Synthesis of carrier ampholyte mixtures suitable for isoelectric fractionation analysis

ANALYTICAL

BIOCHEMISTRY

Synthesis

102, 134- 144 (1980)

of Carrier

Ampholyte Mixtures Suitable Fractionation Analysis1 W. JUST

WILHELM Institut

fuer

for Isoelectric

Biochemie I, Universitaet Heidelberg, D-6900 Heidelberg, Federal Republic

Im Neuenheimer of Germany

Feld

328,

Received April 11, 1979 A new procedure for the preparation of carrier ampholyte mixtures for isoelectric focusing is described. The procedure is based on the reaction of an o-, p-unsaturated carboxylic acid ester and a polyethylene polyamine followed by subsequent hydrolysis of the reaction products. The synthesis procedure is described in detail using acrylic acid methyl ester and pentaethylenehexamine. Due to the high reaction velocity by which acrylic acid methyl ester adds to the amine moiety, it was possible to perform the synthesis in a controlled manner such that the obtained carrier ampholyte mixture exhibits a completely linear pH profile as well as uniform conductivity and buffering capacity without any additional manipulations. Furthermore, the procedure may also be applicable for the direct synthesis of carrier ampholytes covering restricted pH ranges. The high resolution capacity of the synthesized carrier ampholyte preparation is demonstrated by the fractionation of several test proteins.

The technique of isoelectric focusing is distinguished by the fact that a properly charged amphoteric molecule placed within a continuous pH gradient, increasing in the direction of the electric current, migrates under the action of the electric field force toward its isoelectric point (PI) where it condenses in a discrete focal zone due to loss of charge. Thus, isoelectric focusing offers the possibility to fractionate and to characterize a complex mixture of properly charged amphoteres. Two different routes for the generation of continuous pH gradients are known. The one utilizing normal buffer systems is the route by which the principle of isoelectric focusing was first demonstrated by Kolin (1,2). Buffer electrofocusing using either amphoteric or nonamphoteric systems was recently expanded by Nguyen and Chrambach (3,4) and Chrambach and Nguyen (5). These buffer

systems which may be formed according to the isotachophoretic principle provided normal times for pH gradient generation, stability range, and decay of the pH gradient. The pH range, the steepness, and the ionic strength of the buffer systems are extensively susceptible by varying the buffer components. A gradual change of the dielectric constant of a normal buffer solution was utilized by Troitsky et al. (6) to obtain narrow-range pH gradients. The other route, the theory of which was mainly elaborated by Svensson (7-9), utilizes large numbers of low molecular weight amphoteres, each exhibiting a slightly different pl. Upon stationary electrolysis these amphoteres (carrier ampholytes) align in order of their pl values. Since the complete separation of a mixture of two amphoteres requires a third intermediate amphotere (7,10), the complete separation of a multicomponent mixture of amphoteres requires a carrier ampholyte mixture composed of a large number of ampholyte species, each having a different pl.

r This work was reported in parts at the Electrophorese Forum, Technical University Munich, October 25-27, 1978. 134 0003-2697/80/030134-11$02.00/O Copyright All rights

0 1980 by Academic Press, Inc. of reproduction in any form reserved.

CARRIER

AMPHOLYTE

The first synthesis of a suitable aminocarboxylic acid carrier ampholyte mixture was reported by Vesterberg (11). Other laboratories (12- 15) have reported their success in obtaining carrier ampholyte mixtures following Vesterberg’s procedure, which needs fractionation and reconstitution of the synthesized carrier ampholytes. A different synthetic approach was introduced by Pogacar and Jarecki (16) and Grubhofer (17) who synthesized aminosulfonic acid and aminophosphonic acid carrier ampholytes. These ampholytes, however, were not able to cover the pH range 3.5-5.8 without the further introduction of carboxylic acid groups. Blanicky and Pihar ( 18) reported the use of commercial peptone samples for isoelectric focusing. Fawcett (19), however, observed nonlinear pH profiles and a diminished resolution using Bactopeptone as a wide pH range carrier ampholyte preparation. Another type of aminocarboxylic acid carrier ampholyte (Pharmalyte) was recently prepared by copolymerization of epichlorohydrine with amines, amino acids, and peptides (20). The synthesis allowed the preparation of a high number of carrier ampholyte species. Therefore, the pH gradients are distinguished by a rather uniform conductivity and buffering capacity. The synthesis, however, contains a gel filtration step which for laboratory use is very time consuming. The present work describes a new procedure for the synthesis of /3-aminocarboxylic acid carrier ampholytes.2 The procedure is rapidly and easily performed, is well reproducible, and does not require experience with special synthetic techniques. It offers an end product which under stationary electrolysis forms a linear pH gradient of uniform conductivity and buffering capacity prevailing for sufficient time to carry out isoelectric focusing. The uv/visible absorbancy of the synthesized carrier ampholytes is low. The obtained carrier ampholyte ’ U. S. patent

