Text of Isoelectric Focusing and Isotachophoresis applied in
in Protein Chemistry*
It is a great honour for me to address your learned Society today
for the second time in six
years on this occasion of your 26th Anniversary. Some Japanese
friends are active participants to our yearly Colloquia on Protides
of Biological Fluids and among them Professor Hidematsu
Chairman of your Society, is an active and devoted committee
It is my wish that this talk may act as a contribution to an
international scientific understanding
which we all pursue. I therefore wish to apologize for the
references, which are mainly quoted
from our Colloquia and might draw unsufficient attention to the
work done in Japanese laboratories
at this side of the world.
It is not without emotion that I remember the father-figure of
Electrophoresis, Prof. Arne Tiselius, (born in 1902 and died in
1971), who spoke before me on our previous visit to your
I. General Introduction
analysis and 3. Displacement chromatography. The same modes
may be distinguished in electrophoresis (Table 1). These
are 1. Zone electrophoresis, 2. Moving boundary
3. Isotachophoresis. One more development which is not
led in chromatography was introduced and called 4.
focusing. Let us now briefly compare the basic principles of
these different techniques (Fig. 2).
Table 1. Chromatographic and electrophoretic techniques.
* Lecture to The Society of Electrophoresis at the 26 th G eneral
Meeting on October 25, 1975.
** Director of Simon Stevin Institute , Brugge, Eelgium.
1. Zone electrophoresis
In this technique all compartments of the electrophoretic equipment
are filled with one kind of electro-
lyte which is called the background or carrier electrolyte. The
ionized material to be separated is introduced
into the carrier around the center of the separating cell. All ions
in the system-both those of the supporting
electrolyte and of the sample-move with their own velocity
dependent on the experimental conditions such
as pH, conductance and field strength. Separation of anions and
cations occurs simultaneously. Because
the background electrolyte provides a stationary stabilizing
support medium the zones develop further as long
as a field is applied. Reproducibility is improved by the use of an
adequate buffer system whereas diffusion
acts as an important disturbing factor generating shallow and
broadening zones. Adsorption to supporting
materials may cause trailing effects.
Several anticonvectional procedures have been introduced such as
paper, gel and others. Zone electro-
phoresis is commonly carried out in a gel e. g. by Smithies,2)
Katchalsky3) on paper by Grassman and
Hannig4) and by Peeters5) on other porous materials by Consden6)
Due to the swamping effect of supporting electrolytes universal
detection methods such as conductivity,
calorimetry, e. a. can not be used and precise detection and
identification are to be performed after the run
by the use of specific dyes for instance. This type of
electrophoresis is comparable to the elution techniques
in chromatography and probably the most popular technique in
2. Moving boundary electrophoresis
The Tiselius free boundary method bears analogies with frontal
analysis in chromatography and is
carried out in free solutions, the fronts being stabilized by
density differences. A particular about this
method is the fact that the sample is introduced into one
compartment of the system and contains the same
counter ion as the carrier electrolyte. If a separation of anions
is desired the anion of the electrolyte
applied in the other compartment has a net mobility greater than
the other anions of the sample mixture
and in this case the counter ion, being the cation of this
electrolyte, has buffering capacity. A partial
separation is obtained depending on the duration of the run, the
mobilities and concentration of the ionic
constituents, the pH and other parameters. Generally the
experiments are carried out in the so-called
Tiselius cell and detected by means of Schlieren optics. With this
method general detectors can be
applied and detection occurs during the analysis.
Isotachophoresis covers the migration in an electric field of a set
of ion species of the same sign
against a common counter-ion. Isotachophoresis requires following
qualities of the electrolyte system (a) a
sample solution containing the sample ions to be separated and
introduced as a zone between (b) the leading
electrolyte, containing only one ion species, the leading ion,
which bears the same sign as the sample ions
Vol. 20. No. 1. 1976 (5)
to be separated but with a mobility higher than that of the fastest
sample ion and (c) a second electrolyte,
the terminating electrolyte or terminator, which contains only one
ion species of the same sign as the. sample
ions to be separated but with a mobility lower than that of the
slowest sample ion. The polarity of the
electric field shall be chosen so that the separation of the sample
develops into the leading electrolyte.
After an adequate migration time, the system reaches equilibrium
and all ions move at the same speed,
separating into a succession of individual zones in immediate
contact with each other and arranged in the
order of mobilities. The detection can be achieved by using thermal
detectors, conductivity measurements,
measurements of potential gradient with a micro-electrode device,
or by measuring the UV or visible
absorption of samples. These detection techniques can also be
4. Isoelectric focusing
The concept of isoelectric focusing was introduced by the Japanese
chemists, Ikeda and Suzuki7) as early
as 1912. They observed that mixtures of amino acids separated
according to their isoelectric point during
preparative electrolysis. This kind of separation resulted in the
formation of a pH-gradient between anode
Around 1954-1958 the technique was taken up by Kolin811) who
introduced a sucrose density gradient to
reduce the effect of electroendosmosis and other convective forces.
Finally in 1961-1962 Svensson1214)
defined the theoretical requirements for obtaining a stable
pH-gradient in the column. He demonstrated
that the electrolytes used must be ampholytes which retain good
conductivity and buffering capacity at their
isoelectric point. When a sample of an amphipathic substance is
introduced the particles will migrate until
they are separated and have reached a pH zone fitting their proper
pI value assuming the pI values are
sufficiently different. At that moment the effective velocities are
equal to zero. The electric current is
maintained by the buffer ampholytes. Isoelectric focusing is
difficult to compare with any analog in
chromatography. Specific detectors had to be introduced and
detection is usually performed after the
We shall now describe in more detail the historical development of
the principle, the technical develop-
ments and the field of application of two techniques: isoelectric
focusing and isotachophoresis. We shall
conclude by a broad comparison of the advantage of both
II. Isoelectric Focusing
1. Historical development (Table 2)
As mentioned above the concept of isoelectric focusing (IEF) can be
traced back to the pioneering work
of the Japanese chemists Ikeda and Suzuki7~ on amino acid
separation. Due to convective disturbances the
amino acids, separated according to their isoelectric points, did
not develop into a stable pH gradient.
Williams and Waterman15~ used a multichamber device in order to
reduce the effects of electroendosmosis
and other convective forces. The application of this system was
limited because of the variability of the
field strength between anode and cathode. The method was applied by
Williams16~ to the isolation of
vitamins and growth factors, while Du Vigneaud 17) used
multichambers to isolate vasopressin and
Table 2. Developments in isoelectric focusing.
ocytocin from pituitary gland extract. In 1941 Tiselius18)
described the separation of egg-albumin and
haemoglobin by this method.
In the years 1954-1958 the technique of isoelectric focusing
greatly progressed through Kolin's811) work.
He allowed the buffers to diffuse in sucrose density gradients
under an electric current. By this method he
was able to obtain "isoelectric line spectra" in a few minutes.
Unfortunately those gradients were unstable
due to the electrolyte migration of the buffer ions in the electric
field. Isoelectric focusing was further
developed by Svensson. In a series of papers Svensson1214) defined
the theoretical requirements of electro-
lytes for isoelectric focusing and introduced the "carrier
electrolyte concept". These electrolytes must be
ampholytes which retain good conductivity and have sufficient
buffering capacity at their isoelectric point.
