Electrolyte systems in isotachophoresis and their application to … · electrolyte systems in isotachophoresis and their application to some protein separatioi\is proefschrift ter

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Electrolyte systems in isotachophoresis and theirapplication to some protein separationsCitation for published version (APA):Routs, R. J. (1971). Electrolyte systems in isotachophoresis and their application to some protein separations.Eindhoven: Technische Hogeschool Eindhoven. https://doi.org/10.6100/IR43649

DOI:10.6100/IR43649

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ELECTROLYTE SYSTEMS IN ISOTACHOPHORESIS

AND THEIR

APPLICATION TO SOME PROTEIN SEPARATIONS

R.J. ROUTS


AND THEIR

APPLICATION TO SOME PROTEIN SEPARATIONS

R.J. ROUTS


AND THEIR

APPLICATION TO SOME PROTEIN SEPARATIOI\IS

PROEFSCHRIFT

TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE

TECHNISCHE WETENSCHAPPEN AAN DE TECHNISCHE

HOGESCHOOL TE EINDHOVEN OP GEZAG VAN DE RECTOR

MAGNIFICUS DR. IR. A.A.TH.M. VAN TRIER, HOOGLERAAR

IN DE AFDELING DER ELECTROTECHNIEK, VOOR EEN

COMMISSIE UIT DE SENAAT TE VERDEDIGEN OP DINSDAG

9 NOVEMBER 1971 DES NAMIDDAGS TE 16 UUR

DOOR

ROBERT JOHN ROUTS GEBOREN TE BRISBANE

1971

Solna Skriv- & Stenograftjiinst AB, Solna, Sweden

DIT PROEFSCHRIFT IS GOEDGEKEURD DOOR DE PROMOTOREN

PROF.DR.IR.A.I.M.KEULEMANS

EN

PROF.DR. A.J.P. MARTIN, FRS.

P· 81

p. 1 01

P· 117

ERRATA

Fig.4.5~: The leading ion is acetate and

not chloride as indicated. The acetate con

centration is 0.003 M.

The concentration of acrylamide is 3 grams

per 100 ml.

Fig.6.21: The thermostat temperature was

4°C.

\

I I I

ACKNOWLEDGEMENT

This investigation was made at the laboratories of LKB Produkter AB in Stockholm, to whom I am indebted for various kinds of support. For numerous discussions and positive criticism I want to express my gratitude to Dr. Frans Everaerts and Dr. Lennart Arlinger. I also wish to thank Dr. Herman Haglund, Dr. Anders Vestermark and Dr. Hilary Davies for their valuable help. I am very grateful to Mr. Per Just Svendsen for his interest in the investigation and his skillful advice. My thanks are extended to Mr. Berl Larsson for his technical assistance, to Mr. Curt Sivers for his programming work, to Mr. Leo Fitzgerald for linguistic revision of this monograph, to Mrs. Iris Gustafson for the typing of this thesis, to Mr. Gosta Larsson and Mr. Jean Bohman for assistance with the illustrations.

CONTENTS

INTRODUCTION

1. Principle

2. Scope of this monograph

LITERATURE REVIEW

1. The early period

2. The rediscovery of the method

3. The latest developments

II ·ZONE CONCENTRATIONS AND ION MOBILITIES IN ISO

T ACHOPHORESIS

1. Introduction

2. Theoretical model for isotachophoretically moving zones

2.1 The balance of electric current

2.2 The balance of mass

2.3 The electroneutral ity eguations

2.4 Equilibrium equations

3. Application of the equations to some electrolyte systems

3.1 Divalent leading ion and monovalent terminating ion

3.2 Polyvalent electrolyte systems

11

11

13

14

14 16

21

23

23 24 25 28 30 31 31 32 34

4. Limitations of the theoretical model 34 4.1 The influence of diffusion on the zone boundaries 34

4.2 The influence of ion-ion interaction 38 4.3 Constant current density 40 4.4 Electroendosmotic flow 40 4.5 Hydrostatic flow 41 4.6 The influence of the radial temperature gradient on the

shape of the zone boundary 41

Ill CALCULATIONS AND MEASUREMENTS OF ISOTACHO-

PHORETIC ELECTROLYTE SYSTEMS 44

1. Introduction 44

2. pH and temperature in a capillary column 45 2.1 Experimental 46 2.2 The shape of the terminator concentration boundary 47 2.3 Results 49 2.4 Temperature measurements on the capillary tube 51

3. pH measurements in a sucrose gradient 55

3.1 Apparatus 55

3.2 The validity of the theoretical model in a sucrose density

gradient 56 3.3 Results 58

4. Measurements of pH and conductivity in a polyethylene tube 61 4.1 Apparatus 61 4.2 Results 63

5. pH measurements in polyacrylamide gels 66 5.1 Apparatus 66 5.2 Results 67

6. Discussion 67

8

IV CONSIDERATIONS ON THE USE OF THE THEORETICAL

MODEL FOR ISOTACHOPHORETICAL ELECTROLYTE SYs

TEMS 71

1. Introduction 71

2. Selection of electrolyte systems 71

3. Some disturbing phenomena 76

3.1 Disturbance of the boundaries by highly mobile ions 76

3.2 Interrupted pH gradient

3.3 Precipitation during the separation

3.4 Decreasing voltage gradient

V ISOTACHOPHORESIS, A METHOD FOR PROTEIN SEPARA

TION

78 79

80

84

1. Electrophoretic methods in protein chemistry 84

2 lsotachophoresis, an additional electrophoretic method for

protein separation 85

2.1 Classification of isotachophoresis among the electro-

phoretic methods 85

2.2 Comparison of isotachophoresis with other high resolv-

ing. electrophoretic methods

3. Application of the theoretical model to electrolyte systems

for the analysis of proteins

3.1 Leading and terminating electrolyte systems

3.2 Ampholyte mixtures as spacer ions

VI SOME SEPARATIONS OF HEMOGLOBINS AND HUMAN SE

RUM BY ISOTACHOPHORESIS

1. Introduction

86

89 89 90

93

93

9

2 The use of carrier ampholytes and stabilising media for the

isotachophoretic analysis of proteins 94 2.1 UV-detection in capillary tubes 94 2.2 Carrier ampholytes as spacer ions 97 2.3 Stabilisation of the protein zones 100

3. The separation and identification of human serum proteins in

6 mm polyacrylamide gels 105 3.1 Materials and methods 105 3.2 Tris acetate as leading electrolyte 107

4. The choice of the electrolyte systems for human serum

separations 111 4.1 Theoretical calculations on the electrolyte conditions 111 4.2 Cacodylic acid as leading ion for preparative protein

separations 115 5. Conclusion 120

APPENDIX 121

SYMBOLS, INDICES AND ABBREVIATIONS 127

REFERENCES 129

SUMMARY 133

SAMENVATTING 135

CURRICULUM VITAE 137

10

INTRODUCTION

Electrophoresis is a separation principle which continues to gain more and

more importance in biochemistry and clinical chemistry. Although many

principles of the high-resolution electrophoretic methods of the present time

were dealt with at the beginning of the twentieth century, it was not until the

sixties that these principles were rediscovered and became an answer to the

urgent need for separation techniques within the biochemical field.

Electrophoresis is a term which, from the beginning, was used to describe the

movement of charged colloidal particles in an electric field. Later, it was also

used for ions. Although Martin and Synge (6) suggested the more feasible

name ionophoresis for the migration of small ions, the term electrophoresis

was retained, mainly for historical reasons.

One of the newest electrophoretic methods is called isotachophoresis.

Workers in several laboratories developed the technique independently (see

literature review).

1. PRINCIPLE

The principle of the isotachophoresis technique can be described as follows.

Consider three zones, containing the negative ions A, 8 and C respectively

(see fig. 1 ). P is the common positive counter-ion. The mobilities of A, 8 and

C are in the order mA>m8>mc. If an electric field is applied, the ions will

separate and move in consecutive zones in immediate contact with each

other. The velocities of all zones are then equal. The concentrations of 8 and

C will adapt to the concentration of A in the first zone according to the

Kohlrausch regulating function:

11

P+ P+ P+ +-- +--- +--

e Zone- 3 Zonl!' 2 Zone 1 e c- s- A---+ --+ --+

Fig. 1 The ions A-, B- and C- are migrating in separate zones. The mobility of

the ions decreases from A- to C- (mA>mB>mC)

c m (m +m ) A1 = A1 B2 P2 (1)

c m (m + m ) B2 B2 A1 P1

cAl concentration of A in zone 1 molcm-3

CB2 concentration of B in zone 2 molcm-3

mAl mobility of A in zone 1 cm2v-1sec-1

mB2 mobility of B in zone 2 cm2v-1sec-1

mp1 mobility of P in zone 1 am2v-1 sec -l

The first zone, containing ions with the highest mobility, is called the

leading-ion zone or leading electrolyte (fig. 2). The ions with the lowest

mobility migrate as the terminating electrolyte, or terminator. All ions with

intermediate mobilities will move, in the order of their mobilities, between

the leading-ion zone and the terminating electrolyte.

One of the most important properties of the method is the self-restoring

power of the zone boundaries. Convection and diffusion effects, which tend

to destroy the sharp separation of the zones, are counteracted by the

difference in voltage drop between the zones.

12

8 terminating ion C-or 111

terminating electrolyte

buffer ion p+

sample ions

leading ion A-or --+ leading electrolyte

Fig. 2 The electrolyte containing the ions with the highest mobility is the leading

electrolyte. The ions with the lowest mobility are called terminating ions. - - + The separation of the sernple proceeds between the A and C zone. P is

the counter-ion.

2. SCOPE OF THIS MONOGRAPH

Until now isotachophoresis has been applied mainly to the separation of small

ions, e.g. metals, inorganic and organic acids. In this work a set of equations is

developed to calculate the electrolyte conditions for such separations.

Calculations of the electrolyte parameters based on this theoretical model are

checked experimentally. The equations are also used to discuss some

phenomena which can disturb the isotachophoretic migration.

It is shown that the electrolyte conditions for the separation of proteins can

also be computed. A comparison is made between the existing high-resolution

electrophoretic methods and isotachophoresis, with respect to protein

separation. The use of ampholytes as »Spacers>> for protein mixtures is

discussed. Finally separations of proteins in capillary tubes, in 6 mm

polyacrylamide gels, and on a preparative scale are dealt with.

13

Chapter I

LITERATURE REVIEW

1. THE EARLY PERIOD

Experiments by Lodge (1) and Whetham (2, 3) were the basis on which

Kohlrausch (4) developed his theory for ionic displacement Kohlrausch

stated that when two ion zones, separated by a sharp boundary, move in an

electric field, the velocities of these two zones should be identical.

velocity of A in zone 1

velocity of 8 in zone 2

em sec-1

cmsec-1

(1.1)

Such a sharp boundary can only exist when the mobility of the ion species in

zone 2 is smaller than the mobility of the ion species in zone 1. From

equation (1.1) it is easy to derive the Kohlrausch regulating function or, as he

called it, the »beharrlige funktion»:

c A1

m A1

-=--~-m +m c 82 A1 P1

m +m 82 P2 (1.2) m

82

It took until 1923 before the principle of the Kohlrausch moving boundaries

was applied for the first time, by Kendall (7). He succeeded in the separation

of the rare earth metals and some simple acids by, as he called it, the ion

migration method (7, 9). He stated that the ions not only separate but also

adapt their concentrations to the concentration of the first ion zone,

14

according to the Kohlrausch regulating function. Kendall also attempted to

separate Cl35 and ct37 with a mobility difference of 1. 7%, as shown by

Lindemann (8). However, Kendall could not detect any separation of these

two ions, even after very long runs. The fact that other isotopes could not be

separated, either, was very disappointing for him. He concluded that the

Lindemann theory was invalid. (He showed, however, (10, 11) that it was

possible to isolate the radiactive radium from a barium residue of carnotite).

Kendall (10) considered it necessary to be able to follow the separation in a

convenient way. Therefore he suggested the use of a coloured ion which had a

mobility intermediate to the ions of interest. A concentrated coloured band

would then automatically indicate the end of the experiment. Other

detection methods he mentioned were temperature and conductivity meas

urements in the zones. He pointed out that, when analysing metals,

spectroscopic detection was very easily achieved. Finally, in those cases where

radioactive materials were to be analysed, measurement of radioactivities

supplied the necessary information.

The moving boundary method which Macinnes and Longsworth (12) used to

determine transference numbers in 1932, was based on the Kohlrausch

moving boundary theory. In specially designed electrophoresis apparatus,

they ran the ion species of interest as a leading ion. The velocity of the zone

boundary between this ion and an arbitrarily chosen terminating ion was

measured. The voltage was supplied by a constant current source. The

following equation enabled them to calculate the transference number of the

leading ion:

t

T A1

v c F A1 A1

it

transference number of A in zone 1

volume the zone boundary passed

Faradays constant

current density

time

(1.3)

15

Furthermore, they considered the influence of convection and diffusion on

the boundary sharpness and found it to be very small. They measured

transference numbers of K+, Na +, Ag +, H+ and u+ at several concentra

tions and found their results in excellent agreement with the Debye-Huckei

Onsager theory.

Surprisingly enough, Kendalls work was forgotten for a few decades. During

this period other types of electrophoretic methods were developed. Tiselius

(14, 15) published his work on the separation with the free boundary method

in 1925. Today, the free-boundary method is used mainly for the

determination of mobilities and isoelectric points of purified proteins.

2. THE REDISCOVERY OF THE METHOD

Already in their paper on ionophoresis in 1946, Consden, Gordon and Martin

( 16) pointed out that the separation based on mobilities, such as Kendall had

performed, was a field with many possibilities that had not, up to that time,

been explored. In the same year, Martin (17) separated chloride, acetate,

aspartate and glutamate by this method.

Longsworth (13) realized the importance of Kendall's work and continued it

in 1953. He avoided using agar gel, because no optical detection method

could be applied. In a Tiselius boundary apparatus, he introduced a mixture

of metal ions, Ca, Ba, Mg between two ion zones, called the leading solution

(CsCI) and the trailing solution (LiCI). The mobilities of the metal ions

decreased when going from the leading to the trailing solution. Schlieren

scanning patterns showed very clearly the sharpness of the boundaries

between the zones. Because the migration distance in the Tiselius apparatus is

quite short, Longsworth introduced a counterflow of leading solution. The

counterflow was adjusted in such a way that the zones stayed in the detection

region until the separation was complete. Longsworth also showed that when

all components are separated a steady state is reached on passage of a

constant current. When separating acids, and especially amino acids, he

16

stressed the importance of the pH in the trailing solution. Hydroxyl ions can

destroy the steady state situation because of their high mobility. Poulik (1957) (18) was not aware that he was working with systems which were regulated by Kohlrausch's function. He used, as he called it, a »a discontinuous buffer system» of borate and citrate and found improved resolution of his protein separations.

Kaimakov and Fiks (19) reported in 1961 the use of a separation chamber

filled with quartz sand to eliminate convection problems. They filled the cell with »indicator electrolyte» with a higher mobility than their test solutions,

which they introduced on top of the electrolyte. By using counterflow they

obtained the steady state concentrations according to Kohlrausch's law. In this way the transport number and the mobility of H+ were determined as a

function of its concentration. Transport numbers of lithium·chloride and copper(ll)chloride were measured by Kaimakov (20), and Konstantinov and

Kaimakov (21), respectively, in 1962. In their paper on »The use of the Kohlrausch relation for the determination of transport numbers in highly concentrated electrolyte solutions», Konstantinov, Kaimakov and Vashav·

skaya (22) extended the measurements of transport numbers made by

Hartley {23) and Gordon and Kay (24) in dilute solutions {<0.1 N), to very

concentrated solutions. They determined the transport numbers, using the Kohlrausch equation:

cA1 _ TA1 --CB2 TB2 (1.4)

The transport number T 82 was easy to calculate when using a leading electrolyte of known concentration and transport number and determining the c82 by conductivity measurements after the steady state was reached. In principally the same apparatus as that used by Kaimakov and Fiks (19), they measured transport numbers of Cu2+ and Cd2+. The same authors (25)

published measurements of transport numbers in solutions of copper (II) chloride, cobalt chloride, zinc and cadmiumchloride.

17

Konstantinov and Oshurkova (26) published in 1963 an analytical application

of the moving boundary method. Their separation chamber was a capillary

tube with an inner diameter of 0.1 mm and a wall thickness of 0.05 mm. The

basis for the choice of these dimensions was calculations of the diffusion

coefficient and the temperature distribution over the cross section of the

tube. They separated 1 o-7 to 10-8 g of material. Measurements of the

refractive indices of the ion zones by photographic methods gave a

registration of the zone boundaries.

In 1964 Kaimakov and Sharkov (27) reported the use of microthermistors to

detect zone boundaries.

In the same year Konstantinov and Fiks (28, 29, 30) published a work on the

separation of isotopes by »Countercurrent electromigration», in fact the same

method they had published in 1961 (19). They derived some differential

equations describing the separation process and proved that even if a system had not yet reached Kohlrausch's steady state, it could yield enrichment of

certain isotopes. They performed their experiments in a column filled with silica sand. The first isotope of interest was that of lithium (30). Later,

Troshin (31), Fiks (32), Konstantinov and Bakulin (32), Konstantinov,

Kaimakov and Bosargin (34), measured, respectively, the mobility differences

between isotopes of potassium, rubidium, chloride, and uranyl ions.

In 1966 Konstantinov and Oshurkova (35) repeated their 1963 paper on

capillary tube separation. This time they gave a more extended derivation of

the equations for the influence of diffusion, of convection caused by

temperature differences between the zones and of counterflow on the zone

boundary sharpness. One year later they published a paper (36) on the

separation of amino acids in a capillary tube, according to the moving

boundary principle. They claimed the separation of the amino acids as

positive ions, using H+ as leading ion, and the separation of the amino acids

as negative ions, using OH- as leading ion. They worked with very high

ion-concentrations (5N leading ion). It is therefore highly questionable

whether their electrolyte systems were still obeying the Kohlrausch regulating

function.

18

In their articles on »ion focusing», Schumacher and Friedli (37) emphasized

the need of a pH gradient for their experiments. In 1964 Schumacher and

Studer (39) considered therefore the natural pH gradient according to

Svensson (38). They rejected his method because it did not supply them with

the optimal pH distribution. Instead, they proposed to use the Kohlrausch

regulating function for the creation of a pH gradient. Naturally, the pH will

adapt, in the same way as the other concentrations, to the electrolyte

conditions in the leading ion zone. When running a mixture of weak acids,

they obtained a pH gradient from 1.6 to 3.2. They also showed that the

separation method was useful for quantitative work on weak acids.

Ornstein and Davis (40, 41) introduced their l>disc electrophoresis» in 1964.

In fact they were the first ones to apply the Kohlrausch regulating function

to the separation of proteins. They placed a protein mixture between a

terminating ion (glycine) and a leading ion (chloride). Although the proteins

were separated according to their mobilities, it was impossible to detect them,

because their zones were extremely narrow. Therefore, in the second stage of

the procedure zone electrophoresis was used, which allowed every protein to

move at a different velocity. Polyacrylamide gel was used as stabilizing

medium, acting at the same time as a molecular sieve. In this way the

mobilities of proteins could be controlled by varying the pore size in the gel.

Ornstein (40) derived several equations with which it was possible to calculate

the mobilitie!: and pH values of the electrolyte systems he needed in his

experiments. Today, the disc electrophoresis method is among the most

important electrophoretic methods, because of its simplicity, high resolution

and short separation time.

In 1966 Vestermark (42) introduced an electrophoretic method called »eons

electrophoresiS», still another name for Kendall's l>ion migration method».

Vestermark described »electrophoretic experiments resulting in the arrange

ment of compounds in consecutive zones». The separation experiments were

made on thin-layer strips. He showed that the adaptation of all concentra

tions to that of the leading ion, could be extremely useful for the

concentration of dilute samples. His most important contribution to this type

19

of electrophoresis was his »Spacer» technique. Kendall (10) had already stated

that it would be most convenient to have a coloured material with a mobility

such that it would migrate between two ions of interest. Vestermark used

such materials, mainly amino acids, to »Space» proteins in human sera and the

components of red-beet juice.

Later, Westermark (43) described the separation of red-beet juice which had

been incubated with 35s sulfate, using auto-radiography as the detection

method. Vestermark and Wiedemann (44) used methylamine as a spacer for

the separation of sodium and potassium isotopes. -y-counting and densito

meter tracings of autoradiographs gave the necessary information about the

quality of the separation. Eriksson (45) separated insect hemolymph

components on cellulose strips. In 1969 Westermark et al. (46) showed the

application of the technique to the analysis of Hg2+ and CH 3Hg +.

