Vol. 20. No. 1. 1976 (3)

〔Special Lecture〕

Isoelectric Focusing and Isotachophoresis applied

in Protein Chemistry*

By

Hubert Peeters**

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 Hirai,

Chairman of your Society, is an active and devoted committee member.

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 Society.

I. General Introduction

Tiselius1) (Fig. 1) clearly distinguished three modes of chro-

matography, namely 1. Elution or zone chromatography, 2. Frontal

analysis and 3. Displacement chromatography. The same modes

may be distinguished in electrophoresis (Table 1). These techniques

are 1. Zone electrophoresis, 2. Moving boundary electrophoresis,

3. Isotachophoresis. One more development which is not paralle-

led in chromatography was introduced and called 4. Isoelectric

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 General Meeting on October 25, 1975.

** Director of Simon Stevin Institute, Brugge, Eelgium.

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Fig. 2. Basic principles of the electrophoretic techniques.

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 use.

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.

3. Isotachophoresis

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

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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 applied simultaneously.

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

and cathode.

Around 1954-1958 the technique was taken up by Kolin8～11) who introduced a sucrose density gradient to

reduce the effect of electroendosmosis and other convective forces. Finally in 1961-1962 Svensson12～14)

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

separation.

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 methods.

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.

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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's8～11) 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 Svensson12～14) 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.

Vesterberg19～21) 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 stabilization.

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.22～28) 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 materials.

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.

Fig. 3. General formula for the Ampholine carrier ampholytes.

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 electric field.

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

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Vol. 20. No. 1. 1976 (7)

Fig. 4. Principle of isoelectric focusing.

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

components.

A treatment of the theoretical background of isoelectric focusing is to be found in the fundamental

papers of Svensson and Vesteryberg12～14,20.21).

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)

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Fig. 6. Apparatus for isoelectric focusing in gel

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 generation.

B. Preparative procedures

Due to its superior resolving power, isoelectric focusing became a generally applicable and efficient

preparative procedure.

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,

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Vol. 20. No. 1. 1976 (9 )

Fig. 7. Isoelectric focusing column (LKB).

Fig. 8. Multi-membrane apparatus described by Williams and Waterman.15)

Fig. 9. Apparatus for zone convection isoelectric focusing according to Valmet.29)

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allowing purification of several grams of materials.

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)

b. Gels:

Polyacrylamide gels are prepared either by chemical or by photopolymerization. Photopolymerization

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.

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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.

5. Applications

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 point.

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 Valmet.44).

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 monomers.45)

Fig. 11. Density _gradient isoelectric focusing of delipidated BSA

(0.2mol FA/mot BSA).45)

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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 studies.

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 BSA

(9mot FA/mol BSA).45)

Fig. 13. Density gradient isoelectric focusing of relipidated BSA

(2mol FA/mol BSA).45)

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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 the separation.

Fig. 14. Density gradient isoelectric focusing of human apo HDL3

with and without 8M urea.47)

Fig. 15. Density gradient isoelectric focusing

of human apo HDL3 in different pH

ranges.47)

Fig. 16. Density gradient isoelectric focusing

of human apo HDL3 during 23 hr and

72 hr respectively.47)

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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 procedures:

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 focusing

presented on Protides of the Biological Fluids.

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Table 5. Applications of one-dimensional gel isoelectric focusing.

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 constituents63～66) and for the analysis of metalloproteins.67～70) 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

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Fig. 17. Isoelectric focusing of the major HDL apoproteins, apo Lp-AI

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 major band.

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 Alpert.79)

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):

Immunochemical methods can be applied after isoelectric focusing in polyacrylamide or organic gels31～33)

Catsimpoolas82) distinguished three types of immunoisoelectric focusing.

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

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Table 6. Applications of two-dimensional gel isoelectric focusing.

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

electrolytes.

A further development was the introduction of isoelectric focusing-gel electrophoresis.85,86)

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.