This requires access to a family of ampholytes with isoelectric
points scattered over the entire pH range.
Vesterberg1921) succeeded in synthetizing a series of aliphatic
amino-carboxyl acids now being produced
under the trademark "Ampholine", which satisfy the requirements
defined by Svensson (Fig. 3). Vesterberg's
contribution opened the way for isoelectric focusing to become a
method of widespread acceptance. The
pH-gradient reaches a stable equilibrium if the system is
stabilized against convection. This is usually
achieved by means of either a density gradient or gel
Initially, isoelectric focusing with carrier ampholytes was
conducted in sucrose density gradients and
primarily used for preparative purposes. The system was soon
adapted to small-scale analytical procedures,
the most promising adaptation being the introduction of a gel as
anticonvective medium.2228) Gel isoelectric
focusing overcame many of the problems associated with convective
mixing, diffusion and isoelectric precipi-
tation in liquid media and offers considerable savings in time and
Valmet29) introduced the principle of zone convection isoelectric
focusing. In a thin horizontal electro-
lysis cell local density gradients, due to thermal diffusion of the
ampholytes, stabilized the solution against
convection. Stabilization by using a rotating tube has been
reported by Hjerten,30) using his apparatus for
free zone electrophoresis. In addition, new immunochemical
techniques described by the term "immuno-
electrofocusing" have been developed by Catsimpoolas31, 32) and
Riley and Coleman.24) They used antigen-
antibody reaction for identification of protein fractions after IEF
in polyacrylamide gel columns, or in
agarose gels on slides.
2. Theoretical aspects
Isoelectric focusing or electrofocusing, isoelectric fractionation,
isoelectric separation, stationary electro-
lysis, isoelectric condensation and isoelectric analysis are some
of the names given to the phenomena
occurring to ampholytes in a pH gradient under influence of an
The isoelectric point, pI, of any ampholyte or protein is the pH
value at which it carries a zero net
charge (Fig. 4). A protein dissolved in a medium with a pH more
basic than its own pI value becomes
negatively charged and the protein migrates towards the anode in an
electric field. Conversely if the
protein is dissolved in a medium with a pH lower than its pI value,
the protein becomes positively charged
and migrates towards the cathode.
By submitting a mixture of carrier ampholytes to a potential
gradient in a convection free medium a
natural pH gradient is generated by the current itself. Under these
circumstances the mixture of proteins
separates into the pH zones of the system corresponding to the zero
net charge of each protein. Obviously
the focusing will take place at the isoelectric point of each
fraction. Since the diffusion coefficient is
inversely proportional to the molecular weight, better focusing can
be obtained with higher molecular weight
A treatment of the theoretical background of isoelectric focusing
is to be found in the fundamental
papers of Svensson and Vesteryberg1214,20.21).
3. Instrumental aspects
A. Analytical procedures
a. Gel isoelectric focusing in tubes:
Tubes have the advantage of protecting the gel from evaporation and
drying during isoelectric focusing.
The gel electrophoresis equipment of Ornstein and Davies33) can be
adapted to gel IEF. To overcome
heating problems Fawcett25) thermostated the gel tubes in a bath of
an electrolyte solution and in order
to minimize the volume of electrolyte used, he inserted a platinum
electrode directly into a layer of ampho-
line on top of the gel (Fig. 5). Righetti and Drysdale34,35)
developed an apparatus which allows efficient
Fig. 5. Apparatus for isoelectric focusing in
gel tubes according to Fawcett.25)
thin-layers according to Awdeh.28)
cooling of gels by circulating fluid and which contains small
electrolyte compartments . Wrigley36) used the
Davies37) design with a length of the glass tube of either 65 or
120mm, an internal diameter of 5mm and
polyacrylamide as a stabilizing agent.
b. Thin-layer gel isoelectric focusing:
Thin-layer IEF of proteins is a very useful analytical tool. The
assembly described by Awdeh28) is
placed upside down on two carbon electrodes mounted inside a
plastic box (Fig. 6). The cathode and
anode are moistened respectively with a solution of ethylene
diamine and phosphoric acid. The carrier ampholytes are thus
effectively prevented from approaching the electrodes while the
conductive basic and
acidic solutions simultaneously prevent Joule heat
B. Preparative procedures
Due to its superior resolving power, isoelectric focusing became a
generally applicable and efficient
a. Density gradient columns:
Vertical glass columns (LKB) thermostated by an internal and
external cooling water compartment are
available in sizes of 110 and 440ml effective separation volume,
respectively (Fig. 7) . The location of the
platinum electrodes is such that no gas bubbles nor electrode
products interfere with the IEF process. In a
given density gradient a higher total load of protein can be
separated in function of the number of zones obtained.
b. Multi-membrane apparatus:
The multi-membrane apparatus used by Ikeda7) and by Williams and
Waterman15) are limited only by
the total volume and protein concentration which it is convenient
to work with Fig. 8.
To achieve effective cooling stirring is required inside each
compartment. Membrane polarization can
be counteracted by the use of closed compartments as in the
apparatus of Rilbe.38)
c. Gel isoelectric focusing:
For fractionation of proteins which tend to precipitate at the
isoelectric point preparative focusing in
thin-layers or tubes is more suitable than in a density gradient.
For the recovery of the separated proteins
an efficient system was devised by Suzuki39) in which the separated
proteins are eluted electrophoretically
from gel segments with excellent recovery.
Another preparative approach is the continuous flow technique in
thin-layer gels as reported by Faw -
cett40) with elution under an electric field in order to eliminate
the diffusion effects encountered during
sample recovery. Furthermore the apparatus can be run with
continuous sample inflow and recovery ,
Fig. 8. Multi-membrane apparatus described by Williams and
Fig. 9. Apparatus for zone convection isoelectric focusing
according to Valmet.29)
d. Zone convection isoelectric focusing:
Valmet29) adapted the membrane apparatus designed for zone
convection isoelectric focusing (Fig. 9).
The differences in relative density which arise are utilized for
stabilization of the liquid against thermal
convection. The force of gravity leads to convection flow and the
horizontal density gradients are converted
into a vertical density gradient. The special design of the
separation cell prevents the vertical density
gradient from moving along the direction of the current flow which
follows the pH-gradient.
4. Experimental aspects
A. The carrier ampholytes
The carrier ampholytes are delivered at a concentration of 40%
(W/V) and cover the pH range 2.5-11.
Usually an ampholyte concentration of±1% is sufficient but
concentrations up to 10%may be required e. g.
for proteins with low solubility (Table 3).
Corrections for the absorption of the ampholytes at 280nm can be
made by running a control experi-
ment without protein. To obtain the desired diffusion rate for the
ampholytes and to enable their separa-
tion from proteins by gelf iltration the MW of the carrier
ampholytes may not exceed 1,000.