In his papers on »Counterflow ionophoresis» (Gegenstromionophorese), in

1966, Preetz (47) gave a theoretical treatment of counterflow in isotacho

phoresis. In a second article, Preetz and Pfeifer (48) described an apparatus

specially designed for measurements of potential gradient and ion concentra

tion. Preetz also performed experiments in capillary tubes. A further

development (49) of counterflow ionophoresis is continuous counterflow

electrophoresis. Between two glass plates a flow of electrolyte is applied,

perpendicular to the ion migration direction. In 1966 Everaerts (50), not aware of Preetz and Konstantinov's work,

discussed »displacement electrophoresis» in capillary tubes. Mixtures of strong

acids were separated. A thermocouple was used as a detector. Together with

Martin (51) he showed the separation of chloride, nitrate, oxalate, acetate

and hydrocarbonate. The thermocouple detection gave two types of

information:

1) qualitative: each zone having its own specific resistance and therefore

its own temperature;

2) quantitative: the length of the »temperature steps» were an indication of

the quantity of an ion species in a sample.

In his thesis, Everaerts (52), and, later, Martin and Everaerts (53), described

20

the influences of diffusion, electroendosmosis and pH on the separation.

They showed analyses of mixtures of weak acids, metals and some fruit

juices. They also considered the use of non-aqueous solvents for isotacho

phoresis.

Hello (54) described the moving boundary analysis in 1968 and used it for

the separation of H+ and Li+.

Frederikson (55) showed that conductometric measurement, by using

platinum electrodes inserted in the electrophoresis tube, gave very high

resolution of the zone boundaries.

3. THE LATEST DEVELOPMENTS

In 1970 Everaerts et al. (56) applied counterflow in capillary tubes by

creating a difference in the electrolyte level in the electrode compartments.

The level difference was regulated by a plunger in a reservoir connected to the

leading electrolyte compartment. This plunger was operated by the signal of a

thermocouple mounted on the wall of the tube. The advantage of using such

a regulated counterflow was that ions with small mobility differences could

also be separated, because the effective separation length was increased.

In his review article in April 1970, Haglund (57) listed all the names used for

the described technique. Most of the names were either too general or were

wrong in principal. Therefore the name isotachophoresis was introduced. The

Kohlrausch principle of equal (iso) velocities (tacho) of the zones is the

central theory of the method.

Svendsen and Rose (58) introduced preparative isotachophoresis in polyacryl

amide gels. They separated human blood proteins in a pH gradient formed by

carrier ampholytes (»ampholine»), which are used in isoelectric focusing.

These ampholytes acted as spacers for different protein mixtures. They found

that much higher sample amounts could be applied compared to other

electrophoretic techniques.

Arlinger and Routs (60) reported the use of a UV photometer as a detector in

21

capillary isotachophoresis. They proved in this way that the boundary width,

which Konstantinov (35) ~nd Everaerts (52) estimated to be very small, was

less than 1 mm. They also demonstrated the separation of proteins (hemo

globin and ceruloplasmin) using ampholine as spacers.

Vestermark (61) measured pH differences between leading and terminating

ions for univalent electrolyte systems. He performed his experiments in a

sucrose gradient. Everaerts and Verheggen (62, 63) presented new constructions of their

capillary apparatus. They replaced the original straight capillary tube with a

helical tube. The terminator compartment was constructed in such a way that

samples could be applied by a microsyringe. In the other compartment (the

leading electrolyte compartment) the electrode was separated from the

capillary by a cellulose-acetate membrane, to avoid hydrostatic flow. Analysis

of weak acid mixtures showed a reproducibility of 0.5% in this new

apparatus.

Beckers and Everaerts (64) reported the separation of metal and acid ions in

methanol. They showed that metal ions, especially, were easier to separate in

this solvent. Furthermore, they proved that in methanol, hydroxyl ions

migrated in a separate zone between nitrate and formate. Preetz and Pfeifer

(67) also, did experiments with non-aqueous solvents. They separated

osmium chloride and osmium bromide in liquid ammonia. Furthermore, Blasius and Wentzel (66) demonstrated isotachophoresis in non aqueous

solvents. They used a gel of 2.5% cellulose-acetate and 97.5% formamide.

Everaerts and van der Put (65) showed that it was possible to separate some

amino acids in water-formaldehyde mixtures by isotachophoresis in capillary

tubes. They investigated a number of counter-ions, which would be useful for

this kind of separation. They found collidine to be the best one.

Postema and Brouwer (68) described the separation process in isotachophore

sis before the steady state is reached. They attempted to calculate the time

required to reach this state.

22

Chapter II

ZONE CONCENTRATIONS AND ION MOBILITIES IN ISOTACHOPHORESIS

1. INTRODUCTION

In the introduction of this thesis it has already been pointed out that the

mobilities of the sample ions have to be intermediate to those of the leading

and terminating ions in order to obtain an isotachophoretic separation. Only

then will all ion species move in separate zones, with the same speed.

For the separation of metals and strong acids and bases, the literature contains

values of the mobilities, which will give direct information for the choice of

the leading and terminating electrolyte systems.

When dealing with partially ionized material, the problem becomes more

complex. It is the net mobility, rather than the mobility of the totally ionized

molecules, which determines the isotachophoretic behaviour of weak ions.

Consden, Gordon and Martin (16) defined the net mobility as the product of

the mobility and the degree of ionisation.

Since the net mobility of an ion is pH-dependent, it is clear that it is

determined by the ratio of its concentration and the concentration of the

counter-ion. Both concentrations are adjusted to the leading electrolyte

concentration. We can therefore conclude that the net mobility of an ion

species and its place in the isotachophoretic separation are dependent on the

electrolyte conditions in the leading ion zone.

Several authors (4, 40, 52, 53) derived equations to describe isotachophoreti·

cally moving zones. In this chapter a more extended model will be developed

to calculate the concentrations and mobilities of ions in isotachophoretic

23

systems. It contains an parameters involved in the establishment of the final

pH and the net mobilities in the zones. This model should enable us to

compute suitable electrolyte conditions for the separation of any kind of

material.

2. THEORETICAL MODEL FOR ISOTACHOPHORETICALLY MOV

ING ZONES

Because every ion zone adapts its concentration to the concentration in the leading ion zone, it is sufficient to consider only two zones. These two zones

migrate in a steady state. The first zone fig.2.1) contains the negative ions A-, ..... ,Aa- and the second one B-, ..... ,stJ-. The common positive

counter-ions are p+, •.... ,P1r+. Furthermore the influence of the protons

and hydroxyl ions is taken into account.

3 2 1 zone boundary

• 11'+ + + 1t+ + P, •.•. P ,H P, .... P ,H

zone 2 zone 1

Fig. 2.1 lsotachophoretically migrating zones of the ions A •.•• ,AOI:- and B-, p;.. .... ,B •

P + .... ,Pn+ is the buffering counter-ion.

The theoretical model, which correlates the concentrations in the second

zone and those in the leading electrolyte, contains a number of equations

based on the following conditions: the balance uf electric current

The current density is the same in all zones.

24

the balance of mass

The concentration of the counter-ion within one zone is constant in the

steady state. This means that the amount of P transported into a zone is

equal to the amount that leaves the zone.

the electro-neutrality equation

The amounts of positive and negative charge are the same within one

zone.

chemical equilibrium equations

The acid and base equilibrium constants determine the concentrations

of dissociated and undissociated molecules.

From the derivation of the equations below it will become clear that the

balances of current and mass are also applicable to systems with positively

charged ions A and 8 and negatively charged ions P. The electro-neutrality

and equilibrium equations are different for the cationic and anionic systems.

In the derivation of the following equations it is assumed that:

1) the diffusion effects are negligible,

2) the solutions are very dilute, i.e. the activity coefficients are equal to

umty,

3) the area through which the current passes is constant,

4) tile effect of electroendosmosis is negligible,

5) no hydrostatic flow exists,

6) the zone boundaries are straight (there are no radial temperature

differences).

A discussion on the feasibility of these assumptions is given in paragraph 4 of

this chapter.

2. 1 The balance of electric current

The specific electric conductivity in zone 1 (fig. 3) is:

25

(2.1)

x, specific conductivity in zone 1 n-1cm-1

c0H1 concentration of OH- in zone 1 grion cm-3

CAli concentration of ion A in zone 1

with charge i grion cm-3

mH1 mobility of H+in zone 1 cm2v-1sec-1

mAli mobility of ion A in zone 1 with charge i cm2v-1sec-1

z charge

F charge of one mol Cmol-1

a,{3,1t highest ionisation degree for ions A, B and P

For zone 2 we find

A2 = F(cOH2mOH2+ cH2mH2+ i:: ic .m .+ ~ ic .m ) (2.2) i=O 821 821 i=O P21 P21

As the current is the same in zones 1 and 2, and the voltage gradient is higher

in zone 2, the development of Joule heat will also be higher, which means an

increase in temperature. This implies that the mobility values in every zone

have to be corrected for the temperature in that zone. This is discussed in the

next chapter.

Ohms law gives

current density in zone 1

voltage gradient in zone 1

*For the mobilities, the absolute values should be inserted.

26

(2.3)

(2.4)

The balance of current is

Combination of (2.3), (2.4} and (2.5} gives

Insertion of (2.1) and (2.2) in (2.6) results in

G2 cOH1mOH1+cH1mH1+ ~ ic .m .+ ~ ic .mPI't i=O Ah All i=O Ph

- ---------------------------------c m +c m + ~ ic m + ~ ic m OH2 OH2 H2 H2 i=O B2i B2i i=O P2i P2i

(2.5)

(2.6)

(2.7)

According to the principle of isotachophoresis the velocities of zone 1 and 2

are equal;

uA1 = u82

u velocity of the zones em sec-1

Furthermore uA1

= G1mA

1

mA 1 is the net mobility of the compound A. It is defined as:

or

(2.8)

(2.9)

(2.10)

(2.11)

'Z1

total concentration of A

Insertion of (2.11) in (2.9) results in

c* AI

(I{

:E c m i=O Ali Ali

Combination of (2.8) and (2. 12) gives

G 1 £ m c G 2 ~ m .c . i=O A1i Ali= i=O 821 821

c* c* A1 82

Substitution of (2.13) in (2. 7) results in:

(2.12)

(2.13)

c* A1

0:

.I: mAI.cA1" I=O I I c m + c m + 4 ic .m . + £ ic m OH2 OH2 H2 H2 i=O B21 B2• i=O P2i P2i

0: 1T c* 82 cOH1mOH1 + cH1mH1 + i~Jc A1imA1i+ i~O icP1imP1i

(2.14)

This is an extended form of the Kohlrausch regulating function.

2.2 The balance of mass

The boundary between zone 1 and 2 moved with the speed u A 1. The amount

of counter-ion which is transported into zone 2 due to this movement is

uA 1cp1· In the opposite direction there is a migration of P ions which is

equal to up1cp1.

28

The total mass transport of P through boundary 2 is therefore:

Q u c* + u c* P1 Al Pl P1 P1

In the same way as uAl and u82, up1 is defined as:

G1 11'

-~ cP1'mP1' c* 1=0 1 1

P1

Insertion of (2.16) in (2.15) results in:

Q P1

* 11' = u c +G ~ m c Al P1 1 i=O P1i P1i

It!! the same way we find for the transport of P through boundary 3:

11' Q u c* +G ~ m c

P2 = 82 P2 2 i=O P2i P2i

(2.15)

(2.16)

(2.17)

(2.18)

In the steady state (uA1 = u82) equal amounts of Pare transported through

boundaries 2 and 3:

11' u c* + G ~ m c

A1 P1 1 i=O P1i Pli

11' = u c* + G ~ m c

82 P2 2 i=O P2i P2i (2.19)

Substitution of (2.8) and transformation of (2.19) results in:

* * G 11' G 11' c -c =- 1 ~ m c + 2 ~ m c P1 P2 0 i=O P1i Pli u i=O P2i P2i

A1 A1

(2.20)

29

Insertion of (2.12) and (2.13) in (2.20) gives:

1(

~ m c c* -c* =-c* i=O P1iP1i+

P1 P2 A1 ex ~ m c i=O A1i Ali

23 The electro-neutrality equations

The balance of charge for zone 1 is:

c* 82

1r ~ m c i=O P2i P2i

~ m c i=O B2i B2i

ex n c z F+~ c z F=c z F+~ c z F OH1 OH1 i=O A1i Ali H1 Hl i=O P1i P1i

or

a n c + ~ ic = c + ~ ic OH1 i=O Ali H1 i=O P1i

The balance of charge for zone 2 is:

c + £ ic = c + ~ ic OH2 i=O B2i H2 i=O P2i

(2.21)

(2.22)

(2.23)

(2.24)

The equations (2.23) and (2.24) will be different if we turn to a system of

positive ions A and B and negative ion P:

tr a c +~ic =c +~ic OH1 i=O P1i H1 i=O A1i

(2.25)

c +~ic =c +£ic OH2 i=O P2i H2 i=O B2i

(2.26}

30

24 Equilibrium equations

The following equations for A, Band Pare valid:

c i c _ A10 . IT k A1i- (c )i i=1 A1i

H1

c i c = 820 .II k B2i (c

H2 )i j=1 B2j

c (c )i

c = P10 H1 P1i i

II k j=1 P2j

c (c )i

c = P20 H2 P2i i

II k j=1 P2j

(2.27)

(2.28)

(2.29)

(2.30)

For the reverse system with A and B as positive ions, the equilibrium

equations for A and B are of the type (2.29) and (2.30).

3. APPLICATION OF THE EQUATIONS TO SOME ELECTROLYTE

SYSTEMS

An application of the equations to a system with a divalent leading ion and a

monovalent terminator is given. Such an electrolyte system is very useful

when a buffering leading ion with high mobility is required.

Secondly, some remarks are made concerning the application of the model

for polyvalent ion systems in general.

* All k values refer to the acidic equilibrium constant. 31

3. 1 Divalent leading ion and monovalent terminating ion

Consider a system with a divalent, negatively charged leading ion and a

monovalent terminator. The buffering counter- ion is also monovalent. As a

first approximation the influence of protons and hydroxyl ions is neglected.

Equation (2.14) will result in:

c* m c + m c m c + m c A1 A11 A11 A12 A12 821 821 P21 P21

c* 82

m c 821 821

m c + 2m c + m c A11 A11 A12 A12 P11 P11

(2.31)

Application of the electroneutrality principle gives:

c +2c =c (2.32) A11 A12 P11

c = c 821 P21 (2.33)

Combination of (2.31), (2.32) and (2.33) gives

c* m + m m c + m c A1 821 P21 A11 A11 A12 A12

c* 82

m 821

(m + m ) c + 2 (m + m ) c A11 P11 A11 A12 P11 A12

(2.34)

Insertion of the equations (2.32) and (2.33) in the equation for the mass

balance (2.21) results in:

m (c + 2c ) c* -c* =-c* P11 A11 A12

P1 P2 A 1 m c + m c A11 A11 A12 A12

32

m + c* P21

82 m 821

(2.35)

The equilibrium equations are

(c* -c -c I A1 A11 A12 k

c H1

' A1

(c* -c -c ) A1 A11 A12

2 (c I

H1

(c* 82- c821) c - k 821- c . 81

H2

(c* - c I c P1 P11 . c P11 = k H1

P1

{c* -c P21l c P2 . c P21 = k H2

P2

k A1

k A2

(2.36)

(2.371

(2.381

(2.39)

(2.40)

If two of the nine parameters in the seven equations (2.34)-(2.40) are

known, the other seven can be calculated. Thus if the pH and the concentration of the leading ion are chosen, all other ion concentrations including the pH in the terminating electrolyte are fixed. Consequently we are able to calculate the net mobility of the terminating ion from the equation (2.11):

33

c m -~.m (2.41)

82- c* 821 82

3.2 Polyvalent electrolyte systems

The application of the theoretical model to systems of polyvalent ions yields nonlinear sets of equations, especially when proton and hydroxyl ion influences are included. The use of a computer is necessary to obtain all

roots. With increasing nonlinearity the equations will have a rising number of

roots which are physically incorrect solutions for the system considered. The

correct solution is obtained by rejecting all roots containing negative,

imaginary and obviously unrealistic concentrations. In more complicated

electrolyte systems it is possible that two realistic roots will be obtained. In

this case the right solution can be found experimentally.

4. LIMITATIONS OF THE THEORETICAL MODEL

In paragraph 2 of this chapter a number of assumptions were made for the derivations of the equation system. We will now discuss the influence of all these points on the model.

4. 1 The influence of diffusion on the zone boundaries

The sharpness of the zone boundaries in isotachophoresis is counteracted by

diffusion in the direction opposite to the migration of the zones. Therefore

the boundaries will have a certain width, within which the ion concentrations

vary from their zone concentrations to zero. The equations in paragraph 2

will be valid when the width of the zone boundary is very small compared to

the zone length.

34

We will now derive an equation which will enable us to calculate the

boundary width.

The mass flux of a fully ionized compound A (fig.2.2) with a mobility mA

and a charge zA through a certain cross section within the boundary region is

given by the Nernst·Pianck flux equation:

mA dp.A J _ -, c (z FG + -l A-- z F A A dx

(2.42)

A

mass flux of compound A

chemical potential of A

zone I zone 2

.. X

Fig.2.2 Diffusion pattern at the boundary between the isotachophoretically

migrating zones of the ions A and B.

The two terms in equation (2.42) represent the mass transport by migration

and diffusion respectively.

Neglecting the activity coefficient we can write for p.A

(2.43)

*All indices refer to the parameter values in the boundary region. 35

Insertion of (2.43) in (2.42) results in:

J = - m A . c (z FG + RTdlncA) A z F A A dx

(2.44)

A

The distribution of A in the boundary region moves with a constant speed uA

at the steady state. Therefore we can state that

J = u c A AA

Insertion of (2.45) in (2.44) gives

u A

m dine + ...A RT ____.8. + m G

z F dx A A

0

For compound B in this zone boundary we can write

u B

m dine + __!! RT --8 + m G = 0

z F dx B B

(2.45)

(2.46)

(2.47)

Transformation and subtraction of the equations (2.46) and (2.47) gives

z-1 CA

-din A z-1

c B B

uF 1 1 = -- (---)dx

RT m m (2.48)

A B

If the temperature change between the zones is small, we can integrate

equation (2.48):

36

-1 z

CA _A_ z-1

c 8 8

uF RT (

= k.e

m -m A 8

m m )x A 8

z-1 z-1

If the origin of the coordinate system is chosen at c A c A the integration constant k will be equal to unity. A 8

(2.49)

The equation (2.49) is comparable to those which Longsworth (12),

Konstantinov (35) and Everaerts (51) derived for the boundary width. As a

numerical example (2.49) gives x==1.15 mm for a potential gradient of 100

Volts per centimeter, and mobility values of A and 8 equal to 40.4.10-5

cm2v-1sec-1 and 40.10-5 cm2v-1sec-1 respectively. This value is even

decreased when the mobility difference is bigger and when multivalent ions

are considered.

It is clear from {2.49) that disturbance of the boundary by diffusion is

directly counteracted by the potential gradient. An increase in the potential

gradient by a factor of 2 means a decrease of the boundary width by the same factor. On the other hand very high tensions will cause big temperature

differences between the zones, which increase convection.

For weak electrolytes, it is more difficult to integrate the equation (2.49),

because the mobility values are then depending on x. This can be explained

by the fact that between weak electrolyte zones there usually exists a pH difference as a result of the difference in pK values {this will be shown in the next chapter). In negative ion systems the pH is usually rising from the

leading electrolyte to the terminator. Consequently the ions A will obtain a

higher net mobility after diffusion into the second zone. Therefore the zone

boundaries between weak electrolytes may be sharper than their difference in

mobility indicates.

37

4.2 The influence of ion·ion interaction

In equation (2.1) the conductivity of zone 1 is given as a function of

concentration, charge and mobility of the ionic constituents of that zone.

The specific conductivity is, however, not a linear function of the

concentrations when non·ideal solutions are considered.

The ionic cloud around a migrating ion is egg shaped. Due to this fact the

center of charge of the ionic cloud is not the same as the center of charge of

the migrating ion (central ion). This causes an electric force on the central

ion, in the opposite direction to the force of the external electric field.

The electric force due to the shape of the ionic cloud consists of two

components:

1. relaxation force: the dislocation of the two charge centers causes an

electric field opposite to the external field.

2. electrophoretic force: the center of charge of the ionic cloud tends to

migrate in the direction opposite to the central ion.

The Onsager equation (72) describes the influence of these effects on the ion

mobility. For the leading ion we can write:

m = m0 - (A+ Bm0 )c 1/2

A1i A1i Ali Ali

For water at 25°C, A and 8 are given by the following equations:

Where

38

z +z Ali P1i

z z q 8 = 0•783 A1i P1i

1/2 1+q

z z A1i P1i q =

z + z Ali P1i

2

z + z A1i P1i

2

z m0 + z m0

P1i Ali Ali P1i

(2.50)

(2.51)

(2.52)

(2.53)

Very accurate correction of the mobility values is not required, because the

literature data on the mobilities are quite unreliable. Different references give

values varying by up to 3%.

The influence of the concentration dependency of the mobility on the final

outcome of the equations is partly eliminated, because the nominator, as well

as the denominator, in the equations (2.14) and (2.21) should be corrected.