No. 4131534,

other

patents

pending.

135

PREPARATION

mixture may be advantageous particularly in preparative work for which the method is not often used for economic reasons. MATERIALS

Acrylic acid and acrylic acid methyl ester were obtained from Fluka (Buchs, Switzerland). The substances were distilled prior to use, to remove the polymerization inhibitor. Various oligoethyleneimines were obtained by fractional distillation following the reaction of either dichloro- or dibromoethane with ammonia (20). As the starting amine, pentaethylenehexamine, which was obtained as a pale yellow oil distilling between 180200°C at l-2 Tot-r, was mainly used. The average molecular weight of this product was 232 as determined by cryoscopy. Heptaethyleneoktamine was the highest homologue which was obtained by vacuum distillation. Other reagents used were of analytical grade and were obtained from various sources. METHODS

Synthesis of Carrier Ampholytes From the different possible variants of synthesis only the one providing optimal results under a minimum of handlings is described. Other variants are discussed. Stock solutions of 1 M pentaethylenehexamine and 4 M acrylic acid methyl ester in methanol were prepared. Each of the solutions was poured into the reservoirs of a suitable gradient former. The Ultrograd gradient former (LKB, Bromma) was used for these studies; however, any other model should be adequate. If such an apparatus is not available a stepwise gradient could be used. The equipment consisted of the control gear containing the information of the gradient characteristics in a template. The control gear regulates a valve which on the one hand is connected to the reservoirs and on the other hand to a mixing channel.

136

WILHELM

Liquid flow is controlled by a peristaltic pump located behind the channel. Following thorough mixing, the solution was pumped through a reaction coil made from Teflon tubing of 1.5-2.0 mm i.d. which was contained in a water bath maintained at 40°C. The pumping rate was chosen so as to assure retention of the solution within the reaction coil for at least 1.5 h. For IO- 12 m Teflon tubing the flow rate was about 0.4 ml/min. After passage through the reaction coil, the liquid outflow was collected in fractions of 2-3 ml which were allowed to stand for another 2 h at room temperature. Each 1520 successive fractions were combined. The solvent was evaporated at 30-40°C and after dissolution of the residue in toluene the solvent was removed again. This procedure was repeated once more to eliminate unreacted acrylic acid methyl ester. To the oily and slightly yellow residues, 50 ml water was added and the aqueous solutions were placed into a sterilizer for 2 h at about 120°C. Molecules containing high amounts of ester groups do not readily dissolve in water. However, this does not considerably influence their hydrolysis. If a product of low optical density was desired, milder hydrolysis conditions were chosen. Therefore, the aqueous solutions kept under nitrogen were maintained in a water bath at 40-60°C until pH constancy, which was normally reached after 16-20 h. During hydrolysis, discoloration of the product increased. However, a good deal of the absorbancy in the visible and the uv region was removed by catalytic hydrogenation of the combined fractions for 2-4 h at 40°C. Palladium on activated charcoal (Merck, Darmstadt) served as a suitable hydrogenation catalyst. After filtration, which has to be done under exclusion of oxygen, the colorless aqueous solution was lyophilized. Dissolving the residue in ethanol and removing the solvent on the rotary evaporator results in a colorless crystalline but hygroscopic powder.