B. The electrode solutions
The carrier ampholytes are protected from anodic oxidation and
cathodic reduction by respectively an
acid and an alkaline electrode solution which repel them from the
electrodes In practice the anodic solution
consists of±1% phosphoric or sulphuric acid and for the cathode
solutions of 2% ethylene diamine,
ethanolamine or±8% sodium hydroxide are recommended.
C. Zone stabilizing agents
a. Density gradient:
Sucrose used at a convenient concentration of 50% (W/V) is the most
common stabilizer against thermal
convection.11) In case of reaction of the sample with the sucrose
other solutes, such as ethylene glycol and
glycerol are used.41)
Polyacrylamide gels are prepared either by chemical or by
can be inhibited by the sample itself.36)
Catsimpoolas31) and Riley and Coleman24) obtained separations of
rabbit serum proteins and human
serum in 1% agarrose gels. Due to their large pore size and
satisfactory mechanical properties, agarose gels
seem to be an ideal support for IEF, however these gels did not
give stable pH-gradients and equilibrium
patterns were difficult to establish due to the high
electroendosmotic flows. For the same reason paper and
cellulose acetate are unsuitable as support media in IEF.
Table 3. Experimental aspects of isoelectric focusing.
Radola42) performed IEF in granular gels, particularly Sephadex,
while Fawcett40) used Sephadex G-100
and graded particles of polyacrylamide as stabilizing media for
continuous flow TEF.
The main applications of isoelectric focusing are the analytical
and preparative separation of amphoteric
substances, especially proteins. The precision and reproducibility
of the pH slope can be as high as 0.01
pH unit and enables accurate characterization of the isoelectric
A. Density gradient isoelectric focusing
A subfractionation of human serum albumin followed by immunological
characterization of the focused
fractions was first reported by Carlsson and Perlman.43) The
contribution of bound fatty acids to the
microheterogeneity of plasma albumin was investigated by
Our experiments with native, delipidated and relipidated bovine
serum albumin (BSA) were aimed at
an interpretation of the contribution of the fatty acids to the
microheterogeneity of BSA.45) Fig. 10 shows
the IEF pattern of native BSA monomers, which is characterized by
three distinct fractions. Fatty acid
analysis revealed differences in the amount of fatty acids bound to
each of these fractions. After charcoal
Fig. 10. Density gradient isoelectric focusing of native BSA
Fig. 11. Density _gradient isoelectric focusing of delipidated
(0.2mol FA/mot BSA).45)
defatting the IEF pattern shifted almost entirely into the basic
component with disappearance of the most
acidic fraction (Fig. 11). After complete relipidation of BSA (9
mole fatty acid per mole protein) we
obtained the reverse shift to a single peak of pI= 5.07 (Fig. 12).
When however only 2 moles of oleic
acid were added per mole of defatted BSA (Fig. 13) the
heterogeneity of the native sample was reconstituted
regarding both the pI values of the focused fractions and their
relative amounts. Such results indicate the
usefulness of isoelectric focusing in protein-ligand binding
In 1968 we introduced the isoelectric preparative separation of
apoprotein subunits from plasma lipopro-
teins, working without dissociating agents, and demonstrated the
apoprotein heterogeneity within each UCF
class.46) As an example we demonstrate the heterogeneity observed
with human apo HDL3.47) This disa-
ppears almost completely under influence of 8M urea (Fig. 14)
proving the presence of aggregates in the
native apo HDL3.
By pre-isoelectric focusing narrower pH ranges were prepared
starting from the commercially available
pH ranges 3-10 and 4-8. The mostt selective separation of HDL3
apoproteins is obtained in the pH range
Fig. 12. Density gradient isoelectric focusing of relipidated
(9mot FA/mol BSA).45)
(2mol FA/mol BSA).45)
Vol. 20. No. 1. 1976 (13)
5.2-6.7 which was confirmed by subsequent SDS-PAGE and amino acid
analysis of the IEF fractions
(Fig. 15). Fig. 16 demonstrates the influence of the running time
on the fractionation. A more selective separation was obtained
after 23 hours. After 72 hours the IEF pattern became more diffuse.
In our hands
the decrease in amperage enables a good prediction of the end of
Fig. 14. Density gradient isoelectric focusing of human apo
with and without 8M urea.47)
Fig. 15. Density gradient isoelectric focusing
of human apo HDL3 in different pH
of human apo HDL3 during 23 hr and
72 hr respectively.47)
The comparative preparative isolation of the major HDL apoproteins
from man and chimpanzee was
described by Blaton.48)
A set of applications derived from papers presented at our 17 nd
Colloquium on Protides of the Biolo-
gical Fluids has been summarized in Table 4. Howard and Virella49)
demonstrated the heterogeneity of
human IgG by isoelectric focusing with a sucrose gradient. Li and
Li50) utilized the technique of isoelectric
focusing in combination with other separation procedures to isolate
glycosidases and concluded from these
experiments that IEF is particularly valuable at the final stage of
the purification procedure and for estima-
ting the isoelectric point of the protein.
The preparation of pure haemolytic proteins is obtained by using
chromatography on Sephadex followed
by isoelectric focusing as reported by Mollby and Wadstrom.51)
Using isoelectric focusing as a fractionation
method, it has been possible to demonstrate the heterogeneity of
gut glucagon-like immunoreactivity (GLI)
and species differences with respect to the GLI component.52)
Eulitz53) performed a good separation of
gamma G myeloma proteins by IEF. Other important applications
included studies of eye lens proteins,54)
the isolation of the fibrin stabilizing factor from human
plasma,55) of the steroid binding beta-globulin of
human serum,56) the study of proteases57) and the preparative
separation of Bence Jones proteins.58)
These examples demonstrate the successful use of isoelectric
focusing in a sucrose density gradient for
the preparative fractionation of many proteins.
B. Gel isoelectric focusing
When compared with gel electrophoresis, gel isoelectric focusing
shows several advantages: (a) Sample
application is less critical than in gel electrophoresis. (b) The
separation profile remains unaffected by the
structure of the gel. (c) The components become sharply focused at
their isoelectric point and their final
position is independent of the gel pores. a. One-dimensional
Since the development of gel isoelectric focusing (GEF) more than
1,000 papers dealing with this
technique have been published and there is little chance of
summarizing the results (Table 5).
One of the more . important fields of application of GEF in
biomedical research lies in the field of
enzyme chemistry. The carrier ampholytes, because of their
polyvalent nature, afford greater stabilization of
the proteins in solution than inorganic salts. This phenomenon has
been demonstrated for alpha-hemolysin,
protease and a hexosaminidase from staphylococcus ateus.59)
Table 4. Applications of density gradient isoelectric
presented on Protides of the Biological Fluids.
Table 5. Applications of one-dimensional gel isoelectric
Loss of activity during IEF may occur on prolonged exposure of the
enzyme due to unfavourable pH
ranges or to chelation of metal cofactors. Furthermore problems may
arise due to oxidation of methionine
and cysteine residues, which can be avoided by the presence of
thiodiglycol or ascorbic acid as antioxidants.
