obtained by size exclusion chromatography correspond to the solution before concentration, standard sample solution of 100 times the concentration of the original sample solution, and the final concentrate, respectively.
Fe, and Ni) were concentrated in the ion-exchanger layer. After concentration, the ionexchange resin was washed, dried, mixed with charcoal, and analyzed by atomic emission. This technique has been used for determining Co, Cu, Fe, and Ni and other elements in corrosion products of constructional materials. The concentration coefficient was ca lo3. The authors did not discuss the direction of electroosmotic flow. In this arrangement of the cell, the flow velocity should be from cathode chamber to anode chamber. The use of an ion-exchange resin for collecting cations is an attractive idea. Both solution and ions are forced to move to the anode (by electroosmotic flow and electrophoretic mobility, respectively) and are then trapped by the ion-exchange resin before reaching the anode. This geometry can avoid deposition of metal and undesirable electrochemical reactions at the electrodes. The electroosmotic flow can be replaced by pressurized flow (electroosmosis is used solely to transfer the medium to the anode).
3 2 1
13
6
12
11
Figure 12.14. Cell for electroconcentration with electroosmotic flow [ 12.91. 1) Anode chamber; 2) Acrylic plastic beaker; 3) Cathode chamber; 4) Anode; 5 ) Inner cylinder; 6) Cathode; 7) PVC hose for supplying solution continuously; 8) Mariotte vessel; 9) Outlet pipe; 10 Measuring cylinder; 11 Ion-exchange resin; 12) membrane; 13) Outlet cylinder.
244
I2 Concentration Processes in Analytical Chemistry
Figure 12.15. Apparatus for electrodepostion and release in a silver powder column [12.11]. A) Reservoir; B) column, 1cm inner diameter, 30 cm long; C ) Mercury pool; D Dropping mercury electrode; E) Voltage supplier; F) Silver powder; G) Glass wool; H) Silver electrode; I) Rubber tube for connecting column segments; P) Polarograph; S ) Injector.
12.6
Electrodeposition of Metal Ions and their Selective Release
Fujinaga et al. [12.11] devised a novel electrochromatographic column composed of several segments, each with electrodes at either end. These segments are connected to make one column and covered with rubber tubes. A voltage is applied along several segments of the column, in which silver particles (10-16 mesh) are packed (Fig. 12.15). The sample is introduced through a tube in the side of the column. The injected sample flows through the channels between the silver particles, is electrically deposited, and then released by lowering the voltage (Fig. 12.16). A mixture of cadmium, lead, and copper ions is injected under applied voltage 1.8V. Cd2+is eluted under these conditions. The applied voltage was then lowered to l.OV, and PbZ+ eluted. With the applied voltage set at zero, Cuz+is obtained [12.11]. Fujinaga et al. did not report the use of different local potential gradients, but it would be possible devise a new type of electrochromatographic preconcentration mode along these lines. The present method is very effective for the trapping and concentrating of very dilute metal ions in large volumes of solution. In Chapter 4, Nagaoka describes a concentration method with a radially applied voltage. 30-
12.7 Electrochrornatographic Solid-Phase Extraction
DOWNFLOW-
4
c - UPFLOW
- l P 1.3 cm
12.7
245
Figure 12.17. Electrochromatographic solid-phase extraction [12.12]. Retention of drug in serum: voltage off1 upflow direction with pressurized flow. Elution: voltage onldownflow toward negative electrode. Octadecyl-silanemodified silica gel (100mg, 40pm particle size) packed between two 20 pm porous polyethylene frits in a solid-phase extraction cartridge.
Electrochromatographic Solid-Phase Extraction
Solid-phase extraction is generally performed by pressurized flow, and very suitable for extracting a sample from a complex matrix. If we use both electrophoretic mobility and electroosmotic flow for extraction, we can achieve selective and effective sample collection, especially in soft gels. The set-up for electrochromatographic solid-phase extraction is shown in Figure 12.17 [12.12]. Octadecylsilane-modified supports, 100 mg 40 pm particle diameter, in a Supelclean LC-18 l.OmL SPE tube (Supelco) are used as solid-phase adsorbent, packed between two porous polyethylene frits, 20 pm pore diameter. The cartridge is a positioned horizontally between the platinum electrodes, and the negative electrode is sharpended to a point, and attached to the front of the frit. Effective electrochromatographic solid-phase extraction and preconcentration has been demonstrated for the determination of cimetidine in serum in the concentration range 0.2-11 pg/mL [ 141. Cimetidine, N-cyano-N-methyl-N’-[2(5-methyl-lH-imidazol4-yl)methylthioethyl]guanidine, is a histamine H2 receptor antagonist which is used to reduce acid secretion in the treatment of gastrointestinal ulcers [12.13].
12.7.1
Operational Procedure
The cartridge was activated with methanol (1 mL) by syringe suction (downflow, Figure 12.17). The remaining methanol was removed with 1.0 mL water, while the cartridge was conditioned to pH 7.7 with 1.0mL buffer - 10mM tris[hydroxymethyl]aminomethane (tris) - 3.2mM acetic acid. A 0.5mL sample (cimetidine in serum, ca. 1.8 pd0.5 mL) was dispensed by means of a 1.OmL syringe, from the bottom of the cartridge in an upward direction, followed by washing with l.OmL water in the same direction. Finally, water in the cartridge was compensated for in the suction mode with 160 mL of the eluting solvent - tetrahydrofuran buffer, 5 mM tris - 1.7mM acetic acid (p 7.7) (50 :50). An electric field of 150 V was applied as the driving force. Cimetidine, pK, 6.8, may be dissociated in the eluting solvent and has a positive charge (its pK, in the mixture of organic solvent and acid becomes larger than the apparent p H of the eluting solvent as the pH of the mixture is suppressed to the neutral region by the organic solvent. Therefore cimetidine migrates to the negative electrode by its electrophoretic mobility, and is also transported by electroosmotic flow. The eluting solvent around the negative electrode is sucked into 20 pL microcapillary pipets. The electropherogram of the first collection is shown in Figure 12.18A. For comparison, the electropherogram of the collection under pressurized flow is shown in Figure 12.18B. The latter fraction is obtained only by very careful operation, and it is
4
0
5
c----3
TIMWmln
10
i
15
VOLTAGE OFF
-
0
4
c STANDARD
TIMUmln
10
(5
PRECONCEWRATION
wmom
0
c--
D
10
15
BLANK SERUM
ure 12.18. Electropherograms of the first collection of cimetidine eluted from the cartridge 212.121. Sample obtained by electrochromatographic concentration after electrical elution (3.7 mg/mL of cimetidine in serum). 1) cimetidine; 2) ranitie added as an internal standard; P1) unknown serum background component. B) Sample obtained by pressurized flow, with no applied-voltage he last procedure 20 pL of the eluting solvent is sucked with a syringe and collected by a pipet. C) Standard solution without preconcentration. concentration of cimetidine was 11.4 &mL in THF of buffer. D) Blank serum extract after electrochromatographic preconcentration. Elecherograms were obtained by capillary electrophoresis.
1
1
VOLTAGE ON I IZOdI
B
1
12.8 Use of Electrochemical Reactions for Decomposition of Metal Complexes
247
very difficult to obtain reproducible results. The peak height of P1 in A is less than the value in B, and the minor peaks in A are few compared with B. Although cirnetidine can be concentrated by both procedures, electrochromatographic preconcentration is more selective and reproducible compared with pressurized flow [12.12]. Experimental conditions for the electrochromatographic preconcentration of cimetidine include small variations of the pH of sample and eluting solutions. The method is rather new; we hope to learn how to control it better for effective preconcentration.
12.8
Use of Electrochemical Reactions for Decomposition of Metal Complexes
A flow-through cell [12.14] has been developed for the collection of metal ions in water, in which the metal ions generally form complexes with humic and fluvic acids [12.15]. To eliminate the effect of organic compounds, wet and dry ashing and UV irradiation are used, but these methods are lengthy and laborious . Use of applied voltage for this purpose is very effective and the equipment is simple (Fig. 12.19) [12.14]. The electrodes are protected by an ion-exchange resin to avoid direct contact with metal ions, and to reduce the possibility of electrodeposition of metal on the electrodes. As a result of electrochemical decomposition of water at the electrodes, the solution in the anodic compartment falls to pH 1-2, while in the cathodic compartment it rises to p H 11-12. To increase the conductivity of the solution, and to produce the conditions required to generate an oxidizing medium in the anodic compartment, 0.2-0.4gL KCl is added. Chlorine generated at the anode diffuses into the anodic compartment, and both chlorine (or chloride) and acidic medium break down the humic and fulvic heavy-metal complex. The bare metal ions are kept in the anodic compartment, because the anionexchange resin separating the two compartments prevents the transfer of metal ions to the cathodic compartment. The metal ions obtained then analyzed [12.14].
1 3
1
5
4 2
Figure 12.19. Electrochemical flow-through cell for treating natural water [12.14]. 1) Graphite anode; 2) Graphite cathode; 3 ) Cation-exchange membrane; 4) Anion-exchange membrane; 5) Anion-exchange membrane.
248
12 Concentration Processes in Analytical Chemistry
12.9
Conclusions
Electrochemical deposition is a well-known classical method. We have developed several different methods which use dynamic processes for collecting a minor component from very dilute solutions. These dynamic processes include recycling of the solution, continuous electrodialysis, counter-currents in a sample and a medium, use of electroosmosis for transferring the medium or samples, selective deposition on ionexchange resins, etc. The method of in-column focusing described in Chapter 6 is also very effective [12.16]-[12.18]. As concentration enhancement is very important for analytical procedures, dynamic electroconcentration promises to become one of the most important as pretreatment methods in the near future.
l2.10 References [12.1] G. Volland, P. Tschopel, G. Tolg, Anal. Chimicu Actu, 90, 15-23 (1977). [ 12.21 T. Matsui, S. Kamatani, Y. Noguchi, Ekitai Kuromatogurafu kenkyukai (Symposium for Liquid Chromatography), p. 65-68, Kyoto, Jan. 28-29, 1986; Technical booklet of Acylizer, Asahikasei Kogyo Co. (Kawasaki, Japan), Aug. 1993. [12.3] A. Hori et al., Anal. Chem., 65, 2882-2886 (1993). [12.4] H. Okada, T. Tsuda, 1993, unpublished work. [12.5] G. Taylor, Proc. R . SOC.,219A, 186-203 (1953). [12.6] H. Tanaka and T. Tsuda, 1994, unpublished work. [12.7] T.Tsuda et al., J . Chromatogr., 632 (1993) 201-207. (12.81 T. Tsuda, “Control of electroosmotic flow in capillary electrophoresis”, Chap. 22 Handbook of capillary electrophoresis J. P. Lander ed., CRC Boca Raton, 1993. [ 12.91 Yu.A. Zolotov, N. M. Kuz’min, “Preconcentration of trace Elements”, Comprehensive Analytical chemistry X X Y Elsevier, Amsterdam, 1990. [12.10] L. N. Moskvin et al. Zh. Anal. Khim., 31,2396 (1976). [12.11] T. Fujinaga,T. Nagai, S. Okazaki, C. Takagi, Nippon Kagaku Zusshi, 84,941-942 (1963). [12.12] H. Soini, T. Tsuda, M. V. Novotny, J. Chromatogr., 559, 547-558 (1991). [12.13] R. N . Brodgen, R. C. Heel, T. M. Speight, G. S. Avery, Drugs, 15, 93 (1978). [12.14] L. D. Svintsova, A. A. Kalin, T. B. Rubinskaya, N. M. Mordvinova, J . Anal. Chem. USSR,46 (No. 1 Part2), 119-122 (1991). [12.15] T. M. Florence, G. E. Batley, CRC. Cr. Anal. Chem., 9, ( 3 ) , 219, 285 (1980). [12.16] T. Tsuda, Anal. Chem., 60, 1677-1680 (1988). [12.17] T. Tsuda, Y. Muramatsu, J. Chromatogr., 515, 645-652 (1990. 112.181 P. H. O’Farrell, Science, 227, 1586-1589 (1985).
13 Pulse Immunoassay and Pulse Electrovoltage for Cell Manipulation and Analytical Coagulation Processes Eiichi Tamiya
l3.1
Introduction
Immunoassay is based on the specific binding reaction of an antigen and an antibody. Various antigens, including proteins, peptides, drugs, and microorganisms are determined by immunoassay. Immunoreaction results in the formation of antigen - antibody adducts; the amount formed can be measured by visual inspection, turbidimetry, or weighing after centrifugation. When the amount of adduct is too small to be detected by these methods, radioisotopes, enzymes, and fluorescence dyes are used for amplification [13.1, 13.21. However, the antigen - antibody agglutinating reaction proceeds very slowly, sometimes requiring a few hours for completion. Acceleration of the agglutination process is required for rapid immunoassay. The promotion of the agglutinating reaction is to be expected by increasing the contact frequency between antigen and antibody. Mechanical stirring may be ineffective if only a minute amount of reaction solution is available. Increase of temperature is also ineffective for biologically active substances, including antibodies. On the other hand, it has long been observed that conducting or nonconducting particles suspended in various fluids form linear linkages under electric fields [13.3-13.51. These particles may be aluminum powder, carbon powder, potato starch, polystyrene particles, red blood cells, or yeast cells. Electric field effects were considered promising for increasing the contact frequency between antigen and antibody. In the case of antigens of the order of micrometers in length, agglutination is proceeds in two steps; 1)binding of antibody on a particle (antigen), and (2) further binding of another particle to the antibody-bound particle. An electrical pulse probably accelerates the second step and increases the total reaction rate. Similar effects are expected in the case of small antigens on latex beads [13.7, 13.61.
13.2
Pulse Immunoassay for Pathogenic Cells
A novel method using an electrical pulse has been applied to the immunoassay of Candida albicans (in which the antigen has a size of several micrometers). C. albicans is a pathogenic yeast and the determination of its concentration has clinical importance [13.8, 13.91. Since yeast cells are known to form linear linkages in an electric field, the immunoreaction of C. albicans with antibody is expected to accelerate.
250
13 Pulse Immunoassay and Pulse Electrovoltage for Cell Manipulation Pulser
,Cover g l a s s
(A) I
I
I
I
(B) Figure W.1. Reactors for pulse
immunoassay. A) Slit reactor; B) Cuvette reactor.
1mrn
13.2.1 Experimental Procedures Figure 13.1 shows the two reactors used for the immunoreaction. The slit reactor is composed of a two lead foil electrodes (electrode distance, 1mm; electrode thickness, 25 pm) and a glass slide. The cuvette reactor is constructed from glass plates, with two electrodes (3 x 1cm2) placed on the inside surface of the cuvette. The distance between the electrodes was 1mm. After a drop of cell suspension was placed on the slit reactor (Fig. 13.1A), it was covered with a glass, and oberserved through a microscope ( X 200). Electrical pulses were applied with a pulse generator. Reversible agglutination and dispersion of microbial cells were observed on the application of the field and its release. By means of a syringe, 100 pL of C. alhicans suspension and 40 pL of antibody were mixed in the cuvette reactor. Electrical pulses were applied for a certain period. Immediately after pulses were stopped, an aliquot of reaction solution was transferred to a glass slide and a micrograph was taken. As a control procedure, the immunoreaction was performed without electrical pulses. A reaction solution containing C . albicans and its antibody was also placed in the cuvette reactor, mixed with a syringe, and allowed to stand for the same reaction period. Electrical pulses were then applied to the reactor. After a further period, the agglutinations formed were photographed under the electric field. The effects of pulse height and frequency on the agglutination rate were investigated as discussed below. The extent of agglutination observed unter the pulsed electric field or after its release were given by the agglutination rate; A.R. defined as:
n=l x loo(%) A.R.= 7
EN" n=l
where N,, is total number of n-cell agglutinations. The lower limit ( I ) of n in the determination is selected as appropriate to each experiment.
13.2 Pulse Irnmunoassay for Pathogenic Cells
251
Figure 13.2. Homogeneously dispersed C. albicans with no electric field.
Figure 13.3. Linear agglutination of C. albicans under an electric field. Pulse frequency 1/T= 8 kHz; pulse width 7 = 20 ps; pulse heigt H = 100 V.
13.2.2 Effect of Pulse Conditions on Linear Agglutination C. albicans cells are homogeneously distributed (Fig. 13.2). When pulses were applied to the suspension, linear linkages were formed within several seconds (Fig. 13.3). The alternation of patterns following the application and release of the pulsed voltage, as shown in Figures 13.2 and 13.3, are reproducible. Figure 13.4 shows the effects of pulse height on the rate of linear agglutination at 8 kHz. No agglutination is observed below 20V. Above 20V, the extent of agglutination increases with increasing pulse height. Linear agglutination increased with increasing frequency. The higher the pulse frequency, the shorter is the pulse resting time. During pulse resting, cell particles can move randomly due to Brownian motion. Shorter resting times contribute to promotion of agglutinated forms. In this experiment, the pulse height and frequency are 1OOV (1 kV/cm) and 8 kHz.
13.2.3
Agglutination by Immunoreaction in an Electric Field
When the cell suspension is exposed to electrical pulses in the presence of antibody, some agglutinations remain, even after the removal of the field. Immunoreaction performed with or without electrical pulses is shown in Figure 13.5. With no field, A.R.