W. JUS’I

Computation of the Template Pentaethylenehexamine contains six nitrogen atoms in a molecule to which a maximum of eight carboxylic acid ester molecules may be linked. Therefore, the simplest ampholyte is formed by reacting the amine and carboxylic acid ester solutions in a molar ratio of 1: I. Using the stock solutions mentioned above this 1:l ratio is obtained when the pentaethylenehexamine and the acrylic acid methyl ester solutions are mixed in the percentage concentration ratio of 80:20, respectively. This ratio, therefore, represents the starting ratio for the synthesis. The final ratio at which the synthesis is completed is reached at a molar ratio of the amine and the carboxylic acid ester of 1:8, or at a percentage ratio of the stock solutions of 35:65, respectively. Starting from the percentage ratio of 80:20 several individual fractions were prepared changing the ratio gradually until reaching the final ratio of 35:65. The fractions were stored at 35°C for 2 h. The solvent was removed in the vacuum and the residue, after dissolution in water, was hydrolyzed for 2 h at 120°C in a sterilizer. Figure 1 shows the pH of the various fractions plotted versus the percentage ratios of the stock solutions. The pH of a particular fraction approximately indicates the pZ of the ampholytes formed. Therefore, this curve basically served for the entire computation of the template. One additional correction was made. Since the synthesis was performed by adding increasing concentrations of acrylic acid methyl ester to decreasing concentrations of pentaethylenehexamine, the number of ampholyte molecules synthesized per time unit decreases concomitantly since it is obviously dependent on the pentaethylenehexamine concentration. The decrease of N-H equivalents available for the reaction per unit of time or volume is shown in Table 1. To keep constant the amount of material synthesized per time unit, this decrease of N-H equivalents was balanced

CARRIER AMPHOLYTE

137

PREPARATION

I

80

60

40

20

40

60

20 80

For most cases the pH gradient characteristics provided by the synthesized ampholytes were investigated after establishing TABLE OF THE AND

PERCENTAGE 4

M

6

5

the pH gradients in a continuous-flow electrophoresis apparatus as previously described (22-25). An electric field strength of 130 V/cm and a flow-through time of about 60 min was applied. The apparatus allowed the splitting of the pH gradient into 48 fractions. The pH gradients were also developed either in a density gradient column or in a polyacrylamide gel plate using standard procedures. The acrylamide concentration in the l-mm gel was 5% and the crosslinking percentage 3%. The ampholytes were incorporated into the gel at a final concentration of 2%. To demonstrate the generation and stability of the pH gradient the same gel (110 x 240 mm) was prepared 2 mm thick containing the ampholytes at a 2% concen-

of the Carrier Atnphoiytes

VARIATION

7

FIG. 2. Template used for the synthesis of PEHA ampholytes covering the pl range of 3.5- 10. The construction of the template is outlined in the text.

by proportionally increasing the time available for the synthesis of one pH unit. In the entire graph of the synthesis template which is shown in Fig. 2, the total pentaethylenehexamine concentration is represented by the upper area and a balanced synthesis is obtained by providing equal areas for each synthesized pH unit. Characterization

8

PH

% AMINE % AME

FIG. 1. The pH of the hydrolyzed reaction products which were obtained by the reaction of various percentage ratios of 4 M AME and 1 M amine in methanohc solution. TETA = Triethylenetetramine; TEPA = Tetraethylenepentamine; HEOA = Heptaethyleneoctamine. PEHA = Pentaethylenehexamine.

THE

I 9

31,

RATIOS

ACRYLIC

ACID

1 OF

1M

METHYL

(PEHA)

PENTAETHYLENEHEXAMINE ESTER

(AME)

N-H equivalents Percentage PEHA

Percentage AME

N-H equivalents

AME equivalents

AME equivalents

PH

90 80 70 60 50 40 35 30

10 20 30 40 50 60 65 70

7.2 6.4 5.6 4.8 4.0 3.2 2.8 2.4

0.4 0.8 1.2 1.6 2.0 2.4 2.6 2.8

18.0 8.0 4.7 3.0 2.0 1.3 1.1 0.9

10.5 10.2 9.6 8.8 6.7 5.1 4.6 4.5

” The variation is accompanied by a decrease in the N-H equivalents of the amine available for the reaction. The ratio N-H equivalents/AME equivalents indicates the starting and terminating percentage ratios of both reaction components, respectively. The pH of the various fractions was measured after a 2 h hydrolysis at 120°C.

138

WILHELM

W. JUST

100 -.

.

80

.