One of the first demonstrations of the high resolving power or gel
isoelectric focusing of enzymes was the
fractionation of the glycoprotein L-amino acid oxidase.60) Disc
electrophoresis revealed the presence of three
isoenzymes, which, when analyzed by GEF were further fractionated
into 18 enzymatically active molecular
forms. These differences are no artifacts but the result of
differences in primary structure and variations in
the carbohydrate content.61)
The high resolving power of GEF further demonstrated the enormous
heterogeneity of immunoglobulins.62)
Many authors showed that gel isoelectric focusing may provide a
useful alternative for the fractionation and
characterization of membrane constituents6366) and for the analysis
of metalloproteins.6770) Kopwillem71)
analyzed a series of peptides of human growth hormone synthesized
by the Merrifield technique.
Because of the tendency of lipoproteins to precipitate at their pI
value the gel technique avoids this
problem and became a useful analytical fractionation procedure.
Kostner72) pre-stained human serum lipopro-
teins and obtained eight distinct fractions after GEF. Albers and
Scanu73,74) also used GEF for the analysis
of human serum apolipoproteins. Comparing the apoprotein pattern
given by SDS-gel electrophoresis and
gel isoelectric focusing, it was shown that polypeptides of similar
size are still heterogeneous. In a compari-
son of the plasma HDL from man and non-human primates Blaton75)
checked the major apoproteins, apo Lp-
AI and apo Lp-AII isolated on DEAE-cellulose, by means of gel
isoelectric focusing on polyacrylamide thin-
layer. Although apo Lp-AI appeared on SDS-PAGE as a single
component the pattern given by GEF is
considerably more complicated proving a further microheterogeneity
of apo Lp-AI(Fig. 17). Identical patterns
Fig. 17. Isoelectric focusing of the major HDL apoproteins, apo
and apo Lp-AII, on polyacrylamide thin-layers.
could be obtained for man and chimpanzee whereas the baboon showed
an analogous pattern although with
lower pI values. For apo Lp-AII similar GEF patterns were obtained
for the three species studied, although
for human apo Lp-AII a minor fraction was observed next to the
One of the most promising applications of gel isoelectric focusing
is its use for routine clinical chemistry
procedures. Whereas with the conventional electrophoretic
procedures the separation of several hemoglobins
is not achieved, gel isoelectric focusing has proved to be of value
for detecting haemoglobinopathies.76) GEF
was further used for the analysis of human urinary proteins from
normal individuals and from patients with
chronic pyelonephritis77) and for the fractionation of human
salivary proteins.78) The isolation and characteri-
zation of human alpha-fetoprotein in the serum of patients with
primary hepatoma and with embryonal
carcinoma of the gonades was achieved with success by
The technique of gel isoelectric focusing can also provide
information about the interaction between
proteins and other components. Examples of such interaction studies
were published for subunit exchange
between several human hemoglobins,80) concanavalin A-carbohydrate
binding81) and for several enzyme-
substrate or enzyme-coenzyme complexes.35)
b. Two-dimensional procedures(Table 6):
Catsimpoolas82) distinguished three types of immunoisoelectric
In "disc immunoisoelectric focusing" the protein separation is
achieved by combination of isoelectric
focusing and immunodiffusion. This method involves separation of
proteins by IEF on a gel column which
is then embedded intact in the buffered agar gel for
immunodiffusion. Determination of the approximate
Table 6. Applications of two-dimensional gel isoelectric
isoelectric point of separated antigenic components is not possible
with this technique.
"Sectional immunoisoelectric focusing" involves isoelectric
focusing on a polyacrylamide gel column
which is followed after section by immunodiffusion of the sections
into agar gels. This technique is more
laborious than "disc immunoisoelectric focusing" but has the
advantage of correlating pI values and immuno-
diffusion patterns of the separated proteins.
A third variant involves immunoisoelectric focusing directly in an
agarose gel on a microscope slide.
This technique is similar to the immunoelectrophoresis described by
Grabar and Williams83) and modified
by Scheidegger84) with the exception that the buffer is replaced by
the carrier ampholytes and electrode
A further development was the introduction of isoelectric
The obtained "protein maps" are valuable for genetic studies in
assigning phenotypes to the chromosomes
that control their synthesis. Wrigley and Shepherd87) mapped wheat
grain protein from several wheat
varieties. Another variant of the two-dimensional procedures is the
combination isoelectric focusing-electro-
phoresis in gel gradients. Kenrich and Margolis88) modified their
two-dimensional procedure of electro-
phoresis by performing gel isoelectric focusing in the first
dimension and embedding the gel into the top of
a gradient gel slab, followed by electrophoresis. With this
technique both the pI value and molecular weight
of the separated protein can be determined.
An alternative method is isoelectric focusing-SDS gel
electrophoresis.89) After IEF the focused fractions
are subjected to SDS gel electrophoresis in the other dimension.
Barret and Gould90) and McGillivray and
Rickwood91) used this technique for the characterization of
non-histone proteins in chromatin.
From these examples we can conclude that gel isoelectric focusing
will provide a useful alternative
procedure to gel electrophoresis.
The migration of boundaries between salt solutions having one
counter-ion species in common was
described by Whetham92) as early as 1893. The main data in the
development of the technique are summa-
rized in Table 7.
In 1897 Kohlrausch93) published a theoretical treatment of the
conditions prevailing in the migrating
boundaries of such a system and these equations express the basic
theory of isotachophoresis. In 1923
Kendall94) and coworkers described the separation of metal ions by
an ionic migration technique. But it
was not until 1963 that Konstantinov and Oshurkova9" developed a
moving boundary method for micro
analysis of chemical elements based on the isotachophoretic
principle. In order to decrease disturbances due
to convection their separation was carried out in a capillary tube
and the fractions were detected by measu-
Table 7. Developments of isotachophoresis.
ring the refractive index of the zones. Being mainly applied to the
separation of isotopes this system was
extended in 196796) to the micro analysis of amino acids in a
capillary resulting in the separation of 17
amino acids. Vestermark,97) in 1966 further developed the basic
conclusions of Kohlrausch's equation and
pointed out the advantages and possibilities of isotachophoresis in
connection with its zone stabilizing effects
and the concentrating power of the method. He reported
isotachophoretic analysis of biological substances
such as protein samples and red-beet juice. In their disc
electrophoresis technique Ornstein98) and Davies3)
made use of the concentrating effect of a field strength gradient
in the stacking gel and succeeded in
separating complex protein samples into extremely sharp zones in a
polyacrylamide gel. Finally the spacer
technique was introduced by Vestermark99,100) who proposed to use
as spacers the same type of substances
already in use in isoelectric focusing, namely a mixture of
synthetic amino-carboxyl acids called "Ampho-
Several developments were also concerned with detection methods. By
means of thermocouples glued
to the capillary tube Martin and Everaerts101) detected thermic
steps corresponding to the boundary between
each successive fraction. But when components migrate in narrow
zones very closely to each other the
sensitivity of the thermal detection can be lower than the
sharpness of the resolution. Moreover when
spacers are used no defined thermal steps are observed as the
ampholytes are spread over the whole tube.
For that purpose Arlinger and Routs102) and Arlinger103,104)
suggested the use of a UV or visible detector.