252
13
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Pulse Immunoassay and Pulse Electrovoltage f o r Cell Manipulation
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:*
I
I
n
h
$
v
5
4
20
0
40 60 Pulse Height (V)
80
401/ 0
5
10
15
Pulse Application Time (rnin)
20
100
Figure 13.4. Effect of pulse height of linear agglutination. 1/T=8kHz; z = 2 0 p s .
Figure 13.5. Time courses of agglutination. C. albicans 6 x 10’ celllmL, (open circles) with addition of protein antibody (2.9mg/mL) with a pulsed electric field; (triangles) antibody (2.9mg/mL) with no electric field; (full circles) n o antibody, with pulsed electric field; field parameters: l/T=8kHz; t = 2 0 ~ s ; H = 1 0 0 V .
increases gradually to ca. 10% in 20min. In contrast, A.R.increases sharply and reaches 50 % in 5 min with exposure to electrical pulses. Without antibody, A.R.also increases under the electric field, but it was only 10% after 20min. These results clearly show that electrical pulses accelerate immunoreaction. Under an electric field, a certain amount of protein is probably denatured by the heat generated. Since the denatured protein often causes irreversible agglutination, the effect of human serum albumin (HSA)on cell agglutination under an electric field is investigated. When a cell suspension containing 2.9 mg protein in 1mL HSA is exposed a pulsed, field a small amount of agglutination occurs. The agglutination ratio is similar to that obtained without antibody (Fig. 13.5). Therefore, the presence of HSA does not affect nonspecific agglutination.
13.2 Pulse Immunoassay for Pathogenic Cells
0
0
2o
t 0
0
0
1
0
2
3
253
-
4
Antibody Concentration (rng protein rn1-l)
Figure W.6. Effect of antibody concentration on agglutination. C. albicans (6 x lO’cells/mL), (open circles) with pulsed electric field for 5 min; (full circles) 5 min after the electrical pulses; field parameters in Figure 5.
As the reaction rate depends considerably on the relative concentration of antibody and antigen, the effect of antibody concentration on A.R.was examined in the presence of antigen (C. albicans, 6.0 X 107cell/mL).With no electric field, agglutination is not observed below a protein concentration of 2.4mg/mL, but A.R.increases slightly above this concentration (Fig. 13.6). On the other hand, on application of the field, A.R.increases sharply for protein concentration > 2.4mg/mL and reaches 50% at 2.9 mg/mL.
0
Cell Concentration (rnl-l)
Figure W.7. Effect of C. albicans concentration on the agglutination rate. Antibody concentration 2.9 mg/mL, (open circles) with pulsed electric field; (full circles) no field; field parameters and other experimental conditions in Figure 6, except for C. albicans concentration.
254
13 Pulse Immunoassuy and Pulse Electrovoltage for Cell Manipulation
13.2.4 Calibration for C. albicans The method was applied to various concentrations of C. alhicans. Figure 13.7 shows the relationship between A.R.and cell concentration. With no electric field, A.R.is ca. 10% in the range 3.8 x 107-3.6 X 108cell/mL. The value of A.R.increases markedly in the range 106-108cell/mL,and then decreases. This decline may be due to relatively higher concentrations compared with the antibody concentration, i.e. A.R.may depend on antibody concentration. From these results, estimation of C. albicans concentration is feasible in the range 1 x lo7-6 x 107cell/mL [13.10]. This method is applied to the rapid identification of pathogenic cells, rather than quantitative analysis. The present study concerns immunoassay for C. alhicans, which is rather large so that its motion can be observed and analyzed by microscopy, but in the case of immunoassays for proteins, peptides, and drugs, both antigen and antibody are invisible. Therefore, antibody or antigen must be immobilized on appropriate carrier particles several micrometers in size before it is possible to observe their motion.
13.3
Alternating Current Field Enhancing Latex Immunoassay for Human Myoglobin
An alternating current (a.c.) field-induced force on microscopic particles induces alignment of the particles in a “pearl-necklace” or chain, the result of attractive forces between induced dipoles on neighboring particles. Dielectric induced force is expressed (Fig. 13.8) by the equation:
7 35
13
6
28
48 108 33
8
37
2 2 6 7 65
73
67
10
27
6
35 17
8
ia
8
S 3 6 5 7 8 1 6
8
56
40 20
30
16
i s 20
1s
5 1 1 8 0 7
54
AREA OF SINGLE LATEX: 8
(NUMBER OF AGGLUTINATION--FREQUENCn
- 6,
1-12,
2
6
7-2,
-1.
3 -7, 8-
0,
14-1,
NUMBER OFTOTAL LATICES;153 A. R.= (141/153)xlW=S2.2%
Figure W.8. Dielectric induced force between two latex particles. r, radius of particle; E,,, E , , e, dielectric constants; E, electric field,
force.
50
4 -3.
5 -6s
9-2
10-2,
13.3 Alternating Current Field Enhancing Latex Immunoassay for Human Myoglobin
255
where F > 0 refers to an attractive force. Several factors are involved in forming the chain [13.11]: 1) the radius of the microparticle; 2) the dielectric constants of the particle and solution medium; 3) the strength and frequency of the electric field. In latex immunoassay, the agglutination reaction can be used to form a cluster. However, the surface of the latex is usually negatively charged in order to avoid spontaneous agglutination. This electrostatic repulsion slows down the immunoreaction. Chain formation by application of an a.c. field increases the reaction rate. Lower-strength a.c. fields are disruptive to the protein. Therefore, we will examine the effect of an a.c. field on the agglutination of latex beads and its acceleration of a latex immunoreaction. Human myoglobin was used as a model antigen. The protein is composed a single polypeptide chain of 153 residues (molecular weight was 17800) which binds oxygen reversibly, like hemoglobin. Its content in urine or serum can relate to acute necrosis of the myocyte [13.10].
13.3.1 Experimental Procedures An a.c. field is supplied by a waveform synthesizer (FS-2121 Toa Electronics. A fluorescent microscope (Optiphot Nikon) and a CCD camera (NEC) were used to give a CRT image. FDM 98-1 (Photoron) and PC-9801 (NEC) computers with software based on the FDM operating system were used to calculate the area distributions of agglutinated latex beads. After background subtraction, the area of each latex cluster was measured and divided by the area of a single latex bead to obtain the number of beads. The relation between the frequency and the distribution of each bead cluster is shown in Figure 13.9. After one aliquot of 1% fluorescent microspheres in GBS buffer (100mM glycine, 50mM NaCl,O.l% NaN,, pH8.6) was added to one aliquot of the antibody (75 pg/mL), the suspension was gently stirred for 2 h at room temperature. The latex - IgG suspension was washed with GBS - BSA buffer (0.2%, BSA), three times, with centrifuging. The latex - IgG reagent was diluted to 0.5% and kept in a refrigerator until use. A slit reactor was used for immunoreaction. The latex - IgG solution was mixed with its antigen, transferred immediately to the reactor, and an a.c. field was applied for
0
1
2 Time (min)
3
Figure 13.9. Example of a measured image and its data processing for area distribution. Fluorescent latex particles were agglutinated by an a.c. field. The area of each cluster was measured, and the number frequency distribution of latex particles per cluster are shown.
256
13 Pulse Immunoassay and Pulse Electrovoltage for Cell Manipulation
1min or 30 s. After standing for another minute (to allow dispersion of the latex), the number of agglutinated beads was counted by fluorescence image analysis. The agglutination ratio (A.R.), defined as in Equation (l), was measured for even different images and the average value was calculated.
13.3.2 Agglutination Induced by a.c. Fields A mixture of equal volumes of latex - IgG (0.1%) and human myoglobin (100 ng/mL) is placed in an a.c. field (100 kHz, 2 0 V h m ) for 1min to form the chain products and is then left for another 1-2 rnin with no a.c. field, to disperse the latex which has not acquired active functional groups for the immunoreaction. Figure 13.10 shows that the A.R. increases for 1rnin with a.c. the field, and then decreases slightly in the presence of substantial amounts of antigen (100ng/mL) with no a.c. field. However, in the
0 ‘
I
I
II
I
0
1
2
3
Time (min)
Figure U.10. Effect of the a.c. field on the A.R. value. 100kHz, 20 V/mm a x . applied to the solution for 1min, and left to stand for another 1 or 2 rnin with no field wave; reaction solution was one volume of latex - IgG (0.1%) and one volume of myoglobin (100 ng/ ml); A.R. was counted with (squares) and without (circles) antigen.
I vv
80-
60v
u U
40-
20-
0
Figure U.ll. Effect of a.c. field strength. Various potentials of 100kHz a.c. were applied to the solution for 1min and it was left to stand for another 1min. The reaction solution was an equalvolume mixture of latex - IgG (0.1%) myoglobin (100 ndml); (open squares) A.R. at 1rnin with antigen; (circles) A.R. at 2 min with antigen; (full squares) A.R. at 2 rnin without antigen.
t 5
10
20
Electrical potential (V rnm’)
30
13.3 Alternating Current Field Enhancing Latex Immunoassay for Human Myoglobin 100
257
-
a 4oL
O
10
100
1,000
10,000
Frequency (kHz)
Figure l3.12. Effect of a x . frequency field. Various frequencies of 20 V/mm a.c. were applied for 1 rnin and the solution left to stand for another lmin; the reaction solution was equal volumes of latex - IgG (1%) and myoglobin (100ng/ml); (triangles) A.R. at 1 rnin with antigen; (circles) A.R. at 2min with antigen; (squares) A.R. at 2min without antigen.
absence of antigen, the A.R. decreases rapidly. The A.R. in the presence of antigen is about the same as that in the absence of antigen at 1min. Different potentials of the 100 kHz a.c. field were applied to the solution for 1rnin to form the chain, and the solution is left to stand for 1 rnin to disperse the latex. Figure 13.11 shows that with an a.c. field of 20V/mm, about 90% of the latex - IgG beads were agglutinated in 1min. Similar results are reported by Schwan [13.11]. The A.R. curves at 1 and 2 rnin in the presence of antigen show a similar tendency, which means that the cluster formed with antigen is sufficiently stable 1min after removal of the field. Different frequencies of a 20 V/mm a.c. field were applied for 1rnin and the solution was left to stand for 1min. Figure 13.12 shows some variation of the A.R. in the absence of antigen at 2 min. The fluctuation inA.R. observed in the absence of antigen is partially due to nonspecific agglutination and partially to purely physical agglutination which may disperse 1rnin after releasing the a.c. voltage by electrostatic force on the latex particles. As the dielectric constants of latex, buffer, and protein vary with the a.c. frequency [13.11,13.12], the frequency affects the attractive force between particles. If the attractive force is so strong that particles cannot be easily dispersed, then the A.R. may increase without any specific immunoreaction. Different concentrations of latex - IgG were reacted with human myoglobin (100 ng/mL) in a 100 kHz, 20 V/mm a.c. field. The field was applied for 1rnin and then the solution was left to stand for 1min. The concentration of free latex particles also affects the rate of chain formation (the higher the number of latex particles, the shorter the distance between them, making it is easier to form a chain with the same, dielectric attractive force). It is favorable to use a latex concentration > 0.075%. The A.R. in the absence of antigen increases steeply when the latex concentration increases from 0.1% to 0.125%. Sine the concentratoin of latex particles is so high that the distance between them is small, many particles are forced to aggregate. Therefore, the A.R. at 2 min with antigen depends on the value of A.R. at 1min.
258
13 Pulse Immunoassay and Pulse Electrovoltage for Cell Manipulation
t
a b
80-
C
d
60-
e
40-
f
Y
u U
9 h i
20-
0 '
I
I
I
I
0
0.5
1.o
1.5
Time (rnin)
Figure l3.W. Time course of reaction rate. Various concentrations of antigen were used to apply a 100kHz, 20V/mm a x . field for 0.5, 1, 1,5 min, and the solution left to stand for another 1min; the latex - IgG was 0.1% a) A.R. with antigen 250 ng/mL; b) 100ng/mL; c) 50 ng/ml; d) 25 ng/mL; e) 10 ng/mL; f) 5 ng/ml; g) 2.5 ng/mL; h) 1ng/mL; i) 0 ng/ml.
13.3.3 Time Course of Immunoreactions Figure 13.13 shows the time course curve for agglutination. Different concentrations of human myoglobin and 0.1% latex - IgG were reacted under a 100 kHz, 20V/mm a.c. field. The field was applied for 30s, 1min, or 1.5min and the solution left to for 1min. The A.R. increased to a plateau at 1min. Figure 13.14 shows the standard calibration curve obtained from the A.R. at 1min. The minimum detectable amount of myoglobin is 1ng/mL in the case of an a.c. field-enhanced immunoassy, - much more sensitive than turbidimetric measuring. It seems that the sensitivity of fluorescent image analysis is nearly the same as that of automatic particle counters. The rate of reaction enhanced by an a.c. field was compared with that of the reaction with no field, as a control. The standard calibration curve was linear from 1 to 100ndmL in the presence of the a.c. field, whereas there was linearity from 5 to 50ng/mL in the control reaction. Latex immunoassay usually uses submicrometer-sized latex. Larger particles were used in this experiment because it is easier to form chains of these. Another merit of larger particles is improved better resolution of images in the microscope. Figure 13.15 shows that there is a correlation between the rates of chain formation and the immunoreaction (correlation coefficient was 0.98). In the absence of antigen, the A.R. curve increases from the value of 70% A.R. at 1min, because of nonspecific agglutination and of too many latex particles. Latex immunoassay proceeds with two reaction steps. The first step is the binding of latex - IgG to free antigen, and the second is binding of latex - IgG - antigen to another latex - IgG. The second step may be the rate-determining step [13.3]. It may be that the heat generated by the a.c. field does not have a significant effect on the acceleration of the second step, because the temperature jump accelerates Brownian motion and changes the structure of antibody protein, which inhibits an antigen - antibody immunoreaction.
13.3 Alternating Current Field Enhancing Latex Irnrnunoassay for Human Myoglobin
0 ' .1
10
1
259
1000
100
1 Myoglobin concentration (ng rnr )
Figure 13.14. Standard calibration curve of a.c. field-enhanced reaction and the usual incubation immunoreaction. Control reaction was carried out at 37 "C for 20min; (circles) A.R. with a.c. field; (squares) A.R. with no field
loo 80
.-C
E 60
N M
RI
4
40
20
I
I
I 20
I
I 40
I
I 60
I
I 80
I
100
A. R. at lmin
Figure l3.15. Correlation diagram of the rates of chain formation and of immunoreaction. Data from Figure 9, 10, 11; the a x . field was applied for 1min and the solution left to stand for another 1min, thus the A.R. at 1min represents the rate of chain formation and the A.R. at 2min represents the rate of immunoreaction; (squares) A.R. with antigen; (circles) A.R. without antigen.
260
13 Pulse Immunoassay and Pulse Electrovoltage for Cell Manipulation
13.4
A Micromachined Reactor for Latex Immunoassay Enhanced by a.c. Fields
Micromachining of silicon or other planar glass plates provides a means of developing miniatuarized chemical analysis devices, such as capillary electrophoresis or integrated enzyme columns I13.14, 13.151. The combination of photolithography with isotropic or anisotropic etching techniques, as well as controlled thin film deposition or sputtering, allows fabrication of microsized devices [ 13.141. A microreactor using these micromachining techniques has some advantages: 1) the size of three-dimensional structures can be precisely controlled; 2) it is possible to fabricate multiwells in one reactor, which have the same size and shape. It is essential to compare the results of samples with those of standard control sample. In practical terms, an immunosensing system should be rapid and able to treat many samples simultaneously. We have demonstrated the feasibility of multisample analysis using a microreactor designed for latex immunoassay enhanced by an a.c. field.
13.4.1 Experimental Procedures The glass etching process was carried out by Oyama Optic (Tokyo). A glass substrate was masked with a metal layer; followed by lithographic patterning of a metal mask. After exposing the glass to UV light it was etched to create wells of 80 pm depth with an HF-based solution and the mask was then removed. In order to form gold electrodes on the glass plate, a chromium layer (200A) and a second gold layer (2000A) were deposited on the plate by vacuum evaporation (EBH-6, ULVAC), and the layer was photographically etched with photoresist (OFPR-800, Tokyo (Ohka Kougyou). The glass plates were then bonded to a small cover slip by melting I13.41, as follows: the base plate and cover slip were clamped tightly and heated in an electric furnace to 500 "C for 1 h, 550 "C for 0.5 h, 630 "C for 2 h, 550 "C for 1h, and then allowed to cool to room temperature. The image analysis system was the same as described previously.