.-

90

1.20

AA

0

30

60 tme

min

FIG. 3. Velocity of the addition reaction of 4 M acrylic acid (AA) and 4 M acrylic acid methyl ester (AME) to equal volumes of 1 M pentaethylenehexamine (PEHA) in methanolic solution at room temperature. The ordinate represents the percentage decrease of the OD at 260 nm.

tration. Isoelectric focusing was performed at 10 W constant power for up to 7 h at 0°C. At various times the power was switched off and a lo-mm gel strip was cut into 21 successive fractions of 5 mm length. The gel pieces were immediately immersed in 1 ml H,O and carefully homogenized. The homogenate was used for determining the pH and the buffering capacity. All the other physicochemical determinations relate to a 1% ampholyte concentration. Capillary isotachophoresis was carried out using the Tachophor (LKB, Bromma).

= i

g

The leading electrolyte system was 5 mM glutamic acid/T& of pH 7.2 and the terminating electrolyte system was 5 mM glycine/ Tris of pH 9.0 (26). Hydroxypropylmethyl cellulose (DOW Chemical Co. Midland, Mich.) was added to the leading electrolyte buffer at a final concentration of 0.1% to suppress electroendosmosis. RESULTS

In Fig. 3 the velocities of the addition of 4 M acrylic acid and 4 M acrylic acid

_ 6-

3.14*-

0,3-

FIG. 4. pH Profiles of three independently synthesized batches of PEHA-ampholytes (- 0 -, - 0 -, - n -) as well as buffering capacity (-. .-), conductivity (- - -), and OD at 365 nm (-.-.) and 280 nm (. .) of one batch (-0-) following continuous-flow electrophoretic pH gradient development.

CARRIER AMPHOLYTE

139

PREPARATION

-; d(-j E : I : I 3.10

-2

-

PH

0.4-

2.16*0.21-10-*-

o-

o-

OJ, 0

10

20 fractiot!

FIG. 5. pH Profiles, buffering capacity, and OD at 365 and 280 nm (upper curve) of PEHA ampholytes used for isoelectric fractionation of proteins shown in Figs. 9 and 10.

methyl ester to equal volumes of 1 M pentaehtylenehexamine at room temperature in methanolic solutions are compared. The percentage decrease of the concentration of the unsaturated compounds, as measured by the decrease in OD at 260 nm, is plotted versus the time. After 2 h all the acrylic acid methyl ester present in the mixture was readily consumed. Within that period practically no reaction of acrylic acid was observed. After 16 h, 76% of unreacted acrylic acid was recovered. The reaction velocity of acrylic acid was practically the same using either aqueous or methanolic solutions. Figure 4 demonstrates the pH profiles of three independent synthesis batches, exhibiting the high reproducibility of the synthesis procedure. For one of the three batches the OD at 365 and 280 nm was recorded as well as the conductivity and the buffering capacity. The synthesis of carrier ampholytes covering the pH range of 3.5-9, using a commercially available pentaethylenehexamine preparation, is shown in Fig. 5. Except for somewhat higher OD measured at 280 nm this amine was found to be as suitable as the synthesized amine mixture. The formation of the pH gradient and its stability was studied over a time period of 7 h (Fig. 6). During this time 10 W constant

power were applied to the gel. Linearity of the pH gradient was reached between 2 and 3.5 h and remained constant up to 7 h. At that time the pH gradient starts to decay, 10,

~”

*IO

=

1009 600 200 2

4

6

8

FIG. 6. Formation and stability of the pH gradient (O), simultaneous distribution of buffering capacity (0) as well as time course of voltage (x) and current (A) in a 2-mm polyacrylamide gel of 5% T and 3% C and a carrier ampholyte concentration of 2%. To the gel a constant power of 10 W was applied at 0°C (see Methods). Linearity of the pH gradient is reached between 2 and 3.5 h and remains constant up to 7 h. Under these conditions isoelectric focusing was normally finished between 3 and 4 h.