In the case of protein mixtures, run in the presence of spacers,
only the protein components are detected at
280nm. Other developments in the field of detection involved the
contribution of Hello105) who described
a potentiometeric detection method and of Fredrickson106) who
developed an apparatus using conductivity to
detect zones. Mulder and Zuska107) developed an automatic unit for
measuring the time of passage of
successive zone boundaries.
The latest developments in the field of isotachophoresis are
devoted to the theoretical definition of
several parameters such as pH, electrolyte concentration, current
intensity, separation time, etc. and are
mainly due to the team of Martin and Everaerts,108)
2. Theoretical aspects
Isotachophoresiss is based on the equation formulated by
Kohlrausch93) which can be derived in a simple
way from the basic transport equabions in the system (Fig.
Let us consider two ionic compartments containing each a different
anion and a common cation. The
initial conditions are defined by C1, μ1, F1 and C2, μ2, F2,
respectively concentration, mobility and field
strength of the ions in zones 1 and 2. Migration takes place at a
constant current intensity I in each
compartment defined as:
Fig. 18. Isotachophoretic conditions.
According to the principle of isotachophoresis and once equilibrium
is reached, all ions migrate at the same
Moreover the condition of electroneutrality requires that:
C-1/C-2=μ-1(μ-2+μ+)/μ-2(μ-1+μ+)…(4) This last equation (4) is
Kohlrausch's equation and determines the concentration of each ion
in its migrating
3. Experimental aspects
At the application level the basic Kohlrausch equation leads to 4
main consequences which are funda-
mental to isotachophoresis:
1) The sequence of the ions in the electrophoretic equipment is
solely determined by their mobility.
2) The length of each zone has to be directly proportional to the
concentration of the ion in the zone.
This means that if an ion is applied at low concentration a
narrowing of the original zone has to occur.
Fig. 19 shows the starting condition where the concentrations CA
and Cs of the two ions to be separated
have been chosen equal, and where uB<uA. This means that the
starting concentration of B is higher
than the equilibrium concentration given by Kohlrausch's
When the current is turned on, a concentration gradient will
therefore develop at the starting point as
shown on the same figure. This concentration gradient will be
stationary during the experiment. The new,
sharp boundary between A and B moves toward the anode, the
concentration ratio across this boundary
obeying Kohlrausch's equation.
In the case of three electrolytes (Fig. 20), the anions A, B and C
with a mobility of C intermediate
between A and B and a concentration of C low compared to that of A
and B, ion C will, at the start of
the experiment, occupy a considerable length of the tube. However
after equilibrium is reached, the length
of the C-zone should be directly proportional to the amount of C in
the zone and this is obtained by a
concentration of the original zone. This can occur in practice
because of the low conductivity of the dilute
starting zone of ion C, which creates a considerable initial
potential gradient in that region and induces a
higher speed for the C ions than for the leading A-ions.
Consequently the C-ions will concentrate at the
A/C boundary and the conductivity of the C zone will gradually rise
due to this concentration effect.
Concommitantly the potential gradient will decrease until the same
speed is reached for C as for A.
3) The Kohlrausch equation also precludes that, when the mobilities
of two components are too close to
enable sufficient resolution they can be separated by the addition
of a "spacer-ion" with an intermediate
mobility (Fig. 21). By choosing the amount of the added spacer the
distance between C and D can be
4) A self-stabilizing effect is inherent to the principle of the
separation. If through diffusion, an ion
moves into an adjacent zone, the boundary can only be restored by
an active relocation of this ion towards
its original position. The boundary between two zones containing
respectively the A and C-ions, will move
electrophoretically toward lower field strengths. If, through
diffusion, C-ions from the A/C boundary are
brought into the A zone, they will lag behind because their
mobility is lower than that of the A-ions.
They will thus be drawn closer to the boundary again, and diffusion
is counteracted. Through this mecha-
nism the zone boundaries are submitted to a sharpening effect as
long as the current is applied. Actually it
is this phenomenon which is responsible for the high resolution
power of isotachophoresis.
5) The isotachophoretic run is characterized by a thermic step at
the boundaries (Fig. 22). The field
strength varies from zone to zone and is inversely proportional to
the mobility in each zone. Moreover as
by definition the current is constant along the tube, zone
boundaries are characterized by sharp modifications
Fig. 21. Use of spacer in isotachophoresis.
Fig. 22. Principle of isotachophoresis.
in temperature and one can take advantage of this effect to locate
the position of the zone boundaries.
These thermic steps can be located e. g. by means of a
thermo-couple. This also yields information about
the differences in mobility between the separated sample ions. The
height of each step is directly related
to the mobility of the corresponding ion. Therefore if a reference
ion is introduced in the same run the
value of the ionic mobilities can be derived. The length of the
thermic step is proportional to the concent-
ration of the ion in the zone.
4. Instrumental aspects
A. Analytical isotachophoresis
a. Capillary columns:
Originally isotachophoresis was developed in a capillary tube where
convection is reduced due to the
narrowness of the tube and microquantities of samples can be
concentrated in narrow zones (Fig. 23). The
length of such a capillary is about 50cm with an internal diameter
of 0.4mm. No stabilizing agent such
as polyacrylamide or agarose is required, but some compounds may be
added to reduce electroendosmosis,
such as 0.05% polyvinyl alcohol, 0.5% methyl cellulose or Triton X
Let us now assume that a separation of anions is considered.
Initially the separation tube is filled with
the leading electrolyte which contains an anion with a net mobility
larger than any of the sample ions
present. The cation of the leading electrolyte has buffering
capacity in order to keep the pH of the electro-
lyte inside the tube almost constant during the analysis. The
anodic electrode compartment is filled with
Fig. 23. Analytical isotachophoresis in capillary columns.
the leading electrolyte and separated by means of a semi-permeable
membrane from the rest of the tube,
with the intention of decreasing the electroendosmosis and
prohibiting all kinds of hydrodynamic flow.
The cathodic compartment is filled with the terminator solution.
The ions of this electrolyte must have a
net mobility smaller than any of the sample constituents and
consequently smaller than that of the leading
ion. As the concentration of the terminating ion will adjust
automatically during the analysis (isotacho-
phoretic principle) the ionic strength of the terminating
electrolyte is chosen roughly so that its concentra-
tion approaches that of the leading electrolyte. The sample is
introduced by a microsyringe via a septum
between leading and terminating electrolyte while the current is
applied. Depending upon the electrolyte
conditions chosen, a stabilized current from 5 to 100 micro Amp. is
usual. Depending on the length of the
tube and on the nature of the terminating electrolyte the voltage
will vary between 1 kV at the beginning
and about 20kV at the end of the separation.
The isotachophoretic equilibrium is a steady state reached at the
end of the experimental run after a
period of moving boundary migration (Fig. 24). Initially the sample
ions move at different speed until separated in consecutive zones
according to the correct sequence of their net mobilities. When a
state is reached all zones move at an equal speed i. e.
isotachophoresis. No changes in either the sequence
or the composition of the zones will occur assuming that some
operational conditions are fulfilled which can
be summarized as follows (Table 8):
1) The capillary tube has a constant diameter and is uniformly
2) The leading electrolyte has a constant composition.
3) The buffer capacity of the leading electrolyte prevents pH zones
from migrating into the opposite direc-
tion of the migration of the sample zones.
