JOURNAL OF CHROMATOGRAPHY LIBRARY - volume 78 electrophoresis a survey of techniques and applications part A: techniques editor Z. Deyl Physiological Institute, Czechoslovak Academy of Sciences, Prague co-editors F.M. Everaerts, Z. Pruslk and P.J . Svendsen ELSEVIER SCIENTIFIC PUBLISHING COMPANY Amsterdam - Oxford - New York 1979 ELSEVIER SCIENTIFIC PUBLISHING COMPANY 335 Jan van Galenstraat P.O. Box 21 1, 1000 AE Amsterdam, The Netherlands Distributors for the United States and Canada: ELSEVIER/NORTH-HOLLAND INC. 52, Vanderbilt Avenue New York, N.Y. 10017 Library OF Congress Cataloging in Publication Data Main entry under title: Electrophoresis : a survey of'techniques and applica- tions. (Journal of chromatography library ; v. l8- ) CONTEITTPS: pt. A. Techniques. Includes bibliographical references and index. 1. Electropharesis. I. Deyl, Zdenkk. 11. Series. QD79.344345 541' .37 79-22525 ISBN 0-444-41721-4 (pt. A) ISBN 0-444-41721-4( Vol. 18) ISBN 0-444-41616-1( Series) 0 Elsevier Scientific Publishing Company, 1979. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or trans- mitted in any form or by any means, electronic. mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher, Elsevier Scientific Publishing Company, P.O. Box 330, 1000 AH Amsterdam, The Netherlands. Printed in The Netherlands Contributors Dr. L. Arlinger, Development Department, LKB Producter AB, S-161 25 Bromma 1, Sweden Dr. P. Blanicky, Research Institute of Child Development, Faculty of Pediatrics, Charles University, Prague 5-Motol, Czechoslovakia Dr. N. Catsimpoolas, Biophysics Laboratory, Department of Nutrition and Food Science, Massachussetts Institute of Technology, 02 139 Massachussetts Avenue, Cambridge, Mass., U.S.A. Dr. Z. Deyl, Institute of Physiology, Czechoslovak Academy of Science, Bud6jovicka 1083, Prague 4-KrE, Czechoslovakia Dr. F. M. Everaerts, Department of Instrumental Analysis, Eindhoven University of Technology, Eindhoven, The Netherlands Dr. 2. Hrkal, Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague 2. Czechoslovakia Dr. A. Kolin, School of Medicine, Biophysics Laboratory, The Center for the Health Sciences, Los Angeles, Calif. 90024, U.S.A. Dr. W.O strowski, N. Copernicus Academy of Medicine in Krakow, Institute of Medical Biochemistry, ul. Kopernika 7, 31 -034 Krakow, Poland Dr. Z. Prusik, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Flemingovo nam. 2, Prague 6, Czechoslovakia Dr. P. J. Svendsen, Protein Laboratory, University of Copenhagen, Sigurdsgade 34,2200 Copenhagen, Denmark Dr. J. Vacik, Department of Physical Chemistry, Faculty of Science, Charles University, Albertov, Prague 2, Czechoslovakia Preface The separation and characterization of the individual components of mixtures has been a prerequisite for progress in chemistry since the infancy of the science. It is there- fore most fitting that the Dutch word for the entire science of chemistry, Scheikunde, means the art of separation. Until the beginning of this century, the most commonly used separation methods were filtration, distillation and crystallization. Pioneering studies on electrophoresis were pub- lished in 1892 by Picton and Linder, in 1897 by Kohlrausch and in 1899 by Hardy. Tswett’s first work on chromatography was published in 1903. Among the earliest centri- fugation experiments one can mention the attempt by De Lava1 in 1879 to separate cream from milk. Electrophoresis, chromatography and centrifugation have since become the most widely used and effective methods available for the separation and identification of all kinds of substances. Later studies have revealed several fundamental similarities among these three methods. A new electrophoresis method is thus rapidly followed by its counterpart in, for example, the field of chromatography. An example is displacement chromatography, which is analogous to the displacement electrophoresis developed earlier by Kohlrausch. The salient features of both methods are: (1) that the solute zones follow each other like carriages in a train, (2) that the height of a zone (i.e., the maximum con- centration of the solute) is a characteristic of the particular solute making up the zone and (3) that the width of the zone is proportional to the amount of solute present. Further, (4) the leading electrolyte used in displacement electrophoresis corresponds to the medium with which the chromatographic column is equilibrated initially and (5) the terminal electrolyte of the electrophoresis corresponds to the eluting medium in displace- ment chromatography. In order to emphasize the similarities among different methods and thereby to increase our understanding of them, we should try to use analogous terminology as much as possible, for example, displacement electrophoresis (instead of the commonly used term isotachophoresis) and displacement Chromatography. Another illustration of the similarities among electrophoresis, chromatography and centrifugation is the fact that each of these methods can be operated in a frontal (moving-boundary electrophoresis, chromatographic frontal analysis and sedimentation-velocity ultracentri- fugation), zonal (zone electrophoresis, chromatographic elution development and gradi- ent centrifugation) or displacement (displacement electrophoresis, chromatography and centrifugation) mode. When Arne Tiselius did his pioneering work on moving-boundary electrophoresis, his interest was focused mainly on the characterization of proteins, and one should remem- ber that his teacher, The Svedberg, was at the same time engaged in similar studies