140

WILHELM

W. JUST

6-

211 A

IO

20

i0

40

fractions

FIG. 7. pH Profiles ofPEHA ampholytes covering restricted@ ranges. The ampholytes were obtained by utilizing corresponding parts of the template shown in Fig. 3.

which is manifested by the formation of a plateau around neutral pH (27). Concomitant with the pH gradient formation the change in buffering capacity was followed over the same period. In the l- and 2-h graph of Fig. 6

the carrier ampholyte mixture exhibited a rather uniform buffering capacity throughout the gel with a minimum in gel slice number 10 corresponding to a pH 6.0 and 6.5, respectively. With prolonged focusing time this

FIG. 8. Analytical isotachophoresis of three fractions exhibiting the average pH of 5.0 (B), 5.5 (D), and 6.0 (C) following continuous-flow electrophoretic fractionation of PEHA ampholytes (pl3.5- 10). A = Thermal signal as revealed by the boundary between the leading and the terminating electrolyte. The ordinate represents the relative thermal signal.

CARRIER

AMPHOLYTE

w

FIG. 9. Electrofocusing of some test proteins in a l-mm polyacrylamide slab gel (110 x 240 mm, 5% T, 3% C, 2% carrier ampholytes) using the ampholyte mixture characterized in Fig. 5. From the top (cathode) cytochrome c, ribonuclease A, whale myoglobin, horse myoglobin, bovine hemoglobin, conalbumin, /3-lactoglobulin, and bovine serum albumin. The total mixture of proteins additionally contained ferritin (arrow). Protein load was 15-30 pg. Electrofocusing was carried out in the LKB Multiphor at 10 W constant power.

minimum became more pronounced, however, it was still located in slice numbers 9- 10. The distribution of buffering capacity obtained after focusing time of 4.5 and 7 h was almost identical. Figure 6 also shows the simultaneous change of voltage and current during the formation of the pH gradient applying 10 W constant power to the gel. To improve the resolution of the separation of amphoteres, isoelectric focusing offers the use of narrow range pH gradients. Ampholytes generating such restricted pH gradients could be obtained by direct synthesis rather than by electrophoretic fractionation of the complete pl range (23,24) merely using that part of the template responsible for the synthesis of the ampholytes of the desired pl range. It has to be mentioned, however, that the restriction of pH cuts by direct synthesis is limited, especially in the pl range corresponding to the steeper parts of the curves shown in Fig. 1, i.e., around neutrality. Figure 7 shows pH gradients restricted for the regions 8-10, 5-7,

141

PREPARATION

and 4-7 which were obtained by direct synthesis. To get an approximate estimation on the number of individual carrier ampholytes contained in the synthesized mixture, three fractions of average pH 5.0,5.5, and 6.0 obtained by continuous-flow electrophoretic fractionation of the 3.5- 10 pH gradient, were analyzed by capillary isotachophoresis. For the identification of separated ampholyte species a thermal detector was used. Each separated component is recognized by a rise in the thermal step height (Fig. 8) which is indicated by the arrows. A mean of 12 carrier ampholyte individuals per fraction was recorded. This gave the approximate

+ FIG. 10. Electrofocusing of three different commercially available collagenase preparations recommended for the use in tissue dissociation. The samples were layered in the middle of the gel. The lower end of the pixture represents the anodic gel end. The cathodic gel end is not visible. Other conditions were as mentioned in Fig. 9.

142

WILHELM

number of 550 different carrier ampholyte species, corresponding to a mean pl difference of 0.012 pH units between two adjacent ampholytes. Figure 9 shows isoelectric focusing in the pl range 3.5-9 of a test protein system elaborated by Radola (28). The different proteins were chosen to cover the whole pH range of the gradient with their pl values. The focusing pattern obtained indicates an equal resolution capacity throughout the gradient, The insert of Fig. 9 shows, comparatively, isoelectric focusing of the same proteins using LKB AmpholinepZ3.510. Figure 10 demonstrates the complex separation pattern of various commercially available collagenase preparations recommended for tissue dissociation studies. They were useful because most of the contained proteins cover with their pZ values the pH range 5-7 which is known to be the range exhibiting lowest conductivity and lowest buffering capacity (Figs. 4-6). DISCUSSION