13.4.2 Design of Micromachined Reactors Pyrex glass plates contained four channels designed for immunoreaction. The volume of each channel (80 pm deep, 0.5 mm wide, 10mm long) is 0.4 pL (Fig. 16). The depth of 80pm is a compromise distance for obtaining low flow resistance while getting a clear views through the microscope Thin gold films (2000A) were deposited in channels on both sides of the walls as electrodes, and on the top side of the glass plates with lead wires. Films for electrodes and lead wire were deposited at the same time and connected nearly vertically with each other. If the thickness of the deposited gold films was less than 1000A, it was difficult to cover the boundary edges of the two parts. After gold film deposition and etching processes, the products were checked by microscope. Introduction of reaction solutions into these microchannels is performed by capillary action. After the immunoreaction was completed with the application of an a.c. field for 1min, 10 pL of GBS buffer was added by a microspoid dilution adapter. The reaction solution diluted with buffer mixture could then be taken out by attaching the end of spoid to the open end of the channel. Too high a concentration of latex will reduce
13.4 A Micromachined Reactor for Latex Immunoassay Enhanced by a.c. Fields
A
T
261
A'
ETdHED REGION (0.5XlOX0.03) ROOM FOR REACTION BUFFER
I
A-A'
u
Lf
U
t
B
n w
I+-
COVER SLIP
B-L& 1: -
BASE PLATE GLASS
OD
0.5mm
Figure 13.16. Multireactor system for latex immunoassay enhanced an a.c. field.
the accuracy of the measurement by image analysis; nonaggregated particles look as it they have aggregated, and it this causes misestimation of the A.R. of a blank sample; to avoiding this, a dilution step was adopted in this study.
13.4.3 Application to Immunoassay for AFP Alpha-fetoprotein (AFP) is a protein with molecular weight ca. 70000. AFP in the serum of a pregnant woman can be a indicator of neural tube defects in the fetus. AFP can also be a diagnostic aid for liver cancer [13.16]. IgG - latex and 500ng/mL of human AFP were reacted in the microchannels under 100 kHz, 12 V a.c. square-wave field applied for 1min, and the reaction solutions were diluted to 0.02% (wthol) latex concentration with GBS buffer. Figure 13.17 shows that the concentration of latex particles also affects the immunoreaction. As described earlier, the greater the number of latex particles, the easier it is to form a chain, assuming the same attractive force
a
20 1
I
0
Figure 13.17. Effect of latex concentration on the change of A.R. value with (circles) or '
I 0.25
'
I 0.5
b
I 0.75
:
( 1.0
Latex concentration (%)
I
without (squares) ax. field. An ax. wave (lOOkHz, 12V) was applied for 1min; reaction solution contains AFP 500 ng/ ml .
262
13 Pulse Irnrnunoassay and Pulse Electrovoltage for Cell Manipulation
0'
"
"
15
30
0
"
45
Time
"
"
"
60
75
90
Figure W.18. Time course of agglutination with circles or without (squares) AFP. The IgG - latex (1%wt/vol) solution and AFP (500ng/mL) were used with an a.c. wave (100 kHz, 12 V) applied for 15 , 30, 1min, and 1.5 min
(sec)
-ds h
a
,001
.01
.I
1
10
100
AFP concentration (nglml)
1000
Figure W.19. Calibration curve for AFP. The conditions of reaction enhanced by an a.c. field (squares) were the same as those in Figure 18, except the field was applied for 1min; control reaction by incubation at 37 "C for 20 min (circles).
[13.13]. The a.c. field was applied for 15 s, 30s, I min, or 1.5 min. After dilution with 10 pL of GBS buffer, the A.R. was determined. Figure 13.18 shows the time course of the agglutination reaction; the A.R. increases up to 1min and levels off after that. Figure 13.19 shows a standard calibration curve. Different concentrations of human AFP and IgG - latex (1.0% , wt/vol) were reacted under 100 kHz, 12 V of a.c. field for 1min. The lowest detectable amount of AFP was 50pg/mL, which is more sensitive than our previous data-based on a laser scattering method, and an enzyme-linked immunoassay, and that of automatic particle counting measurements [13.17]. Linearity was obtained from 50pg/mL to 500ndmL AFP by image analysis, which is a much wider range than that of particle counting measurements [13.17]. In Figure 13.19, the rate of reaction enhanced by the a.c. field was compared with that of the reaction with no field (the control reaction was carried out by the usual 37 "C incubation method for 20min, with gentle stirring). The lowest detectable amount of AFP was around 5 ng/mL in the control reaction. The concentration of AFP is ca. 10ng/mL in normal serum and increases to 100-5000ng/mL if the fetus has a neural defect [ 13.171.Therefore, a wide detection range is desirable in AFP determination, as well as good sensitivity with high accuracy.
13.6 References
13.5
263
Conclusions
A novel immunoassay has been developed by the use of a pulsed voltage or an a.c. field. The phenomenon of “pearl-necklace’’ chain formation enhanced by an a.c. field has the effect of increasing the collision frequency, making this assay rapid. This assay uses larger particles, such as pathogenic cells, and latex beads linked with IgG. The rate of immunoassay depends on the rate of chain formation, which varies with the strength and frequency of the electric field and the concentration of particles. The agglutination of particles in an immunoreaction can be accelerated by pulsed voltages or a.c. fields. A microreactor has been fabricated for a multisample-microimmunoassay using micromachining technology. This method can be applied for determination of Cundidu ulbicuns as pathogen and myoglobin and alpha-fetoprotein as diagnostic antigen.
13.6
References
[13.1] R. S. Yalow, Methods in Radioimmunoassay of Peptide Hormones, Amsterdam, North Holland, 1976. [13.2] G. D. Jonson, E. J. Holborow, J. Dorling, Handbook of Experimental Immunology, Oxford Blackwell Scientific Publications, 1978. [13.3] M. Saito, H. P. Schwan, Biological Effects of Microwave Padiation, NewYork, Plenum Press, 1960. [13.4] A. A. Furedi, R. C. Valentine, Biochim. Piophys. Acta, 56, 33-42 (1962). [13.5] U. Zimmerman, J. Vienken, G. Piloat, Z. Naturforsch, 36C, 173-177 (1981). [13.6] E. Tamiya, N. Watanabe, H. Matsuoka, I. Karube, Biosensors, 3, 139-146 (1988). [13.7] E. Tamiya, I. Karube, “Electric Pulse Accelerated Immunoassay“, in Electrochemical Sensors in Immunological Analysis, T. T. Ngo (ed) Plenum Press, 1987. [13.8] T. E. Kiehn, Science, 206, 577-580 (1979). [13.9] M. A. Peris, J. C. Burnham, Appl. Environ Microbiol., 42, 364-369. [13.10] H. Matuoka, E. Tamiya, I. Karube, Anal. Chem., 57, 1998-2002 (1985). j13.111 H. P. Schwan, L. D. Sher, 1. Electrochem. SOC. 22C, 116-125 (1969). [13.12] E. H. Grant, G. P. South, Adv. Mol. Relaxation Processes, 3, 355-365 (1972). [13.13] M. I. Song et al., AndChirn. Acta, 282, 193-198 (1993). [13.14] D. J. Harrinson et al. Anal. Chem., 64, 1926-1932 (1992). [13.15] Y. Murakami et al.,Anal. Chem., 65, 2731-2735 (1993). [13.16] G . I. Abelev, Adv. Cancer Res., 14, 295-358 (1971). [13.17] D. Collet-Cassart et al., Clin. Chem., 27, 64-67 (1981).
This Page Intentionally Left Blank
14 Molecular Orientation of Organic Compounds in Electric Fields: Organized Photochemistry Katsuhiko Takagi
14.1
Introduction
There have been many studies on organized photochemistry, employing various sorts of applied or spontaneous electrostatic fields. This is largely because photochemists seek to mimic the bioreactions occurring in organized micro-heterogeneous fields in order to achieve clean and efficient photochemical processes. For example, photosynthesis by green plants occurs in the cell membrane which constructs an organized set of solar energy harvesting systems, including photosensitizers (light-absorbing species), electron transfer mediators, and the combined redox system; otherwise no net reaction takes place. Applied electrostatic fields are generated in nonfluid matrices, such as polymer films externally charged by strong electric fields. Polarizable or zwitterionic guest molecules are forced into parallel alignment by using an external electric field generated by an apparatus such as that shown in Fig. 14.1. One aim of this work is to produce a noncentrosymmetric array of organic molecules to yield nonlinear optical materials. Spontaneous electrostatic fields involve an interface formed by self-assembled molecular aggregates with oppositely charged double-layers several hundred Angstroms thick. A typical example is the micellar surface of aggregated ionic surfactant molecules. Such fields occur not only in vesicle, bilayer, or multilayer systems (including Langmuir-Blodgett films), but also in ionically charged interlayers of lamina1 materials. In general, ionically charged guest organic compounds tend to be adsorbed electrostatically at interfaces, forming organized molecular aggregates characteristic of the type of host interface.
Figure 14.1. Apparatus for corona charging thin films on a hot plate.
266
14 Molecular Orientation of Organic Compounds in Electric Fields
14.2
Photochemical Reactions
14.2.1 Applied Electrostatic Fields Polarizable organic compounds, when crystallized or dissolved, are generally aggregated so that their microscopic polarizabilities cancel out. Such materials may be affected by an external electric field, resulting in geometrically controlled orientation. Organic and polymeric materials are promising candidates for nonlinear optical devices [14.1]. They can be dispersed in thin films, making them very useful in integrated optics. Unfortunately, some compounds with high microscopic polarizability p crystallize in a centrosymmetric fashion giving zero or negligible bulk polarizability x. Organic materials dispersed in thin films formed by spin or dip coating techniques remain centrosymmetric. A number of molecular alignment processes have been tried to overcome this difficulty by aligning suitable chromophores in a noncentrosymmetric array, including utilization of spontaneous electrostatic fields [14.2-14.41. One of the most fascinating methods is ordering of polarizable species by a strong electric field (several hundreds kilovolts per centimeter) [14.3]. The poled polymer film technique consists in assembling organic dyes with large second-order nonlinear optical susceptibilities in polymer glasses and subjecting them to an electric field. A typical example is the treatment of thin films (ca. 4 pm) of the azo dye, 4-[N-ethyl-N-(2-hydroxyethyl)lamino-4'-nitroazobenzene dispersed in poly(methylmethacry1ate) (PMMA). On applying an intense electric field of 200-600 kV/cm while heating the film above Tg (glass transition temperature) and subsequent cooling, second harmonic generation (SHG) was observed (Fig. 14.2). The intensity of SHG is a function of the angle 0between the incident fundamental beam and the film [ 14.3al. Molecules containing conjugated n electronic systems with charge asymmetry exhibit an extremely large value of p. Maximum values are obtained when the molecule contains substituents that lead to low-lying charge transfer resonance states. A disadvantage is disordering of dye molecules with the progress of time in nonfluid matrices. In order to overcome this, organic dye molecules have been covalently bonded to polymer chains. Table 14.1 summarizes a number of organic dyes dispersed in polymer matrices, or their analogs covalently bonded to polymer chains.
-
07
t 3 z
-
a U
5 -
1 3
1
e (DEG.)
I
I
,
,
Figure 14.2. Intensity of polarized fundamental and second harmonic vs. incident angle, a poled film of azo dye in PMMA.
267
14.2 Photochemical Reactions Table 14.1. Organic dyes oriented in polymer matrices by means of intense electric fields No.
Dye
Polymer matrix
Reference
[14.3a]
[14.3b]
\
[ 14.3f]
(14.51
268
14 Molecular Orientation of Organic Compounds in Electric Fields
N , N-Dimethylaminostilbazolium ions and their analogs (STZ') are good candidates as dyes on account of their reasonably high /3 values, and are reported to exhibit SHG when incorporated in Langmuir-Blodgett films. However, no attempt has been reported to align STZ+ions in a poled polymer film and to estimate quantitatively how much they are ordered. Apart from nonlinear optical devices, electrical poling has been utilized to produce geometrically organized STZ' aggregates in our laboratory. Photodimerization of STZ' organized in heterogeneous fields such as micelles and inorganic interlayers has been studied, and found in our laboratory to form stereoselective cyclodimers, reflecting their orientation in the field. We have studied the alignment of STZ' ions by an intense electric field and their stereoselective photocyclodimerization in poled polymer glasses [14.7]. Thin film samples were prepared by casting an aqueous ethanolic solution of stilbazolium poly(styrenesu1fonates) ( l a and 2a), prepared by mixing stilbazoles (20 mg in 10 mL ethanol) with appropriate amounts of aq. 65 mM poly(styrenesu1fonic acid) (PSS) f14.81on IT0 glass plates (40 X 40 mm) heated on a hot plate at ca. 60-80 "C. The resulting films, pale yellow (Lax = 334 nm for la) or red (Lax = 470 nm for 2a), were 10-50 mm thick, and somewhat fragile. Neutralization of the remaining protons of l a and 2a by laurylamine remarkably improved their mechanical strength. However, the laurylammonium ion is flexible in structure and similar to STZ' in size, causing homogeneous mixing with STZ'. Thus, it is suspected that addition of laurylamine may suppress the interaction of an excited STZ' with a neighboring STZ' in the film to lower the efficiency of photodimerization.
l a , R = H; 2a, R = N(CH&
A
HOzC-CH
FcoF +HN
R
H02C-CH CH- C02H
X"
A strong excimer fluorescence was observed at ca. 500 nm on irradiating a film of l a at 350 +_ 2 nm; its intensity was decreased but the monomer fluorescence at 430 nm was increased by adding excess laurylamine. Monomer-excimcr fluorescence ratios (I&) were examined as a function of the STZ' content. Figure 14.3 shows that the monomer fluorescence disappears at STZ' in content 10 % . This indicates that an effective interaction leading to the photodimerization is possible when more than 10% of PSS anionic sites are occupied by STZ' ions.
14.2 Photochemical Reactions 30
269
I a
*
o
0
* * I
O Y
10
1
[Stilbazol] / [PSSH]
X
100 (%)
-
Figure 14.3. Monomer-to-excimer fluorescence ratios of l a (Im&) against stilbazol content in PSS. Open circles [laurylamine] + [stilbazol] = [PSSH]; full circles, without laurylamine.
As an alternative polyelectrolyte, a copolymer of poly(ethy1ene and maleate) (PEcoMA, Aldrich) [14.7] was studied. Thin films (2b) of up to 8.2% 4-(N,Ndimethy1amino)stilbazolium ions based on the carboxylic protons of PEcoMA copolymer were found transparent and suitable for the following poling and irradiation procedures. The thin films ions were poled by corona charging at 105-106 V/cm for 10 min at 117 k 1 "C [4.18] and kept at the same voltage until the film had cooled to room temperature (Fig. 14.4). The poled films were irradiated for 8 h by a 150 W Xe lamp without a filter at room temperature, dissolved in H 2 0 , and analyzed by HPLC equipped with a ODS column, eluting with ethano1:water (270:230 mL) including 1 mL conc. ammonium hydroxide. Product distributions were confirmed by NMR analysis of the reaction mixtures in comparison with the pure samples. The isomeric cyclodimer distributions are summarized in Table 14.2. H H :HT ratios of photocyclodimers were increased significantly by the poling treatment in 4-(N,N-dimethylamino)stilbazoliumions (2) (Films 2 and 8, or 6 and 13) but were little changed with the unsubstituted one (1) (Films 1 and 7). That is, the more polar olefins tended to undergo more stereoselective photodimerization, suggesting that they are oriented more efficiently by intense electric fields. This implies that corona charging induces an alignment, predicted by the Boltzmann distribution law, in which the incident beam is polarized parallel and perpendicular to the polar axis, indicating that not all the poled olefins are oriented parallel to the axis, but distributed over a wide range, from random to parallel, to produce the bulk polarizability [14.9]. It is intersting to note that corona charging of 2a in PEcoMA copolymer enhances HH:HT cyclodimer rations at 4-5-fold. On the contrary, PSS showed a somewhat
S n 0 7 Electrodc
Figure 14.4. Schematic representation of high electric field poling.
270
14 Molecular Orientation of Organic Compounds in Electric Fields
Table 14.2. Photodimerization of la, b and 2b dispersed in poled and unpoled thin films
Film STZ'
Supported polymer
Adsorption of dodecylamine
Conversion, Selectivity of product, YO
HWHT
Yo syn-HH syn-HT cis
1 2 3 4 5
6 7
A) Before poling la PSSH None Ib PSSH None PSSH 30 Yo PSSH 50 Yo 2b PEcoMA 4.1%' PEcoMA 8.2 %" cf. la PSSH (aq. solution)
57a 57 55 24 72 71
14 4 9 17 3 3
70 12 7 21 5 4
5 84 84 62 92 93
0.20 0.33 1.3 0.81 0.60 0.75
70
4
48
13
0.09
65" 25 16 20 42 43 55
12 12 25 25 36 23 18
75 20 12 15 12 5 6
<1 68 63 60 52 72 76
0.16 0.60 2.1 1.7 3.0 4.6 3.0
B) After poling 8 9 10 11 12 13 14
la lb 2b
PSSH PSSH PSSH PSSH PECOMA PEcoMA PEcoMA
None None 30 % 50 % 4.1%" 6.4 Yo" 8.2 Yo"
'
"Anti-HH dimer was formed (11 YO); molar ratio of olefin to copolymer; anti-HH dimer was formed (12 Yo).
lower effect, i.e., at most within a factor of 2 (cf. Films 2, 3, 4 and 8, 9, 10 of Table 14.2). The strong acid polymer PSSH forms a polymer salt with STZ in a nearly quantitative way, and the molecular motion of STZ' is hindered on account of the highly charged polymer salt. On the other hand, the weaker acid copolymer PEcoMA interacts less with STZ', and hence the STZ' ions can be reorientated by the poling process. Finally, a considerable decrease in the photochemical conversion was noted after the poling treatments. This is presumably because the poling procedure roughens the film surfaces, reducing their transparency.