using XI1 PREFACE the ultracentrifuge. Tiselius’ laboratory notebooks reveal that he had accomplished a zone separation of proteins in a gelatin gel as early as 1927. Had he not underestimated the importance of these experiments and therefore declined to publish them - a misjudge- ment which he contemplated with regret in a later biography the big breakthrough in - biochemistry would have come much earlier than it did. As it was, more than 20 years elapsed before he and other research groups began to study zone electrophoresis in earn- est and finally developed this efficient and mild method for the purification of biologic- ally important molecules. In order to perform a meaningful investigation of the properties or function of a sub- stance, it is often of decisive importance that the substance is pure. In all branches of chemistry one must therefore have access to high-resolution separation methods for both analytical and preparative purposes. In biochemistry and other biological sciences, separa- tion work is often complicated by the structural lability of the substances of interest, which might prohibit large variations in pH or ionic strength. Further, the crude starting material is often extremely complex with regard to both the number and the nature of the components present and the interactions among them, and may contain only a few micrograms of the substance that one wishes to isolate. Isolation work is thus very often the “bottleneck” in research projects within the biological sciences. Increased knowledge of different fractionation methods can greatly facilitate the choice of an appropriate method for the isolation or analysis of a particular substance and can provide the basis for many valuable inferences regarding the properties of a substance as a “bonus” to the iso- lation work. In connection with structural studies, one often needs to know the,principle on which the separation is based. For example, knowledge of the sieving properties of polyacrylamide gels was a prerequisite for their use in the determination of the nucleotide sequences of DNA molecules. Many papers on various biopolymers are of dubious value because the studies were carried out with heterogeneous material, and it is often not a straightforward matter to infer the properties and activities of individual components from the properties of the mixture. It is therefore understandable that modern biochemical, medical and biological research training includes exercises in separation methodology. As the treatment is often hasty and superficial, a book such as this can serve the very useful purpose of providing a good theoretical background to various electrophoresis methods and illustrating their practical applications. As a teacher in the annual Uppsala Separation School on modern biochemical separa- tion methods I am often asked the question, “Which separation method is the best?” There is unfortunately no simple answer to this question. One method might be superior for a particular separation problem, whereas an entirely different method might be prefer- able in another situation. One is often obliged to use a series of methods based on differ- ent separation parameters. If the substance of interest is particularly labile, electro- phoresis has several advantages over other methods, especially when applied in free solution. Electrophoresis, particularly in gels, affords a higher resolving power for bio- polymers than any other method, and is therefore universally used for the analysis of protein preparations. Electrophoresis for preparative purposes has not enjoyed the same popularity, despite the fact that the resolution is nearly as good as that obtained on the analytical scale. Chromatography is considerably more popular than electrophoresis for PREFACE XI11 the isolation of biological material, although its resolving power is usually lower. This is due to the fact that preparative electrophoresis is much more complicated than chroma- tography or analytical electrophoresis as regards both the design and construction and the operation of the apparatus. Preparative electrophoresis will not receive the attention that it deserves until easily operated equipment becomes commercially available. Publishers could also help by offering frequently revised bibliographies of electrophoresis literature so that the latest methods could become rapidly known (a loose-leaf file with pages up- dated every year or two might be useful). If preparative electrophoresis on a laboratory scale has received little attention, the situation is even worse regarding the application of the method on an industrial scale. This is mainly due to the inherently low capacity of the method. However, displacement electrophoresis might be useful for the preparation of valuable enzymes on a limited scale, because the concentration in a zone can be extremely high, in the range 5-20% (w/v) under ordinary experimental conditions. However, intensive research must be carried out in order to solve some of the problems that hinder the practical application of the method. For example, one must find some means of preventing precipitation of proteins at the high concentrations attained. I believe that displacement electrophoresis would enjoy wide- spread success if a series of well defined spacer molecules with small, known differences in mobility became comniercially available, so that one could simply select the appropriate spacer(s) for each particular experiment. Most electrophoresis experiments are carried out in ordinary buffers. There