Table Of ContentJOURNAL 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.
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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.