14.2.2 Spontaneous Electrostatic Fields The following sections describe the effects of spontaneous electric fields in micelles, reversed micelles, vesicles, membranes, polyelectrolytes, and inorganic solid surfaces (silica, clays, and zeolites).
14.2.3 Micelles Aqueous micelle solutions are easily accessible and excellent media for electrostatic interfaces. Numerous examples have been reported of organized photochemical reactions in conventional aqueous micelles [14.101. The ionic surfactant molecules form molecular aggregates (micelles), in water at concentrations higher than their critical micelle concentrations (CMC). As typical examples, sodium dodecylsulfate (SDS) and
14.2 Photochemical Reactions
UptoafewA
271
Figure 14.5. A simplified picture of a spherical ionic micelle. ow\,surfactant molecules; x, counter-ions.
cetyltrimethylammonium bromide (CTAB) form anionic and cationic micelles above CMC of 8.1 and 0.92 mM, respectively. CH~(CHZ)IIOSO~NB
SDS
CH3(CHz)15N(CH3)+3Br CTAB
A simplified model for ionic micelles [14.11] is useful for their visualization (Fig. 14.5). According to the model, micelles are roughly spherical and contain 50-200 surfactant molecules. Their radii are close to the lengths of the hydrocarbon chains of surfactants, 10-30 A.The micelle has a hydrocarbon core and a polar surface; it resembles an oil droplet with a polar coat. The head groups of ionic micelles and associated counterions are located in the compact Stern layer. Some of the counter-ions are bound on the surface, but most are located in the electric double layer (up to a several hundred Angstroms thick). Organic guest molecules are solubilized in the core or the interface of micelles, depending on their hydrophobicityhydrophilicity.The volume of the aggregate is estimated as 3.99 x lo4 A3 [14.12], half of which is occupied by the inner hydrophobic core and the rest by the Stern interface layer.
14.2.4
Energy and Electron Transfer
Most photochemical studies utilizing micelles have been concerned with the effects on excitation energy and electron transfer reactions. Micelles are suitable for constructing energy (Eq. la) or electron transfer (Eq. lb) systems in which donor (D) and acceptor (A) pairs can be confined in micelle particles (asterisks denote electronically excited states). Selective energy transfers between D* and A in SDS and CTAB micelles have been noted on account of the high local concentrations of D and A [14.13].
272
14 Molecular Orientation of Organic Compounds in Electric Fields Energy transfer
r
D'
+
A
1
- D
+ A *
(la)
Electron transfer
*
D+ +
A-
(1 b)
A marked micellar effect is observed in the case of electron transfer between D*and A to give a pair of charge-separated species, D+ and A- (Eq. lb). The resulting chargeseparated species react rapidly (Eq.2), if no other reactions of D+ and A-occur. D+ + A - + D
+A
(2)
In order to utilize the charge-separated species for chemical reactions, it is necessary to suppress the facile reverse electron transfer [14.14].The ionic interface of micelles has been found to be effective for this purpose. A micelle surface forms an electric doublelayer with a steep potential gradient over a few hundred Angstroms. Two plausible mechanisms are envisaged for the stabilization of charge separated states in Figure 14.6I14.151.When either of the charge-separated species is repelled from the similarly charged surface, an oppositely charged species interacts strongly with the surface, resulting in an effective charge separation (Fig. 14.6A). In an alternative case (Fig. 14.6B)an isothermal electron relay proceeds along the acceptor series, Al,A2,A3, .., , stabilizing D'x and A-x by their distance apart.
ZnTPPS: M = Zn; X = S03Na; Y = C ZnTPP: M =Zn; X = H; Y = C ZnTPyP: M = Zn; X = CH3; Y = N+ ZnTPSPy: M = Zn; X = CH2CH2S03-;Y = N-
A typical example is the dramatic effect of SDS micelles on the photochemical charge separation between zinc(I1) tetraphenylporphyrin (ZnTPP) and sodium 2,6-disulfonatoanthraquinone (3, R = SO,Na), eventually yielding sodium 2,6-disulfonatoanthranol (4, R = S0,Na) [14.16].The reverse electron transfer is effectively suppressed by the electrostatic repulsion of anion radical 1-X from the anionic micelle surface. A similar effect is also noted in the photochemical charge separation between excited Ru(bipy)?+ and N , N-dimethylaniline [ 14.171 or Nbutylphenothiazine [14.18,14.191.A remarkable effect of micelles has been reported for the charge separation between anthraquinone-2-sulfonate (3, R = H) and hydroxide ion; the addition of CTAB micelle, or benzylhexadecyldimethylammonium bromide (BHDB) reversed micelle, accelerates the photochemical one-electron transfer
14.2 Photochemical Reactions
273
Figure 14.6A, B. Tow possible mechanisms for charge separation.
from hydroxide ion to the excited quinone 3*, yielding its anion radical (3-x) or dianion (3”) 114.201. Another effect of the CTAB micelle was reported in the electron transfer between a neutral ZnTPSPyP sensitizer and anthraquinone 3 (R = S0,Na) [14.21]; the electron transfer becomes possible by dissociating the ZnTPSPyP-quinone complex via the anionic micelle.
Na03S
0
OH
3
4
A crown ether surfactant ( 5 ) forms a micelle, even in concentrations of lo9 x 10” M [14.22]. Such a micelle efficiently accommodates silver ion in its crown ether core. Irradiation of cyanine dye (6) in the presence of 5-Ag’ micelle leads to a rapid electron transfer within 1.5 ns, producing a micelle containing metallic silver [14.23]. In this case, the oxidized cyanine dye ( 6 + ~is) separated from the reduced silver metal by micelle aggregate.
5-Ag+
6
5-Ag
An efficient photoinduced electron transfer has been observed from Nmethylphenothiazine (7) to copper(I1) ion incorporated in crown ether micelles [ 14.241. The charge-separated state is produced by expelling the resulting cationic 7’x from the micelle surface. The reduction product in metallic Cu(0) which is stabilized by the crown ether, as in the case of 5-Ag(0).
274
14 Molecular Orientation of Organic Compounds in Electric Fields H
CH3
7
8
Efficient electron transfer reactions can be realized by utilizing redox reagents as a counter-ion of anionic micelles [14.251. Copper(T1) dodecylsulfate forms a micelle with cupric ion on the surface. Irradiation of dihydroindolo-[3, 2-blcarbazole (8) dissolved in copper(I1) dodecylsulfate micelle resulted in an effective electron transfer from 8 to cupric ion. Triarylmethane dyes (9) bound to polyelectrolytes, on irradiation, undergo photochemical electron transfer to alkyl viologen (RV"). Addition of CTAB micelles to this system increased the population of an excited triplet, a precursor of the electron donor, by a heavy atom effect of the micelle [14.26]. Viologen with long alkyl chains (RV", R = (CH2)&H3) is an excellent electron carrier between aqueous and oil phases: RV2+is hydrophilic, but its reduced cation radical is hydrophobic. The irradiation of Zn(I1)TPP or Ru(bipy)?+ in CTAB micelles including the carbon-chain-containing viologen RV2+resulted in quite stable charge separation; i. e., the reduced viologen RV'. had a lifetime of several milliseconds [14.27] and migrated from the bulk water to the micelle interior. Stable charge separation occurred between the excited donor and the electron carrier.
RV2+
RV+'
One-electron oxidized (D') and/or reduced species (A-) stabilized by micelles can be used for further redox reactions. We have found micellar effects on the photochemical debromination of 2,3-dibromocinnamic acid (10) giving 11 with a suitable reductant . and sensitizers [14.28]. Zinc(I1) tetraphenylporphyrin (ZnTPP) was used as a hydrophobic sensitizer and its sulfonate (ZnTPPS) as an anionic one. EDTA of triethanolamine (TEtOA) was used as reductant. C&i&HBrCHBrC02R
C&jCH =CHC02R 11
10
(a, R = H; b, R = Na)
The debromination was shown to be initiated by a one-electron transfer from the sensitizer to 10 by detecting a transient ZnTPP(S)+x by laser flash spectroscopy. In addition, the following characteristics were noted in the reaction: (i) radical species were involved, since a controlled reaction in toluene gave diphenylethane and benzyl bromide at the expense of cinnamic acid, (ii) the quantum yields for formation of 11 were greater than unity, implying the occurrence of radical chain reactions. Table 14.3 shows the effect of CTAB on the debromination efficiencies of 10. The most effective was system S + S where both ZnTPPS and 10b are adsorbed on the anionic micelle interface, and the least effective was system I + I where ZnTPP and
275
14.2 Photochemical Reactions
Table 14.3. Debromination of 2,3-dibromocinnamic acid (10) sensitized by ZnTPP or ZnTPPS in micelles Donor
Acceptor/reductant
Surfactant
System
Quantum yield of formation of 12
ZnTPP ZnTPP ZnTPP ZnTPPS ZnTPPS
l0aEDTA lOa/TEOA 10WEDTA 10dEDTA 10WEOA
CTAB SDSmea CTAB CTAB CTAB
1-34 1 4 1-s
0.04 0.01 0.28 0.36 1.39
S+I
s-4
~
SDSme: a mixture of SDS (10 mM), amylalcohol (61 mM), and dodecane (16 mM); notation as in the text.
10a were in the micelle interior (S and I refer to the surface and the interior of the micelle). System S + S was ca. 14 times as efficient as I + I. Intermediate efficiencies were observed for S + I or I + S, where the sensitizer and dibromide (10) were separately dissolved in either the interface or the interior [14.29]. Thus, there is a considerable difference in the efficiency of photochemical redox reactions depending on the solubilization sites for sensitizer and dibromide. Overall quantum yields for debromination are correlated with the efficiencies of net charge separation between sensitizer and 10. The superiority of S -+ S over other systems can be explained by the following points: (i) The reverse electron transfer is slowed down by the charge separation of ZnTPP(S)+x and 10-x in the electrostatic field of the CTAB micelle; (ii) the electrostatic attraction is weakened by the neutralization of net charge by the anionic surfactant. Another method to separate an oxidized species from a reduced one is shown in Figure 14.2B. In a typical example, Ru(bipy)?+ sensitizes cis-trans isomerization of stilbazolium ions (12) adsorbed on SDS micelles, with quantum yields exceeding unity [14.301. The cis-trans one-way isomerization has been concluded to proceed through pyridyl radicals formed by photochemical one-electron reduction. Noteworthy here was a reasonably high quantum yield for the isomerization in the presence of SDS. Table 14.4 summarizes the quantum yields related to the type of micelle. The most interesting observation is that the resulting quantum yields were practically the same as the aggregation numbers n A of the anionic micelles. Thus, all the molecules adsorbed on a micelle undergo the cis-trans isomerization by a single photon. This indicates an electron relay chain reaction through a radical 13 on the micelle surface (Fig. 14.7). Table 14.4. Limiting quantum yields (@c+t),,,ax systems
for the isomerization of cis-12 in various surfactant
Surfactant ~~
(@c-tr)max
Aggregation no.
0.58 f 0.03 64 k 2 (63) 49 k 1 86 f 4 98 f 9 0.73 f 0.05
62 50 82 100 50
~
Sodium dodecyl sulfate (SDS) Sodium decylsulfate (SDeS) Sodium tetradecylsulfate (STS) SDS microemulsion Dodecyltrimethylammoniumbromide (DTAB)
70 60 113
276
14 Molecular Orientation of Organic Compounds in Electric Fields
0
: Ru(bpy)32t
Q
: cis-12
Figure 14.7. A schematic picture for the electron relay chain reaction.
% R+N\3
R+N\
\
/
d
cis -12
/
%NR trans -12
13
Quinolium salt (14) is an excellent mediator for the photoreduction of keto carboxylates [ 14.311. The photochemical reduction of methyl benzoylformate (15) to ahydroxyphenylacetate (16) was achieved by using ZnTPPS sensitizer and quinolinium amide 14 as the electron mediator. Irradiation of a mixture of 14, ZnTPPS, and 15 in the presence of CTAB micelles resulted in the formation of a-hydroxyacetate 16. In this case, 14, a mediator, was solubilized in the hydrophobic core of CTAB micelles, but anionically charged ZnTPPS was present in the bulk water because of the electrostatic repulsion of the micelle surface. ~ C 0 N H C H 2 C H 2 0 H
0 It
OH I
C ~ H S C C O ~ C HC~~ H S C H C O ~ C H ~ I
CH3 14
15
16
Photochemical electron ejection has been observed in the case of surfactantized phenothiazine (17, R = (CH2)12S03Na).The surfactant molecules, on UV irradiation, ejected electrons to give the radical cation 17+x, which was significantly stable with a lifetime of a few days [14.32]. R I
17 R = (CH2)12S03Na
14.2 Photochemical Reactions
277
14.2.5 Miscellaneous Reactions Aqueous micelles provide ionic media which organize organic guest compounds, but have the drawback that the solubilities of guest molecules are not always sufficient, and product isolation is quite tedious. Therefore, most photochemical studies utilizing micelles tend to be directed toward physicochemical processes, e. g., photosensitized electron transfer. In order to make use of micelles for preparative-scale experiments, sufficient amounts of guest molecules must be solubilized. Guest molecules may be accommodated as counter-ion in an ionic micelle. Thus, N, N-dimethyllaurylammonium hydroxide forms a salt with various guest carboxylic acids, to form a micelle in water. For the work-up after photolysis, simple extraction with organic solvents, e. g., diethyl ether from HC1-acifidied photolysate solution, is sufficient for separation of reacted carboxylic acids. It is intriguing to inquire how guest molecules incorporated in micelles are conformationally organized in the structured micelle environment. In order to monitor the properties of micellar aggregates, probe molecules exhibiting fluorescence or absorption spectra characteristic of the solubilization sites may be used. A typical example is pyrene as a fluorescence probe. Kalyanasundaram and Thomas 114.331 found a profound polarity effect on the ratio of the first (373 nm) and the third peak (385 nm) intensities of the structured fluorescence spectrum of pyrene. For example, the solubilization sites for pyrene molecules in SDS micelle were deduced to be significantly polar and close to those for ethanol. The intensity ratios are linearly correlated with the absorption maxima of CTbands between stilbenes and methylviologen (RV”, R = CH,) in aqueous anionic micelles [14.34]. Ionically charged surfactants such as CTAB and SDS adsorb oppositely charged substrates electrostatically. For example, cationic micelles bind carboxylate and phenolate anions, and anionic ones bind quaternary ammonium ions, including protonated amines. Since these ionic substrates are hydrophilic, it is not clear whether they are adsorbed on the micelle surface or not. A fluorescence probe to investigate the solubilization sites can be applied on the basis of the quenching efficiency by guest molecules [14.35-14.371. Thus, the fluorescence of zinc(I1) tetra@-su1fonate)phenylporphyrin (ZnTPPS) is quenched efficiently by 1,2-dibromocinnarnate in the presence of CTAB, but not SDS, micelles [14.29]. As discontinuity occurs in the plot of fluorescence intensity and dibromide concentration in CTAB micelle solutions. Since the discontinuity appears at equimolar concentrations of CTAB and cinnamate, it is apparent that one surfactant molecule adsorbs just one cinnamate ion and the excess cinnamate ions in the bulk water. The discontinuity determines the maximum solubilization ability of the micelle. Usually, ionic micelles are capable of accommodating oppositely charged organic ions equal to its aggregation number nA. The structure of the rigid, immobile SDS micelles at 77 O K in an ethylene glycol-water mixtures has been characterized by luminescence quenching studies [14.38]. These rigid micelles provide a unique electrostatic field which fixes guest molecules in a known geometry. Dimerizations. Photochemical [2 +21 cyclodimerizations are a useful probe to investigate the alignment of olefin molecules, organized or not, in heterogeneous fields. Various kinds of organized media have been employed for the cyclodimerizations. The photochemical cyclodimerization of 9-methyl [14.391and 9-hydroxymethylanthracenes [14.40] is known to give head-to-head dimers, indicating their parallel orientation in CTAB and SDS micelles. In contrast, the cyclodimerization of acenaphthene was shown to be governed by the solvent polarity. A linear relationship between log([syn-
218
14 Molecular Orientation of Organic Compounds in Electric Fields
dimer]l[anti-dimer]) and the polarity is also observed with SDS and dodecyltrimethylammonium iodide micelles [ 14.41, 14.421. This indicates that acenaphthene molecules are organized, in contrast to the anthracenes, by the electrostatic fields of micelles. An interesting result was obtained for the photodimerization of surfactantized stilbazolium ions (trans-12, R = Cl6H3.,) dissolved in CTAB micelles. The stereochemistry of the resulting dimer was essentially syn-head-to-head (syn-HH, 18), which is in sharp contrast to the dimer of syn-head-to-tail (syn-HT, 19) in relatively concentrated solutions. The preferential formation of syn-HH dimer in CTAB micelles clearly reflects the fact that surfactant stilbazolium ions are aligned parallel on the CTAB micelle surface [14.43].