In the present work it is shown that following hydrolysis of the reaction products, the addition of acrylic acid methyl ester to an amine such as pentaethylenehexamine results in an /3-aminocarboxylic acid carrier ampholyte mixture suitable for isoelectric focusing. The procedure outlined above is still far from optimization, i.e., so far no effort was made to increase the number of individual carrier ampholyte species. This might easily be done either by increasing the heterogeneity of the amino moiety or by using other CY-,p-unsaturated carboxylic acid esters in addition to acrylic acid methyl ester. A high number of carrier ampholyte species might be essential, particularly in using restricted pH gradients which are extended over longer distances. Several difficulties were noted using Vesterberg’s procedure (10) for the synthesis of carrier ampholytes. For example, the

W. JUST

continuous decrease of the pH of the reaction mixture, caused by the addition of acrylic acid to the amine solution, led to a change in the charge pattern of the amine nitrogens which, therefore, may become less susceptible for the addition reaction. As a consequence, an extended reaction time was needed and relatively high amounts of unreacted acrylic acid were found to be present in the reaction mixture and were not easily removed. Furthermore, some ampholytes may be present in the reaction mixture at much higher concentrations than others. It is, therefore, necessary to fractionate the reaction product electrophoretically prior to recomposing the desired carrier ampholyte mixture. The use of acrylic acid methyl ester may provide several advantages over the use of the free carboxylic acid. First of all, the ester adds to the amine moiety with a remarkably higher reaction velocity than the free acid (Fig. 3). The half-life times of the (Y-, p-unsaturated compounds calculated from Fig. 3 were 3.5 min and >30 h, respectively. The higher reaction velocity exhibited by acrylic acid methyl ester facilitates the maintenance of the reaction parameters ensuring a reproducible synthesis product (Figs. 4 and 5) and allowing the controlled synthesis of carrier ampholytes covering restricted pH ranges. During the addition reaction, the amine nitrogens are preserved in their uncharged state and may, therefore, retain their nucleophilic force. Since acrylic acid methyl ester is uncharged and volatil its excess is easily removed by distillation in vucuo. The reaction is favorably carried out in an organic solvent where there are no side reactions with atmospheric oxygen; in aqueous solutions these lead to an undesired discoloration of the reaction product. Finally, the controlled performance of the synthesis eliminates the need for fractionation and reconstitution of the carrier ampholyte mixture (10,12,14-16). On the other hand, the use of acrylic acid

CARRIER AMPHOLYTE

methyl ester instead of acrylic acid may favor amidation of some amine groups, thus contributing to an additional heterogeneity of the ampholyte mixture. Although this aspect was not investigated in detail, it is believed to be of minor influence since IR spectroscopy of the reaction products either using acrylic acid methyl ester or acrylic acid gave an identical distribution of the C=O signals (results not shown). The quality of carrier ampholyte preparations is particularly dependent on the number of individual carrier ampholyte molecules present in the mixture (7,s). The total number of carrier ampholyte species in commercial preparations, however, is still a controversy (29-31). Actually, it is rather difficult to fractionate the complex ampholyte mixture. The number of synthesized carrier ampholyte species as determined by analytical isotachophoresis, 550, may still be an approximate number. Prefractionation of the pH gradient into 48 fractions by means of continuousflow electrophoresis (22-25) simplified the heterogeneity of the fractions finally subjected to analytical isotachophoresis. However, the degree of resolution as revealed by the rather insensitive thermal signal is not quite clear. On the other hand, “bad” carrier ampholytes (31) distributing broadly within the pH gradient may appear in several fractions after the prefractionation procedure. Isoelectric focusing of test proteins and of several commercially available collagenase preparations shows that the synthesized carrier ampholyte mixture has a high resolution capacity throughout the pl range 3..510 which was rather identical to that obtained by the use of LKB Ampholines (Fig. 9). ACKNOWLEDGMENTS Parts of this work were done at the Max-PlanckInstitut fur Hirnforschung (Executive Director Prof. R. Hassler) and were supported by the Max-PlanckGesellschaft. The author is greatly indebted to Prof. R. Hassler for his continued interest in this work and to Prof. H. Schimassek and Dr. D. Jeckel, Biochemical Institute, Univ. Heidelberg, for valuable comments upon reading the manuscript.

143

PREPARATION

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79,462-469.