4) The electroendosmosis is sufficiently suppressed.
5) The hydrodynamic flow is preventend.
As all zones move at an equal speed the potential gradient is
increasing from the front zone towards
the rear zone and is constant per zone. The zone boundaries are
remarkably sharp and unaffected by diffu-
Fig. 24. Schematic representation of the different steps
during isotachophoretic analysis.
Table 8. Operational conditions in isotachophoresis.
Table 9. Detectors in analytical isotachophoresis.
sion as duration of separation increases. The amount of heat
generated is specific for each zone and cons-
tant if a stabilized current is applied.
A variety of detectors can be used for determining quantitatively
the ionic species of the sample.
For example a thermal detector, or a conductivity detector using a
microsensing electrode or a potential
gradient electrode as shown in Table 9. Detectors based on the
absorption of UV or visible light yield
more specific information about each boundary. A combination of two
detectors, e. g. thermic and UV-
proved useful for component identification (Fig. 25).
b. Gel isotachophoresis:
Gel isotachophoresis can be performed either in tubes or on
thin-layer plates (Table 10). A tube
Table 10. Gel isotachophoresis.
isotachophoresis technique has been proposed by Ornstein,98)
Davies,37) Griffith113) and Catsimpoolas and
Kenney.114) It is based on the use of conventional equipment for
polyacrylamide gel electrophoresis with
different buffers, with a leading and terminating electrolyte at
the extremities of the gel . The ampholytes
are usually added to the sample. This technique enables the
prefractionation of the protein in the stack
gel. In a subsequent step the migration occurs throughout the tube.
The main advantage of this method
is an increased sharpness of the bands and a high resolution power.
Chrambach and Skyler115) made a
theoretical study of the electrolyte, pH, ionic strength, etc.
conditions for an optimal resolution of various
The thin-layer technique described by Vestermark and SSodin116)
made use of cellulose acetate spread on
a glass plate. Rayon-silk cellulose was used in the wicks
connecting the thin-layer to the buffer troughs.
The plate was cooled from underneath with sprinkling water and
detection was performed after the run
using either stains or autoradiography.
Uyttendaele117) has developed a similar technique using agarose as
supporting agent. Detection of the
fractions after the run occurs by staining and identification by
B. Preparative isotachophoresis
a. Column isotachophoresis:
For preparative purposes isotachophoresis can be carried out with
the same column equipment used for
preparative isoelectric focusing. In the latter method separation,
carried out in a sucrose gradient, resolves
only a few mg per cm2 cross-section area, and precipitation of
concentrated protein bands at their isoelectric
point has to be prevented. The separation capacity can be increased
by a factor 5-10 using polyacryl-
amide gel as a stabilizing agent with the disadvantage of requiring
recovery by elution of the sliced gel .
On the other side in preparative isotachophoresis, based on the
"steady-state stacking" technique intro-
duced by Ornstein,33) concentration of protein and carrier
ampholytes as a stack of discs in polyacrylamide
gel is a sereous advantage. This stack of discs located between the
leading and terminating ion migrates
throughout the column and can be eluted directly from the gel.
Moreover this migration occurs in a buffer
having sufficient ionic strength to prevent precipitation of the
Vol. 20. No. 1. 1976 (25)
A vertical column electrophoresis equipment118) with PAA as
supporting medium is commonly used
(Fig. 26). Generally the gel is polymerized in the presence of
ampholyte and the column consists of 2
parts, the lower containing buffer gel only, the upper containing
buffer gel+ampholytes. On top of the
gel the terminating electrolyte is layered connecting the system to
the upper electrode chamber. A buffer
of the same composition is used in the elution chamber in the lower
electrode compartment isolated from
the column by an electrically permeable membrane.
The protein sample mixed with the terminating electrolyte
(tris-glycine buffer) is layered on top of the
column. To facilitate the application of the sample solution its
viscosity is increased by addition of 3%
sucrose. The migration takes place between the leading ion, mostly
acetate and the terminating ion, mostly
glycine, in the order of their mobilities which corresponds to the
order of their pI value. Therefore the
migrating ampholine zone resembles a pH-gradient and the separation
of the protein is similar to that
taking place in isoelectric focusing with however a main advantage
of keeping the proteins away from their
isoelectric points. Such a column electrophoresis equipment is
commercially available from LKB (Fig. 27)
and with such equipment 250mg of protein can be successfully
b. Counter flow isotachophoresis:
Column efficiency can be increased by taking advantage of the
counter-flow technique (Fig. 28). This
involves the application of aa flow of leading electrolyte in the
direction opposite to the isotachophoresic
migration usually at a rate equal to the speed of migration so that
this migration is completely counter-
acted.119) This procedure is especially valuable for the
preparative isolation of minor constituents of a
protein sample. In this technique all or most of the compounds
other than those of interest will be broa-
dened to such an extent that they will migrate out of the column
towards the electrode compartment.
Furthermore, if the leading and terminating ions were chosen so
that their mobilities are close to that of
the sample ions of interest the other ions will migrate according
to ordinary zone electrophoresis. This
technique was applied by Preetz and Pfeifer119) to the separation
of lithium isotopes.
Fig. 26. Schematic view of the column for
adapted for preparative isotachophoresis.
Since its introduction, and especially in the last years,
isotachophoresis (ITP) has found applications in
various branches of chemistry. In isotachophoresis one can
distinguish 4main types of application (Table
1) The analytical separation of ions, especially of proteins,
peptides, nucleotides, phosphates, acids and
2) The preparative separation of samples on a large scale. Analysis
of gram samples and even larger have
3) The use of the concentration power for the analytical separation
of very dilute samples.
4) The determination of the mobilities in an accurate way.
These different types of application require a different equipment.
The use of capillary tubes is
recommended for the analytical separations and for the mobility
determinations. The thin-layer technique
can be adapted to an analytical scale and for the concentration of
dilute samples. Column isotachophoresis
seems very useful for the preparative analysis and also for the
concentration of samples.
A. Isotachophoresis in free solutions (capillary column)
The separation of inorganic ions was successfully demonstrated by
Konstantinov and Oshurkova120) and
Table 11. Fields of application of isotachophoresis.
Vol. 20. No. 1. 1976 (27)
by Preetz and Pfeifer,119) who achieved a considerable resolution
in the separation of isotopes with very
Everaerts and Verheggen121) applied ITP for the analysis of fruit
juices and obtained a separation of
both strong and weak acids and their salts.
Arlinger and Routs102) fractionated weak acids and the proteins
haemoglobin and ceruloplasmin in capi-
llary tubes. In order to achieve a record of the true separation of
the protein zones, they developed an
Nucleotides were successfully separated and the results are
comparable with zone electrophoresis .122) For the separation of
complex mixtures of nucleotides the use of a counter-flow of
electrolyte and/or a combina-
tion of more systems may be necessary.