18 (syn-HH)
19 (syn-HT)
20 (anti-HH)
A contrasting result was obtained in the photolysis of 3-alkylcyclopentenone (21) in potassium dodecanoate (KDC) micelles. The regioselectivities of HT and H H dirners were reversed by solvents, i.e., benzene vs. KDC micelle [14.44]. The HH dimer was predominant in the micelle system. A preferential formation of HH dimers in micelles is also noted in the photodimerization of isophorones [14.45] and coumarines 114.461. The regiochemistry of the dimers is reasonably assumed to reflect the mutual organized alignment of olefin molecules in micelles. Spherically structured aggregates as normal or reversed micelles (see Fig. 14.1) suggest similarly aligned molecules, and seem to explain the observed regiochemistry of cyclodimers. Analogous stereochemistry of cyclodimers can been explained in terms of parallel alignment of guest molecules [14.47, 14.481. Norrish Type I Reactions. Photodecarbonylation of dibenzylketone (22, R = H) to give dibenzyl and carbon monoxide is a typical Norrish type I reaction [14.49, 14.501. The photolysis of unsymmetrical dibenzyl ketone (22, R = CH3) in solution gave two kinds of homo (AA and BB) and crossed bibenzyl (AB) in the ratio 1:1:2.
22
This ratio was significantly changed by the presence of CTAB micelles. The statistical ratio was observed when the surfactant concentrations were much smaller than that of the starting ketone, but when the surfactant concentration was close to that of ketone or CMC, only the crossed dimer was produced. This is because the micelles act as a solvent cage which confines the resulting radical pair.
279
14.2 Photochemical Reactions 22
hv
- co
AA
A0
f
+
00
Turro et al. [14.51] reported an interesting observation of enrichment of 13Cin 22 (R = H) during the photodecarbonylation of I3C-labeled22. The triplet radical pair (23) formed in CTAB micelles either dissociates into free radicals, forming bibenzyl and carbon monoxide, or undergoes intersystem crossing (isc) to yield the singlet radical pair (24), regenerating the starting ketone 13C-22.The enrichment of I3C in the recovered ketone 22 is explained by the enhanced isc of the triplet radical pair 23 containing 13Catoms.
Addition of micelles is known to suppress the formation of radical-derived products in the Norrish type I reaction. Typical examples are the photolyses of dimethyldeoxybenzoin [14.52, 14.531 and adamantylphenylketone [14.54, 14.551.
The Barton Reaction. The Barton reaction of alkylnitrile (25) produces oxime (26) via 0-NO homolysis to yield 27, intramolecular hydrogen abstraction, radical coupling, and isomerization to the oxime. This reaction was found to be inhibited by micelles. There may be several explanations, including the polarity of the medium, but no dramatic change in the photolysis was observed in a variety of solvents, polar and nonpolar [14.56]. Potassium dodecanoate (KDC) micelle retarded the Barton reaction involving a cyclic transition state and hydrogen abstract ion on account of its microviscosity [14.57]. Instead, the resulting alkoxy radical undergoes disproportionation, giving alcohol (28A) and ketone (28K). 0
0
NOH OH
OH
ON0
OH 26
0
25
OH 27
0 OH
OH 28A
v 0
28K
280
14
Molecular Orientation of Organic Compounds in Electric Fields
Cycloadditions. Photochemical cross-additions of olefins are likewise controlled by electrostatic fields. For example, the regiochemistry of the photoaddition of olefins to cyclopentenone, yielding 29 and 30, was dependent on the reaction medium or the presence of micelles [14.58]. The polar carbonyl groups are directed to the outer interface of the KDC micelle, and the hydrophobic carbon chains to the inner core. This orientation effect explains the observed regiochemistry of the adducts. 0
0
29
30
Hydrogen Abstraction. Excited triplet ketones abstract hydrogen atoms from solvent molecules to yield alcohols and/or 1,2-diols. Photolysis of a mixture of SDS and benzophenone-4-carboxylateleads to the insertion of the benzophenone carbonyl into the alkyl chain of SDS [14.59, 14.601. Detailed product analyses showed that the excited carbonyl showed a random attack on the entire chain of SDS from C-5 to C-11. This suggests extensive coiling and folding of the detergent chains in the micelle.
14.2.6 Reversed Micelles Some amphipathic materials from molecular aggregates in hydrophobic solvents, e. g., hexane, benzene, and chloroform. These are called a reversed (or inverse) micelle in comparison with the normal micelles in water. Typical surfactants for reversed micelles are sodium bis(2-ethylhexyl)sulfosuccinate (AOT), dodecylammonium propionate (DAP), and didodecyldimethylammonium chloride (DDDAC). They form linear aggregates at lower concentrations but yield (especially in the presence of water droplets) spherical reversed micelles at higher concentrations [14.61].
Amphiphilic ionic pairs (31 and 32) can easily be prepared by mixing SDS with Nalkylpyridinium halides or CTAB with sodium alkylcarboxylates, respectively, followed by extraction with hydrophobic solvents such as CH2C12and benzene.
31
14.2 Photochemical Reactions
281
The amphiphilic ion pairs obtained form micelles, and sometimes vesicles, which are characteristic of self-associating reaction fields. Ionic substrates, e. g., carboxylates protonated amines, or pyridines with long alkyl chains, are capable of forming micelles or premicelle aggregates. Aggregation numbers of amphiphilic ion pairs and surfactant substrates have been measured by NMR spectrophotometry [14.62, 14.631, or fluorescence probes [14.64, 14.651 as well as directly by light scattering [14.66]. The resultant 1 : l aggregates 31 ( R = -CH=CHPh) and 32 ( R = -CH=CHPh) have nA= 3-7 in cyclohexane, and nA = 63, respectively, which are close to the values in aqueous SDS micelles.
Dimerizations. Equimolar mixtures of protonated stilbazolium ions (trans-12, R = H) with aerozol OT (AOT) anions form reversed micelles which are soluble in organic solvents, e. g., chloroform, carbon tetrachloride, and benzene. Aggregation of stilbazolium ions was evidenced by the fact that the micelle system exhibits a strong excimer fluorescence, whereas a homogeneous solution emits only monomer fluorescence at similar concentrations. The ion pair in the reversed micelle yielded mainly the syn-HH (18) dimer along with minor fractions of syn-HT (19) and anti-HH (20) dimers t14.671. This reaction system is convenient procedure for product isolation. The reactions mixture after irradiation is treated with conc. HCl aq. followed by extraction with dichloromethane. Reversed micelles include water droplets of various size in their core. Larger droplets tend to lower the stereoselectivity of syn-HH dimer 18, implying a loosening of molecular packing in the reversed micelle. Cinnamic acids form ion pairs with laurylamine which are soluble in hydrophobic solvents as small aggregates consisting of several ion pairs. Irradiation of ion pair (32) resulted in the formation of syn- and anti-HH dimers (33 and 34) in a ratio of ca. 4: 1 [14.68]. The observed stereochemistry of the ion pair aggregates was quite different from that in solid state, where dimer 33 was formed, reflecting the packing of the crystal structure. The quantum yield for syn-HH dimer formation was 0.15, over 20 times higher than the value in the absence of laurylamine.
Another interesting point is a solvent effect on the stereoselectivity of the photodimerization of laurylammonium 2-indenecarboxylate (35) [14.69]. A preferential dimer formation from syn-HH (36) to anti-HH dimers (37) has been observed with increasing solvent polarity. It is apparent that polar solvents may change the molecular alignment in reversed micelles in organic solvents.
282
14 Molecular Orientation of Organic Compounds in Electric Fields
36(~yn-H H)
37(anti-HH)
38(anti-HT)
14.2.7 Vesicles Vesicles are aggregates, much larger than micelles, having a spherical or ellipsoidal closed bilayer structure [14.701. Aggregates from phospholipids are called liposomes. The polar head groups of the lipids are exposed to the aqueous phase on both sides of the bilayer, and the hydrocarbon chains align themselves in the inner core, forming the bilayer. When guest molecules are included in such a molecular aggregate, it is interesting to know how they are organized and how their molecular conformation affects photochemical reactivity. Lipid vesicles and liposomes are known to have phase-transition temperatures, T, at which the alignment of surfactant molecules in the bilayers is changed. As explained previously for micelle systems, the relative intensities of vibronic bands in pyrene fluorescence, 1373/1385, can be used to study the polarity of the solubilization sites. Pyrene was found to be solubilized in a rather polar region near the polar head groups below T,, whereas it migrates into the nonpolar hydrophobic core region above T, [14.71]. At least two different solubilization sites exist in the vesicles. Solubilization sites of guest molecules are also dependent on the properties of the guest molecules. Dipalmitoyllecithin (DPL) vesicles, for example, are reported to accommodate 4,4’-disubstituted transstilbenes (39-41) on their polar interfaces or in the organized bilayer region, depending on the chain length of the alkyl substituents; i.e., they are solubilized in the polar interface region in the case of shorter chains, but in the bilayer core in longer chains [14.72]. Further evidence of the solubilization sites was also produced by measuring the rates of bromination of substituted stilbenes in DPL vesicles, where the bromination obeyed a two-component, first-order rate equation [ 14.731, Triplet decay rates of 4-nitro-4’methoxystilbene are quite sensitive to polarity, viscosity, and pH of its microenvironment. Triplet decay is divided into two components, i. e., slower and faster decays, which are interchangeable, depending on the relationship of reaction temperature to T, [14.74].
41
14.2 Photochemical Reactions
283
Isomerizations. Photoisomerizations between cis- and trans-olefins are among the most important photochemical reactions. The process is closely related to an excitation of the visual nerve in animals, via the stereoselective isomerization of 11-cis-retinal into the all-trans form of rhodopsin. Cis-trans photoisomerizations of olefins are useful for the preparation of cis-olefins, and the reaction media sometimes affect the isomerization, on account of their polarity, viscosity, or solubility. Hence, microheterogenous fields such as micelles, vesicles, mono- or bilayer membranes would be expected to function as organized media controlling the photoreaction pathways. Substituted stilbenes (39-41) were subjected to photochemical cis-trans isomerization in SDS micelles, DPL and sodium dicetylphosphate (DCP) vesicles [14.72]. The substituted stilbenes are solubilized at different sites in these heterogeneous fields. Suddaby et al. have concluded that the inner phase of SDS micelles is similar to homogeneous solutions, and the micelles have no effect on the photochemical isomerization. In contrast, DPL or DCP vesicles provide an organized environment, in which the structural change of adsorbed stilbenes is restricted and exhibits a discontinuity at T,.
No example is available of the utilization of vesicle system for preparative-scale photochemical reactions. However, vesicle aggregates provide an excellent system for photochemical charge separation in the aqueous compartments insulated by bilayers. Interstingly, photochemical electron transfer across vesicles is closely related to natural photosynthesis in thylakoid membranes. As described later, the essential process is charge separation on organized interfaces after photochemical electron transfer between a photosensitizer and an electron acceptor. A large number of papers have been published concerning electron transfer in such systems.
14.2.8
Mono- and Multilayer Membranes
When surface active agents are spread on water, they form a monolayer film, directing their hydrophobic alkyl chains toward the atmosphere. Successive deposition of monolayers on appropriate supports produces a multilayer. Mono- or multilayers transferred from the air-water interface to glass slides are called Langmuir-Blodgett (LB) films and offer possibilities for controllable photochemical molecular devices [14.75]. Energy and Electron Ikansfer. The great advantage of mono- or multilayer assemblies is based on the artificially controllable arrangement of surfactant aggregates. Layered assemblies are suitable to investigate photochemical energy or electron transfer processes between organized donors and acceptors, indicating the importance of intermolecular alignment. Though some stereoselective photodimerizations have been investigated [ 14.431, LB films are not suitable, for preparative-scale photoreactions; they are often unstable, and scale-up is quite difficult. An elegantly organized energy transfer has been studied by Kuhn and Mobius [14.75, 14.761. They constructed multilayer assemblies consisting of oxacyanine (42) and thiocyanine (43), insulated by pure arachidic acid layers. Energy transfer from
284
14 Molecular Orientation of Orgatlic Compounds in Electric Fields
excited 42 to 43 was confirmed at a distance of 50 A by the observation of sensitized yellow fluorescence from 43*. Energy transfer was not observed at 150 A.
I
Y
I
C18H37
Cl8H37
42 (X = 0); 43 (X = S)
A systematic study has been reported on the dependence of energy transfer efficiencies on the distance between D* and A (Eq. la). The sophisticated multilayer assemblies are composed of Ru(II)-(2,2'-bipyridine),(2,2'-bipyridine-4,4'-dicarboxylic acid)2+ donor layer (D) and l,l'-dioctadecyl-4,4'-carbocyanine acceptor layer (A) insulated by arachidate spacer layers. The effect of spacer distance d on the relative luminescence intensities, of D*, was studied, where I d and I, are the intensities of the ruthenium luminescence in the presence and the absence of A [14.76b]. Since the fluorescence maximum of D overlaps the absorption spectrum of A, Forster-type resonance energy transfer is operative [14.77, 14.781. An interesting photochemical electron pumping system utilizing alkyl viologens as electron carrier has been constructed by Tabushi et al. 114.791. It is interesting to note that the efficiency of electron transfer was dependent on the chain length of alkyl viologens, reflecting the hydrophobicity of its reduced form (Fig. 14.8) [14.80]. When dioctylbi yridinium ion (C,V") was reduced to its radical cation (cgv'x) by excited Ru(bipy)!+, CgV+*was shown to disproportionate to yield a neutral diradical (C,V) which is quite soluble in the organic phase. 2C&+'
z=== C8V2+ +
CSV
By using a water-oil two-phase system, the two-electron reduced viologen was used to reduce 1,2-dibromides [14.81]. A bilayer membrane of dipalmitoyl-D,L-phosphatidylcholine(DPL) may adsorb Ru(bipy):+ and octadecyl viologen (C,,V") on the inner and outer interfaces divided by the hydrophobic bilayer. Electron transfer occurred through the bilayer membrane to yield a pair of oxidized and reduced species, Ru(bipy)?+ and c,gv+X ,separated on both sides, the quantum yield being as high as 0.027 [14.82]. A similar charge separation was reported for ZnTPyP' and viologens in lipid vesicles [14.83, 14.841.
Aqueous Phase I
Oil
Aqueous Phase II
Q ~J:~,RR
0 FMN
Figure 14.8. Photochemical electron pumping using alkyl viologen as mediator.
14.2
I
Photochemical Reactions
285
AQDS D : ZnTPyP
Bu
a)$;"
A
EDTA
0
DBA
bilayer
Figure 14.9. Two-step photochemical electron transfer across a bilayer membrane.
Concurrent excitation of ZnTPyP adsorbed on both sides of ribosome bilayers interfaces (Fig. 9) is an elegant model system for electron transfer in photosynthesis [14.85], [14.86]. In the photochemical electron transfer across the bilayer membrane, excited ZnTPyP was reduced by EDTA (ethylene diamine tetraacetic acid) in the inner aqueous phase and, the electron was transferred through DBA as a mediator to another ZnTPyP on the opposite interface. The second step is photochemical reduction of anthraquinone disulfonate (AQDS) to AQDSH2 [14.87].
Norrish Q p e I and I1 Reactions. Photolysis of carbonyl compounds (44) results in homolytic a-fission and/or y-hydrogen abstraction (Norrish type I, and I1 reactions). 0
hv
C6H5
Norrish type I
*
). C6H5
+
.CH2
44
C & j C H O + CH3(CHz)zR, etC.
Cyclization
C6H5
Cleavage
+ CH;?=CHR
It appears that the Norrish type I1 reaction is dependent on the reaction medium because of the stereochemical requirement for intramolecular hydrogen abstraction via a six-membered transition state. Hence, the Norrish type I1 reaction via the cyclic transition state would be unfavorable. The quantum efficiencies for the photochemical reaction of butyrophenone (44, R = H) suggest that butyrophenone, when incorporated in a LB film, is constrained and unfavorable for the cyclic transition state [14.88]. Almost equal efficiencies in micelles and standard solvents show that there is no significant viscosity difference between them. This agrees with an observation that the micelle interior is disordered and not so viscous as tert-butanol [14.89]. When butyrophenone is incorporated in the LB film of arachidic acid, neither type I nor type I1 reaction takes place at all. In the LB film, butyrophenone is assumed to be organized as rigid molecular aggregates, and hence the radical pairs once formed tend to recombine, giving the original ketone.
286
14
Molecular Orientation of Organic Compounds in Electric Fields
Similar to the micelle system, the stereochemistry of the cyclodimer reflects the organized aggregates of olefins accommodated in bilayer membranes [14.90, 14.911.