In the equilibrium equations (2.27-2.30) the activity coefficients are not

taken into account. The Debye-Huckel limiting law may be useful for the

calculation of the influence of these coefficients on the theoretical model:

. 2 1/2 log f = Az I (2.54)

Ali A1i

f activity coefficient for the ion A in zone 1 with charge i Ali

A constant

ionic strength of the solution

For an equilibrium of a univalent acid A- + H+~ AH the equilibrium

constant is given by (721:

f f c} K= A11 H1

1-a

* . c A1

(2.55)

ionisation degree

T,he ionisation degree depends on the electrolyte concentration, according to

the following equation, which is a combination of Arrhenius and Onsager's

law: A

(2.56) a - --------rno:--A -(A+ BA ) (c* ) l/2

o o A1

39

equivalent conductance of the electrolyte n-1 cm2

equivalent conductance of the electrolyte

at infinite dilution

The Onsager correction of the Arrhenius law on the dissociation constant.

and the Debye-Huckel activity coefficients, balance each other to a certain

extent. For acetic acid, a deviation of 2% from the pK value at infinite

dilution was measured for an acid concentration of 0.0035 M. The variation

in the pK data in literature can be far more than 2%.

4.3 Constant current density

A constant current density is easily maintained by running experiments in

tubes or layers with a constant cross section area.

4.4 Electroendosmotic flow

The influence of electroendosmosis on an electrophoretic separation is

already described by many authors. The velocity of the electroendosmotic

flow u0

(neglecting the flow profile in the electric double layer) is given by

the Helmholtz equation (69).

u = GO 0 41T11t

(2.57)

D dielectric constant of the bulk liquid A sec v-1cm-1

~ zeta potential v

'11 viscosity of the bulk liquid gcm-1sec-1

From this equation it can be seen that there are several ways to decrease the

40

influence of electroendosmotic flow. Everaerts (52) increased the viscosity of

the bulk liquid by adding a polymer. Hjerten (70) introduced a counterflow

of electrolyte to compensate this flow. He also treated the walls of his quartz

tubes with methylcellulose to decrease the zeta potential, which can also be

decreased by using various forms of electrostatically inactive plastics, instead

of glass and quartz.

4.5 Hydrostatic flow

A hydrostatic flow can be used to increase the effective length of the

separation tube (see lit. review) or to compensate the electroendosmotic flow

as indicated above. One must, however, be aware of the fact that the

parabolic profile of a hydrostatic flow can destroy the theoretically straight

zone boundaries.

4.6 The influence of the radial temperature gradient on the shape of the

zone boundary

During any electrophoretic experiment there exists a radial temperature

gradient in the tube. Since the mobility is dependent on the temperature, the

velocity of the ions in the tube center will be different from their velocity

closer to the wall. Hjerten (70) derived an equation for the velocity in the

tube:

velocity of the ions at a distance r from

the center of the tube

velocity of the ions at the tube wall

constant, equal to 2400 °K

em sec-1

(2.58)

41

temperature in a zone at a distance r

from the center of the tube

temperature of a zone at the tube wall

temperature of the cooling liquid

Hjerten also proved that this difference in mobility causes a parabolic shape

of the zone boundary. He derived the following expression:

R

'A

K

r

2 2 2 Bi (R -r )

2 4 2 4'A1T R KT0

radius of the tube

electric conductivity

thermal conductivity of a zone

current

(2.59)

em

s:r1cm-1

J -1 -1 oK-1 sec em

A

radial distance from the center of the tube em

If the current, the wall temperature and the thermal conductivity (variation

0.2% per centigrade) are equal in every zone, it is clear that the curvature of

the zone boundaries is merely dependent on the electrical conductivity. This

conductivity decreases from the leading electrolyte to the terminator. The

Fig. 2.3

42

leading electro(!)

e- A-

migration direction

The influence of the radial temperature gradient on the shape of the zone boundary. The ions D-, E- and F- have low mobilities compared to A-, B-and C-.

boundaries of the zones near the terminator will therefore be more curved

than the boundaries of the high conductive zones (fig. 2.3).

Since the parapolic shape can only to a low degree be surpressed by low

temperature cooling, the only way to straighten the fronts is to reduce the

field strength. The use of a counterflow to restore the boundaries of the

terminating zones includes the danger of destroying the straight fronts of the ·

leading ion zones.

43

Chapter Ill

CALCULATIONS AND MEASUREMENTS OF ISOTACHOPHORETIC ELECTROLYTE SYSTEMS

1. INTRODUCTION

In order to check the validity of the theoretical model derived in the

preceding chapter, the results of calculations based on this model will be

compared to values of the pH and/or conductivity, experimentally found in

four different kinds of apparatus.

For the theoretical calculations the computer program »lsogenl was used,

which contained the equations derived in paragraph 2 of chapter 2. In this

program no corrections were made for the temperature and concentration

influence on the mobility. In case theoretically exact results are required, e.g.

for mobility measurements, all corrections should be taken into account. An

estimation of the magnitude of the influence of concentration and tempera

ture on the mobility values (and thus on the theoretical model) is given in paragraph 6 of this chapter.

The experimental measurements were made in different types of equipment

in order to exclude any incidental influence on the separation by the shape of

the column, and to study the validity of the equations in several types of

stabilizing media:

1. a PTFE capillary tube with an inner diameter of 0.45 mm.

2. a glass column with a cross section at area of 5 cm2. As a stabilizing

medium, a sucrose gradient was used.

3. a polyethylene tube with an inner diameter of 1 mm.

*»lsogen» program is listed in the appendix.

44

4. glass tubes with inner diameter of 6 mm. Cross-linked polyacrylamide

gel was used as supporting medium.

The diffusion at the boundary between the leading and terminating ion was

decreased, either by applying high voltage gradients (apparatus 1 and 3) or by

decreasing the diffusion coefficient using stabilising media (apparatus 2 and

4).

Electroendosmotic flow was counteracted by reducing the zeta potential at

the wall by using electrostatically inactive plastic tubes (apparatus 1 and 3),

or by increasing the viscosity of the electrolytes (apparatus 2 and 4).

Hydrostatic flow was avoided by blocking the separation chamber at one side

(apparatus 1, 3, 4), or by levelling the liquids in the electrolyte chambers.

The procedure for the experiments in all apparatus was the same. The

separation chamber was filled with a leading electrolyte of known concentra

tion and pH. A terminator of a certain concentration was brought in contact

with the leading ion zone. During the experiment the terminator migrated

into the separation chamber and replaced the leading electrolyte. After the

leading electrolyte had left the separation chamber, the terminating ion zone

was collected. The pH and/or conductivity was measured and compared with

the theoretical values.

2. PH AND TEMPERATURE IN A CAPILLARY COLUMN

Several authors (26, 35, 48, 53) reported the use of capillary tubes for

isotachophoretic experiments. Everaerts (52) and Routs (73) showed some

separation of weak acids and attempted to give a quantitative interpretation

of the results: In this kind of analysis it is necessary to know the pH-values of

the zones, in order to be able to calculate the net charges and therefore the

net mobilities of the acid ions.

45

2 1 Experimental

A capillary apparatus was constructed to measure the pH differences between

the leading and terminating zones. This apparatus is depicted in Fig. 3.1. It

consists of six parallel capillary tubes, which are coupled to specially

constructed electrode vessels. The inner/outer diameters of the capillary tubes

were 0.45/0.75 mm. The cathode compartment consisted of a plexiglass

block (Fig. 3.1a), containing six reservoirs (2) for the terminating electrolyte.

Fig. 3.1

A 8 C D

I I I a}

TOWARDS ANODE

) I I I /,\\

A 8 C D I I I I

TOWARDS CATHODE

The electrode compartment of the capillary apparatus.

b)

a Terminator block: 1. cathode. 2. reservoir for terminating electrolyte. 3. capillary

b Leading electrolyte block: 4. anode. 5. polyethylene tube. 6. membrane.

7. compartment for leading electrolyte 8. capillary

The anode compartment (Fig. 3.1bl was a reservoir for the leading

electrolyte. To prevent hydrostatic flow and to diminish the influence of

electroendosmotic flow a cellulose acetate membrane (6) was placed over the

anode vessel. The membrane was stretched by an exactly fitting polyethylene tube (5).

46

A Baird-Atomic voltage supply, model 1512, delivered a constant voltage of

5 kV. A constant voltage meant, however, a decrease of the current when the

terminator migrated into the capillary. Therefore the voltage drop per em in

the leading ion zone decreased and as a consequence the zone velocity also decreased. This fact made it difficult to estimate how far the zone boundary

had migrated in the capillary. In each experiment one of the terminator

reservoirs was, therefore, filled with 0.02M picric acid. The yellow colour of

this compound gave a visible indication of the zone boundary.

When the terminator had moved into the leading electrolyte vessel, the

current was switched off and the liquid from two capillaries was collected and

its pH measured. Then the pH of the content of the next two tubes, and

fin~.tiiY of all five tubes together, was determined. The results were averaged,

together with those of a second identical experiment. The standard deviation

was found to be less than 0.03 pH units. The pH in these experiments was

measured by a digital pH meter (Philips).

22 The shape of the terminator concentration boundary

Approximately 5% of the capillary content at the terminator side was

discarded bofore the pH-measurement was made. The reason was that the

concentration boundary existing between the two »parts» of the terminator,

the one in the electrolyte compartment and the one in the capillary, is neither

sharp nor immobile. This in turn is due to three reasons:

a) During the filling procedure of the terminator compartment there will

always occur some mixing with the leading electrolyte in the capillary. When

the terminator reservoir was filled with a solution of dyestuff to estimate the

magnitude of this effect, the leading electrolyte in the capillary mixed with

the dyestuff over a distance of 3 to 7 mm.

b) Terminating ions will diffuse from the electrode compartment into the

capillary during the experiment. This effect can be estimated with the

Einstein-Srnoluchowski equation:

47

2 <x > = 2Dt (3.1)

2 cm2 <x> mean square distance of diffusion

D diffusion coefficient cm2sec-1

t time sec

Assume that the concentration of the terminator in the capillary is much

smaller than in the reservoir. If Dis 10-5 cm2 sec-1 and the analysis time is

two hours, < x2> is equal to 0.14 cm2. The root-mean-square distance is

0.12cm.

c) Electrophoretic migration of the terminating ions will cause the

concentration boundary to move, for which Macinnes and Longsworth (12)

derived the following equation:

T' -T" I B B f e= . -.

c' - c" F s (3.2)

8 8

'a the migration distance of the zone em

boundary

T' B transport number of 8 in the cathode

reservoir

T" B transport number of B in the capillary

c' B concentration of Bin the cathode reservoir molcm-3

c" B concentration of B in the capillary mol cm-3

f quantity of charge through the capillary c F Faraday's constant c s cross-sectional area cm2

Assume that the terminating ion B and the counter-ion P have the mobilities

48

40. 10-5 cm2v- 1sec-1 and 20. 10-5 cm2v-1sec-1, respectively, at

infinite dilution. If the concentrations of Band P in the terminator vessel are

0.01M and in the capillary 0.001M, Ti:J and Ts will be 0.685 and 0.672,

respectively, in accordance with equation (2.50). An analysis time of two

hours with a current of 100 pA will give a value of le equal to 0.07 mm. The

boundary between the leading electrolyte and the terminator has, in the same

time interval, covered a distance of 3.6 meters. It is clear that 18 is negligible

compared to the migration distance of th,e isotachophoretic front.

2.3 Results

In the first series of experiments a solution of 0.014M histidine and 0.01M

HCI was used as a leading electrolyte. Eleven different weak acids were used

as terminators. They are listed in Table 3.1 together with their pK and

mobility values at infinite dilution;:.. (first six columns). The pH-values of the

terminating zones were measured and compared to the theoretically

calculated values (last two columns).

In the next series of experiments the leading electrolyte consisted of 0.01M

HCI and, as counter-ion benzidine at a pH of 3.35. The reasons for the choice

of benzidine as counter-ion were:

1. the possibility to check the equations for a divalent counter-ion

2. the pK values of most weak acids are within the buffering region of

benzidine

3. the fact that benzidine is buffering at low pH and therefore can be used

to test the validity of the equations, even when a relatively large

amount of the current is carried by H+.

On the other hand benzidine is not very stable, is only slightly soluble in

water and is poisonous.

The experimentally determined pH values in the benzidine system (Table 3.2)

do not agree with the theoretical values as closely as in the histidine system,

*Most of the pK and mobility data are taken from literature (88-91) 49

TABLE 3.1

The theoretical and experimental values of pH and the calculated net mobility (m821 in an isotachophoretic system with

histidine-HCI as leading electrolyte. The experimental values were obtained from metiSUrements in a capillary apparatus at 25°C. The

pK and mobility data used for the theoretical calculations are listed in the first columns.

ION SPECIES pKB21 pKB22 pKB23 mB21 mB22 mB23 mB2 pHtheor. pHexp.

cm2v- 1sec-1 • 105

chloride 78 78 5.75. 5.75

oxalate 1.23 4.19 40 73 72 5.76 5.78

tartrate 2.98 4.34 39 64 62 5.79 5.80

formate 3.75 56 56 5.79 5.81

citrate 3.08 4.74 6.40 38.5 55 70 56 5.82 5.81

succinate 4.16 5.61 40 60 52 5.85 5.81

malonate 2.83 5.69 40 58 51 5.85 5.83

acetate 4.75 41 39 5.89 5.87

a-hydroxybutyrate 3.65 39 36 5.89 5.87

phosphate 2.12 7.21 12.67 38 55 69 34 5.87 5.87

carbonate 6.37 10.25 44.5 72 23 6.39 6.41

but in most cases the deviation is not very large. The discrepancy between

theory and practice is largest for the low conductive zones, such as acetate,

a-hydroxybutyrate and carbonate.

TABLE 3.2

Theoretical and experimental values of the pH and the calculated net mobility (m821 of

different terminators in an isotachophoretic system, with benzidine-HCI as leading

electrolyte. The experimental values were obtained from measurements in a capillary

apparatus, at a temperature of 26°C.

ION SPECIES pHexp.

chloride 3.35 3.35 oxalate 3.56 3.42 formate 3.97 3.85 succinate 3.65 3.62 tartrate 3.60 3.75 phosphate 3.60 3.70 citrate 3.78 3.74 malonate 4.16 4.00 acetate 4.47 4.20 a:·hydroxybutyrate 4.44 4.19 carbonate 5.36 4.80

24 Temperature measurements on the capillary tube

mB2

cm2v-\ec-1 x 105

78 46 35 35 33 32 28 21 15 14 4

As Everaerts (52) pointed out, the heat production is different in each zone

as a result of the different voltage gradients. The evolution of heat in a zone,

and thus its temperature (73), has a linear relationship with the specific

resistance of the zone, when the current is constant throughout the system.

The temperature difference between the zones can be detected by a thermocouple. An example is given in Fig. (3.2). The step-height of an acid is

51

Fig. 3.2

Recorder I response glutamat~

Detection of a number of isotachophoretically moving weak-acid zones by

a thermocouple in a capillary tube. The leading electrolyte was 0.02 M

histidine and 0.01 M HCI at pH 6.1. Glutamic acid was used as terminator.

The sample was 0. 7 #A of a 0.05 M solution of oxalic, citric and adipic acid. The current was 70 IJ,A. The thermostat temperature was 25°C.

a measure of the temperature and therefore of the specific resistance of the

zone.

All the experiments with the histidine-HCI buffer mentioned in section 3.2

were repeated in the apparatus described by Everaerts and Verheggen (63).

The capillary tube was thermostated by winding it around an aluminium heat

sink which was kept at 25°C. It was only at the detector that the capillary

had no contact with the block. The reference temperature of the thermo

couple was the block temperature.

The total concentrations of terminator and buffer, and the net mobility of

the terminator are given in Table 3.3, columns 1-3, as they were calculated

52

TABLE 3.3

Theoretical concentrations net mobility, resistivity and step-height in different terminator zones. The leading electrolyte is

histldine-HCI at 25°C. The step-height (temperatures) measurements were made in a capillary tube apparatus by

thermocouples.

ION SPECIES CB2 cP2 mB2 p h

cm2v- 1sec-1 x 105 llcm.10-3 mm

chloride 0.0100 0.0145 78 1.12 0 oxalate 0.0050 0.0143 72 1.20 20.1 tartrate 0.0048 0.0142 62 1.38 47.2 formate 0.0094 0.0139 56 1.50 51.5 citrate 0.0043 0.0139 56 1.51 60.0 succinate 0.0052 0.0138 52 1.61 68.0 malonate 0.0055 0.0137 51 1.64 72.1 acetate 0.0087 0.0132 39 2.03 109.4 a-hydroxybutyrate 0.0085 0.0130 36 2.16 125.2 phosphate 0.0077 0.0127 34 2.27 140.4 carbonate 0.0089 0.0133 23 3.55 187.3

from the theoretical model. From these values it is possible to calculate the

specific resistance of the zones (column 4). In the last column the

experimental step heights of all zones are listed. In figure 3.3 the specific

resistance of the acids is plotted against the step-height. There is a linear

relationship for the first seven acids. The resistance of the zones 8-11

becomes so high that the thermocouple signal is no longer linear with the

temperature inside the tube. This is due to the temperature dependence of

the heat transport from the electrolyte zone, through the capillary wall, to

the thermocouple.

Fig. 3.3

54

o~~~i_~~-L-L-L~~~~~~~i_~~~-L-L-L~~

30 0 50 100 150 200 ~

The theoretical specific resistances of eleven isotachophoretically moving

acid zones plotted against the signal amplitude of a thermocouple, which

measured the temperatures of the zones. The leading electrolyte was

histidine-HCI at pH 5.75.

1. chloride 2. oxalate 3. tartrate 4. formate 5. citrate 6. succinate 7. malo

nate 8. acetate. 9. Q.hydroxybutyrate 10. phosphate 11. carbonate

3. PH MEASUREMENTS IN A SUCROSE GRADIENT

3. 1 Apparatus

An isoelectric focusing column (LKB 8100) was used for measurements on a

larger scale. Part of the platinum wire of the anode (fig. 3.4) was removed to

increase the migration distance. A constant flow of 1 0 ml leading electrolyte

per hour was applied around the anode to maintain a constant histidine

concentration at the electrode and to prevent migration of electrode products

into the column. The bottom part and the inner part of the column were

filled with leading electrolyte in a 40% sucrose solution. A sucrose gradient

from 40% to 10% was then layered on top of this solution. Finally the

terminating electrolyte was introduced. The temperature in the cooling jacket

Fig. 3.4 Column apparatus

A. cathode B. inner cooling jecket C. outer cooling jecket D. annular

separation chamber E. anode F. valve G. outlet H. buffer circulation pump.

55

was kept at 25°C by a water-thermostat. Two power supplies (LKB 4471 D)

were connected in series and delivered a constant voltage of 1600V. The

current decreased from 10 to 2 mA during the time of the analysis, which was

3 hours. After the experiments the electrolyte was pumped out of the

column, collected in 2 ml fractions and the pH was determined for each

fraction. Fig. 3.5 shows the result of such an experiment. The pH was

measured by a Radiometer (PHM4c), using an electrode of the type

GK 2322c.

Fig. 3.5

pH

L.o

5.0

4.0

3·0 ol--+-----,,~o--*=,s---:f:2o=----:f2s=----:!3':-o -----=3~5-___,&._40

Fraction number ____,...

Variation of pH through an isotachophoretic system containing chloride

and cat:bonate in a sucrose gradient. The buffering counter- ion was benzidine.

3.2 The validity of the theoretical model in a sucrose gradient

As indicated in section 3. 1, the solutions in the column are stabilized against

convection by a sucrose gradient. Because the mobility is dependent on the

56

viscosity of the solution, the conductivity, and thus the voltage gradient, will

increase when the sucrose concentration is rising. Edward (74) proved

experimentally the validity of the following equation for the mobility:

z n

r

m = 1.602.10-12 z n1Tr'l'}

charge of ion

constant, the value of which depends on the

radius of the ion compared to the radius of

the water molecule. For organic ions with

radii greater than 3A but less than 2500.&,

n is equal to 5.

viscosity

radius of the ion

(3.3)

For non-spherical ions, r has to be multiplied by a factor, which is equal to

the ratio of the friction coefficient of a spherical molecule of the same

volume to the actual friction coefficient

Edward proved the validity of the equation(3.3):) for small organic ions. For

small inorganic ions, hydration and superfluidity cause deviation from the

theory.

For one ion species, equation (3.3) can be written as:

1 m = c · :;; , where c is a constant (3.4)

If we assume that the viscosity is constant over a narrow interval..:b:1 and .ax2 (fig. 3.6) of the column, the combination of (3.3) and (2.1) gives:

(3.5)

and

(3.6)

57

Fig. 3.6

Visjuy/

~r--~ I I -column length r 1 r _.; .. ;oo .;..,.;'"

terminator : leading electrolyte zone b'ound<lry

Viscosity profile in a sucrose gradient. The viscosity is considered to be

constant over small regions Ax1 and Ax2 in the column.