are very few reports on the use of mixtures of water and organic solvents, because the substances of interest are usually water-soIuble and might even be denatured by solvents such as alcohols. However, during recent years there has been a boom in research on the structure and function of cell membranes. As the lipids and many of the proteins that make up the membranes are not water-soluble, it is obvious that membrane research would be greatly served by the availability of electrophoresis buffers that are capable of solubilizing these strongly hydrophobic substances. One might even expect that such buffers would be less denaturing than ordinary aqueous buffers. as they more closely resemble the natural hydrophobic environment of the membrane proteins. Of course, one can solubilize niem- brane coniponents by the use of deteigents, but tlie resulting micelle formation greatly coniplicatcs the interpretation of the experimental data. Since the time of Tiselius, research at tlie Institutc of Biochemistry in Uppsala has centred on the development of new separation methods and the improvement of existing methods. Tiselius often pointed out that the methodological work usually proceeds best when it is carried out in close collaboration with someone who has a particular separation problem to solve, which mininiizcs the risk that a poor niethod might be developed for its own sake and ensures that practical ‘‘snags’’ will be detected and hopefully overcome at an early stage. When a new niethod is to be publishcd, it is important to aim the paper at investigators who might have little interest in the method as such but who might find it useful in connection with some current separation problem, as well as at specialists in methodology. In the recently founded Jounial of Biochenzical and Biophysical Methods every paper contains a section devoted to a popular treatment of the method and some areas of application. In all areas of present-day research we use more or less sophisticatcd equipment, XIV PREFACE including that involved in separation work. Although this is a condition for the solution of various research problems, it also involves considerable risks if we do not thoroughly understand the principle on which the apparatus is based and its relation to the data that the apparatus provides. There is also the risk that the accessibility of sophisticated “black boxes” might undermine our faith in manual experimental skill and thus suppress the most straightforward approach to the solution of an experimental problem. One can easily become bound to a particular apparatus even when there are easier and better ways of obtaining the desired information. For example, many electrophoresis experiments can be carried out with very simple apparatus if one has the basic knowledge of electrophoresis contained in this book. In this connection I might remind the reader of E dF ischer’s fundamental contributions to the chemistry of amino acids, peptides and proteins, despite the primitive separation methods available to him. Experimental skill and ingenuity in the planning of experiments are just as necessary now as in Emil Fischer’s day. I have only touched upon possible future developments in electrophoresis. To specu- late about the future is easy, but experience shows that such speculations seldom hold up, owing in no small part to the dynamic character of research. I have therefore carefully avoided great visions, which are better reserved for an afterdinner talk - where they are promptly forgotten. Institute of Biochemistry, STELLAN HJERTEN University of Uppsala, Biomedical Center, Uppsala (Sweden) Introduction The present volume is the first one of a two-volume project devoted to electromigration techniques and their applications. It was the intention of the Editors to summarize general aspects of these techniques in the first volume in the proportion in which they currently are used in laboratories and at the same time trying to emphasize perspective ones. Applications of these techniques were deferred to the second volume. In order to keep the first volume consistent, it was inevitable to show the capacity of individual types of electromigration techniques by separating various classes of compounds or by using some types of separations as demonstrative examples. Thus some applications are discussed here to a limited extent. On the other hand, we tried to go into more details regarding instrumentation and some prescriptions that are more or less generally applicable. We tried to arrange mathematical and physicochemical background, in such a way so that it also could be easily understood by non-professionals using the electromigration techniques in daily routine work. It is the sincere hope of the Editors that such knowledge could help to abolish trivial problems that we are frequently witnessing in applicatory work, and which are fundamental in unsuccessful separations. Prague, August 1979 ZDEN~KDEYL Chapter 1 Theory of electromigration processes J . VACIK CONTENTS Introduction ................................................. 1 Equilibria in electrophoretic systems ................................... 2 Equilibria in solutions of electrolytes. ................................ 3 Weak acids and bases ........................................ 3 Ampholytes ............................................. 4 Phase boundary equilibria ....................................... 5 Adsorption at the liquid-solid interface ............................. 5 Electrical double layer ....................................... 