III. Isotachophoresis

1. Historical development

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-

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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-

lines".

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) Everaerts109～111)and Routs.112)

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. 18).

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:

I=I-1+I+1=I-2+I+2… (1)

where:

I-1=C-1 μ-1 F1

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Vol. 20. No. 1. 1976 (19)

Fig. 18. Isotachophoretic conditions.

I+1=C+1μ+F1

I-2=C-2μ-2F2

I+2=C+2μ+F2

From (1) it follows that:

(C-1μ-1+C+1μ+)F1=(C-2μ-2+C-2μ+)F2…(2)

According to the principle of isotachophoresis and once equilibrium is reached, all ions migrate at the same

velocity: v1=v2

with v1=μ-1F1

v2=μ-2F2

Introducing this into equation (2) we obtain:

C-1μ-1+C+1μ+/μ-1=C-2μ-2+C+2μ+/μ-2…(3)

Moreover the condition of electroneutrality requires that:

c+1=C-1

C+2=C-2

and this leads to:

C-1(μ-1+μ+)/μ-1=C-2(μ-2+μ+)/μ-2

or:

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

zone.

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

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Fig. 19. Zone concentration in isotachophoresis

(two electrolytes).

Fig. 20. Zone concentration in isotachophoresis

(three electrolytes).

than the equilibrium concentration given by Kohlrausch's equation.

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

manipulated.

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

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Vol. 20. No. 1. 1976 (21)

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 100.

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

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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 steady

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 cooled.

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-

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Vol. 20. No. 1. 1976 (23)

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

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Fig. 25. Dual detector system in isotachophoresis.

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

electrolytes.

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 immunoelectrophoresis.

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 proteins.

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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 separated.

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

preparative isotachophoresis.

Fig. 27. The LKB Uniphor column equipment

adapted for preparative isotachophoresis.

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Fig. 28. Apparatus for counter-flow isotachophoresis.

5. Applications

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

11):

1) The analytical separation of ions, especially of proteins, peptides, nucleotides, phosphates, acids and

metal ions.

2) The preparative separation of samples on a large scale. Analysis of gram samples and even larger have

been reported.

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.

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Vol. 20. No. 1. 1976 (27)

by Preetz and Pfeifer,119) who achieved a considerable resolution in the separation of isotopes with very

close mobilities.

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

UV-absorption detector.

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 even better.

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 22nd Colloquium

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 obtaineda 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 presented

on Protides of the Biological Fluids, 22, 1974.

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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 electrolyte.

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.

Table 13. Influence of lipids on the isotach'ophoretic

analysis of lipoproteins.

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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 disease.

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

peptide separation.

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

of metabolites.

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-

ing.

B. Isotachophoresis in gel tubes

Catsimpoolas and Kenney114) separated human serum proteins on polyacrylamide gel tubes using ampho-

line spacers.

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 HDL-

phospholipid complexes.

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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 pH unit.

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 were studied.

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-

gents.

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.

C. Isotachophoresis in gel thin-layers (Table 14)

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 on

Protides of the Biological Fluids, 22, 1974.

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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-

ted fractions.117)

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

lines.

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 isotachophoresis.117)

Table 16. Source and quantity of sample and quantity of spacer

used in isotachophoresis on agarose thin-layers.117)

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Beside the presence of albumin and transferrin a lipid carrying protein of undetermined nature was also

detected (Fig. 33).

Fig. 31. Isotachophoretic analysis on aggrose of human albumin-transferrin.

Fig. 32. Isotachophoretic analysis on agarose of urinary samples.

Fig. 33. Isotachophoretic analysis on agarose of human sweat.

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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.

Table 17. Comparison of isotachophoresis, isoelectric focusing

and zone electrophoresis.

Advantages linked to isotachophoresis

Some of these advantages can also be found in isoelectric focusing.

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

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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 concentration.

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

medium.

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 narrow zone.

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

acid.

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.

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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 meetings.

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