Furthermore, combination of (3.4) and (2.13) results in:

a: c G _ 2: m ~

1 111 i=O A c* A

£ m CB i=O B c*

B

(3.7)

When we combine the equations (3.5), (3.6) and (3.7), the viscosity terms are

eliminated and we obtain the Kohlrausch regulating function (2.14). In the

same way it can easily be derived that equation (2.21) is independent of

viscosity. It is therefore clear that the pH which is calculated for the terminating zone is not dependent on the viscosity.

3.3 Results

In the sucrose gradient, the same electrolyte systems were used as in the capillary experiments. The results are given in table 3.4. The experimental

results show good agreement with the theoretical results.

The influence of the pH of the originally applied terminating solution on the

pH of the isotachophoretically migrating terminator zone was studied in a

58

en CD

TABLE 3.4

Theoretical and experimental values of the pH of different terminators in two isotachophoretic systems, one with

histidin~HCI and the other with benzidin~HCI as leading electrolyte. The experimental values were obtained from

measurements in a sucrose density gradient (25°C).

Leading Electrolyte Leading Electrolyte

histidine- HCI benzidine - HCI

ION SPECIES pH Chloride pHtheor. pHexp. pH Chloride pHtheor. pHexp. zone terminator zone terminator

oxalate 5.88 5.89 6.00 3.88 4.09 4.06 formate 5.88 5.92 6.92 3.78 4.19 4.22 succinate 6.10 6.16 6.21 3.70 4.36 4.34 tartrate 6.11 6.13 6.19 3.88 4.12 4.12 phosphate 5.93 6.04 6.14 3.72 3.87 4.10 citrate 5.91 5.97 6.10 4.20 4.36 malonate 5.85 5.94 5.90 3.81 3.89 4.00 acetate 5.78 5.92 5.91 3.80 4.65 4.62 cc·hydroxybutyrate 5.86 5.98 6.05 3.70 4.58 4.28 carbonate 5.89 6.43 6.40 3.70 5.52 5.52

few experiments. There was no difference in the experimental pH values, as

shown in table 3.4, irrespective of whether histidine oxalate at pH=5.1, or

oxalic acid at pH=1.95 was used as original terminating electrolyte. Also,

carbonate solutions at pH=9.1 and 7.0 gave the same pH values.

Vestermark (61) reported pH measurements of isotachophoretic systems in

sucrose gradient from 10 to 50%. The electrolyte concentrations were 0.1 M.

This means that the Debye-Huckei-Onsager approximation is no longer valid.

Therefore it cannot be expected that the results of his measurements fit our

theoretical values exactly. He ran glycinate, carbonate and lactobionate as

terminators. In table 3.5 Vestermark's results are listed, together with the

calculated pH values.

TABLE 3.5

Theoretical and experimental values of the pH of three different terminators, with

0.1 M tris chloride as leading electrolyte in a sucrose density gradient. Glycinate,

carbonate and lactobionate are used as terminators. The experimental data are according to Vestermark (61 ).

pH pH pH pH

chloride zone glycinate zone carbonate zone lactobionate zone

theor. exp. theor. exp. theor. exp.

7 8.92 8.8 7.41 7.5 7.1 7.43 7.6 7.18 7.5 7.2 8.94 8.8 7.29 7.6 7.3 8.95 8.8 7.5 8.97 8.9 7.59 7.95 7.7 7.81 8.0 7.9 9.05 8.9 7.99 8.35 8.0 9.08 9.0 8.09 8.40 8.1 8.17 8.35

60

4. MEASUREMENTS OF PH AND CONDUCTIVITY IN A POLY

ETHYLENE TUBE

4. 1 Apparatus

As separation chamber, a polyethylene tube of 1 m length and an inner/outer

diameter of 1/1.4 mm was used. When a moderate current (60J.LA) is used, the

temperature steps between the zones in such a tube can be kept low, thereby

avoiding the need for an agent which will stabilise against convection.

According to Wagener and Bilal (75), the difference between the cooling

temperature and the temperature in the tube can be calculated to be not

more than 0.6°C for a specific zone conductance at 1 o....:4n-1 em - 1. The

electrolyte content of this wider tube has sufficient volume to allow

measurement of the conductivity.

The ends of the tube (fig.3. 7) were connected to the teflon-lined valves (2)

Fig. 3.7 Apparatus used for pH and conductivity measurements in isotachophore

tically migrating zones in a polyethylene tube (inner/outer diameter

1/1.4mm,length 1m).

1. electrode 2. valve 3. polyethylene tube. 4. valve 5. leading electrolyte reservoir 6. leading electrolyte compartment 7. membrane 8. plug

61

and (4). The terminator reservoir (1) was made out of plexiglass. The leading

electrolyte vessel (6) was connected to the tube via a semi-permeable

membrane (7). A reservoir (5), containing 20 ml of leading ion, was placed

between the tube and the leading electrolyte vessel. The function of this

reservoir will be explained below.

To fill the tube and the compartment (5) with leading electrolyte, valve (2)

was disconnected from the terminator vessel and the plug (9) was loosened.

The liquid was pumped into the apparatus via valve (2). After the filling

procedure, the valve (2) was connected again and the vessel (1) was filled with

terminating electrolyte.

The voltage was supplied by a constant current source with a maximum

voltage of 26kV. The temperature of the thermostated water around the tube

was 25°C.

When the experiment was finished, i.e. when the terminating ion zone had

passed valve (4), the current was switched off and the valves (2) and (4) were

closed. The valves were disconnected from the electrode vessels and the

contents of the tube were collected.

The function of the reservoir (5) was to act as a buffer compartment for the

H+ front caused by the membrane (56). If this precaution was omitted the

pH front would enter the polyethylene tube, and the pH and conductivity

measurements were valueless. Fig. 3.8 shows how the pH varied in the leading

ion zone, when no buffer reservoir was present. The experiment was allowed

to proceed during half of its usual analysis time.

The conductivity of the leading and terminating electrolytes was measured

with a conductolyzer (LKB 53008). The measuring cell was a modified,

commercially available, flow type (LKB 5311 B) with a cell constant equal to

0.053. The spiral tube, which was coupled to the cell to make sure that the

sample liquid had the same temperature as the thermostat bath, was removed.

In this way, the necessary sample volume was only 100J,.tl. The temperature

during the conductivity and pH measurements was 25°C. The pH was

measured by a radiometer of the type described in section 3.1.

62

4.5

4.4

pH

4.3

4.2

4.1

4.0

3.9

3.8 0

Fig. 3.8

20 40 60 so 100 distance trom terminator compartment

em

The pH-profile in the polyethylene tube for a system with 0.002 M NaCI at

pH 4.13 as leading electrolyte and oxalic acid as terminator. The extra

reservoir (6) (fig. 3. 7) was omitted in this experiment.

4.2 Results

The first series of experiments was done with sodium chloride as leading

electrolyte. The concentrations were 0.002M hydrochloric acid and 0.0019M

sodium hydroxide with a pH to 4.13. Sodium was chosen as a counter-ion to

check the validity of the equations for a non-buffering counter-ion. In table

3.6 the experimentally measured and theoretically calculated pH-values are

listed, together with the specific conductivities when weak acid were used as

terminators. All measurements were made twice and the average values are

listed in Table 3.6. The standard deviation did not exceed 0.03 pH units or

0.06.1o-4n.-1cm-1. Good agreement between theory and experiments is

shown. The fact that the pH, as well as the conductivity, in the malonate

zone were too high indicates that a systematic error is probably introduced in

the pK and/or mobility values of this compound.

63

TABLE 3.6

Theoretical and experimental values of the pH, net mobility lm 82 l and the specific conductivity of the

terminator zones, with 0.002 M sodium chloride as leading electrolyte.

The experiments were made in a polyethylene tube with an inner diameter of 1 mm at 25°C.

ION SPECIES pHtheor. pHexp. mB2 Atheor. X exp. \t.eor./corr.

cm2v-1sec-1x 105 !"r1cm-1x 104

chloride 4.13 4.13 78 2.66 2.59 2.55 oxalate 4.41 4.41 61 2.07 2.04 1.95 tartrate 4.48 4.51 53 .1.80 1.67 1.70 formate 4.64 4.63 48 1.65 1.64 1.57 citrate 4.53 4.54 44 1.50 1.34 1.42 malonate 4.47 4.62 39 1.34 1.41 1.29 succinate 4.94 4.96 37 1.26 1.14 1.19 carbonate 6.91 6.93 35 1.18 1.16 1.11 acetate 5.33 5.34 33 1.11 1.10 1.07

In a second series of experiments the theory was tested with positive leading

and terminating ions. The leading electrolyte was 0.002M potassium acetate

at pH=4.8. Some metals and amines were used as terminators. The results are

listed in Table 3. 7. The standard deviation was 0.025 pH units and

4.10-6n-1 em - 1. The deviation in the pH value in the thorium zone is

probably due to the high value of the activity coefficient of this ion. If the

theoretical conductivity values are reduced by 5% to allow for the

concentration influence on the mobility (see paragraph 6), the same type of

systematic error as dealt with above can be found for UO~+ , hydrazinium

and quinine. For sodium, the experimental pK and conductivity values show

deviations in the opposite direction.

TABLE 3.7

Theoretical and experimental values of the pH, net mobility (m82l and the specific

conductivity of the terminator zones for a positive ion system with 0.002 M potassium

acetate as leading electrolyte.

The experiments were made in a polyethylene tube with an inner diameter of 1 mm at

25°C.

ION SPECIES pHtheor. pHexp. mB2 \t,eor. A exp.

cm2v- 1sec-1x 105 W 1cm-1x 104

K+ 4.80 4.80 73 2.26 2.17 Na+ 4.17 4.77 50 1.54 1.39 u+ 4.63 4.65 39 1.19 1.09

Thriethylamine 4.57 4.55 33 1.02 0.99 uo2+ 4.56 4.63 32 0.98 0.97 2 Th4+ 4.48 4.04 27 0.83 0.87

Hydrazinium 4.45 4.20 26 0.79 0.71

Quinine 4.43 4.50 27 0.82 0.88

65

It is obviously possible to apply the theoretical model also to positive ion

systems.

5. PH MEASUREMENTS IN POLYACRYLAMIDE GELS

5. 1 Apparatus

The apparative set-up for the gel experiments was very simple. A glass tube of

6 mm diameter was filled with a histidine-HCI buffer solution containing

3.5 g acrylamide, 0.175 g bisacrylamide and 0.25 mg riboflavin per 100 ml.

The polymerisation took place in UV light for 1 hour, after which the

polymerisation was considered to be finished. The tube was then placed

between two reservoirs, one containing the leading electrolyte (fig. 3.9) and

the other containing the terminator.

Fig. 3.9

66

0

0

The apparatus set-up for the test of isotachophoretic electrolyte systems In

polyacrylamide gel.

1. terminating electrolyte compartment 2. pyrex tube li.d. 6 mml contain

ing 3.5% polyacrylamide gel 3. leading electrolyte compartment

The voltage was supplied by a constant voltage source (LKB 4471 D). The

applied voltage was 200 V. The current varied in accordance with the

conductivity of the terminator in the reservoir. As an indicator of the zone

boundary, tetrasulfonated indigo was used. When the blue band had covered

two thirds of the distance through the tube, the experiment was stopped. The

gel was taken out of the tube and the part containing the terminator zone was

eluted with carbondioxide-free water for 12 hours. Then the pH of the

terminating zone was determined.

5.2 Results

As indicated in section 3.2 all molecules will be retarded by the same factor,

when migrating in a medium of uniform viscosity. The conductivity of the

zones is influenced by the gel concentration but the pH is not. This statement

is only valid if the ions are much smaller than the poresize of the gel.

The leading electrolyte in the gel experiments was histidine-HCI at pH=-6.1.

The chloride concentration was 0.01 M. The same series of weak acids were

used as terminators as was the case in section 2.3. The results of the

experiments are listed in Table 3.8. Each experiment was made three times

and the results were averaged. The standard deviation was 0.02 pH units.

Duimel and Cox (76) also described isotachophoretic experiments in

polyacrylamide gels. Their gel concentration was 5.5%. Their experimental

results are in good agreement with the results of calculations according to the

theoretical model in chapter 2 (Table 3.9).

6. DISCUSSION

The results of the experiments seem to confirm the theoretical model. The

use of the same leading electrolyte in three different types of apparatus shows

clearly that the influence of the separation chamber on the isotachophoretic

separation is minimal.

67

TABl.E 3.8 Theoretical and experimental values of the pH in some

terminating weak acid zones, with histidine HCI as leading

electrolyte. The measurements were made in a 3.5%

polyacrylamide gel with 5% crosslinking.

ION SPECIES

chloride

oxalate

tartrate

formate

citrate

succinate

malonate

acetate a·hydroxybutyrate

phosphate

carbonate

pHtheor. pHexp.

6.07 6.07

6.08 6.09

6.09 6.11

6.11 6.13

6.11 6.16

6.13 6.12

6.13 6.08

6.16 6.18

6.13 6.16

6.20 6.18

6.51 6.54

TABLE 3.9

Theoretical and experimental values of the pH in the

glycinate terminating zone. Trisphosphate was the leading

electrolyte. The experimental data are taken from ref. 73.

5.5% polyacrylamide gel was used as stabilizing agent.

pH of the phosphate zone pH of the glycinate zone

pHexp. pHtheor. pHexp.

5.50 8.90 8.95

7.00 8.95 8.95

7.20 8.92 8.96

8.50 9.24 9.27

The theoretical data listed in the tables 3.1 to 3.9 can be further refined by

correction of the mobility values for the temperature and concentration

influence and by introduction of the activity coefficients in the equilibrium

equations.

The temperature dependence of the mobility is given by the following

equation (71 ):

T m

A 25

m A

mobility of ion A at T°C

mobility of ion A at 25°C

a, (j constants specific for ion A

(3.8)

For small temperature ranges (15°C-35°C), the /3-term can be neglected. The

temperature coefficient a is, for most ion species, close to 0.02 at 25°C,

except for protons and hydroxyl ions. This means that the temperature

influence on the mobility can be eliminated in the equations (2.14) and

(2.21) in a pH range where the contribution of the protons and hydroxyl ion

to the conductivity can be neglected. The total terminator and buffer

concentrations will therefore be independent of the temperature differences

between the zones. The pH in the terminator zones, calculated from these

concentrations, will also be temperature independent over small temperature

ranges (3-4°C), where the pK values can be considered to be constant. The

specific conductivity in the zones, however, will rise by about 2% for every

degree centigrade. The temperature increase from zone to zone can be

strongly reduced by intensive cooling, as in apparatus 1, 2 and 3.

Apart from the temperature, the mobility of an ion is also dependent on the

ionic strength in the solution (equations (2.50) -(2.53)). To estimate the

influence of this effect on the model, the theoretical specific conductivities in

table 3.6 are corrected according to the Onsager equation, using the method

of successive approximation. The primary input data for the mobilities in the

69

computer program »lsogen» were the mobility values at infinite dilution.

From the total concentrations and the pH, which were calculated with the

computer program, the corrected mobilities were obtained using the equation

(2.50). These corrected values, in their turn, were used as input data for a

second approximation, which gave new mobility values, differing less than 1%

from the first corrected values.

When dealing with divalent and trivalent electrolyte systems, which are partly

ionised, the application of equation (2.50) became more difficult. In such

cases, the following procedure was chosen: From the first computations

(using the net mobility at infinite dilution) the average charge and net

mobility of the terminator were obtained. From these values the constants A

and 8 were calculated with the equations (2.51) and (2.52), respectively.

Then, one could calculate the corrected mobility of each ion species in the

terminator zone.

The results of the correction are listed in the final column of table 3.6. The

specific conductivities are, on average, 5% lower than the originally calculated

values. The experimental results in general show better agreement with the

corrected theoretical values than with the non-corrected ones. If this same

correction of 5% is applied to the conductivity values listed in table 3.7, they

will likewise come closer to the experimental results. The corrected pH values

of the terminator zones differed, at the most 0.01 pH units from their

originally calculated values.

As already pointed out in chapter 2, section 4.2, the influence of the activity

constants is partly counterbalanced by the Onsager correction of the

ionisation degree. Furthermore for most weak acids the corrections for the

influence of the activity coefficients is well within the variations of the

literature data on the pK values (89) within a certain concentration range

(<0.01 Ml.

70

Chapter IV

CONSIDERATIONS 01\1 THE USE OF THE THEORETICAL MODEL FOR ISOTACHOPHORETICAL ELECTROLYTE SYS· TEMS

1. INTRODUCTION

After it has been shown in the previous chapters that the theoretical model

gives good agreement with the experimentally obtained results, the practical

usefulness of the model will now be discussed.

Firstly, it is probable that the model will be used mainly for the calculations

of the optimal separation conditions for mixtures of ions. The leading and

terminating electrolytes appropriate for any separation can be selected

theoretically.

Secondly, the model contributes to the explanation of certain phenomena

which tend to disturb the separation picture, e.g. interrupted pH gradient and

falling voltage gradient.

2. SELECTION OF ELECTROLYTE SYSTEMS

The selection of leading and terminating electrolyte systems for the

separation of certain samples is of course dependent on the available

information about the composition of the samples. The case of a sample with

a known composition will be dealt with first Frequently, enough pre-infor·

mation about the sample mixture is available, once the stage has been reached

where isotachophoresis is to be used as one of the last links in a separation

chain.

71

It has been shown above that the electrolyte conditions in the leading-ion

zone determine all parameters in the succeeding zones. The selection of a

leading electrolyte system for a sample consists, firstly, of the choice of a

leading-ion with a mobility, or a net mobility value, higher than any of the

sample ions. Literature data on mobilities and equivalent conductances will

usually supply this information. Secondly, a proper counter-ion has to be

chosen. The pK of the counter-ion has to have such a value that it ensures a good buffer capacity in the pH interval, within which the separation is taking

place. The zones will then possess good stability against pH disturbances. In

section 4.4 details will be given of how the use of a non-buffering counter-ion

can lead to peculiar side effects.

The pH of the leading zone has to be selected in such a way that the net

mobilities of the sample ions will not be equal or very close to each other.

When there is little difference between the mobilities of two ions (1-2%,

chapter IV, section 4) diffusion at the zone boundary can counteract the

separation power to a high degree. The resolution of narrow zones will then

be diminished and long separation times will be required.

In order to find the right conditions for the separation, the »lsogen» program

can be used to scan a certain pH interval in the leading electrolyte. An output

of such a scan is shown in the appendix. Fig. 4. 1 shows, in a diagram, the

result of a pH scan. The leading ion was 0.01 M chloride, with three different

counter-ions because of the large pH interval. For the eleven succeeding weak

acid zones, the net mobilities were plotted against the pH in the leading

electrolyte. From this diagram it is easy to select, in the leading electrolyte

zone, a pH which produces a good separation of the succeeding ions. The

curves in the diagram are based on average literature data on the mobility.

The variations of 3-4% in the literature data should be taken into

consideration.Fig.4.1 also illustrates clearly the risk of random selection of the

electrolyte conditions. There are many cross-over points of curves, which

represent pH values at which it is impossible to separate the components

which are involved.

The same diagram can also supply information about the selection of the

terminator ion. Carbonate or diethylbarbiturate would suit very well as

72

\

'• 88

~ 1 ~

70

e z ~ z i a: ... .... > .... :::i ii 0 ::& t; z

40

Fig.4.1

COUNTER-tON

Cl

ox

ta

fo

ci ma ph

hy su

at

CREATININE HISTIDINE TRIS

CHLORIDE

OXALATE

CITRATE

TARTRATE

SUCCINATE

MALONATE

FORMATE

PHOSPHATE

CARBONATE

ACETATE

4-HYDROXYBUTVRATE

DIETHYLSARBITURATE

8.0 9.0 pH LEADING ELECTROLYTE

The net mobility of 11 weak acids as function of the pH in the leading

electrolyte. The leading ion is 0.01 M HCI. The choice of the counter-ion

species and concentration depends on the pH in the leading electrolyte:

creatinine for the pH interval 4.0-5.4, histidine for 5.4-7.0, tris for

7.0-9.0.

terminating ion for most mixtures of ions shown in fig. 4.1.

Some experiments were made to support the validity of the diagram. A

sample mixture of tartrate, citrate and succinate was analysed in a capillary

apparatus according to Everaerts and Verheggen (63). A thermocouple was

used as detector. In fig. 4.2a the separation is shown with histidine-HCI at

pH=5.4 as leading electrolyte. The current was 70 J.I.A and the temperature

25°C. Fig. 4.2b is a recording of the same mixture with tris HCI at pH=8.20

in the leading ion zone. Note that the migration order of citrate and tartrate has changed. Finally, in a third experiment histidine HCI at pH=6.6 was used.

Citrate and tartrate move together in one zone.