5. Chrambach, A., and Nguyen, N. Y. (1977) in Electrofocusing and Isotachophoresis (Radola, B. J., and Graesslin, D., eds.). pp. 51-58, de Gruyter, Berlin, New York. 6. Troitsky, G. V., Zav’yalov, V. P., Kirjukhin, I. F., Abramov, V. M., and Agitsky, G. J. (1975). (1975) Biochim. Biophys. Acfa 400, 24-31. 7. Svensson, H. (1961) Acra Chem. Stand. 15, 325-341. 8. Svensson, H. (1962) Acta Chem. Stand. 16, 456-466. 9. Svensson, H. (1962) Arch. Biochem. Biophys. Suppl. 1, 132-138. 10. Vesterberg, 0. (1968) SY. Kern. Tidskr. 80, 213225. 1I. Vesterberg, 0. (1969) Aria Chem. Stand. 23, 2653-2666. 12. Vinogradov, S. N., Lowenkron, S., Andonian, M. R., Bagshaw, J., Felgenhauer, K., and Pak, S. J. (1973) Biochem. Biophys. Res. Commun. 54, 501-506. 13. Righetti, P. G., Pagani, M., and Gianazza, E. (1975) J. Chromntogr. 109, 341-356. 14. Gianazza, E., Pagani, M., Luzzana, M., and Righetti, P. G. (1975) .I. Chromatogr. 109, 357-364. 15. Righetti, P. G., Balzarini, L., Gianazza, E., and Brenna, 0. (1977) J. Chromatogr. 134,279-284. 16. Pogacar, P., and Jarecki, R. (1974)in Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel (Allen, R. C., and Maurer, H. R., eds.), pp. 153, de Gruyter, Berlin, New York. 17. Grubhofer, N., and Borja, C. (1977) in Electrofocusing and Isotachophoresis (Radola, B. J., and Graesslin, D., eds.), pp. 111, de Gruyter, Berlin, New York. 18. Blanicky, P., and Pihar, 0. (1972) Collect. Czech. Commun.

37, 319-325.

19. Fawcett, J. S. (1977) in Electrofocusing and Isotachophoresis (Radola, B. J., and Graessling, D., eds.), pp. 59, de Gruyter, Berlin, New York. 20. Williams, K. W., and Soderberg, L. (1979) Znr. Lub. January/February 45-53. 21. Hutchinson, W. M., Collett, A. R., and Lazzell, C. L. (1945) J. Amer. Chem. Sot. 67, 19661968.

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22. Just, W. W., Leon-V., J. O., and Werner, G. (1975) in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.), pp. 265, North Holland, Amsterdam. 23. Just, W. W., and Werner, G. (1977)in Cell Separation Methods (Bloemendal, H., ed.), pp. 129, North Holland, Amsterdam. 24. Just, W. W., and Werner, G. (1977) in Electrofocusing and Isotachophoresis (Radola, B. J., and Graessling, D., eds.), pp. 481, de Gruyter, Berlin, New York. 25. Just, W. W., and Werner, G. (1979) in Electrokinetic Separation Methods (Righetti, P. G., Van Oss, C. J., and Vanderhoff, J. W., eds.), Elsevier, Amsterdam/New York. 26. Everaerts, F. M., Beckers, J. L., and Verheggen, Th. P. E. M. (1976) in Isotachophoresis: Theory, Instrumentation, and Applications, (Everaerts, F. M., and Becker, J. L., eds.),

W. JUST Journal of Chromatography Library: Vol. 6, Elsevier, Amsterdam/New York. 27. Baumann, G., and Chrambach, A. (1975)in Progress in Isoelectric Focusing and Isotachophoresis (Righetti, P. G., ed.), pp. 13, North Holland, Amsterdam. 28. Radola, B. J. (1973) Biochim. Biophys. Acta 295, 412-428. 29. Brown, R. K., Lull, J. M., Lowenkron, S., Bagshaw, J. C., and Vinogradov, S. (1976) Anal. Biochem.

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30. Radola, B. J., Tschesche, H., and Schuricht, H. (1977) in Electrofocusing and Isotachophoresis (Radola, B. J., and Graesslin, D., eds.), pp. 97, de Gruyter, Berlin, New York. 31. Righetti, P. G., (1977) J. Chromatogr. 138, 213215. 32. Righetti, P. G., and Chrambach, A. (1978) Anal. Biochem.

90,633~643.