Everaerts and Verheggen123) described the use of ITP in capillary
tubes with an apparatus modified
from earlier experimental models. The new equipment proved to work
well and separations of metal ions
and of weak acids were obtained with a reproducibility of 0.5% or
Some theoretical and practical aspects of isotachophoretic analysis
of anions, cations, nucleotides and
fatty acids were reported by Everaerts.110) The advantage in
analyzing fatty acids with ITP is that
no extraction is needed from the original sample. Due to the low
solubility of some fatty acids in water , methanol was used as the
solvent. Isotachophoresis was also one of the main topics at our
on Protides of the Biological Fluids (Table 12).
Everaerts124) introduced an apparatus for the conductometric
detection of the separated components
present in fruit juices. The detection of some components, at this
time not yet determined, was possible by the combination of a
UV-detector with their conductometric detector.
For the isotachophoresis in the absence of any supporting medium
Hjerten used his neutral gravity
apparatus previously utilized for conventional zone electrophoresis
and for isoelectric focusing .12s) Stabiliza- tion against
convective disturbances is achieved by a slow rotation of the
electrophoresis tube . He obtained a good separation of low
molecular weight substances such as nucleotides, utilizing succinic
and citric acid
as spacers. An increased resolution for human plasma proteins was
obtained by mixing the plasma with
spacers in the form of carrier ampholytes. Large particles, such as
viruses and bacteria, are unable to
migrate electrophoretically on a supporting medium, so that the
only alternative in such case is the isota-
chophoresis in free solution. In this way Hjerten isolated
satellite tobacco necrosis virus in a pure form . Furthermore, he
submitted whole cells to ITP. At the same Colloquium Bier126)
presented the poten-
tial use of ITP under zero gravity in space because it appeared to
be a high resolution technique potentially
applicable to living cells.
Previously, analytical ITP in capillary tubes has mainly been used
for the separation of relatively low
Table 12. Application of isotachophoresis in capillary columns
on Protides of the Biological Fluids, 22, 1974.
molecular weight components. This limitation was due to
disturbances caused by gravity and electroendos-
mosis. However by adding methylcellulose to the leading electrolyte
in order to increase the viscosity and
to reduce the electroendosmosis a good stabilization is obtained.
Arlinger127) studied haemoglobin as a
model system for protein separation using methylcellulose in the
We presented the isotachophoretic separation of the plasma HDL
apoproteins in order to confirm the
data obtained by means of other electrophoretic and/or
chromatographic separation methods.128) The influ-
ence of phospholipids on the apoprotein mobility was also
investigated by comparing the patterns obtained
with delipidated and relipidated high density lipoproteins (HDL).
The apo HDL separated into two main
fractions as shown by the thermal signal line, whereas the
UV-signal resolved several subcomponents (Fig.
29). This figure points out that the main part of the protein was
stabilized between the leading and the
terminating electrolyte. The small UV tailing however suggested
that a minor part is separated zone
electrophoretically since this had a lower mobility than the
terminating ion. In order to evaluate the
influence of the phospholipid binding on the apoprotein mobility,
the thermal step height was calculated for
the different samples and summarized in Table 13.
Fig. 29. Isotachophoretic analysis and SDS-PAGE
of human apo HDL.
analysis of lipoproteins.
Vol. 20. No.1. 1976 (29)
These results demonstrate that the apo HDL separations are
reproducible and that the apoprotein mobi-
lity was significantly decreased by its lipid load. The first
fraction appeared to be more sensitive to lipid
binding and its mobility became twice as high in the relipidated
sample as in apo HDL, while the mobility
of the other fraction was less affected (Fig. 30). The identity of
the two fractions was checked against
isolated apo Lp-AI and apo Lp-AII fractions.
From these data we can conclude that ITP of apo HDL resolves the
two main apoproteins and that a
selective decrease in the apoprotein mobility is observed after
relipidation of apo HDL. Therefore isota-
chophoresis might become a useful tool in the study of selective
relipidation of the apoproteins and for
detecting abnormalities in lipid transport by lipoproteins in
Synthesizing peptides with the solid-phase Merrifield technique
requires great efforts in the purification
of the material in order to obtain a single pure peptide. For this
reason Kopwillem129) used ITP to follow
the purification steps of an undeca peptide and different
electrolyte systems were tested to optimize the
Dunn and Kemp130) reported on isotachophoretic studies of adenosine
phosphates from perfused mouse
liver cells at different levels of divalent cations.
Isotachophoresis was also used as a new technique for the
determination of tissue metabolite concentra-
tions. Sjodin131) concluded from their experiments on tissue
samples such as of human skeletal muscle
biopsies, that ITP gives equal or better possibilities than other
methods to obtain quick and accurate analysis
Kopwillem132) applied analytical ITP to the qualitative and
quantitative analysis of serum phenylalanine
for the detection of the metabolic disorder phenylketonuria. The
method is extremely simple, does not
require expensive reagents or lengthy sample manipulations, is not
based on a colorimetric reaction, and
gives results comparable to those obtained by standard techniques.
This method however can only be
applied for the confirmation and quantitation of phenylketonuric
samples already detected during mass-screen-
Catsimpoolas and Kenney114) separated human serum proteins on
polyacrylamide gel tubes using ampho-
Svendsen133) demonstrated the reproducibility of preparative ITP
and the effect of increasing the amount
of carrier ampholytes for the separation of human haemoglobin on
polyacrylamide gel columns. The power
Fig. 30. Isotachophoretic analysis of HDL, apo HDL and apo
of preparative ITP is considerably increased by performing two or
three consecutive experiments at different
molarity ratios of leading ion and counter-ion and that the carrier
ampholytes must be chosen so that the
pH range coincides with the pK value of the protein to within±0.5
Griffith113) reported on some important factors affecting
analytical separation of proteins; in poly-
acrylamide gels with carrier ampholyte spacers. For the separation
of human haemoglobin, beta lactoglobulin
and soybean trypsin inhibitor, the influence of the most important
variables, such as the effect of carrier
ampholyte concentration, pH range of ampholytes, concentration of
leading ion, pH of leading electrolyte,
nature of leading ion, duration of the run and the field strength
A preparative ITP separation method using carrier ampholytes as
buffer and spacer substances was
described by Svendsen and Rose.118) The separation takes place in a
carrier ampholyte gradient migrating
isotachophoretically in a polyacrylamide gel column. In comparison
to isoelectric focusing in a sucrose
gradient, this method allows a larger amount of protein to be
separated and the proteins migrate electrically
charged in a buffer having sufficient ionic strength to reduce the
risk of precipitation.
A set of applications was demonstrated at our 22nd Colloquium in
1974 (Table 14). Bog-Hansen134>
demonstrated the preparative ITP of human erythrocyte membrane
proteins, totally solubilized by
sodium dodecyl sulphate, and indicated some possibilities of
introducing an interacting component in the
electrophoretic system. However, the results obtained with
non-ionic detergents were more promising.
In another paper the same authors135) described technical details
of the procedure using non-ionic deter-
Preparative isotachophoresis was also proved to be an excellent
method for the purification of enzymes ,
with protection of enzyme activity and high recovery, especially
when combined with biospecific interaction
for the removal of impurities.136) Chrambach and Skyler115)
described a practical application of selective
stacking of the isohormones of human growth hormone. Selective
stacking permits the introduction of
charged molecules into the automated procedures of analytical and
preparative "qualitative PAGE" at an
optimized pH. It is universally applicable without regard to
initial macromolecule concentration. In the
work of Kopwillem137) it has been demonstrated that amino acids and
peptides can act as discrete spacers,
separating human serum proteins into distinct sub-groups.
