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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
Document status and date:Published: 01/01/1971
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ELECTROLYTE SYSTEMS IN ISOTACHOPHORESIS
AND THEIR
APPLICATION TO SOME PROTEIN SEPARATIONS
R.J. ROUTS
ELECTROLYTE SYSTEMS IN ISOTACHOPHORESIS
AND THEIR
APPLICATION TO SOME PROTEIN SEPARATIONS
R.J. ROUTS
ELECTROLYTE SYSTEMS IN ISOTACHOPHORESIS
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.
The conditions are as in fig. 6.2.
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
The conditions are as in fig. 6.2.
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.
The conditions are as in fig. 6.2.
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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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
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