Apart from the possibility of choosing an optimal leading and terminating

electrolyte, diagrams such as fig. 4.1 also give information about the way to

analyse sample mixtures with one component in excess. Assume that we are

interested in the citric acid content of a mixture containing at least 99%

succinate. In a conventional isotachophoretic experiment it would take a long time before the two acids had separated properly. The use of counterflow of

leading electrolyte (56) is one solution to this problem. However, it is also

possible to modify the terminating electrolyte conditions in such a way that only the citrate ion will move isotachophoretically. When, for example,

tris HCI at pH=8.2 is chosen as leading electrolyte and tartrate as terminator, the succinate ions will move slower than the terminator, which implies a

quicker separation. Instead of tartrate, succinate itself could also be used as

terminator. When a sample contains large quantities of very fast ions, another

procedure is chosen. Assume that the component of interest is again citrate,

but now in an excess of chloride. In this case a leading ion slower than chloride, such as oxalate, should be chosen. During the analysis, chloride will

move away from the citrate into the· leading zone. The risk that the chloride ions will disturb the leading electrolyte and in this way the

isotachophoretic conditions, can be counteracted by using a counterflow of leading electrolyte. Also, chloride itself could be used as leading ion.

The procedure for the determination of leading and terminating electrolyte

systems for samples of unknown composition is partly based on theoretical

74

calculations and partly on trial and error. One usually chooses a leading ion

and a terminator with a very wide mobility interval. More information about

Fig. 4.2

Recorder response

recorder response

recorder resp<?f1S&

-;-------Acetate

a -Chtoride

b

Acetate

Succin' Tartrate Citrate

c --Chtoride

em

time

em

lime

em

time

Separation of a mixture of 0.03 M, tartrate, citrate and succinate in a

capillary tube at 25°C . .The terminator was acetate. The electropherogram

represen1S the recording of a thermocouple mounted on the capillary wall.

The current was 701J.A.

a leading electrolyte: 0.01 M HCI and 0.012 M histidine at pH==5.4

b leading electrolyte: 0.01 M HCI and 0.044 M histidine at pH==6.6

c leading electrolyte: 0.01 M HCI and 0.02 M tris at pH=S.18.

The chart speed was 1 em/min.

75

the sample can also be obtained by adding an ion of known mobility and pK

value to the sample. This could give enough information about the net

mobility range of the components in the sample and the next choice of the

leading electrolyte.

lsotachophoresis can also be used to determine the pK and mobility values of

unknown ions.. Suppose a sample of unknown composition is separated on a

relatively large scale. Then, the separated zones are collected and their pH and

conductivity are measured. From the conductivity it is possible to calculate

the voltage gradient in the zones. Using equation {2.12) it is easy to calculate

the net mobility of the sample ions. Several experiments at different pH

values in the leading electrolyte can supply enough information for the

calculation of an approximate value for the pK and mobility of an ion.

3. SOME DISTURBING PHENOMENA

3. 1 Disturbance of the boundaries by highly mobile ions

Although the development of the theoretical model included the influence of

protons and hydroxyl ions on the conductivity its validity range is dependent

on the pH. When a relatively large part of the current is carried by H+ or

OH-, the basic condition of isotachophoresis is no longer fullfilled. Two

zones migrate with the same velocity, because a region between them which is

without charge is forbidden by the electroneutrality rule. At extreme pH

intervals, however, two anionic zones, for example, will be separated from

each other by hydroxyl ions and the conditions for isotachophoretic

migration are no longer fullfilled. The pH at which the hydroxyl influence

becomes noticeable, depends on the concentration and mobility of the

sample ions.. Generally, the pH interval between 4 and 10.5 can be considered

as a »Safe» region.

76

Hydrocarbonate ions frequently cause the same kind of problems. Since

carbondioxide is present in the air, it is difficult, although possible, to prevent

its entrance in the terminator vessel, especially in a preparative apparatus. If

the electrolyte systems possess a pH between 6 and 10, the migration of

hydrocarbonate ions from the terminator reservoir, through zones with a

lower mobility than the hydrocarbonate itself, can disturb the stability of the

zone boundaries in the same way as hydroxyl ions. Precipitation of the

hydrocarbonate .in the terminator electrolyte with barium hydroxide gives a

solution to this problem, provided that the terminator itself does not react

with barium h_ydroxide.

In fig. 4.3 the influence of hydrocarbonate on the zone boundary is shown.

Fig. 4.3

rKOrder response

glycocholic acid

time

The influence of hydrocarbonate ions on experiments at elevated pH. The

leading electrolyte was 0.01 M chloride and 0.0172 M tris at pH=S and

glycocholic acid was the terminator. The curve represents an analysis where

hydrocarbonate ions from the terminator compartment migrate contin

uously through the zone boundary. In the second experiment (dotted line)

the hydrocarbonate ions were removed from the terminator by barium

hydroxide. The current was 40/JA and the thermostat was 25°C. The chart

speed was 1 em/min.

77

The curve represents the hydrocarbonate and glycocholic acid temperature

step, when tris HCI at pH 7.5 was used as leading electrolyte. The hydrocar

bonate step was caused by the hydrocarbonate ions present in the leading

electrolyte and also those from the terminator compartment. The dotted curve b represents the same analysis, when 0.002 M Ba(OH)2 had been added

to the terminator solution. It is clear that in the latter case a much sharper

zone boundary was obtained.

3.2 Interrupted pH gradient

The results of the calculations and experiments in chapter Ill may suggest

that in an anionic system the pH rises from one zone to the next according to

the net mobilities in the zones. This, however, does not invariably happen.

There are exceptions. Especially in systems which contain both partially and

completely ionised electrolytes with comparable net mobilities, a pH drop

from one zone to the other can be expected. This is clearly shown in

table 4.1, for sample mixtures of phosphate and picrate. Tris-chloride is the

leading electrolyte and glycine is the terminator.

At pH 7.5 in the leading electrolyte, phosphate will migrate in front of

picrate, but at a higher pH. When the pH in the leading electrolyte is

decreased (table 4.1 b) the pH gap between phosphate and picrate is even

larger. The net mobility data may show that the picrate and the phosphate

ions will separate in this system, but in reality this is impossible. Assume that

a steady state would be reached, as described in table 4.1 b. When a

phosphate ion diffuses into the picrate zone, it enters a lower pH region. At

pH 7.08, phosphate would possess the same net mobility as picrate and the

phosphate ions could not possibly migrate back into their own zone. After

some time the boundary between both ions would be very diffuse. In order to

test this reasoning, an experiment was made, under the conditions mentioned,

in the sucrose gradient column described in chapter Ill, section 3.1. Instead

of the short, sharp, yellow zone of picrate which one theoretically should

expect, a long and very diffuse yellow zone was obtained.

78

TABLE 4.1

a pH and net mobility values in zones of phosphate, picrate and glycinate. The leading electrolyte consisted of 0.01 M HCI and 0.123 M tris at pH 7 .50.

b identical to a, but the leading electrolyte consisted of 0.01 M HCI and 0.0107 M

tris.

ION SPECIES

chloride

phosphate

picrate

glycinate

pH

7.50

7.65

7.57

8.97

a Net mobility

cm2v-1sec- 1

78.10-5

48.10-5

38.10-5

5.110-5

3.3 Precipitation during the separation

pH

7.00

7.37

7.08

8.94

b Net mobility

cm2v-\ec- 1

78.10-5

45.10-5

38.10-5

4.7 10-5

The computer output always contains the total concentration in each zone.

When the sol:.~bility of a component is low, e.g. for fatty acids, cholic acids,

the theoretical total concentration should be compared with solubility data

of the component In case the degree of ionisation is low, the risk of

precipitation is especially increased. When an acid precipitates during an

analysis, it will remain immobile - provided it has a zeta potential equal to

zero - and it will be overtaken by the next zone.

Because the pH in that zone is usually higher, and the ionic strength lower,

the precipitate will dissolve again and move back into its own zone. However,

the solvation process takes time, especially when aggregates of precipitate are

formed. The result is that there will always I be a certain quantity of these

slightly soluble acids in the succeeding zone.

79

3. 4 Decreasing voltage gradient

In isotachophoresis the voltage gradient is different in each zone. In order to

give low mobility zones the same speed as zones containing ions with higher

mobility, the voltage gradient in the low mobility zone has to be higher than

in the high mobility zone. If one deliberately selects a terminator with a

higher mobility than the leading ion, the terminator will migrate into the

leading ion zone. However, we have seen in chapter Ill that each concentra

tion boundary is also a pH boundary. This pH boundary can create the

peculiar situation that ions with a higher mobility than the leading ion

migrate as a terminator zone. Fig. 4.4 depicts such a situation. Sodium

Fig. 4.4

Migration direction

HCOj Na+

0 pH:6.66 pH-5.07 0 msz=29.5

The net mobility and pH values in two succeeding zones of acetate and

hydrocarbonate. The leading electrolyte consisted of 0.003 M acetic acid

and 0.0019 M sodium hydroxide.

acetate is chosen as the leading electrolyte at pH 5.07, and hydrocarbonate as

the terminator. The result of the calculations according to the theoretical

model is, that a pH of 6.66 is created in the terminator zone, which results in

a net mobility of hydrocarbonate which is higher than net mobility of

acetate. Consequently, hydrocarbonate ions will try to overtake the leading

ion zone. However, as soon as a hydrocarbonate ion enters the leading ion

zone, it will experience the low pH and its velocity will decrease with a factor

of at least 10. Therefore, the pH boundary keeps the two zones separated and

forces them to move with the same velocity. The higher net mobility in the

terminator zone, and the fact that the zones migrate with equal velocity,

80

implies that the voltage gradient in the hydrocarbonate zone must be lower.

An experiment was made in a capillary apparatus to verify the theory. The

leading ion was a mixture of 0.002M acetic acid and 0.0021 M sodium hydrox·

ide. The resulting pH was 5.07. Sodium carbonate was used as terminator. The

thermo-detector output is shown in fig.4.5.a.lt shows clearly a decrease in the

temperature as a result of the reduced voltage gradient in the hydrocarbonate

zone. Fig. 4.5b shows the hydrocarbonate zone again, but this time as a zone

0)

em chloride

hydroc<~rbonQtt /

time

Fig. 4.5 a Thermal detection of a hydrocarbonate zone migrating behind a leading

electrolyte, consisting of 0.003 M acatic aeid and 0.0021 M sodium hydroxide. The current through the capillary was in all experiments 40 PA and the thermostat temperature 25°C. The chart speed was 1 em/min.

b Sodium hydrocarbonate was injected between the leading electrolyte, as in a, and diethylbarbiturate.

c Sodium hydrocarbonate was injected between the leading electrolyte, as in

a, and cacodylate.

81

migrating between acetate and diethytbarbiturate for the same leading ion

conditions. When cacodylic acid was used as terminator, the hydrocarbonate

zone disappeared (fig. 4.5c). The explanation of this fact is easily found in

table 4.2.

TABLE4.2

pH and net mobility values in zones of hydrocarbonate, cacodylate and diethylbarbiturate. The leading electrolyte consists of 0.003 M acetic acid and 0.0021 M sodium hydroxide.

lon species pH net mobility cm2v-1sec-1·105

acetate 5.07 28

hydrocarbonate 6.66 29.4

cacodylate 6.41 23.2

diethylbarbiturate 7.61 23

It is clear from this table that acetate, hydrocarbonate, and diethylbarbiturate

form a stable system. However, using cacodylate as terminating ion, the

hydrocarbonate is moving ahead of a lower pH zone. This results in mixing of

the two zones (section 3.2) and consequently the electropherogram shows

only two zones.

When carbonate and cacodylate were used as intermediate ions between

acetate and diethylbarbiturate, the situation got more complex. Fig. 4.6

shows separations of samples of hydrocarbonate and cacodylate at different

concentration ratios. When only a small amount of cacodylic acid was present

(fig. 4.6a), there was still enough hydrocarbonate left to keep up its separate

zone. When the sample got richer in cacodylate, the temperature of the

cacodylate zone increased and the length of the carbonate zone decreased

(fig. 4.6b).

82

recorder respon""

Fig. 4.6

diethylborbiturote

Recorder response

diethylborbiturate

b

em

time

A sample of cacodylic acid and hydrocarbonate migrating between acetate

and diethylbarbiturate. The conditions were as in fig. 4.5a. a the sample was 0.8 /A 0.01 M NaHC03 and 0.2 /A 0.01 M cacodylic acid.

b the sample was 0.5 /A 0.01M NaHC03 and 0.5 /A 0.01 M cacodylic acid.

The basic reason for these unwanted phenomena is that the counter-ion is not

buffering. When a counter-ion with pK 5 and mobility 25.10-5cm2v-1

sec -l is used, the pH and net mobility values in the hydrocarbonate zone will

be 6.08 and 15.10-5cm2v-1sec-1 respectively. Then, the normal situation

is once more obtained.

83

Chapter V

ISOTACHOPHORESIS, A METHOD FOR PROTEIN SEPARA· TION

1. ELECTROPHORETIC METHODS IN PROTEIN CHEMISTRY

As pointed out previously, there are many application fields for isotacho

phoresis. The separation of weak acids and metals, which has been dealt with

in previous chapters, is one important application. In protein chemistry,

electrophoresis has always been one of the most important separation

methods with respect to analysis and isolation. It started with Tiselius' free

boundary electrophoresis ( 14). The main use of this method today is for the

determination of the mobilities and isoelectric points of proteins. However,

new methods have been developed, which replace this rather laborious

technique. Zone electrophoresis is the most common electrophoretic method.

This method can be divided into two main parts: free zone electrophoresis,

and zone electrophoresis in stabilizing media. The use of free zone

electrophoresis is limited, because stabilization against convection and

diffusion in free solution is difficult, especially at high protein concentra

tions. However, Hjerten (70) developed an electrophoresis apparatus with a

rotating separation tube, which provided the needed stabilization.

Zone electrophoresis in stabilizing media is more commonly used than free

zone electrophoresis. Many kinds of carriers have been developed. Paper (77),

cellulosa acetate (78) and agar (79) were the ones which were mainly used in

the beginning. Starch (80) and polyacrylamide gel do not merely stabilize the

protein zones, but give an extra dimension to the separation because of their

molecular sieving properties. Polyacrylamide gel electrophoresis has gained in

84

importance, especially since Davis (41) and Ornstein (40) introduced disc

electrophoresis. The resolving power of this method is very high.

Another high resolving method, isoelectric focusing (81), which in theory was

already developed in 1960 by Svensson, was introduced in 1966 and has

already gained an important position among the electrophoretic methods.

Most of the electrophoretic methods can be used in combination with

immunochemical analysis. This technique is called immunoelectrophoresis.

Grabar and Williams (82) allowed the antiserum to diffuse perpendicularly to

the direction of migration. Laurel! (83) and later Clark and Freeman (84)

forced the proteins to migrate into a gel containing antiserum.

Recently, isotachophoresis was introduced as a method for the separation of

proteins (58, 60). The place of isotachophoresis among modern high resolving

techniques will now be discussed.

2. ISOTACHOPHORESIS, AN ADDITIONAL ELECTROPHORETIC

METHOD FOR PROTEIN SEPARATION

2 1 Classification of isotachophoresis among the electrophoretic methods

lsotachophoresis is a new electrophoretic principle. Until now, protein

separation by electrophoretic methods was based upon the difference in

charge and size of the molecules in a buffered solution. The migration

velocity of a protein is determined by these two properties and will be

different for every protein. This is illustrated by Fig. 5.1. Here, the velocity

of the protein ions in an electrophoretic separation is plotted against the pH

(85). The line a) represents the group of zone electrophoretic methods and

also the moving boundary electrophoresis. In the latter case we must consider

the velocity u as the velocity of the zone boundaries. Line b) in Fig. 5.1

represents discontinuous zone electrophoresis, as used, for example, in disc

electrophoresis.

lsotachophoresis and isoelectric focusing (lines c) and d)) are based on

85

Fig. 5.1

pH

d

u

Classification of the electrophoretic techniques. The velocity of the protein

zones is plotted against the pH in the zones.

a zone electrophoresis and moving boundary electrophoresis

b discontinuous zone electrophoresis

c isoelectric focusing

d isotachophoresis

principles which are »perpendicular» to the principles of the techniques dealt

with above. In isoelectric focusing (line cl pH-axis) the proteins are separated

according to their isoelectric point. In a natural pH gradient they migrate to

pH regions which are equal to their isoelectric points. Then they will remain

immobile (u=o, pH-axis).

It is clear that isotachophoresis closes the ring of possibilities. All zones move

with the same velocity, but at different pH. It is a method which can supply

new information about protein mixtures, which previously have been

analysed by other electrophoretic techniques.

22 Comparison of isotachophoresis with other high resolving electro

phoretic methods

What is the advantage of isotachophoresis over other techniques, such as disc

electrophoresis and isoelectric focusing? To answer this question we could

86

compare the analytical and preparative value of the technique with respect to[

the separation power and time of analysis.

One advantage of isotachophoresis over disc electrophoresis is that a steady

state is reached once the separation is completed. If two components, with

mobilities which are quite near to each other, have to be separated, the analysis time can be extended. No harm is done to the separation. On the

contrary, the separation gets better until the steady state is reached.

(Konstantinov (30) even succeeded in the enrichment of Li·isotopes). A disc

electrophoretic analysis, however, can only be performed during a limited

time because of diffusion effects. The stacking procedure in disc electro

phoresis is in fact a concentration procedure to obtain a sharp starting protein

zone. During that time, however, one performs an isotachophoretic separa

tion, which in the second phase of the procedure is destroyed. In this second

phase the proteins move into a very tight gel, which decreases their mobility

to such a degree that the terminator passes the protein zones. The proteins

then move zone-electrophoretically. This implies that the larger distance the

zones migrate, the more diffuse they get. This limits the separation power of

the technique.

lsotachophoresis may have the advantage of a steady state and concentrated

protein zones, but in practice it will be very difficult to detect, or for

preparative purposes collect all the different protein zones. In fact the

second phase in disc electrophoresis was one answer to this problem, although much of the primarily obtained separation was destroyed. Spacing of the

different zones, as already described by Kendall (10), is another solution. The

protein sample is mixed with compounds which have a mobility range

covering that of the proteins. Vestermark (42) used amino acids for this purpose. Svendsen and Rose (58)

and also Routs (60) showed the usefulness of mixtures of ampholytes as

spacers for isotachophoretical analysis. However, the addition of such

ampholytes, which create a mobility gradientdoes also cause diffusion of the

protein bands, because part of the ampholytes have mobility values equal or

very close to the mobility values of the proteins. Contrary to what happens in

87

disc electrophoresis, this diffusion is independent of time, when the steady

state is reached.

Disc electrophoresis is a fast and cheap analytical method with a high

resolving power. It is easy to handle. This is not the case with isotach~

phoresis. When one wants to separate a certain sample one has to decide

about concentration and pH in the leading zone, mobility of the terminator,

the properties of the counter-ion and so forth. For fast information about the

composition or homogeneity of a protein sample the biochemist will

therefore probably prefer the disc technique.

For routine analysis it could be advantageous to run isotachophoretic

separations in capillary equipment. The detection procedure in isotach~

phoresis is simpler. As all zones move with the same velocity, the detector can

be fixed on one spot, while in disc electrophoresis the use of a scanning

detector is required.

Success has never really been achieved in maintaining the high resolving

power of disc electrophoresis in preparative runs. Overloading effects disturb

the separation. Loads of almost 3 mg/cm2 protein are allowed. In isotach~ phoresis the sample amount should not, theoretically, influence the separa

tion. More sample increases the length of the zones, but does not affect the

protein concentration in the zones. Svendsen (86) already separated sample

amounts of up to 50 mg per cm2.

lsoelectric focusing has a very high resolving power. Vesterberg (59) showed

separations of myoglobines with a difference in pi of 0.02 units. The

diffusion in isoelectric focusing is much less because the zones are immobile.

There is equilibrium between the force of the electric field and the diffusion

force. Fewer parameters are involved in isoelectric focusing than in

isotachophoresis. The only parameter one has to select is the pi range of the

carrier ampholytes creating the pH gradient.

Analytical experiments are performed in polyacrylamide gels. The carrier

ampholytes are mixed with the monomer before polymerization. This

inclucies the risk that part of the ampholytes will react during the

88

polymerization procedure and in this way become fixed in the gel. This

causes difficulties when staining the gel.

Preparative isoelectric focusing is performed in sucrose density gradients. The

sample amounts are limited by the bearing capacity of the gradient.

Furthermore, many proteins tend to precipitate at pH values near their

isoelectric points. In an isotachophoretic separation the proteins migrate at a

pH which is quite remote from their pl.

Because disc electrophoresis, isoelectric focusing and isotachophoresis are

based on different separation principles they will supply different informa

tion about a sample. lsotachophoresis seems to be a technique which can

supply new, additional information for analytical purposes. As a preparative

method it is very promising, because the factors which tend to disturb

preparative separations in disc electrophoresis and isoelectric focusing, are

counteracted by the isotachophoretic principle.