Quantitative immunoelectrophoresis of fractions
obtained from polyacrylamide gel columns showed the complete
resolution of eight human serum proteins
into three sub-groups using threonine and glycine as spacers. The
maximal capacity of the columns was
not fully investigated, but an amount of 120mg of protein applied
seemed to be far below the separation
capacity of the system.
Vestermark and Sjodin116) applied isotachophoresis and zone
electrophoresis separately or in combination
for the separation of various glucose metabolites. For the
isotachophoretic experiments, cellulose acetate
Table 14. Applications of isotachophoresis in gels presented
Protides of the Biological Fluids, 22, 1974.
Vol. 20. No. 1. 1976 (31)
strips and for the zone electrophoresis, cellulose thin-layer
plates were used. The portion of the isotachopho-
retic strip containing the zones of interest was cut out and placed
on the cellulose thin-layer. If there is
incomplete separation during ITP, the separation in the first
dimension has to be rerun, the cellulose acetate
strip containing the zones of interest being used for a second
isotachophoretic separation. This rerunning
procedure is similar to that used by Kendall and Crittenden138)
many years ago in an attempt to separate
isotopes. At our 22nd Colloquium on Protides of the Biological
Fluids we introduced agarose as a suppor-
ting medium in ITP thus opening the possibility for immunological
analysis of the separated and concentra-
The experimental conditions for gel isotachophoresis of proteins
are shown in Table 15. The samples
used, their quantity and the amount of spacer are given in Table
16. Feasibility studies were performed
with albumin-transferrin solutions. The isotachophoretic pattern
presents two well-separated protein bands
which are identified against anti-transferrin and anti-albumin
(Fig. 31). The precipitation lines demonstrated
the homogeneity of the separated proteins and proves the
sensitivity of the method.
Isotachophoretic patterns of urinary samples are shown in Fig. 32.
Normal urine contains traces of
albumin. Orthostatic albuminuria has a distinct albumin and
transferrin line. Urine samples of nephrotic
proteins show±eight distinct fractions identified as albumin,
transferrin, IgG, IgA and four unidentified
Electrophoretic protein patterns of human sweat are very difficult
to obtain. Due to the simultaneous
concentration and fractionation power of ITP we could readily
detect different proteins in human sweat.
Table 15. Experimental requirements for gel
Table 16. Source and quantity of sample and quantity of
used in isotachophoresis on agarose thin-layers.117)
Beside the presence of albumin and transferrin a lipid carrying
protein of undetermined nature was also
detected (Fig. 33).
Vol. 20. No. 1. 1976 (33)
IV. Evaluation of Specific Electrophoretic Methods
Let us now make a general overview of isoelectric focusing and
isotachophoresis and attempt an evalua-
tion and a comparison of these methods with regard to zone
electrophoresis. The general outlines of this
comparison have been summarized in Table 17. We shall give comments
considering isotachophoresis as
the preferential method.
and zone electrophoresis.
Advantages linked to isotachophoresis
Some of these advantages can also be found in isoelectric
1) Concentration of the substances after separation are determined
solely by their physical properties and
the starting conditions in the apparatus. They are independent of
the initial concentration in the sample
and of the separation time.
2) A concentration effect occurs simultaneously with the
separation. Even when a dilute sample is applied,
its final concentration will be comparable to that of the starting
electrolyte. By contrast, in zone electro-
phoresis, a substance originally present in low concentration
becomes more dilute as the separation proceeds. 3) The sharpness of
the boundary between a given pair of substances is function of the
properties of these
ions and of the experimental conditions but independent of the
original sample concentration. Diffusion is
constantly counteracted so that the sharpness of the boundary is
not altered while separation evolves. This
again is in contrast with zone electrophoresis where a longer
separation aimed towards a theoretical impro-
vement of the resolution is limited by increasing diffusion.
4) The use of spacers with intermediary mobilities enables
separation of ions with close mobilities. The
distance between two zones is a function of the spacer
5) General detection methods based on conductivity or potential
measurements can be applied. Thus any
material present in a sufficient quantity to occupy about 1mm
length of the tube after separation can be
detected. Moreover specific detectors, such as UV or visible
absorption will identify the substances.
6) Use of various solvents is possible. The separation can be
carried out either in aqueous or in organic
7) Columns have a high capacity and fractions are eluted in a sharp
band and in a very high concentra-
tion. Up to 1g of sample can be applied.
8) The method is very gentle to proteins as it does not require to
act at the isoelectric point as with IEF.
This also means that large concentrations can be carried in a
BiFadvantages linked to isotachophoresis
1) It is necessary to select leading and terminating electrolytes
whose mobilities are respectively higher
and lower than that of the sample ions. For low molecular weight
compounds such as organic or anorganic
salts, isotopes, fatty acids, amino acids, etc. a number of ions
are convenient. However separation of high
molecular weight compounds such as peptides or proteins require the
use of special ions such as cacodillic
2) Electroendosmosis must be reduced in the column by increasing
the viscosity of the medium. Various
substances have been proposed such as Triton X 100,
methylcellulose, polyvinylalcohol. Interference with
the sample might however occur.
One requires careful cooling and thermostating, specially if the
zones are located by thermal detection.
3) Resolution is limited by mobility differences between the
components. The use of spacers should
theoretically improve the separation but at the same time it
induces a dilution of the sample fractions.
Moreover in the presence of spacers only the UV detection is
meaningful as the thermal signal presents
only shallow boundaries.
Novel Possibilities for Electrophoresis
All possibilities of the electrophoretic principle have not yet
been exhausted. Performing experiments
under zero gravity could provide better separation systems and in
this way the separation of heavy particles
such as cells could be improved.
Through the absence of gravity the viscosity parameter can be
handled in isolation from the gravity effect
of concentration (Table 18). By the addition of some uncharged
molecules to the carrier buffer the viscosity
parameter can be modified either as a gradient or stepwise without
interference from the concomittant den- sity problems occuring at
gravity one. The viscosity parameter could thus be introduced into
any free flow
uni or two-dimensional system and in combination with an
independent pH-gradient. In preliminary inves-
tigations on the effect of carrier solvent viscosity on protein
solutions we demonstrated that a promising
field of investigation is to be found in the manipulation of this
parameter during separation.139)
Table 18. Electrophoretic mobility as a function of gravity.
Vol. 20. No. 1. 1976 (35)
It follows that refined separation conditions obtainable only at
zero gravity may yield refined fractiona-
tion of some classes of molecules or cells considered to be
homogeneous with the existing methods . There-
fore the handling of the viscosity parameter present in the
fundamental law of electrophoretic displacement
opens a new avenue in electrophoresis. This could be achieved at
zero gravity under the weightless condi-
tions of a space flight or simulated on earth under neutral gravity
conditions such as can be realised in the
rotating tube technique described by Hjerten.30)
I hope to be able to describe these results at one of your next
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