14.2.9 Polyelectrolytes Polyelectrolytes interacts intimately with oppositely charged surfactants [14.92-14.951. For example, micelle clusters were formed on dispersal in water, when sodium poly(styrenesulfonate) (PSSNa) forms a complex with CTAB. This means that cationic substrates may be adsorbed on polyelectrolyte aggregates. In fact, thousands of stilbazolium ions can be effectively adsorbed in one strand of PSSNa, which has been found to have micelle-like clusters. The polyelectrolyte PA-18K2forms a compact oil at lower pH and an extended rod or coil at higher pH. The polyelectrolyte can bind cationic or anionic pyrene derivatives on its micelle-like compact coil. Thus, PA-18K2 with molecular weight 10 000 forms a polymer micelle composed of ca. 25 monomer units, as confirmed by pyrene fluorescence [14.96]. Similarly, poly(acry1ic acid) exists as a coil at lower pH and in expanded form at higher pH, which is supported by the lack of pyrene excimer fluorescence at high pH [14.97]. Addition of CTAB to poly(methacry1ic acid) solution yields micelle-like aggregates in which ca. 100 CTAB molecules are accommodated. Quenching studies with pyrene excimer probes have been successful in determining nAvalues of polyelectrolyte complexes in combination with 1-dodecylpyridinium chloride (DPC) as quencher [ 14.981. Poly(styrenesu1fonate) also forms a micelle-like cluster with stilbazoliurn ions; with n A ca. 100 [14.30]. C02K I fCHCH-CHCH)I; (cH2)15
COpK
CH3 PA-18K2
The structure of hydrophilic aggregates of polyethylene oxide-propylene oxide-ethylene oxide block copolymer (EPE) was investigated by three fluorescence probes [14.991: an indole detergent (In), pyrene (P), and pyrene-3-carboxaldehyde (PA). Fluorescence maxima of In and PA, and the intensity ratio 1373/1385of pyrene fluorescence show that the hydrophobicity of EPE increases with increasing pressure and with decreasing temperature. As a fluorescent probe for polyelectrolytes, the dansyl group [14.100, 14.1011 and diphenylanthracene [14.102] have also been reported.
In
P
PA
14.2 Photochemical Reactions
287
Polysoap, poly(viny1benzo-18-crown-6) (P18C6) adsorbs pyrenylbutylammonium ion and other species [14.103]. The pyrenyl groups are inserted between adjacent crown ether ligands with the -NMe,+ group protruding into the aqueous phase.
Poly(styrenesu1fonate) anion provides a polyanionic field with 100-5000 anionic sites, which are expected to form much larger molecular aggregates of cis-stilbazolium ions. In fact, sodium poly(styrenesu1fonate) with a degree of polymerization of 5000 was shown to gather ca. 4300 molecules of cis-12 on its polymer chain. However, the observed quantum yields for the isomerization were not as high as those expected from the degree of polymerization [14.98], being always around 100, irrespective of the size of the aggregates. This almost constant quantum yield suggests formation of clusters, as in micelles. The electron relay would be confined in one cluster with nAca. 90.
14.2.10 Inorganic Solid Surfaces Solid surface of inorganic materials, e. g., silica, clay minerals, and zeolites, possesse anionic or cationic sites adsorbing charged guest molecules electrostatically. These inorganic minerals are photochemically inert, in contrast to the known photochemical activities of metal oxide semiconductors. Silica Surfaces. Silica colloids consist of negatively charged spherical particles with diameter 50-500 A. The surface is covered by an electric double-layer which is made up of SiO- and OH-, and is usually neutralized by sodium ions. The sodium ions are easily exchanged with organic cations [14.1041. In water, nearly complete substitution with organic cations often results in precipitation of substituted colloids, which are sometimes soluble in organic solvents. It is interesting to note here that nonexponential decay curves were observed for the fluorescences of azobenzene, triphenylmethane dyes, and thioindigo adsorbed on alumina or silica [14.105]. The decay was discussed in terms of different aggregation sites on the polarizing surface, and of interference between directly emitted and scattered fluorescence. When the silica surface is porous, the size of the cavity controls the approach of substrates, affecting photochemical reactions. Porous here means spongelike or containing microscopic holes [14.106]. The specific surface area of porous silica usually reaches 1000 m2/g as determined from Nz adsorption. It is known that cavities with diameter 20 A are effective as adsorption sites and that silanol groups are responsible for the attractive interaction with guest molecules.
288
14 Molecular Orientation of Organic Compounds in Electric Fields
Miscellaneous Reactions. Photocyclodimerization occurs on the surface of inorganic solids. Acenaphthene (45) on silica surfaces gave cis- and trans-cyclodimers (46) [ 14.1071. Singlet excited acenaphthene forms an excimer with a neighboring molecule, leading to the formation of cis-dimer, whereas the excited triplet 45 results in the formation of the trans-isomer via a stepwise radical pathway.
/
/
45
46
47
The excited singlet S1, on account its short lifetime (ca. 1 x W 9 s ) , forms a complex with the nearest olefin on1 On the other hand, the excited triplet acenaphthene may diffuse approximate 300 within its lifetime, 1 x lo4 s. These factors are clearly reflected in the increasing cis:trans dimer ratios (46:47) with the increasing numbers of olefin molecules on the SiOz surface. The rapid diffusion of some aromatic molecules on the surface has been measured by time-resolved laser flash spectrophotometry, using a diffuse reflectance technique [14.108]. Excited triplet benzophenone has been shown to be dynamically quenched by naphthalene on silica gel with a diffusioncontrolled rate [14.109]. In the hindered ketone (48) adsorbed on silica gel, photochemical cross-addition occurs, preferentially on the more hindered side. The stereochemistry is explained by the selective hydrogen bonding of carbonyl with silanol on the silica gel surface from the less hindered side, leaving the more hindered side of the ketone open for attack by cyclopentene [14.110, 14.1111. Aromatic ketones covalently bound on silica surfaces undergo a forced photopinacolization [14.112], while the reaction in micelles affords the crossed adduct between ketones and solvent alcohols.
1
In addition, optically active sensitizers covalently bonded to silanol groups on silica surfaces, have been used in attempts to establish the chirality of N,N-dimethyl-1phenethylamine quenchers [14.113].
14.2 Photochemical Reactions
289
The anionic surface of silica gel is also effective in charge separation between an excited Ru(bipy)?+ and a sulfonate viologen (51). The sulfonate substituents neutralize the cationic charges on viologen nitrogens, and reduce the electrostatic attraction between 51 and the silica gel. When 51 is reduced, the resulting radical (51'X) is anionically charged and repelled from the surface. Reverse electron transfer is 102-103times as slow as that in the absence of silica gel [14.114].
51
Photochemical electron transfer quenching of excited pyrene by N, "-dimethylaniline (DEA) adsorbed on silica surfaces is completely ineffective below the DEA monolayer value. Such DEA inertness is interpreted in terms of its strong interaction with surface silanol groups. Beyond monolayer coverage, the slope of the Stern-Volmer plot increases markedly [14.115].
Clay Interlayers. Clays have layered structures and are readily suspended in aqueous solutions [14.116]. Montmorillonites are composed of units made up of two silica tetrahedral sheets and one alumina octahedral sheet. Their intersheet layer includes exchangeable metal ions (e. g., sodium) neutralizing the negative charge due to partial substitution of Si4+by A13+at the tetrahedral sites. This is why montmorillonites tend to adsorb cationic species and to be readily suspended in water. On the contrary, some clays, e. g., hydrotalcite, include exchangeable negative ions, leading to the effective adsorption of negatively charged guest molecules. The properties of typical clays are summarized in Table 14.5. The numbers of exchangeable ions in clay minerals determine the amounts of organic guest ions intercalated between the clay layers. For example, montmorillonite clay is capable of adsorbing guest cations according to the magnitude of its cation exchange capacity (CEC), which is expressed as miliequivalents of anionic sites per gram of clay. Hydrotalcite accommodates guest anions according to the magnitude of its anion exchange capacity (AEC), expressed as miliequivalents of cationic sites per gram of clay. Intercalation efficiencies are dependent on the type of clay and guest molecule [ 14.117-14.1191. Adsorption on clays is accomplished by adding equimolar ionic substrates aqueous colloidal clay solutions, sonication, and sometimes precipitaTable 14.5. Properties of some typical clay minerals Clay
Layer structure
Exchangeable ion
Ion-exchangecapacity (medl00 g)
Montmorrilonite Saponite Kaolin Hydrotalcite
[SiO4]/[A10,] = 2/1 = 2/1 [Si04]/[A104](orMgOJ = 111 [MgO,]octahedral
Na', Ca" Na', Ca2+
60-80 60-80 2-10 = 400
OH-, CO;
290
14
Molecular Orientation of Organic Compounds in Electric Fields
tion. In most cases, intercalated clay colloids can be homogeneously dispersed in hydrophobic solvents such as benzene, chloroform, and dichloromethane. A longchain alkyl ammonium ion, e. g., hexadecyltrimethylammonium ion (CTAC), forms its double-layer on laponite clay surfaces [14.120]. Nonionic materials may also be intercalated in clay interlayers [14.118, 14.1191. Fluorescence probes can be used to study adsorption on clay interlayers. The emission maximum of Ru(bipy);+ on clay minerals shifted linearly to longer wavelength depending o n the negative charge of the lattice and the average particle size [14.121]. This was interpreted in terms of the distribution of the ions over the external surface (i. e., edge sites) and the interlayer surface (i. e., planar sites). DeSchryver et al. have reported a fluorescence probe comprised of pyrenes with ammonium groups, the conformation of which changes depending on the degree of intercalation and the presence of coadsorbents [14.122]. Hydrophobic pyrene itself was shown to be adsorbed as a monolayer on dry smectite clay and laponite [14.123]. Desorption of pyrene was accomplished simply by adding water to give microcrystals of pyrene. The micropolarity in the region of pyrene molecules was increased by eliminating water, changing the ratio of pyrene fluorescence intensities 1373/1383 from 0.9 to 1.80.
PnN ( n = 1 - 3 )
The conformation of a pyrene-labeled poly(acry1ic acid) adsorbed at the water-alumina solid interface was demonstrated by using a pyrene excimer fluorescence probe, indicating a coiled structure at low pH, but an extended structure at high pH [14.124].
Miscellaneous Reactions. Application of clay minerals to organized photochemical reactions has revealed an interesting aspect due to the spatial confinement in clay interlayers: (i) not only cationic or anionic substrates, but also nonionic polar ones can be efficiently accommodated in the interlayers; (ii) long alkyl chain guest molecules such as CTAB can be arranged in a uniform manner, similar to those of LB films; (iii) intercalation of some surfactants, e. g., CTAB, makes the clay particles soluble hydrophobic solvents such as benzene, dichloromethane, or carbon tetrachloride; (iv) clay colloids adsorbing organic guests from essentially transparent films by simple casting or filtration in vacuo. Clay interlayers are also quite useful electrostatic fields for photochemical cyclodimerization. Stilbazolium ions intercalated on montmorillonite interlayers were shown to undergo a selective cyclodimerization to yield syn-HT dimer C14.125, 14.1261. The high regioselectivity for syn-HT dimer clearly indicates the predominant antiparallel alignment of stilbazolium ions, alternatively adsorbed on upper and lower layer surfaces of the clay (Fig. 14.10). It is known that clay minerals may form a bilayer film of intercalated alkylammonium ions [14.127]. Interestingly, only excimer fluorescence was observable from clay-intercalated stilbazolium ions over a wide range of coverage (0.1-20 %). This indicates that the strong excimer fluorescence, even at 0.1 % coverage, is due to the formation of a cluster of olefin molecules [14.128]. Cluster formation
14.2 Photochemical Reactions
291
Figure 14.10. Schematic representation of stilbazolium ions (rods with positive charges) in the interlayers of saponite clay.
is supported by the fact that the preferential formation of syn-HT dimer is always observed, regardless of the degreee of coverage, but the adsorbed stilbazolium cations depend on an adsorption equilibrium with the external bulk solution [14.129]. Hydrotalcite, an anion-exchanging clay, adsorbs anionically charged olefins, such as cinnamate or stilbenecarboxylate anion, in its interlayers. The adsorption equilibria depend on the type of anion. For example, ca. 37 % of cinnamate anion was intercalated from an equivalent mixture of the clay and cinnamate anion, but stilbenecarboxylate was adsorbed quantitatively. The intercalated carboxylates were photodimerized to give the corresponding syn-HH dimers exlusively [14.126]. Noteworthy here is the absence of E-Z photoisomerization, in contrast to the reaction in homogeneous solutions. The selective formation of syn-HH dimers also indicates the close packing of olefin molecules. The same cis-trans isomerization of cis-12 also takes place on silica colloid surfaces with a quantum efficiency above unity. Since the adsorption of olefins caused the precipitation of colloids, it was impossible to adsorb the olefins sufficiently to induce an efficient electron relay reaction [ 14.1301. Clay interlayers are sometimes used for photochemical charge separation [14.131, 14.1321. Ru(bipy):+-sensitized electron transfer occurs in the presence of methylviologen, MV2+,triethanolamine, and clay colloids; reduced viologen radical cations donate electrons to protons, yielding molecular hydrogen. Efficient charge separation was observed when the sensitizer and methylviologen were adsorbed separately on different interlayers [14.133]. The separate adsorptions may be depend on the different ions densities on different layers [14.134]. Hydrogen generation was also observed with Cu2', Eu2+,nitrobenzene, and dimethylanilinium ion as electron acceptors [14.135]. These acceptors diffuse into the clay interlayers at rates close to those observed in homogeneous solution [14.129]. A remarkably enhanced decomposition of water was observed on irradiation of a mixture of Ru(bipy)?+-adsorbed sepiolite clay and Eu2'-coated aluminum hydroxide in water, where RuOz and platinum colloid are coated on the sepiolite clay [14.136, 14.1371. Zeolite Cavities. Zeolites [14.138] are porous inorganic materials made from tetrahedral metal oxides, A104 and Si04,forcing guest molecules into three-dimensional steric confinement. Zeolite cavities like clay interlayers, include alkaline metal cations exchangeable with guest ions. Zeolites are classified into hydrophilic and hydrophobic types, calles zeolite X andY, and silicalite, respectively (Table 14.6). The structure of X- or Y-type zeolites consists of a 13 A diameter cavity (which is called super-cage) linked by a path diameter 8-10 A,which may accommodate hydrophilic guest molecules. Silicalite possesses an inner cavity of diameter 5-6 A and absorbs hydrophobic guest molecules.
292
I 4 Molecular Orientation of Organic Compounds in Electric Fields
Table 14.6. Representative molecular sieve zeolites Zeolite ~
5Pe ~
A
x
Y L
ZSM-5 Silicalite Mordenite
[AIO,]/[SiO,] ~
faujasite chabazite pentad zeolite
~~
~~
111 112.2- 113 112.6 1/1 1/31 = 1120 = 115
Channel, ~~~
4 8 8 3.1 5.4 3 x 6
A
Cavity diameter, 8, ~~~
11 13 13
7
Organic molecules included in zeolite cavities exhibit characteristic emission properties. Several aromatic ketones, such as acetophenone, benzophenone, and pphenylpropiophenone in zeolites show readily detectable phosphorescence at room temperature with a lifetime of 0.18-0.30 ms [14.139].Thus, the lifetime of excited triplet pphenylpropiophenone at room temperature is enhanced by about five orders of magnitude upon inclusion into the silicalite [ 14.1401.This is attributed to the restricted mobility in the cavity. On the basis of the nonexponential phosphorescence decay of the ketone triplet, silicalite must have at least two distinct inclusion sites. The pyrene fluorescence intensity ratio 137J1385 indicates a very polar medium for pyrene in zeolites X (0.45) andY (0.74). There is an intrinsic difference between zeolite cavities and clay interlayers; the capacity of zeolite cavities is not sufficient to accommodate more than three guest or reagent molecules, which implies no intermolecular reactions or photochemical electron transfer. However, unique unimolecular reactions can be expected in such a spatially confined reaction field. These aspects are in sharp contrast to the case of clay interlayers, where intermolecular reactions are easily accessible because of the intercalation of many guest molecules. Dimerizations. Photocyclodimerization of acenaphthene was found to be susceptible to spatial restriction in zeolite cavities. When the olefin was irradiated in a Y-type zeolite with a super-cage diameter of 13 A, the cis-cyclodimer (diameter ca. 8 A) was formed through exciplex formation in the cage. The trans-cyclodimer (ca. 14 A) is too big to be accommodated in one cavity and is believed to be formed in the cavity linking two super-cages 114.1411.The size of metal cations in the super-cage was found to affect the ratio of cis- and trans-dimers by changing the free volume of the cage. Isomerizations. An interesting selective formation of trans-stilbene was observed in silicalite. Irradiation of cis-stilbene was carried out in the presence of silicalite using 3,4dimethylbenzophenone as sensitizer. Trans-stilbene (trans-52) once formed is intercalated selectively in the 6 A diameter cavity, resulting in the irreversible formation of pure trans isomer [14.142].
cis-52
trans52
14.2 Photochemical Reactions
293
Norrish 'Qpe I and I1 Reactions. The ratio of cyclization to @fission in Norrish type I1 reactions is dependent on the rigidity of the reaction medium. The Norrish type I1 reaction is rigorously inhibited by the restricted reaction fields in inorganic zeolites; silicalite-intercalated valerophenone (44, R = CH3), on irradiation, reacts exclusively by 6-elimination [14.143]. However, on intercalation in X-type zeolites with a larger space volume (13 A), cyclization to cyclobutanol occurs to an extent comparable to pelimination [14.72].The increasing size of alkali metal ions from Li' to Cs' affects the ratio of cyclization to 6-fission in zeolites [14.144]. Singlet and triplet excited state lifetimes depend on the identity and accessibility of the cation present within the supercage of X-type faujasite zeolite (Li, Na, K, Rb, Cs, Tl). The phosphorescence-tofluorescence intensity ratio for acenaphthene in X-type zeolites with Cs' is ca. 1000 times that with Li' [14.145]. The photolysis of isomerically pure d, 1-, or meso-2,4-diphenylpentan-3-ones(53) adsorbed on X- and Y-type zeolites resulted in diastereoselective formation of 2,3diphenylbutanes (54) with preference for d, 1-over meso-isomers [14.146]. This is in sharp contrast to the reactions in homogeneous or micellar solutions with quite poor diasteroselectivity [14.1471.