6 Processes in electrophoretic systems ................................... 8 Transport processes in solutions of electrolytes ........................... 9 Diffusion ............................................... 9 Migration ............................................... 10 Convection .............................................. 12 Electroosmotic flow ........................................ 12 Heat conduction and heat flow .................................. 13 Transport phenomena in stabilizing media .............................. 14 Spatial effects of the inner structure of capillary systems ................... 15 Distribution function and the constant RE ........................... 15 Electroosmosis in capillary systems ................................ 16 Evaporation of the electrolyte and sucking flows ........................ 17 Electrode reactions and transport phenomena ............................ 17 Mathematical description of the electrophoretic process ........................ 18 References .................................................. 20 INTRODUCTION Electromigration separation processes are. in general. based on differences in the mobilities of electrically charged particles in an electric field . The term electrophoretic separation processes is commonly used . The system studied consists of (a) liquid phase (the basic electrolyte system including the compounds to be separated). in which sepa- ration takes place. (b) solid phase. which is in contact with it (i.e., walls of separation column. stabilizing porous environment. etc.), and (c) gaseous phase. which is in equi- librium with the liquid phase (in methods in which a porous carrier of the liquid phase is placed in the so-called wet chamber. or with the gaseous phase originating during elec- trode reactions); such a system will be referred to as the electrophoretic system . The term separation medium will be applied to electrophoretic systems that do not yet contain substances to be separated . It is essential that the separation medium is in thermodynamic 2 THEORY OF ELECTROMIGRATION PROCESSES equilibrium prior to the experiment. This condition is fulfilled when all components have the same chemical potential at all sites in the system, the same electrical potential or the same temperature; mechanical forces also have to be equilibrated. These conditions are first changed by adding the sample to a certain location in the separation medium. The equilibrium is further impaired by the application of an electric field to the system with the subsequent flow of electric current. During the gradual separation of individual components of the sample, this imbalance further increases. Thus, simultaneously with the separation induced by the outer force (electric field), transport phenomena that tend to equilibrate the subsequent concentration, temperature and other gradients begin to arise. In electrophoretic separations, these spontaneous processes (disturbances) usually occur to a greater extent with increasing time. Time is therefore a very important factor, and the proper choice of the time interval can be used to optimize the results of sepa- rations. With some electrophoretic methods a steady state is established that does not introduce any further changes with the advancing electrophoresis. The result of the separation is no longer time dependent after the steady state has been achieved. EQUILIBRIA IN ELECTROPHORETIC SYSTEMS It is a prerequisite of the electromigration separation principle that the liquid phase of the separation medium is formed by a solution of electrolytes. Substances present in the solution must occur at least partially in the form of charged particles (ions). Ions originate in the solution usually from electroneutral molecules by electrolytic dissociation*. Dis- sociation of strong electrolytes is complete; all molecules in the solution are dissociated to ions. Weak electrolytes are dissociated only partially; in the solution they are present both in the form of ions and as neutral molecules. From this point of view of transport phenomena, any type of particle should be considered as a separate component. Thus, all types of ions, non-dissociated molecules of the solvent and weak electrolytes and non- dissociated molecules of the solute can be considered as separate components of the liquid phase. Dissociated ions and their non-dissociated counterparts of a particular compound are referred to as a substance. A collection of electroneutral molecules of the electrolyte (e.g., component of the substance with zero charge) and ions derived from this electrolyte by electrolytic dis- sociation (e.g., components of the substance with a charge z where jzI is a number of elementary charges of the component, the sign designating the polarity of the ion) are considered as a single substance. It holds that ci = ci,=,w here ci is the concentration of the ith substance and c ~ar,e c~on centrations of the component with charge z belonging to the ith substance. The concentration c of all substances in the solution is then deter- mined by c = 8 ci = ? Z ci,=.A lso, the solvent is considered as a separate substance, = I 1. and ions originating from its dissociation are not included among components of other substances, in spite of the fact that they contribute to their ionic equilibria. Concen- tration is defined by the amount of substance of a given component niazi n unit volume V. * The common term dissociation is applied, even if a protolytic reaction of an electrolyte with a sol- vent is in fact involved.