3. APPLICATION OF THE THEORETICAL MODEL TO ELECTRQ..

L YTE SYSTEMS FOR THE ANALYSIS OF PROTEINS

3. 1 Leading and terminating electrolyte systems

Ornstein reported (40) that it was possible to calculate appropriate

electrolyte systems for the steady-state stacking phase in disc electrophoresis.

He used tris chloride as leading electrolyte and glycine as terminator. He

stated that all serum proteins would migrate between these two zones. The

choice of the leading and terminating electrolyte systems for the separation

of proteins is determined by the isoelectric points and the mobility values of

the protein molecules. In an anionic system for example, the pH in the

terminating zone must necessarily be higher than the isoelectric point of any

sample proteins. Otherwise the protein will be immobile or migrate in the

89

opposite direction. As the proteins of interest in our experiments are mainly

blood proteins, the isoelectric points will be between 2.5 and 7.5.

The net mobility of the terminator must be so low that even very

low-mobility protein molecules, such as gammaglobulins, will migrate in front

of the terminator zone. The free mobility values of the serum proteins are

between 7.6 and 1.2•10-5cm2v-1sec-1 at pH 8.6 and at 25°C. As already

indicated in chapter IV, the net mobility of the terminator is determined by

its pK value, mobility and by the composition of the leading electrolyte.

It is not possible to calculate the pH value in the leading-ion zone for optimal

separation of proteins, as was done in chapter IV for weak acids. Protein molecules have many acidic and basic groups with pK values close to each

other. The ion·ion interaction, in this case mainly protein-protein interaction,

cannot be neglected in the derivation of the equations. Correction of the

mobility values with the Onsager equation is meaningless, because Onsager's

approximation is only valid for ions with a maximum of three charges. There

is no longer any agreement between the theory and the experiments for ions

of higher valency.

Mobility values of proteins obtained by other electrophoretic techniques, and

values of pi and pK from titration curves, will usually supply enough

information to determine roughly the order of the different proteins in

isotachophoresis.

3.2 Ampholyte mixtures as spacer ions

In section 2.2 of this chapter the use of mixtures of ampholytes as spacers for

protein zones was mentioned. In isotachophoresis the proteins are concentrat·

ed in very narrow bands and detection of the zones is difficult. Dilution of

the leading electrolyte would result in wider protein zones. However, one has

to use very low leading ion concentrations (0.0001M) to create protein zones

of detectable length. This requires the use of very high tensions. Furthermore

many proteins tend to precipitate at low ionic strength. Another possibility

to make the protein zones detectable, is to increase the sample amount.

90

However, in many cases the sample contains such a low percentage of the

protein of interest, that the sample amount has to be increased 10-100

times. This is disadvantageous, especially for analytical applications.

Spacers would separate the protein bands from each other (Fig. 5.2) and

Fig. 5.2

migration direction

Protein zones P 1• P2, P3 and P 4 which are spaced by ions with a mobility

intermediate to the mobility of the proteins.

make them detectable. The spacer ions which will be used are mixtures of

polyamino-polycarboxylic acids which are used as carrier ampholytes in

isoelectric focusing. Svendsen and Rose (58) were the first to show their

usefulness for isotachophoresis. These ampholyte mixtures are commercially

available in several pi ranges (ampholine LKB 8100). Their general structural

formula is given in Fig. 5.3.

Fig. 5.3

- CH2 - N- (CH2lx- N - (CH2)x- NR2 I

(CH2)x R

x =2or3

R = H or- CH2 - CH2 -COOH

The structure of the polycarboxylio-polyamino acids (carrier ampholytes),

which are used as spacers.

91

For every protein separation one has to select an ampholyte mixture with an

appropriate pi range. It is difficult to give a standard procedure for the

selection of these mixtures. The carrier ampholytes are designed in such a

way (59) that the differences between the basic and acidic pK value is not

more than one pH unit. This means that for an ampholyte mixture with a pi

interval 7-9, the acidic pK values of most components will be between 6.5 and 8.5. If we assume a mean mobility of 30·10-5cm2v-1sec-1·10-5 on all

ions it is possible to calculate roughly the net mobility gradient which is

obtained for a certain leading electrolyte. The proteins have to fit in this

mobility gradient. In the optimal situation all proteins should be spaced by

ampholytes.

92

Chapter VI

SOME SEPARATIONS OF HEMOGLOBINS AND HUMAN SERUM BY ISOTACHOPHORESIS

1. INTRODUCTION

The versatility of isotachophoresis provides us with many possible electrolyte

systems for the separation of proteins. It is clear that for every specific

separation problem, there must be an electrolyte system which effects

optimal separation. It is not the objective of this thesis to describe electrolyte

conditions for many kinds of protein mixtures. A few systems will be dealt

with for the separation of human serum and some hemoglobins. For the

choice of the conditions for the separation of other proteins, reference should

be made to the computer program »lsogen», which is listed in the appendix.

Before the actual separation and identification of proteins in isotachophoresis

was made, it was found necessary to study the carrier ampholytes with

respect to their spacing properties and their absorption of UV light.

Furthermore, the stabilisation of the protein zones against gravity forces had

to be discussed. Experiments to study these phenomena were made in

capillary tubes.

As isotachophoresis is a new electrophoretic approach to the separation of

proteins, it was necessary to make an identification of the protein bands in

the separation pattern. The isotachophoretic analyses for these experiments

were made in polyacrylamide gels with a diameter of 6 mm and a length of

10 em. These dimensions were chosen to facilitate staining of the protein

bands and further identification by immuno-electrophoresis.

93

Finally, experiments identical to those in the 6 mm gels were made in a

preparative column with a diameter of 25 mm, to study the possibilities for

doing the analytical separations on a larger scale.

2. THE USE OF SPACER IONS AND STABILISING MEDIA FOR THE

ISOTACHOPHORETIC ANALYSIS OF PROTEINS

Protein zones are very often thinner than 1 mm (40, 41, 58). Since Everaerts

(52) calculated that the thermal boundary width of the zones is 12 mm for

80% of the total thermal step between two successive zones, thermal

detection will be of little value for protein analysis. Therefore, before the use

of carrier ampholytes and stabilising media for protein analysis is discussed, a

new type of detector is introduced, which is based on the UV light absorption

of the proteins (60).

2 1 UV-detection in capillary tubes

The capillary apparatus, which is depicted schematically in fig. 6.1, is

basically the same as that described by Everaerts and Verheggen (62}, with a

thermocouple glued to the wall as a heat detector. In addition to the

thermocouple, a UV photometer (modified Uvicord L KB 8301 A) was used as

detector. The slitwidth was decreased to 0.3 mm. A wavelength of 280 nm

was used.

Fig. 6.1

94

I I I I L _________ _

Block diagram of the capillary apparatus.

The need for UV detection can easily be understood when we consider

fig. 6.2. It shows an electropherogram of a sample of 0.2J.II ceruloplasmin at a

concentration of 10% 'w/v, injected between a leading electrolyte consisting

of 0.01 M 2-amino-2-methyl-1,3 propanediol (ammediol) and 0.007 M HCI

and a terminating electrolyte ~consisting of 0.01 M phenol and 0.002 M

barium hydroxide. The pH of the leading electrolyte was 8.4. The current was

Fig. 6.2

em

lime

UV- and thermodetection of an analysis of 0.2 f..ll 10% w/v human

ceruloplasmin. The leading electrolyte consisted of 0.01 M ammediol and

0.007 M HCI at pH 8.4. The terminator was phenol. The current was

60 IJA, the starting voltage 3 kV. The thermostat temperature was 25°C,

chart speed 1 em/min.

95

60 JlA and the temperature was 25°C. The starting voltage was 3 kV, the final

voltage 18 kV. This experiment shows clearly the shortcomings of the

thermal detector, which did not reveal the protein zone.

The thermocouple is, however, a more general detector than the UV

photometer, which is limited to the detection of UV absorbing materials.

Fig. 6.3 shows an analysis of 21ll albumin at a concentration of 12% w/v. The

electrolyte conditions were the same as in fig. 6.2. The difference between

Fig. 6.3

96

0 " .<::

0 c ..

.<:: a.

time

c .2 Q. 0 .. .0 0

UV and thermodetection of an analysis of 21A 12% w/v human albumin.

The conditions are as in fig. 6.2.

the properties of the two detectors is obvious. The selectivity of the

UV detector can turn out to be advantageous. In fig. 6.3 it shows clearly

which thermal zone is the albumin zone.

22 Carrier ampho/ytes as spacer ions

In chapter V, section 3.2, it was already mentioned that the carrier

ampholytes, used in isoelectric focusing, are useful as spacer ions in

isotachophoresis. A requirement is that the ampholyte mixtures will form a

mobility gradient which is as linear as possible, to ensure equal spacing at

each point of the gradient. To check the linearity of the gradient the

following experiments were made. In the ammedioi-HCI system, mentioned

above, 11.!1 of»Ampholine» 12% w/v and pi range 4-6 was injected. The

result is depicted in fig. 6.4. The first step in the thermodetection curve

J

Fig. 6.4

time

UV and thermodetection of 1 f..ll12% w/v carrier ampholytes pi range 4-6.


97

represents a zone of hydrocarbonate ions present in the sample. The

discontinuity A indicates the end of the mobility gradient formed by the pi

range 4-6. The thermosignal does not rise steeply to the terminator

step-height, because the commercial »Ampholine» mixtures contain a certain

amount of ampholytes with a pi range 6-10. Apparently the spacers have

very little UV absorption. The same types of curves were found for

ampholytes with pi ranges 6-8 and 7-9.

In order to study the spacing properties of the carrier ampholytes the

following experiments were made. In one case 0.2111 of a 10% w/v solution of

human ceruloplasmin (AB KABI preparation) was injected as a sample

between the leading and terminating electrolytes described in section 2.1

(fig. 6.5a).

Fig. 6.5

0 z

~

l!l § " v

b

~

.., 0 z « 0 9 ;:

0 ~

~

UV detection of an analysis of 0.2/A 10% w/v ceruloplasmin, when

a. no carrier ampholytes are added

b. 0.4% w/v carrier ampholytes are added


In a second experiment the sample also contained 0.4% w/v carrier

ampholytes (4-8) (fig. 6.5b). The current was 60 11A, the temperature 25°C.

To ensure a good separation, the effective column length was increased by

applying a counterflow of leading electrolyte of 60 111/h during the first 1 1/2

hours of the analysis. The total analysis time was two hours. Figure 6.5b

98

shows the existence of at least two main fractions in the ceruloplasmin

sample. In disc electrophoresis three main protein bands were visible. The

strongest contained 60% of the total protein load, the others contained 20%

each.

Fig.6.5a and b show clearly that more resolution can be obtained when

spacers are added to a sample. However, one cannot apply an unlimited

amount of spacer. In the mobility gradient established by the ampholytes

there will always be a certain number of individual ampholytes, which have

the same or nearly the same mobility as the proteins of interest. Therefore

the protein bands get less focused when the spacer-protein ratio is increased.

This is shown in fig. 6.6. All electrolyte conditions were as described in

Fig. 6.6

a Q) c "0 0 ·;::: ·~ 0 :c 0 (,) 1:l

«)

em

time

c Q) 0 :2 ·.;::

0 1: :c 0 (,) 1:l

ro

em

UV detection of an analysis of human albumin together with carrier

ampholytes

a. 2/A solution containing 10% w/v albumin and 2% w/v carrier

ampholytes 4-6

b. 2/A solution containing 6% w/v albumin and 6% w/v carrier ampholytes

4-6.


99

section 2.1. Fig. 6.6a shows an injection of 2 ~I of a solution of 10% w/v

human albumin and 2% w/v carrier ampholytes 4-6. In fig. 6.6b the

ampholyte-protein ratio was increased: 2 ~I of 6% w/v human albumin and

6% w/v carrier ampholyte 4-6. From these figures it is obvious that the

protein bands get more diffuse by the addition of more ampholytes, even

though a better separation from the terminator is obtained in fig. 6.6b.

A serious problem is the binding of the spacer ions to the protein. Using

radioactive 'Ampholine' (C 14), Bakay (87) demonstrated that there were still

spacer molecules bound to the proteins even after prolonged dialysis and gel

filtration on sephadex G-100.

Vestermark (88) suggested the use of acids with long alkanic chains as

spacers. In the event that these acids have low pK values (<3), they would

form a mobility gradient based purely on molecular shape instead of on both

shape and pK value. This system would be more stable against pH

disturbances. However, the danger with this type of spacers is that it may

cause a discontinuous pH gradient such as was dealt with in chapter IV,

section 3.2.

23 Stabilisation of the protein zones

Protein zones adapt their concentration to the concentration of the leading

ion, according to the Kohlrausch regulating function. Because the protein

concentrations in the Kohlrausch equation are expressed in molarities and

their molecular weight is very high (30,000 -1,000,000), the density of the

protein zones will be higher than the density of the leading and terminating

electrolyte. Under the conditions discussed above the protein concentrations

will reach values between 5% and 10% w/v. Fig. 6.7 shows the shape of the

boundaries of a hemoglobin zone in a capillary tube. When the viscosity of

the leading electrolyte is increased, the voltage gradient might be able to

counteract this effect to a certain extent. One will however never be certain

whether the resulting concentration in the protein zones is the one according

100

Fig. 6.7

terminating

0 electrolyte

hemoglobin zone

leading electrolyte 0

The shape of a hemoglobin zone after being concentrated between the

leading and terminating electrolyte.

to Kohlrausch, or if it is some concentration resulting from an equilibrium

between the gravity forces and the force of the electric field. Polyacrylamide

gel was therefore introduced as a stabilising medium. An additional advantage

of using such a gel as polyacrylamide is that diffusion and convection effects

are much lower.

The composition of the gel solution was:

0.3 g acrylamide

0.1 g bisacrylamide

0.5 mg riboflavin

50 Jll TMED

(BDH)

(BDH)

(sigma)

(Eastman)

This was dissolved in 100 ml of leading electrolyte. The electrolyte system

according to Svendsen and Rose (58) was chosen, i.e. tris acetate as leading

electrolyte at pH 4.5. The acetate concentration was 0.03 M. The monomer

mixture was exposed to UV light during a period of one hour, after which the

polymerisation was completed and a gel was obtained.

In some preliminary experiments the gel was pressed into a PTFE capillary

with inner/outer diameters of 0.5/0.8 mm. When separating hemoglobin it

appeared that much shorter separation times were required in this type of

stabilising medium than in free solution. After migration over a distance of

5-8 em, the zones were sharp and migrated in a steady state.

On the basis of these experiments, an apparatus was constructed as shown in

fig. 6.8. It consisted of a quartz capillary tube 1, with inner/outer diameter of

0.4/0.6 mm. It was connected to the electrode compartments 5 and 6, which

were made of perspex. The capillary was thermostated by water, flowing

101

around it in a cooling jacket 2. The jacket was also made of quartz and had

inner/outer diameters of 1/1.4 mm. The capillary was filled with gel through

the valve 3, during which time valve 4 was kept closed. The sample was

injected at point 7, then valve 3 was closed and 4 was open.

Fig. 6.8

0

0

The capillary apparatus used tor isotachophoretic analysis of proteins in

polyacrylamide gels: 1. quartz capillary 2. quartz cooling jacket 3. inlet

valve for gel and sample 4. valve 5. terminator reservoir 6. leading

electrolyte reservoir 7. injection point.

In this apparatus, an analysis was made of freshly prepared solution of 2%

w/v chicken hemoglobin. 1 111 of this sample was injected, together with 1 111

of a solution of 5% w/v 'Ampholine ', pi 7-9. The thermostat temperature

was 3°C. A photograph of this separation is shown in fig. 6.9. To test the

reproducibility of this analysis a Uvicord was built around the outer cooling

jacket. The slit width was 0.4 mm. Fig. 6.10 shows the results of two

102

Fig. 6.9 Separation of 1 f.J of a 2% w/v fresh chicken hemoglobin solution

containing 5% w/v carrier ampholytes 7-9. The stabilising medium was a

3% acrylamide gel with 3% crosslinking containing the leading electrolyte:

0.03 M acetic acid and 0.011 M tris at a pH of 4.5. The terminator was

{3-alanine at pH 9. The thermostat temperature was 5°C.

identical analyses of the chicken hemoglobin for the conditions described

above. Comparison of these two recording shows that there is satisfactory

reproducibility.

In fig. 6.11 the UV recording of an isoelectrofocusing analysis on the same

sample is shown. The experiment was performed in a sucrose gradient column

( LKB 8100). The gradient contained 2 ml protein and 1 ml40% 'Ampholine'

7-9. The experiment was allowed to proceed for 72 hours at a voltage of

600 V. There is a clear resemblance with the isotachophoretic analysis.

However, the first peak of the recordings depicted in fig. 6.10 does not

appear in the isoelectric pattern. lsotachophoretic analysis without any

sample showed that this absorption peak was due to impurities of the gel.

It is very difficult to remove these impurities from the gel. Eluting the gels for

24 hours with the leading electrolyte did not result in any improvement. In a

103

Fig. 6.10

em

-time

em

-time

c: .2 ti. 0 1l "

UV recordings of two experiments identical with the one shown in fig. 6.9.

The chart speed was 1 em/min.

Uniphor column (LKB 7900) an attempt was made to remove the impurities

by isotachophoresis. Glycine was used as terminator, with tris acetate as

leading electrolyte under the described conditions. The UV recording of the

eluate of the column showed a strong absorption peak of the gel impurities

preceding the terminator. Then, the gel was taken out of the column and

soaked for three days with the leading acetate buffer. Even after this

procedure there were still impurities left in the gel, which had a higher net

mobility than glycine (according to capillary experiments). The only

104

explanation for this feature is that the impurities consisted of some

polyacrylic acids, which were gradually released from the gel.

Unfortunately, the impurities of the gel coincide with the proteins in the

alpha region of human serum. This makes it difficult to give an interpretation

of the UV pattern of human serum, especially since the character and the

amount of impurity is different for every new polymerisation.

Fig. 6.11 lsoelectric focusing of the same hemoglobin sample as in fig. 6.9. The

experiments were performed in a sucrose gradient containing 1 ml 40% w/v

carrier ampholytes 7-9 and 2 ml protein. The separation proceeded for

72 hours at 600V. The thermostat temperature was 4°C, the rate of

elution 30 ml/h and the chart speed 12 em/h.

3. THE SEPARATION AND IDENTIFICATION OF HUMAN SERUM

PROTEINS IN 6 MM POLYACRYLAMIDE GELS

3. 1 Materials and methods

The experiments described in this paragraph were made in glass tubes with an

inner diameter of 6 mm and a length of 10 em. Six of such tubes were

105

connected in parallel to two electrode reservoirs. The set·up is, in principle,

the same as shown in fig. 3.9. The sample of 'Ampholine' and protein was

applied on top of the gel with a Hamilton syringe. Because no constant

current supply was available to deliver the necessary current (3-6 rnA), a

constant voltage supply was used.

The general procedure for an analysis was as follows. The tubes were washed

with a 0.2% v/v solution of a surface active agent (Berol) to diminish adhesion

of the gels to the glass wall. Then the tubes were filled with a monomer

solution, which was identical for all experiments. Two stock solutions were

made:

Stock solution 1

7.0 g

0.35 g

Stock solution 2

1 mg

(per 100 ml of leading electrolyte):

acrylamide (BDH)

bisacrylamide (BDH)

(per 100 ml of leading electrolyte):

Riboflavin (Sigma)

Equal volumes of both solutions were mixed and poured into the tubes after

thorough de-aeration (5-10 min). Subsequently, they were exposed to UV

light for 1-4 hours, depending on the pH in the leading electrolyte. Finally,

the tubes were connected to their electrode reservoirs.

As can be seen from the recipe for the stock solutions, no TMED was used as

a catalyst. It appeared that polymerisation could be obtained without the

addition of TMED. The polymerisation without TMED is slower and results

in a lower polymerisation degree, as can be concluded from the rigidity of the

gels. However, in isotachophoresis it is of interest to omit as many extra ions

as possible. During some separations of hemoglobin it was observed that a

boundary was formed between the counter-ion and the TMED ions, when

TMED was used in a quantity of 0.005 M. When this boundary met the

protein bands a dramatic change of the separation pattern took place.

The following procedure was chosen for the staining of the gels. The gels were

left overnight in a 125% solution of trichloroacetic acid to fix the protein

bands and to let the carrier ampholytes diffuse out of the gel. After being

106

washed with water 2-3 times, they were stained in a mixture of 45% ethanol,

45% water, 10% acetic acid and 0.2% coomassie brilliant blue (Sigma) for

about one hour. Then they were de-stained with a mixture of 45% ethanol,

45% water and 10% acid during 24 hours.

The identification of protein bands was made by Laureii-Freeman immuno

electrophoresis (83, 84). The stabilising medium for this technique was

1% w/v agarose gel, cast on glass plates 100 mm wide and 1.5 mm thick. As a

buffer, sodium diethyl barbiturate was used at a pH of 8.6 and an ionic

strength of 0.075. The glass plates were covered to 1/5 of their length with an

antibody-free gel. The remainder of the gel contained an antibody solution at

a concentration of 83.3 ~1/cm3. The antisera which were used were rabbit immunoglobulines against human serum {Dakopatts A/S). Half of a longi

tudinally sliced polyacrylamide gel was applied on that part of the agarose gel

which dit not contain any antibodies, to avoid preliminary precipitation.