53
54
Zeolite cavities discriminate between molecular sizes, and control product selectivity. The photolysis of o-methyldibenzyl ketone (55) in hydrophobic zeolite (ZSM, cavity diameter 6 A) gave only symmetric dibenzyls (56 and 58), while the p-substituted ketone yielded asymmetric dibenzyl (e.g., 57). Turro et al. have explained that the o-substituted benzyl radical, but not the p-substituted radical, is too large to be accommodated in the cavity and therefore diffuses into the bulk [14.148]. Unsubstituted and p-methyl substituted benzyl radicals dimerize in the cavity, but o-substituted radicals dimerizes only in the bulk solution.
\
CH3
55
CH3
59
60
294
14 Molecular Orientation of Organic Compounds in Electric Fie1d.y
A large number of radical pair photorearrangements are known in homogeneous organic solvents. It is intriguing to see that molecular movements may be affected by organized media, controlling the rearranged products. Zeolites are crystalline aluminosilicates with cages and channel systems that can host a variety of organic transformations. This intracrystalline space is akin to a solvent and can be described in terms of solvatochromic indicators. For example, a faujasite zeolite includes a solvatochromic indicator in its cage as a zwitterion stabilized by Lewis acid (Na+) and base (the oxygen of the framework) [14.149]. Irradiation of 55 in X- or Y-type zeolites with a larger cavity of diameter of ca. 13 8, was subject to photorearrangement, to form 59 and 60 [14.150]. In the restricted space of the cavity, radical pairs have a longer lifetime, enough to rotate freely leading to the observed rearrangement. Exchangeable cations in zeolite cavities significantly affect product distributions t14.1511. On going from Li+ to Na' to K', rearranged products (e. g., 59 and 60) are increased at the expense of formation of 57. This correlates to the steady decrease in the amount of void space in a zeolite on going from Li' to Na' to K' . The product distribution from dibenzylketones was found to be dramatically dependent on the loading percentage of the starting ketone in zeolites. Increasing amounts of loaded ketone result in an increase of rearranged products and a decrease in diphenylethanes, which is explained on the basis of rapid diffusion of the resulting radical pairs [14.152].The photochemistry of zeolite-intercalated reactants is very sensitive to simple coadsorbates such as water, benzene, cyclohexane, and hexane [14.153]. Inclusion of organic molecules in the zeolite super-cage causes a significant decrease in void space, and will restrict the diffusional and rotational motion of radical pairs produced by photolysis of 55. As a result, the formation of 56-58 was suppressed, but the yields of 59 and 60 were increased. The addition of water causes quite a different result [ 14.151bl. Preferential adsorption of water molecules at the cavity surface induces displacement of 55 to the external surface. Hence, the radical pairs diffuse freely, drastically decreasing the formation of 59 and 60 [14.150-14.1541. The homolysis of cyclic ketone (61) in zeolite cavities is reported to give ringexpanded ketones (62). This contrasts to the results in solutions (good yields of alkenols) [155]. The molecular size of 61 with n > 15 is too large to be intercalated in the cavity. However, the ring-expanded ketones (62) were formed in the cavity on irradiation in the presence of X-type zeolite. Radical pairs formed by the photolysis of 61 can now enter the cavity and cyclize at the para-position of the phenyl [14.156].
Electron Tkansfer. Zeolites are used as templates for organizing photochemically active molecules in a photochemical electron transfer involving Ru(bipy);+, and MV2+. On irradiation of trapped Ru(bipy):+, MV' x was formed in neighboring cages, which was stable for several hours [14.157]. As an improved system for photochemical electron transfer, the diad system, D-S-A, has been developed [14.158].
14.3 References
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Index
a.c. fields, protein disruption 255 a.c. voltages, water-in-oil emulsions 223 acceptors, electron transfer 271 Acilyzer, ion-exchange chambers 234 ADI: voltage-programmed electrochromatography 44 adsorbents - annular columns 128 - solid-phase 245 - two-dimensional chromatography 126 adsorption - inner surface modification 53 f - potential-dependent 80 AFP (a-fetoprotein), latex immunoassay enhancement 261 agglutination rate, cuvette reactor 250 agglutination reaction, immunoassays 7 agglutinations, a.c. fields induced 256 alignement, molecular 266 alkali metal ion separation, crown ether 80 alteration, column support 35 alternating current field, latex immunoassay enhancement 254 f b-alumina 155 alumina, ceramics forming 156 alumina powders, zeta potentials 155 aluminum ogives 156 AMP, voltage-programmed electrochromatography 44 ampholite, counter-current electroconcentration 2 4 1 ampholite buffer, counter-current electroconcentration 242 amphoteric electrolyte, pressurized flowdriven electrochromatogy 31 anhydrous ERF 179 anion separation, conducting polymers 83 anions, inorganic, ion-exchange chromatography 41 annular bed, adsorbent, two-dimensional chromatography 126 annular columns, adsorbents 128 antibodies, pulse immunoassay 249 antigens, pulse immunoassay 249 antivibration mounting, ERF 181 apparent mean linear flow velocity, electrochromatography 14 f apparent viscosity, ERF materials 174
applications - car production processes 6,171 - concentration 7,229 ff - electro-rheological fluids 171 ff, 181 - immunoassay 7,229 ff - industrial, electroosmotic dewatering 147 f - industrial processes 5,131 ff - molecular orientation 7,229 ff - solvent extraction 6,185 ff - water-in-oil emulsions 6,205 ff applied electrostatic fields 266 f aqueous micelles, photochemical reactions 277 aqueous solutions, inorganic particles 154 f asparagine, PTH-derivatives 34 asphaltic materials, oil industry 215 ATP, voltage-programmed electrochromatography 44 axial mixing coefficient, electrostatic liquid-liquid extractor 195 axial molecular diffusion, electrochromatography 14 azo dye, poled film 266 band broadening, electrochromatography 13 f Barton reaction 279 beds, two different, mixture separation 4 bentonite clay 137 benzoic acid - chromatographic variation 35 - eluent composition 39 bilayer membrane 284 Bingham flow 173 biomolecular analysis, electrochromatography 4,91 ff biomolecules 92 ff biosciences 91 ff Blue Dextran, mixture separation 128 boundary layer, thin diffusional 191 bubbles - electroosmotically driven chromatography 72 - pressurized flow-driven electrochromatography 31
302
Index
CACE (counteracting chromatographic electrophoresis) 105 Candida albicans - calibration 254 - pulse immunoassay 249 capacitance factor - electrochromatography 14 - electrosorption 82 capillary columns - octadecylsilane-modified 65 - open-tubular 56 - pressurized flow-driven electrochromatography 29 - slurry-packed 52,70 capillary electrophoresis (CE) 108 f capillary gel electrophoresis (CGE) 111 capillary tubes, zone profiles - rectangular 21 - round 26 capillary zone electrophoresis 109 car production processes 6,171 carbon, electrodeposition 78 carbon powder, ion separation 77 carboxylic acid, ion-exchange chromatography 42 cation exchange capacity (CEC) 289 cell manipulation, pulse immunoassay/ electrovoltage 249 ff central portion, zone front 24 ceramic sheet, continuous, electrophoretic forming 166 ceramics. forming 153 ff - electrophoretic 5 CGE (capillary gel electrophoresis) 111 chain formation, water-in-oil emulsions 208 chambers, ion-exchange, electrodialysis 233 f charge, effective, electroosmotic dewatering 138 charge density - electroosmotic dewatering 141 - silica gel surfaces 53 charge separation, photochemical reactions 272 charged double-layers 265 charged drops - pendant, solvent extraction 193 - solvent extraction 186 charged inner surfaces 27 f charged molecules, flow profiles 27 f chelate resin 175 f chromatographic behavior, pressurized flowdriven electrochromatography 32
chromatography - electro- see electrochromatography - electroosmotically driven 59 f - two-dimensional 126 f cimetidine, solid-phase extraction 245 circulations, hybrid, solvent extraction 190 clay interlayers 289 clays - ceramics forming 158 - colloids, intercalated 290 - electroosmotic dewatering 133 - spontaneous electrostatic fields 270 coagulation processes, analytical, pulse immunoassaylelectrovoltage 249 ff coalescence - electrostatic liquid-liquid extractor 195 - water-in-oil emulsions 209 coalescer - continuous-flow electrostatic 221 - electrostatic, oil industry 214 collection, selective electric 233 colloids, clay, intercalated 290 column chromatography 2 column electrochromatography 2 column support alteration 35 columns - capillary, slurry-packed 70 - electrode 76 f - electroosmosis 66 f - micro-, electrochromatography 108 f - packed, electroosmotic flow velocity 56 - preparative 97 - pressurized flow-driven electrochromatography 29 compositions, eluents, flow velocities 39 compressible solid-liquid mixtures 137 f compressive pressure distribution, electroosmotic dewatering 139 concentration - electric fields applications 7,229 ff - electrochromatography zones 17 concentration enhancement 240 concentration processes, analytical chemistry 231 ff conducting particles, pulse immunoassay 249 conducting polymers, anions separation 83 conductive stationary phase 75 consolidation, electroosmotic dewatering 145 continuity equation 140 continuous annular electrochromatography 128 continuous electrodialysis 248
Index
continuous incolumn sample focusing 117 f continuous injection, two-dimensional chromatography 126 f continuous paper electrochromatography 2 continuous preparative electrochromatography 128 continuous samples, electrochromatography zones 17 continuous sample introduction, in-column sample focusing 118 continuous-flow electrostatic coalescer 221 contractors 196 - mixer-settler type 185 - spray 185 convection flow, band broadening, electrochromatography 19 corona charging, organized photochemistry 265 correction factor, electroosmosis 52 counter-current, electrochromatography 4, 117 ff counter-current electroconcentration 236 f - apparatus 239 counter-ions exchange 234 counteracting chromatographic electrophoresis (CACE) 105 critical void ratio, electroosmotic dewatering 141 crown ether - alkali metal ion separation 80 - carbon 78 crudeoil 205 current density, electroosmotic dewatering 142 cuvette reactor, pulse immunoassay 250 cyclic voltammograms 84 cycloadditions 280 cylindrical capillary 52 d.c. voltages, water-in-oil emulsions 223 decomposition, metal complexes, electrochemical reactions 247 f dehydration - oil industry 213 f see also dewatering dehydrating crude oil, electrostatic technology 205 dehydrator, filter-press electroosmotic 148 demulsification 6 desalting - electrodialysis 233 f - oil industry 215 - oligosaccarides 234
303
desalting time, pH-dependence 237 dewatering - electroosmotic 5,133 ff - progresses, electroosmosis 137 diameter - effective, electroosmotic dewatering 136 - particles, ceramics forming 160 dielectric constant - ceramics forming 156 - eluent composition 40 dielectric induced force, a.c. field 254 dielectric powder suspension in oil 171 dielectrophoresis, water-in-oil emulsions 206 f dieletrophoretic movements, water droplets, computer simulations 208 diffuse double layers - electrochromatography zones 21 - electroosmosis 48, 57 diffusion, molecular - electrochromatography zones 19 - electroosmosis 61 diffusion coefficient, electrochromatography 18 diffusion processes, band broadening, electrochromatography 14 f dimerizations, photochemical reactions 277 dipole chain 208 dipoles, electric, water-in-oil emulsions 206 disodium fluorescein, charged molecules flow profiles 28 dispersed phase 175 f dispersion - electrostatic, solvent extraction 187 - liquid 192 f - sample, pressurized flow, electrochromatography 19 disruption, solvent extraction 192 dissociation constant, silanols 38 DNA 92 - counter-current electroconcentration 240 DNA fragments - double-stranded 100 - electropherograms 104 donors, electron transfer 271 double-layers - charged 265 - diffuse, electrochromatography zones 21 - - electroosmosis 57 - elephant 167 - thickness 51 f
304
Index
double-stranded DNA fragments 100 drams, ceramics forming 168 drop coalescence, solvent extraction 187 droplet chain, water-in-oil emulsions 207 droplet-droplet coalescence - electrostatic 194 - uniform electric field 210 drops, solvent extraction 187 f dust, electroosmosis 47 dynamic recycling electrolysis cell 231 dynamic spring constant, ERF 184 eddy diffusion, electrochromatography 14 effective charge, electroosmotic dewatering 138 effective diameter, electroosmotic dewatering 136 effective electric charge density, electroosmotic dewatering 141 electric current, pressurized f low-driven electrochromatography 32 electric dipols, water-in-oil emulsions 206 electric field effects, pulse immunoassay 249 electric field poling, organized photochemistry 269 electric flux density, electroosmotic dewatering 134 electrical conductivity, ceramics forming 156 electrical double layer - electroosmosis 48 - electroosmotic dewatering 133 electrical forming, low water content 161 electrical potential, electroosmosis 48 electrical pulses, cuvette reactor 250 electrical resistance, specific, electroosmotic dewatering 141 electrical resolution, water-in-oil emulsions 206 electro-rheological fluids (ERF), applications 171 ff electro-rheological effect 171 electroactive metals, redox separation 79 f electrochemical reactions, metal complexes decomposition 247 f electrochemical separation, ionic substances 3 electrochemistry, ion separation 76 electrochromatographic preconcentration 247 electrochromatography 1f, 11 ff, 47 ff - electroosmotically driven 59 f, 109
- pressurized flow driven - sequential 97 - thin-layer 96
111
electrochromatography/mass spectrometry 123 f electroconcentration 231 - dynamic, analytical chemistry 7 electrode columns 76 f electrodeposition - carbon 78 - metal ions 244 f - slip 153 ff electrodes - ceramics forming 164 f - micromachined reactor, latex immunoassay enhancement 260 electrodialysis, desalting 233 f electrodialysis apparatus, multiple ionexchange chambers 235 electrodynamic extractor 199 electrofractionation (EF), biomolecular analysis 101 f electroinactive species separation 83 electrolysis cell, dynamic recycling 231 electrolytes 232 - amphoteric, pressurized flow-driven electrochromatography 31 electromigration, counter-current, electrochromatography 120 electron carrier 274 electron relay chain reaction 275 electron transfer - membranes 283 - photochemical reactions 271 f - zeolites 294 electroneutrality, electroosmosis 48 electroosmosis 3,19, 47 ff, 133 ff - ceramics forming 158 - counter-current electroconcentration 241 f - theory 50 electroosmotic dehydrator, filter-press 148 electroosmotic dewatering 133 ff electroosmotic flow - band broadening, electrochromatography 13 - dewatering 134 - liquid chromatograpphy 72 - theory 50 electroosmotic flow velocity - packed columns 56 - pH 37 electroosmotic forming, ceramics 153 ff, 168
Index
electroosmotic mobility
51
- open-tubular capillary columns 56 - packed microcapillary columns 58 electroosmotic velocity, electrochromatography 14 electroosmotically driven chromatography 61 f electroosmotically driven electrochromatography 59 f, 109 electrophoresis - capillary 108 f - capillarygel 111 - gel 96 - paper 94 electrophoretic flow velocity, pH 37 electrophoretic forces, water-in-oil emulsions 206 electrophoretic forming, ceramics 5,153 ff electrophoretic mobility 2 - band broadening, electrochromatography 13 electrophoretic partition 2 electrophoretic velocity, electrochromatography 14 electrorheological fluid (ERF) 6,171 f electrosorption - carbon 78 - neutral organic compounds separation 82 electrostatic coalescer, oil industry 214 electrostatic dispersion, solvent extraction 187 electrostatic drop-drop coalescence 194 electrostatic fields - applied 266 f - photochemical reactions 8 - spontaneous 270f electrostatic force, solvent extraction 186 electrostatic interaction 75 electrostatic liquid dispersion 192 f electrostatic liquid-liquid extractor 195 f electrostatic phase separation 219 electrostatic pseudo-liquid membrane extractor 200 electrostatic repulsion, charged molecules flow profiles 28 electrostatic spray column 195 f elephant, ceramics forming 166 eluents, compositions, flow velocities 39 elution ti me - electrocromatography 14 f - potential dependence 81 emulsion breaker, oil industry 217 emulsion liquid membrane process 221
305