After cross electrophoresis for 20 hours at a voltage of 3 V /em, a precipita

tion pattern was obtained. Using specific antiserum, several of the peaks in

the pattern could be identified.

3.2 Tris acetate as leading electrolyte

Svendsen and Rose demonstrated the separation of human hemoglobin on a

preparative scale using tris acetate at pH 4.5 as leading electrolyte (58). The

acetate concentration was 0.06 M. The terminating ion was glycine.

In fig. 6.12 the net mobility of glycine is plotted against the pH in the acetate

buffer. As can be seen from the diagram, the net mobility interval between

the terminating and the leading ion is quite narrow. At pH 4.5 in the leading

electrolyte, the glycinate ion will migrate with a net mobility of 2.0.10-5cm2v-1sec-1 at a pH equal to 8.6. This means that the gammaglobulines having a mobility of 2.0 to 0.5.10-5cm2v-1sec-1 at pH

8.6, will not migrate isotachophoretically in this electrolyte system. /3-Aianine was theretore chosen as a terminating ion. Its higher pK value (10.3) resiJited

107

Fig. 6.12

Fig. 6.13

108

~ 30

1 ;; c 20

10

acetate

~glycinate ~-alaninate - 6.0 pH

leading eltoctrolyte

The net mobilities of acetate, glycinate and f}.alaninate as a function of the

pH in the leading acetate zone. The concentration of acetate was 0.03 M

and the counter-ion was tris. The temperature was 25°C.

Analysis of 25 f.1l (left) and 50 f.1l (right) of a mixture containing 1% w/v

chicken hemoglobin and 10% w/v carrier ampholyte 7-9 on a 6 mm gel.

The leading electrolyte was 0.03 M acetate and 0.011 M tris at a pH of 4.5

in a 3.5% polyacrylamide gel with 5% cross linking. The terminator was

!).alanine. The picture was taken 2 hours after the sample was applied. The

constant voltage was SOV, the start current 0.5 rnA.

in a net mobility of 1.2.10-5cm2v-1sec-1, which means that the igG and

igA will migrate in front of the terminator.

As a test for this system, a freshly prepared sample of chicken hemoglobin

was analysed. The electrolyte system was as described above, except for the

acetate concentration, which was 0.03 M. The left-hand part of fig. 6.13

shows an analysis of 25~od of a mixture containing 1% w/v chicken

hemoglobin and 10% w/v carrier ampholyte with a pi range 7-9. The tube on

the right·hand side was an analysis of 50 pi of the same sample. The voltage

was kept at 80 V to avoid excessive heating. The resulting start current was

0.5 rnA. The picture in fig. 2.13 wastaken after an analysis time of two hours.

In the same electrolyte system, an analysis was made of freshly prepared

human serum. 10 pi of human serum was applied directly on the gel, after

centrifugation. 10 pi of carrier ampholyte with a pi range of 5-6 and a

concentration of 40% w/v was used. The applied voltage was 100 V. The

result is shown in fig. 6.14. The result of the identification of the bands,

Fig. 6.14 Separation of 10 J.ll of fresh human serum using 10 J.ll 40% w/v carrier

ampholyte 5-6 as spacer. The experiment was performed on a 6 mm gel.

The conditions were as in fig. 6.13. The migration direction is downwards.

109

made by Laureii-Freeman immunoelectrophoresis, is shown in fig. 6.15. The

numbered peaks are identified with the aid of specific antibodies. Fig. 6.15

shows clearly that four major groups of peaks are obtained: orosomucoid (1),

alphaglobulin region (2,3,4,5), betaglobulin region {6,7,8), and the gamma

globulins (9, 10). The orosomucoid seems to be completely separated from

the remainder of the protein zones. This could be due to the action of the

hydrocarbonate ions, which create a pH boundary with acetate, as is dealt

with in chapter IV, section 3.4.

Fig. 6.15 lmmunoprecipitin pattern of the separation shown in fig. 6.14. 1. orosom

ucoid, 2. prealbumin, 3. a;-1-antitrypsin, 4. albumin, 5. ceruloplasmin,

6. haptoglobin, 7. transferrin, 8. a;-2-macroglobu!in, 9. igG, 10. igA.

100 p.g of transferrin was applied, together with 10 p.l carrier ampholyte 5-6 at a concentration of 40% w/v. Transferrin was separated in at least five

110

Fig. 6.16 a Analysis of 100 iJg human transferrin together with 10 J1l 40% w/v carrier

ampholyte 5-6.

b Analysis of 10 J1l of a mixture of 4% w/v human albumin and 2.5% human

gammaglobulins, together with 10 J1l 40% w/v carrier ampholytes 5-6.

The conditions were the same as in fig. 6.14. The migration direction is

downwards.

bands, which are, probably genetic variants (fig. 6.16a). Fig. 6.16b shows an

analysis of 10 ,ul of a mixture of 4% w/v albumin (AB Kabi) and 2.5% w/v

gammaglobulin {AB Kabi) together with 10 ,ul 40% w/v ampholine 5-6. This

analysis shows clearly that the gammaglobulins migrate isotachophoretically.

4. THE CHOICE OF ELECTROLYTE SYSTEMS FOR HUMAN SERUM

SEPARATIONS

4. 1 Theoretical calculations on the electrolyte conditions

The theoretical model, which is dealt with in chapter 2, enables us to

calculate electrolyte systems for the separation of proteins. In the intro

duction to that chapter it was already pointed out that there exist a very large

number of possible combinations of electrolyte parameters, which will result

111

in a separation of a protein sample of interest. In this paragraph we will deal

with one set of electrolyte systems.

Consider fig. 6.17. The leading electrolyte is tris citrate at a citrate

concentration of 0.01 M. The pH and net mobility of 8 acids in zones

succeeding the citrate zone, are plotted against the pH of the leading citrate

buffer. Assume that a pH of 5.0 is chosen in the leading electrolyte. When a

line is drawn vertically through this particular pH value, the points of

intersection with the curves represent the net mobilities and the pH values in

respective succeeding zones.

Instead of citrate we can choose acetate as the leading ion, at a pH and

concentration corresponding to its point of intersection. Exactly the same

electrolyte conditions in the succeeding zones 3-9 would be obtained. Also,

any other ion shown in the diagram can serve as the leading ion for all zones,

which contain ions with a lower net mobility than this ion.

The mobility of the serum proteins is, maximally, 8.0.10-5cm2v-1sec-1

{chapter V, section 3.1 ). This means that it is not necessary to choose citrate

as a leading ion. When cacodylic acid, PIPES, TES or diethylbarbituric acid

are used, the mobility interval would be narrower and big temperature and

pH steps are avoided between the leading ion and the succeeding zones. The

swelling of the gel, which very often causes curved zones, is also strongly

reduced by choosing small pH intervals. Cacodylic acid and TES were chosen

as test substances for the leading ion. Carbonate was omitted for practical

reasons. Oiethylbarbiturate was not used, because it has a high UV

absorption, which interferes with the detection of the protein zones. One

additional advantage in using cacodylic acid and TES as leading ion is that

Fig. 6.17

112

The pH and the net mobility of a number of acid zones succeeding a

leading citrate zone, as a function of the pH in the citrate zone with tris as

counter-ion. The citrate concentration is constant, 0.01 M. The calcula

tions are made for 25°C.

1. citrate; 2. acetate; 3. carbonate; 4. cacodylate; 5. PIPES; 6. TES; 7. ver<r

nal; 8. glycine; 9. ;3-alanine.

'i > "e u .. 0 ... Ill .E E .. Ill ... ~ 40

:a ~ 30 ... Ill c

5.0 6.0 7.0 8.0 pH leading electrolyte:

Citrate

113

they confine the pH gradient within the buffering area of the tris ion, which

makes the system much more stable agaii)St pH disturbances.

For the separation shown in fig. 6.18. the leading electrolyte consisted of

0.012 M tris and 0.013 M cacodylic acid at a pH of 7.02. These are the

conditions according to the intersection point in the diagram between the

cacodylate line and the line pH citrate=5. Tris-/3-alanine forms the terminator

solution. A sample of 10 pi fresh serum was applied on a 6 mm gel, prepared

as described in section 3.1. 15 pi 40% w/v carrier ampholyte 5-8 was added

to the protein.

When the intersection point with the TES curve was chosen, the adjusted

concentrations were 0.01 M tris and 0.011 M TES, which resulted in a pH of

7.65. In fig. 6.19a and b the analyses are shown of 10 pi fresh human serum

together with 15t.LI and 20 pi respectively of 40% w/v carrier ampholytes

5-7.

Fig. 6.18

114

Separation of 10 pi fresh human serum using 15 J1l 40% w/v carrier

ampholytes 5-8 as spacer. The leading electrolyte was 0.013 M cacodylic

acid and 0.012 M tris at a pH of 7.02. The terminating ion was /3-alanine.

The voltage was 300V and the start current 1 mA. The migration direction

is downwards.

Fig. 6.19

a b

Separation of 10/A fresh human serum using 15/.ll (a) and 20PJ (b) 40%

w/v carrier ampholytes 5-7 as spacer. The leading electrolyte was 0.011

TES and 0.01 M tris at a pH of 7.65. The terminating ion was ~alanine.

The voltage was 300V and the start current 1mA. The migration direction

is downwards.

4.2. Cacodylic acid as leading ion for preparative protein separations

In preparative columns the bottom of the polyacrylamide gel is usually

supported by a membrane {57). When the sample has migrated through the

gel and the membrane, the protein zones are recovered from the column by

washing the membrane with a so-called elution buffer (fig. 6.20.). Svendsen

and Rose (58) used glycinate to elute all proteins from the column. This

however includes the risk that glycinate molecules will diffuse upward into

the gel and increase the pH at the bottom of the gel. When hemoglobin was

separated in an acetate system at pH 4.5 using tris glycinateas elution buffer,

it appeared that the red protein zones, which were very sharp from the

115

Fig. 6.20

~ 1o UV. detector

The elution of protein zones from a column. After migration through the

membrane. the protein is transported by a stream of buffer (elution buffer}

to a fraction collector.

beginning, turned diffuse, when meeting this pH shift at the bottom of the

gel (92). It is, however, impossible to elute with the leading electrolyte in this

case. If the hemoglobin bands then reach the bottom of the gel, they will be

immobilised, because the pH of the elution buffer is below their isoelectric

point In an effort to elute a serum separation from the column using citrate

at pH 5 as leading ion, the same observation was made for the gammaglobu

lins.

It is clear that one has to use an elution buffer which has a pH value above, or

very close to, the isoelectric point of all proteins present in a sample mixture.

Furthermore, it would be advantageous to use the leading electrolyte as

elution buffer, because the introduction of a »foreign» ion zone in the leading

electrolyte implies a disturbance of the electrolyte conditions. The use of

cacodylic acid is an answer to most of these demands. The pH of 7.02

(according to the intersection in the diagram mentioned above) is not higher

116

than all the isoelectric points in human serum, but it is high enough to elute

most of the proteins from the column.

TES was not used for preparative experiments, because it was quite

expensive. For the preparative separations of human serum in the Uniphor

column (LKB 7900) cacodylic acid could therefore be used as leading ion.

The concentration and pH were as described in section 4.1. The following

experiments were made to show the influence of the elution buffer on the

separation. 100 ml leading electrolyte containing monomer was poured into a

Uniphor column with a cross section area of 5 cm2. After the polymerisation,

the electrode compartments, together with the elution stopper, were

Fig. 6.21

time

UV recordings of preparative separations of 0.5 ml human serum using

0.5 ml 40% w/v carrier ampholytes, 5-7 as spacer on a Uniphor column

(LKB 7900). The leading electrolyte consisted of 0.013 M cacodylic acid

and 0.012 M tris. The terminator was 13-alanine. The current was constant,

10 mA, the start voltage was 220V, the temperature was 25°C. The

proteins were eluted at a rate of 25 ml/h, The chart speed was 2 em/min.

a the leading electrolyte was used as elution buffer.

b the terminating electrolyte was used as elution buffer. 117

mounted on the column (93). A sample of 0.5 mi. carrier ampholytes 5-7

was applied on the gel. When the ampholytes had migrated into the gel,

0.5 ml fresh human serum was applied. This procedure was chosen to avoid

precipitation of the proteins caused by the high ionic strength of the

ampholyte mixture. A constant current of 10 rnA was applied, which resulted

in a start tension of 220 V and a final tension of 430 V. The cooling-water

temperature was 4°C. The eluate was pumped out of the column into a

fraction collector via a UV monitor at a rate of 25 ml/h. Fig. 6.21a and b

show the UV absorption patterns of the elution liquids, when leading and terminating electrolytes, respectively, were used as elution buffer. It can

easily be seen that the elution with the leading electrolyte gives sharper

elution patterns.

Fig. 6.22

118

lmmunoprecipitin pattern of the fractions collected from the experiment

in fig. 6.21 a.

1.orosomucoid, 2. prealbumin, 3. G-1-antitrypsin, 4. albumin, 5. ceru

loplasmin, 6. haptoglobin, 7. transferrin, 8. a-2-macroglobulin, 9. igG.

The 2.5 ml fractions collected from the separation shown in fjg. 6.21a were

analysed by immunoelectrophoresis. To be able to apply the eluate on the

agarose gel, holes were sucked in the gel (58). After application of 5 ~d of

every alternate fraction on the plate, an immunopattern was obtained as

shown in fig. 6.22. Comparison of this analysis with the one in fig. 6.15.

shows that a separation is obtained, which is different from the one using tris acetate as leading electrolyte.

110

Fig. 6.23

0

c: 0

:;::; 10 e-

0 "' .D t1)

20 c: c: :2 c: ~ ·e 'B. 0 ·e ·;:

(.) ::l ~ ::l ::l ::l .D +-' E ..a 0. 30 15 :;::; 0 ~ .§

c: c: "' .E s: e 0. 0

~ 6 40 "' em N c 6 f: ...

so 100 90 so 70 60 so 30 20 10

fraction number

Identification of the UV recording, according to the experiment shown in fig. 6.21a.

With the aid of the immunoprecipitine pattern an interpretation could be

made of the UV absorption curve. Fig. 6.23. shows the result.

After the separation was completed the gel was treated with trichloroacetic

acid and coomassie brilliant blue solution, to find out if everything had been

eluted from the gel. No protein band could be detected at the end of the gel. The top of the gel contained some precipitate of lipoproteins.

119

5. CONCLUSIONS

It is possible to separate proteins by isotachophoresis in capillary tubes with

the aid of carrier ampholytes as spacers. With the use of spacer gradients some

difficulties are aggravated, such as creation of diffuse zones at high

spacer-protein ratios, and also the binding of the ampholytes by proteins.

Some form of stabilising medium is required. In our case, polyacrylamide was

chosen, but other choices are possible, such as sucrose density gradients and

powders. The impurities present in the gel make it difficult to give an

interpretation of the UV absorption pattern of the protein separation.

6 mm polyacrylamide gels can be used for isotachophoresis with respect to

analytical separations and identification. These analytical experiments can

then be used to design the conditions for preparative separations.

The choice of the electrolyte system is important, not only for the type of

separation desired, but also for the recovery of the highly-resolved protein

zones from the preparative column. The most favourable elution buffer is not

the terminating buffer, but the leading electrolyte. If the leading electrolyte is

chosen, then all ion zones will keep their electrolyte conditions adjusted to

the leading electrolyte during the complete elution procedure.

120

APPENDIX

THE »ISOGEN» PROGRAM

The computer program, »lsogen», shown on the next two pages, is based on

the equations which are dealt with in chapter II. The program is written in

Algol language for a GE265 time sharing setvice computer.

The input data are all mobility and pK values involved in the electrolyte

system considered. From these data the program will calculate the total

concentrations of all compounds in the electrolyte system, the net mobility

of all compounds except the counter-ion and finally the specific conductivity

in the zones.

The data can be fed into the program on the lines 15G-220 in the following

way:

150 mH,moH,

160 1, (for the separation of negative ions) or

-1, (for the separation of positive ions)

170 number of zones involved in the separation, (maximally 20),

180 1T,pKP1•PKP2• · · · · • PKp1T' mP11•mP12• · · · · ,mP11T'

190 a,pKA1• ......... ,pKAa• mA11• ........ ,mA1a-

191 t maximal ionisation degree, pK and mobility values

I in the succeeding zones .

.j,

220 p,pKB1• · · · • · · · · · ,pKsp•mB21' · · · · · · · · ,m82J3

121

100 I Hl 1!!0 130

11143

QtGIN INT~GF:P f,.J,L,$fG\tT,..Nl,N!.!viAXJ ~£AL Y.H,:-41)H,PJoiS,?~.tCAT'I•.CL\T,

CTT,NMA,SJ,$?,~3,S,FM-~•0t A~H-Y P~.~~0:20,Jt20iJ INlEGfR A~W-Y ALFA/;0:201,1 fll'l'L'l:AN fll>HS I

I 40 ISO 160 I• I 70 6•

HAT<\ l<IIC>ATA:= 314.!73.

I 80 190 191 19? 193 I 94 I 95 ?30 2 .. 0 "5(1 ?1>0 ?70

t .. 6. J .~s. J, ... 1Q 11 7R, p,f.?,3,4·19,40.73• P.,2.9R,4.34.39,64, I ,:;l-7~,. Sf;, 3.3.08,4.74.~.40·3~-5.55.70 .. 1,A.75,AJ I

REAL PRl'Cf'.lllfl!': F'( "H) J VI'LUE PH 1 IlEAL PH l RF.GIN RFAL AS,JASo~I'S•I~A~.T~>IfS•MTS•IMT~•C~.C~H.CAOoCTO.SJ,sP,

CTOSIS.F'KSI ?J;t) P'tl1Cf0iJRF.: SltMSCJ,pH,A~,fA\S,M.4~.tl~f.l~.);

!>90'VAUJF.: J.PHI!:>ITEGE>< JJ'l~:AL PH>'IS•l"~•~AS,DIII~l 300 REGIN 310 REAL AIJ INT!GF~ lo!HJ 320 j>Jt•AS:•I J IASI•MAS:=IMAS:=O I IH:•ALFAIJl I 330 F'l'>'l 1:•1 STE" 1 Uli<TIL Iii Dl' 340 o:lEGI'Y 3 SO AI I•A I• Fl"'C Sl GN< J- • Sl ·~ tt;;.T*O'ii-t•K:.\J, 1.\)) l 360 j>S:•AS+'II J 370 IAS:=IA~+I*'IIJ 3R0 MAII•MA!+MXJ.IA•AI 390 IMAR t=lMAS+I•M~Joli*AI l 400 ENO I 4 l(l [NO SIJMS I 4?0 Jl30 P~l'CEJ)IJ<:!I:: Sii"'CAL (JoPHll VAI.IIF: J•f'Hl !NTE:G;:;; Jl RF:M. PiH Jl40 "!':GIIII 4 50 l'<U!>!S(J,PHoAS, I ""•:.!"'~• !-">ASH S!Jl'lS((J, PH• 'H'• l TS• :<T'>•lMTSl J 4~0 CHt=IO.,(-t'H)l C~Ht=,-14/CHJ 470 llt=<TS+A~*"'TS/MAS)I 4 !lO ~':NO SliMCAL ; 4'>0 500 !l£AL 1''ll'C'!:OIJRJ;: F.-<1 510 F'KI=MAS/(AS•<IMTS•CTn+IMAS•CAO+C~H*M~H+CH•M~)) J 520 530 IF q?HS THF.N 5 40 1:1!::0 IN 550 Cl'MMENT CALCIIL'ITil'NS F<"' REGit\\J t 1 l 560 SIJI'.CAL( I•I'HSll 570 CAOI•CATS/AS I 5 80 CTOr =<I AS•CAO+>' 1 GNTH C<'K-CH)) /I TSt 590 CTOSIS:=CTO*SII 600 ;;"KS:=FKJ 6!0 Rt'HS:=FAL~E I <!-20 fNi) I 630 Ct'MM!':NT CAI..CIILI\Th':.IS F'l''< ><F:Gll'N ? 1 1 1\40 s:IMCAL(N7.,PHH 1\ 50 CTO I =CTOS !.'\/~II 660 CAO:=CIT~•CTO-SIG~T•<C~H-CH))/l'ISI 6 70 CAT:= CAO•ASI CTTI=GTO•T<; I 6~0 N~At=~li$/AS I 6 SS ~ o•(l"'AS•CAO+IMTS•CT!l+CCC•~1H+C~'H*Mc'~ l •'l!>o "'R1 1•5 1 690 F:=F~S/F~·II 7 00 .: .. o. 710 7 ?.0 F'Rl'C£0.fh'£ Z!:·~t'CF'~X').-.Hl• 1·"~:.:t'~G,F"~L•fi?.-:-.:o)J 730 VALliE )(~,;H,H~I

141l RF:AL l'l"c'C~~IItN!: Fltl"4L ~!;,;iJ•""•XOI "I o;o 'll'l'Li':IIN Yl'·h''l<ll LA'~':!. l'n. I

760 ':.l~(ift\ ~~At. X1.-;-\~~.F),;';).·;:;l..•t.'t .. ;;:.A·v >11

770 XI:•XSIFI:•F(,IJIA:•F•L~F; 7~0 Lit XPI•XI+HIIF~:•F(~?)t 790 IF I'I*(F?.•I'I)>O l'"< '<"l''<G frlf;,\ ROO l'lF:GIN IF " THEN Gl'Tl' rF:LI 'II: •·H II t'il'Tl' I.!' I 810 !;'!Iii) I 1'?.0 IF Fl*FP>O THf.!li 830 ~F:GIN Xt:•~?J Ft::f?l 1'40 L?: l'l:•T•.:!JI': I I (il'T'' 1.11 RSO ~NO 1 ~~0 C~MM~NT PART ?.: I R70 IF FI>O TH~N RRO AFGlN SI•XliXI:•XRIXP:•~I~:=FllFI:•F?.IFP:•!I ~90 END I 900 L~: IF ~AS(XI•KPJ>A~SCHPJ T~FN 910 BEGIN XSt•(~I+X?.JIPI S:=F(~S)I 920 IF 'i>O THf.)>l 930 B~GlN XP:=X~IF?.:•SI 940 !!:NO E:LSF: 9$0 AI!:GIN XI :=XSI'l I"S 960 END I 9 70 Gl'Tl' L3J 980 END J 990 XO:•XP•FP*CX2•XI)I(F2·FI)I I 000 END ZERl' I 1010 1020 ~F:AI)ATACINDATA,MH.M0H.SIGNT,~?.MAX)I I 030 Fl'R I 1 •0 S n:? I !INTI L NZMAX fll' I 040 !lEG IN 1050 REAOATA<fNDATA,J) I ALFA~IA::J l OliO l"l'P. LP•I STEP I 'JNT!L J Ol' 1070 RF.AOATACINOATA,PK~I.LA> 1

I 0!'!0 Fl''l L:•l STEP I UNTIL J Ol' 1090 READATACINDATA•M'l•LA )J I 100 ENOl I I 10 11?0 Ll t P'li!IIT("CATS.PHS;'<TA~T.:HI':P.~Tc'P"l) 1130 RF.AI)ATA(TI'.LF.:TYPF.:oCATSo!'\t.S2•!"3JJ t 140 PR1NT< .. PH"•"CAtu, .. NMA0 •"CTf 11 •"0u) 1 I 150 S3:=S3+o000001*S?.J I 160 Fl'ii PHS: =S I STEP 52 I INTI L 53 Dt' I 170 l'lF.:GIN I I '-'0 FMA>o =OJ N7.: = 1J 1190 Prit=PiiSJ'lPHS::TfWF.I 1?.00 L4f SI=F<PH)J 1?10 IF A~S<S)>F~AX THE~ FMAX:=~I 1220 ?~INHf'H,CI\T,!\1'\A,CT'f,f.l) I I 225 Gl'TO LSI 1?.2'1 F>:L:Pfi!NT<N7.,"PHYSICAI.I..Y !>~?t'SSl'lLO::"H 1230 L5: IF N7.<NZMA)( i'HF:N 1240 qF,GIN NZI=NZ.+II 1250 ZER0(F,?H••I•CAT<O 0R CTT•D•F~L••~IoPHll I :?60 Gc'Tl' L41 121!0 F:ND ; I 290 PRINT<"FMI\X="•F"'IIXH 1300 PRINT(" "H 1310 ENO P0P ?liSI 1320 Gt'H' L11 I 330 ENiJ c'F >'R\'GR6M;

123

EXAMPLE

The leading ion is 0.01 M chloride. The counter-ion is histidine. Oxalate,

tartrate, formate, citrate, and acetate migrate in the succeeding zones. The

input data are as follows:

15'1 3!"1.17~. I t\:1 l• I 71l ~~

I ~0 1•<'>-l•~~. 1 ?~ ~~-10.7'-<, 191 ?,J.~J .. I'hlq,.lj('l.,7'J, l 9?. ?,?•9P.,4•34.-39,A4.~t 193 t .. a.?s,t;r,,. 194 3,3.oA,4.7~ .. 6.~0 .. ~R.s,ss,?o, '9S 1•4•75,.41 9 3!) J ~ 4·:)

The pH region which will be scanned is 5.5-6.1, with intermediate steps of

0.2 pH unit:

When the program is started it will demand the following data: total concentration of leading electrolyte (CATS), the start pH, the intermediate

step, and the final pH for the scanning (PHS: start, step, stop).

The output of the computer is given below. The pH, the total concentrations

of the leading succeeding compounds (CAT) and of the counter-ion {CTT),

the net mobility {NMA) and the specific conductivity of the zones (Q) is

printed on one line for each zone. If the calculations are finished for the first

pH value of the leading electrolyte, the program starts with the next pH.

Finally the program asks for a new leading electrolyte concentration and pH

range. For physically impossible electrolyte systems, which result in negative

concentration values, »Physically impossible» will be printed.

124

I !'L'Gi':l'< 11:39 STrtrl!•~ 30/7rl1

CATS,PHStS1a~J,~lf~,·;T0P

? .'JJ,s.s.-.P..-~:t•l

f'l' CI>T Nt<~;{\ CF " s.s • o I 7f~ • J -~5()71.} 5-•? <i0.946~~ S-4 s. 5::!~!16 4.96619 hJ 71• S">:l!l l-~~3Jqy s-~ '~• ),2.4·~5 , ... So54731 4oRI919 ~-:! (,}>. 5;>96 I• 1'11?1 ~-2 7.97.01? ~-4 5o56945 'h 127"3 ~-3 .,~. 1 t ... Ja loll\3>'<2· ~-9 7.034~3 .1'•4 5oS991f 4.4:)796 ~-1 "iSoO'>P:> l•IM\04 £-:> 7.1}?0"~ $•4

5o1M!1? 1<.?.0173 f-•3 37o:>!55 1·<111?? <;-;> 4o1451:1 ~-.~. I' MAX: l'!o4'>03~ 1; .... ~)

!'h7 ·01 7ih 1·397'!3 $•2 9. Ci~ii~i7 ~ s-.o 5·71761 4.949.4 ~-~ 7:;!.04~9 lo 377!<?. 1•? 'J.1~5l'1~ ~·.Q

So7'3457 4o79~67 ~-:! IS3o:lR7 I• 34':JJP $-?. k..(l~Jt~3 ~-4

o;. 7537 5 9oi2R23 S•3 ss- • .o511:l t.:;tQ~S ~-2 7•06901 'r•4 5o7766f' 4o?492:l !':•3 56· S:l7 1·3?196 ~-2 7. 907 57 ~-Ls

s.~75Rll '1·2015 S•:l 3Sol .. 53 l•PIRP7 .~-? iloHI\?91 $•.0

rt'AX= 2oF<?377 1>•6

5·9 oOI '1F!· lo63(175 $-2 ·»·94169 $•<i

5o91?.29 4o9:3R19 $-3 7'>.o3R6! 1·61314 ~-~ 'h2PI\16 $•4 5o9:?.fl47 4· 77"19?. 5•3 63·3695 lo579(1J S•P R • 07 6~ ~ 1•4 5o94638 9·12864 ~-3 55o6<16 1•5AJ75 f•?. 7oQ9~51 !F-•4 5o9MWI 4oOR.S23 S-1 S!;.lh24 I• 56?41 ~·P 1· 41(171 5·4 6·0:1516 ~hP0182 ~-3 38·9785 I• 45117 !f•2 ... '16811 r-14

FMAX: :?.·9429<>: !•6

6ol oOI 7~. 1. 99984 $•?. 9·94114 ~-4

6oi097R 4·93093 $ ... ~ 71!.6079 l•qSi?.R.2 S•2 9o?.SJ?f.' S•4 6oi?SS5 4o767Q7 S·3 63o596? 1·94'!59 ~-2 ~. l'l471'; S-A 6ol42'16 9o12R9P $•3 5'\o71.0:J 1·'<12'1? s-:?. 7·10794 $•4 6ol5116 :lo92116 $•3 59oP.7611 oOI9:l'l3 7ol\3f)7il S•4 6•?1::!56 !'loi"O??.I $•3 :l9o63~'< lo!'<?O:?:l 5-~ '>•05()9'1 $•4

FI'AXe lo~'ll4 $•6

CATS.PHS:START.STEP,ST~F ?

126

SYMBOLS, INDICES, ABBREVIATIONS

symbols

c ion concentration molcm-3

c* total concentration mol cm-3

D dielectric constant A sec v-1cm-1

f activity coefficient

F Faraday's constant Cmol-1

G voltage gradient Vcm-1

current density Acm-2

I ionic strength grion cm-3

J mass flux mol cm-2sec

k equilibrium constant

K equilibrium constant

migration distance em

m mobility cm2v-1sec-1

mo mobility at infinite dilution cm2v-1sec-1

q constant

a mass flux mol cm-2sec-1

r radial distance em

R radius em

R gas constant J mol- 1 oK-1

s cross sectional area cm2

t time sec

T temperature OK

T transport number

u velocity em sec-1

v volume cm3

127

X

z a

indices

distance from origin

charge

maximal degree of ionisation ion A

maximal degree of ionisation ion B

zeta potential

viscosity

thermal conductivity

specific electrical conductivity

equivalent conductivity

equivalent conductivity at infinite dilution

chemical potential

chemical potential at infinite dilution

maximal ionisation degree ion P

em

v g cm-1sec- 1

J sec-1cm-1 oK-1

!r1cm- 1

s:r1cm2

n-1cm2

J mol-1

J mol-1

A 1 i parameter concerning ion A in zone 1 with charge i A 1 parameter concerning ion A in zone 1

P22 parameter concerning ion Pin zone 2 with charge 2

H2 parameter concerning protons in zone 2

OH2 parameter concerning hydroxyl ions in zone 2

abbreviations

ammediol

pHexp

pHtheor

pHtheor/corr PIPES

PTFE

TES

TMED

128

2-amino-2-methyl-1 ,3 propanediol

experimentally measured pH

theoretical pH

theoretical pH corrected for ion-ion interaction

piperazine-N,N'·bis(2·ethane sulfonic acid)

polytetrafluoroethylene

N-tris(hydroxymethyl)methyl-2-amino ethane sulfonic acid

N,N,N',N'·tetramethylethylenediamine

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130

59 Vesterberg 0., Acta Chern. Scand. 23, 2653 ( 1969) 60 Arlinger L. and Routs R.J., Sci. Tools 17, 21 (1970) 61 Vestermark A., Sci. Tools 17, (1970) 62 Everaerts F.M. and Verheggen T.P.E.M., Sci. Tools 17, 17 (1970) 63 Everaerts F.M. and Verheggen T.P.E.M., J. Chromatog. 53, 315 (1970) 64 Beckers J.L. and Everaerts F.M., J. Chromatog. 51, 339 (1970) 65 Everaerts F.M. and van der Put A.J.M., J. Chromatog. 52,415 (1970) 66 Blasius E. and Wenzel U., J. Chromatog. 49, 527 (1970) 67 Preetz W. and Pfeifer H.L., J. Chromatog. 41, 500 (1969) 68 Brouwer G. and Postema G.A., J. Electrochem. Soc. 117, 873 (1970) 69 Abramson H.A., Moyer L.S., Gorin M.H., Electrophoresis of proteins,

Reinhold Publishing Corp., New York, 1942 70 Hjerten S., Free Electrophoresis, Thesis, Uppsala, 1967 71 Glasstone S., An introduction to Electrochemistry, D. van Nostrand

Company, Princeton, 1960 72 Davies C.W., Electrochemistry, George Newness Ltd, London, 1967 73 Routs R.J., Graduation Report, Eindhoven University of Technology,

1969 74 Edward J.T., Sci. Proc. Roy. Dublin Soc. 27, 273 (1956) 75 Wagener H. and Bilal B.A., Z. Naturforschg. 21a, 1352 (1966) 76 Duimel W.J.M. and Cox H.C., Sci. Tools, 18, 10 (1971) 77 For a review: Lederer M., Paper Electrophoresis, Elsevier, Amsterdam,

1955 78 For a review: Kohn J., Smith 1., Chromatographic and Electrophoretic

Techniques, lnterscience, New York, 1960 79 For a review: Wieme R.J., Studies on agar electrophoresis, Thesis,

Brussels, 1959 80 Smithies 0., Biochem. J. 61, 629 (1955) 81 For a review: Haglund H., Sci. Tools 14, 17 (1967) 82 Grabar P., Williams C.A., Biochim. Biophys. Acta 10, 193 (1953) 83 Laurell C.B., Ann. Biochem. 10,350 (1966) 84 Clarke H.C., Freeman T., Protides of Biological Fluids 14, 503 (1966) 85 Svendsen P.J., to be published 86 Svendsen P.J., private communication 87 Bakay B., private communication 88 Handbook of Chemistry and Physics, The Chemical Rubber Co,

Cleveland, 1968-1969

131

89 KortUm G., Vogel W., Andrussow K., Dissoziationskonstanten organischer sauren in wassriger 16snung, London, 1961

90 Landold-Bomstein, Zahlenwerte und Funktionen, 6 Aufl. Bd. II, Teil 7, Springer Verlag, Berlin, 1960

91 International Critical Tables of Numerical Data, Physics, Chemistry and Technology, McGraw-Hill, New York and London, 1933

92 Davies H., private communication 93 Bergrahm B., Sci. Tools 14, 3 (1967)

132

SUMMARY

lsotachophoresis has been proved to be an electrophoretic method with a

high resolving power. The principle of the technique is that ion zones are

separated according to their net mobilities and will migrate with the same

velocity.

A theoretical model is developed which is the basis for the calculation of the

electrolyte conditions for isotachophoretic separations.

Measurement of several parameters in the zones, such as pH, specific

conductivity and temperature seemed to confirm the theoretical model. The

experiments for these measurements are made in several types of columns in

different stabilizing media.

The theory is used to calculate the optimal separation conditions for a

number of weak acids. It appeared that random selection of the electrolyte

systems was not advisable. The net mobilities of several ion species could turn

out to be equal and thus it would be impossible to separate them. The

possibility of being able to calculate the optimal electrolyte system

beforehand made trial and error investigations unnecessary. Secondly, the

theoretical model has contributed to the explanation of certain phenomena

which tended to disturb the separation picture. The influence of hydroxyl

ions and hydrocarbonate ions, originating from the carbondioxide in the air,

could be estimated. Disturbances of the pH- and tension gradients between

the leading electrolyte and the terminator can be avoided.

The place of isotachophoresis among other electrophoretic techniques is

discussed with respect to the separation of proteins. The theoretical model

has made it possible, also, to select the right conditions for the separation of

proteins. Moreover, the versatility of isotachophoresis provided us with many

133

possible electrolyte systems for the analysis of proteins. A few systems are

dealt with for the separation of hemoglobins and human serum.

Because it appeared that a stabilisation medium was necessary, the separa

tions were made in polyacrylamide gels. With the aid of carrier ampholytes,

used in isoelectric focusing, it was possible to obtain good separations on the

analytical and preparative scales. For some electrolyte systems, part of the

proteins in the separation pattern was identified by Laureii-Freeman

immunoelectrophoresis.

The function of the model for preparative separations of proteins was not

only the calculation of the optimal electrolyte conditions, but was also the

basis for the choice of the buffer, used for recovery of the proteins from the

column.

134

SAMENVATTING

lsotachoforese is een nieuwe electroforetische techniek met hoog oplossend

vermogen. Een theoretisch model is beschreven ter berekening van elektroliet

systemen voor deze scheidingsmethode. Dit model is gekontroleerd door

series experimenten, welke plaats vonden in scheidingskolommen van

verschillende afmetingen en in diverse soorten gestabiliseerde media.

Het model heeft het mogelijk gemaakt de optimale voorwaarden te

berekenen voor de scheiding van ionen. Bovendien gaf het informatie over

faktoren, die het vervagen van scherpe scheidingsgrenzen tussen isotachofo

retisch migrerende zones tot gevolg zouden kunnen hebben. Het toonde aan

dat de aanwezigheid van ionen met een hoge mobiliteit. zoals hydroxyl ionen,

en van onderbrekingen in de pH- en spanningsgradient invloed hadden op de

vorm van de zonegrenzen.

Het bleek dat het theoretische model ook gebruikt kon worden voor de

selektie van elektroliet systemen voor de scheiding van proteinen. Het aantal

keuzemogelijkheden met juiste scheidingsvoorwaarden was echter zeer groot.

Een beperkt aantal systemen werd gebruikt voor de scheiding van haemo

globines en menselijk serum in polyacrylamide gels. De »Carrier ampholytes»,

die in de elektrofokusserende technieken gebruikt worden, werden aange

wend om de proteinen banden gescheiden van elkaar te Iaten migreren. Het

model werd ook gebruikt om voor preparatieve scheidingen de juiste

samenstelling van de buffer te vinden, die benodigd was om de proteinen van

de kolom te elueren.

135

CURRICULUM VITAE

Robert John Routs werd op 10 september 1946 geboren te Brisbane,

Australia.

Het lager onderwijs werd genoten te Roermond, waar hij nadien aan het

Bisschoppelijk College de gymnasiale opleiding volgde.

Het einddiploma gymnasium·B werd behaald in 1964.

In hetzelfde jaar ving hij zijn studie aan voor scheikundig ingenieur aan de

Technische Hogeschool te Eindhoven. Het kandidaatsexamen werd afgelegd

in mei 1967. Gedurende het akademische jaar 1967-1968 was hij gedurende

enkele dagen per week werkzaam als leraar aan het Bischoppelijk College te

Roermond.

Na het behalen van het ingenieursexamen in juni 1969, werd begonnen met

het werk, dat tot het samenstellen van dit proefschrift leidde. H ij verrichtte

zijn onderzoek in de laboratoria van LKB Produkter AB te Stockholm in

samenwerking met Prof. S. Bergstrom van het Karolinska lnstituut.

Op 1 oktober 1971 trad hij in dienst van het Koninklijke/Shell Laboratorium

te Amsterdam.

137

STELLINGEN

1. Voor zijn berekening van de albumine concentratie tij

dens de "steady state stacking" neemt Ornstein aan, dat de albumine zone dezelfde pH bezit als de termi

nerende glycine zone. Dit is onjuist.

L. Ornstein, Ann.N.Y.Acad.Sci. 111 1 321 (1964)

2. De door Konstantinov en Oshurkova uitgevcerde schei

dingen van metaalionen in capillaire buizen zijn onbetrouwbaar. Konstantinov B.P. and Oshurkova o.v., Sovjet Phys.

Tech.Phys.11,693 (1966)

3. In hun berekeningen van de pH gradient, die gevormd

wordt door een aantal isotachophoretisch migrerende zones, verwaarlozen Schumacher en Studer ten onrechte de

invloed van de mobiliteit.

Schumacher E.J. and Studer T., Helv.Chim.Acta 47,957 (1964)

4. Vergelijking van de kalorimetrisch bepaalde enthalpiewaarden met die, berekend met behulp van de vergelij

king van van het Hoff, kan informatie geven over het reaktiemechanisme van konformatieveranderingen in ma

kromolekulen. Tseng T·.Y.,Hearn R.P.,Wrathall D.P.,Sturtevant J.M.,

Biochemistry,1,2666 (1970)

5. Tegen de methode van Roos ter bepaling van de aktivi

teit van alkalis.ch fosfatase zijn bezware,n aan te voeren. Roes. K. ,Sc. ,J.Cl.Inv.j2, 233 (1963)

6. Het gelijktijdig gebruik van twee golflengten bij photo

roetrische titraties verdient meer aandacht.

7. Het door Finlayson en Crarobach beschreven "plateau" ef

fekt in de isoelektrisch fokusserende technieken kan

sterk verminderd worden door het gebruik van een hogere koncentratie "carrier ampholytes".

Finlayson G.R.,Crarobach A.,Anal.Biochem. 40,292 (1971)

8. De meeste onderzoekers, die zich bezig houden met het ontwikkelen van nieuwe instrumenten voor eigen gebruik,

zijn geneigd de waarden van hun vindingen voor anderen

te onderschatten. Zij worden teveel door hun eigen pro

blemen in beslag genomen. Tiselius A. ,Sci. Tools 41 (1968)

9. Goede faci1iteiten tot het volgen van onderwijs in de nederlandse taal zouden de kontaktrooeilijkheden van de buitenlandse werknemer in ons land aanmerkelijk ver

minderen. Een onderwijssysteem naar zweeds voorbeeld is aanbeve

lenswaardig.

R.J. Routs'

9 november 1971

Documents

Electrolyte systems in isotachophoresis and their application to … · electrolyte systems in isotachophoresis and their application to some protein separatioi\is proefschrift ter