emulsion phases, hydraulics 218 emulsions - electric fields applications 6 - water-in-oil 205 ff energy transfer, photochemical reactions 271 f enhanced mass transfer, solvent extraction 187 f equilibria, phases, band broadening, electrochromatography 13 ER effect 172 ERF (electro-rheological fluids) 6, 171 - anhydrous 179 excimer f luorescences, organized photochemistry 268 extraction, solid-phase 245 FAB interface 123 feed stream, two-dimensional chromatography 126 feldspar, ceramics forming 158 a-fetoprotein (AFP), latex immunoassay enhancement 261 filter-press, electroosmotic dewatering 147 f flat channel, electroosmosis 52 flow - electroosmotic, dewatering 134 - hybrid, solvent extraction 187 flow channel 27 f flow path, electroosmotic dewatering 136 flow pattern - hybrid, solvent extraction 191 - solvent extraction 187 f - Taylor 189 flow profiles - charged molecules 27 f - electrochromatography 18 f - electroosmosis, open tube 19 - observations, electrochromatography zones 22 - open-tubular capillary columns 61 flow resistance parameter, electroosmosis 67 flow-through cell, metal complexes decomposition 247 flow velocity - apparent, mean linear, electrochromatography 14 f - electroosmotic, packed columns 56 - pH 37 fluorescent image 22 fluvic acid, metal complexes decomposition 247
306
Index
focusing - columns 104 - electrochromatography 4,117 ff forces, water-in-oil emulsions 206 forming, ceramics 153 ff fraction collector, counter-current, electrochromatography 121 frequencies, alternating current field 257 friction, electroosmosis 51 fused silica capillary tubes - pressurized flow-driven electrochromatograph 29 - zone front profiles 24 gel electrophoresis 96 gels, biomolecular analysis, electrochromatography 4 glass plates, cuvette reactor, pulse immunoassay 250 glass transition temperature 266 glass tube, Vycor 3 gradient - focusing 120 - potential, induced 1 - - radial 27 gradient elution, electrochromatography/ mass spectrometry 124 gravitational fields 191 grinding period, electrical forming 161 guest molecules 265 Guinea Green B, counter-current, electrochromatography 122 H-v relations, electrochromatography 16 heat dissipation, pressurized flow-driven electrochromatography 29 height equivalent-to-theoretical plate, electrochromatography 16 Helmholtz plane, electroosmosis 50 Helmholtz-Smoluchowski equation, electroosmotic dewatering 135 hemoglobin - mixture separation 128 - trypsinized 96 high-performance electrophoresis chromatography (HPEC) 99 high-voltage supplies 171 hot water injection, oil recovery 213 HPEC (high-performance electrophoresis chromatography) 99 human myoglobin 256
humic acid, metal complexes decomposition 247 hybrid circulations, solvent extraction 190 hybrid flow, solvent extraction 187 f, 191 hydraulics, emulsion phases 218 hydrodynamic recycling, metal ions 231 f hydrodynamic resistance - electroosmotic dewatering 133 - specific, electroosmotic dewatering 138 hydrogen abstraction 280 hydrometallurgy, solvent extraction 6 image analysis, latex immunoassay enhancement 258 iminodipropionic acid, dispersed phase 175 immunoassay - electric field enhancement 7 - electric fields applications 7, 229 ff - pulse, cell manipulation 249 ff immunoreaction, agglutination 251 industrial applications, electroosmotic dewatering 147 f industrial processes 5,131 ff industry, oil 213 f inner core, vesicles 282 inner surfaces - charged 27f - chemical modification, adsorption 53 f inorganic anions, ion-exchange chromatography 41 inorganic particles, electrophoretic behavior 154 f inorganic salts, oil industry 215 inorganic solid surfaces 287 instrumentation, pressurized f low-driven electrochromatography 29 f insulators, conducting polymers 83 interaction, electrostatic 75 intercalated clay colloids 290 interfaces - electrochromatography/mass spectrometry 123 - polar 282 interfacial renewal, solvent extraction 187 inverse micelles, photochemical reactions 280 ion-exchange chambers, electrodialysis 233 f ion-exchange chromatography 40 f ion-exchange membranes 233 f ion-exchange resin, counter-current electroconcentration 242 ion separation 75 ff
Index
ionic guest molecules 8 ionic solutes, zone front profiles 28 ionic substances, electrochemical separation 3 irreversible uptake, anion separation 87 isoelectric focusing, gel electrophoresis 96 isoelectric points - focusing 104 - oxides 154 isomerizations 283 Isradipin, electroosmotically driven chromatography 72 jet milling, electrical forming
161
kaolinite, ceramics forming 164 Kozeny constant 137 Langmuir-Blodgett films 283 lapse time, water-in-oil emulsions 209 latex-IgG beads 257 latex immunoassay enhancement 254 f latex particles, a.c. field 257 layers - boundary 191 - electroosmosis 48 LCIMS 123f linear agglutination 251 liquid chromatography system, tiny 72 liquid dispersion, electrostatic 192 f liquid electrochromatography 1 liquid junction interface 123 liquid-liquid extractor, electrostatic 195 f liquid-liquid partition, counter-current, electrochromatography 120 liquid membrane separation 219 liquid mixing, solvent extraction 187 liquid pressure, electroosmotic dewatering 139 lithium polymethylacrylate, ERF 171 local electric field 1 local velocity, electroosmosis 51 loculi, counter-current, electrochromatography 120 low water content, electrical forming 161 macromolecules, electrofractionation 103 malonic acid, eluent composition 39 mass spectrometry, electrochromatography 4, 117 ff, 123 f
307
mass transfer - electrochromatography 14 - solvent extraction 185 - solvent extraction, enhanced 187 materials - asphaltic, oil industry 215 - nonplastic, electrical forming 161 mean hydraulic depth, electroosmotic dewatering 136 MECC (micellar electrokinetic capillary chromatography) 111 mediator, photochemical reactions 284 membranes 283 metal complexes decomposition, electrochemical reactions 247 f metal ion separation, pretreated carbon 80 metal ions - electrodeposition 244 f - hydrodynamic recycling 231 f cis-N-methyl-4-j3-styrylpyridium iodide, electrochromatography 34 micellar electrokinetic capillary chromatography (MECC) 111 micelles - photochemical reactions 280 - spontaneous electrostatic fields 270 microamachined reactor, latex immunoassay enhancement 260 f microcapillary columns, packed 66 f - electroosmotic mobility 58 microcavity, organized photochemistry 7 microcolumns, electrochromatography 108 f - pressurized flow-driven 29 f mine residues, electroosmotic dewatering 147 mixer-settler type contractors 185 mixing/coalescing/settlingbehavior, drops 219 mobility - electroosmotic 51 - - open-tubular capillary columns 56 - - packed microcapillary columns 58 - electrophoretic 2 - - band broadening, electrochromatography 13 - - inorganic particles 154 modification, chemical, inner surface 53 f molecular aggregates, micelles 270 molecular alignement 266 molecular diffusion - axial, electrochromatography 14 - electrochromatography zones 19 - electroosmosis 61
308
Index
molecular evolution 92 molecular orientation - electric fields applications 7, 229 ff - organized photochemistry 265 ff molecules, charged, flow profiles 27 f monolayer membranes 283 morphine alkaloids, voltage-programmed electrochromatography 44 multilayer membranes 283 multiple injection, charged components 117 f multireactor, latex immunoassay enhancement 261 myoglobin, latex immunoassay enhancement 254 f 2-naphthalenesulfonic acid 34 Naphtol yellow S, counter-current, electrochromatography 122 natural electric field, electroosmosis 53 Navier-Stokes equation, electroosmotic dewatering 134 necking process, solvent extraction 193 net solid volume, electroosmotic dewatering 138 Newtonian fluids 172 noncentrosymmetric array 266 nonconducting particles, pulse immunoassay 249 nonlinear optical devices 266 nonplastic materials, electrical forming 161 Norrish type I reactions 278,293 Norrish type IT reactions 285, 293 nucleic acids, biomolecular analysis 92
octadecylsilane-modified capillary column 65 ODs-silica gel, flow velocities 38 oil-in-water emulsions 205 ff oil industry 213 f oil recovery, hot water injection 213 oligo-nucleotides, synthetic, purification 100 oligosaccarides, desalting 234 open-tubular capillary columns, electroosmotic mobility 56 operational factors, pressurized flow-driven electrochromatograpy 31 optical devices, nonlinear 266 organic acids, oil industry 215 organic compounds separation, electrosorption 82
organic dyes orientations, polymer matrices 267 organic solutions, inorganic particles 155 organic solvents, electrodeposition 155 organized photochemistry - molecular orientation 265 ff - organic molecules orientation 7 orientation, molecular 7, 229 ff - organized photochemistry 265 ff - organic molecules 1 oxides, isoelectric points 154 packed columns, electroosmotic flow velocity 56 packed microcapillary columns 66 f - electroosmotic mobility 58 paper electrochromatography 2 paper electrophoresis, biomolecules 94 parabolic pressurized flow, counter-current electroconcentration 238 parallel-plate electrode extractor 197 particle diameter, ceramics forming 160 particles, pulse immunoassay 249 particulate phases 179 partition, electrophoretic 2 pathogenic cells, pulse immunoassay 249 pendant charged drop, solvent extraction 193 permeability characteristics, electroosmotic dewatering 141 Petreco cylectric desalter 217 petroleum plants 205 PH - electroosmosis 55, 242 - elution time ratio 35 phase separation, solvent extraction 187 phases - conductive stationary 75 - dispersed 175 f - emulsions, hydraulic 218 - particulate 179 phases equilibria, band broadening, electrochromatography 13 phenol, chromatographic variation 35 phenylthiohydantoin (PTH) derivatives, asparagine 34 photochemical reactions, electrostatic fields 8, 266 ff photochemistry, organized, organic molecules orientation 7 photocyclodimerization 288 photocyclodimers 269 photoinduced electron transfer 273
Index physical properties, electroosmotic dewatering 141 f plate height - reduced, electroosmosis 63 - theoretical, flow profiles 61 plug flow, electrochromatography zones 20 plug flow profile, electrochromatography 14 Poiseuille flow 14, 18, 20 polar interfaces 282 polarizable guest molecules 265 poled polymers 268 polyacriamide gel, solidified 72 polyaniline - carbon 78 - ion-exchange 83 polyelectrolytes 286 polypeptides, liquid chromatography 100 polypyrrole - carbon 78 - insulator, anion separation 83 - irreversible uptake 87 Ponceau SX, counter-current, electrochromatography 122 porosity, electroosmotic dewatering 136 porous media, electroosmotic dewatering 136 porous silica frit, counter-current electroconcentration 242 potential, zeta 154 potential gradients - induced 1 - radial 27 - two, in-column focusing 120 potential-dependent separation 80 f preconcentration, electrochromatographic 247 preconsolidation, electroosmotic dewatering 137 preparative columns 97 preparative electrochromatography, continuous 128 preparative-scale electrochromatography 122 pressurized flow - counter-current electroconcentration 237 - electrochromatography 3,14 - liquid chromatography 99 - profiles, electrochromatography 18 f pressurized flow-driven electrochromatography 111,124 - microcolumns 29 f pretreated carbon, metal ion separation 80
309
procedures - alternating current field 255 - latex immunoassay enhancement 260 - pulse immunoassay 250 - solid-phase extraction 245 protein separation, gel electrophoresis 96 proteins, biomolecules 93 pseudo-liquid membrane extractor 200 PTH, phenylthiohydantoin 34 pulse electrovoltage 249 ff pulse immunoassay 249 ff pulsed d.c. voltages, water-in-oil emulsions 223 pyridine 155 quantum yields 275 radial field, electroosmosis 47 radial potential gradient 27 radially applied voltage, electrochromatography 3,53 random walk model, band broadening, electrochromatography 14 raw materials, zeta potential 164 recycle, column focusing 5 redispersion, electrostatic liquid-liquid extractor 195 redox polymer, ion exchange 83 redox separation, electroactive metals 79 f refinery, water-in-oil emulsions 205 renewal, interfacial, solvent extraction 187 residues, mine, electroosmotic dewatering 147 resistance-to-mass transfer, electrochromatography 14 resolution, electrical, water-in-oil emulsions 206 retardation factor, electrochromatography 14 reverse parabolic flow profile 25 reversed micelles, photochemical reactions 280 Reynolds number 189 Rhodamine 590 21 RNA 92 rotating cylinder, two-dimensional chromatography 127 salts, oil industry 215 sample dispersion, pressurized flow, electrochromatography 19
310
Index
sample transfer, electroosmosis 242 f samples, continuous, electrochromatography zones 17 saponite clay interlayer 291 SDS-PAGE, gel electrophoresis 96 second harmonic generation, organized photochemistry 266 sedimentation, dispersed phase 175 selective electric collection 233 selective release, metal ion electrodeposition 244 f semisolid, electroosmotic dewatering 137 separation - liquid membrane 219 - phases, solvent extraction 187 - potential-dependent 80 f - protein, gel electrophoresis 96 - redox 79f - two-dimensional, electrochromatography 4,117 ff sequential electrochromatography 97 shear stress, ER effect 173 Sherwood number 191 silanol groups 47 f - flow velocities 38 silica frit, porous 242 silica gel surfaces, charge density 47 f, 53 silica microspheres, viscosity control 178 silica surfaces 287 silicon glass plates, latex immunoassay enhancement 2 60 silicon oil, dispersed phase 175 silver particles, electrodeposition 244 f slip, electrodeposition 153 slip plane, electroosmotic dewatering 135 slit reactor, pulse immunoassay 250 slurry-packed capillary columns 52,70 sodium montmorillonite, electroosmotic dewatering 137 solid compressive pressure, electroosmotic dewatering 139 solid-phase adsorbents 245 solid-phase extraction 245 solidified polyacrylamide gel 72 solvent extraction 6,185 ff split-sample injection, spWreservoir 2 9 spontaneous electrostatic fields 270 f spray column, electrostatic 195 f spray contractors 185 square-wave field, AFP 261 stability, operational, counter-current electroconcentration 241 stationary phase, electric field, electrochromatography 13
stereoselective photodimerization 269 Stern layer, electroosmosis 50 stilbazoles 268 f stress, tangential, solvent extraction 188 suramin, electrochromatography/mass spectrometry 125 surface charge, silica gel 47 f surface-active reagents 53 surfaces - charged, inner 27 f - inorganic solid 287 - silica 287 - volumetric specific, electroosmotic dewatering 136 surfactants, electrochromatograms 68 suspension, dielectric powder in oil 171 tangential stress, solvent extraction 188 Taylor flow pattern 189 thickness, double-layer, electroosmosis 50 thin diffusional boundary layer 191 thin films, organized photochemistry 269 thin layer electrochromatography 2, 96 time course, immunoreactions 258 time lag, pressurized flow-driven electrochromatography 34 tiny liquid chromatography system 72 torque, viscosity control 172 tortuos channels, electroosmosis 58 tortuousity, electroosmotic dewatering 137 trimesic acid, chromatographic variation 35 trypsinized hemoglobin 96 tubes - capillary, round, zone profiles 26 - open, flow profiles, electroosmosis 19 - rectangular capillary, electrochromatography zones 21 - Vycor glass 3 turbulences, solvent extractions 6 two-dimensional chromatography, continuous injection 126 f two-dimensional separation, electrochromatography 4,117 ff uracil, pressurized flow-driven electrochromatography 34 vehicles, ceramics forming 156 velocity - electrochromatography 14 - local, electroosmosis 51
Index
vesicles 282 - spontaneous electrostatic fields
270
vibration frequency, ERF 184 vinylferrocene/maleic anhydride copolymer 78 viscosity control, electric fields 6,171 ff void ratio, critical, electroosmotic dewatering 141 voltage-programmed electrochromatography 42 f voltages - radially applied 53 - water-in-oil emulsions 223 voltammograms, cyclic 84 volume, net solid, electroosmotic dewatering 138 volumetric specific surface, electroosmotic dewatering 136 Vycor glass tube 3 Walden product, electroosmosis 53 f water drop formation, solvent extraction 194 water droplets, computational movement simulations 208
water injection, hot, oil recovery 213 water-in-oil emulsions, electric fields applications 6, 205 ff water-in-oil-in-water emulsions 205 waxes, oil industry 215 yield stress, ER effect 173 zeolite cavities 291 zeolites, spontaneous electrostatic fields 270 zeta potential 154 - raw materials 164 - eluent composition 40 zigzag motion, electrostatic pseudo-liquid membrane extractor 200 zinc drams, electroosmotic forming 168 zone front profiles - electrochromatography 18 - fused silica capillary tubes 24 - ionic solutes 28 - rectangular capillary tubes 21 zones, electrochromatography 17 f zwitterionic guest molecules 265
311
Process Scale Liquid Chromatography edited by G. Subramanian
1995. XVI, 225 pages with 84 figures and 18 tables. Hardcover. DM 178.00. ISBN 3-527-28672-1 This book provides the industrial chromatographer and production scientist with a comprehensive account of process scale liquid chromatography. The basic theory is presented, guiding the reader through system design, simulation and modelling techniques, giving due consideration to economic aspects, as well as safety and regulatory factors.
A thorough, up-to-date survey of current techniques and media stresses their advantages and limitations in such a way as to faciliate their application to real-life problems. In view of the rapid rate of progression in industrial chromatography, one chapter provides an assessment of future developments. The chapters are written by acknowledged experts from Europe and the United States.
