Study of ADP binding to mitochondrial ATPase by
isotachophoresis
Citation for published version (APA):
Wielders, J. P. M. (1978). Study of ADP binding to mitochondrial ATPase by isotachophoresis. Technische Hogeschool Eindhoven. https://doi.org/10.6100/IR147397
DOI:
10.6100/IR147397
Document status and date: Published: 01/01/1978 Document Version:
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STUDY OF ADP BINDING TO MITOCHONDRIAL ATPASE
BY
ISOT ACHOPHORESIS
STUDY OF ADP BINDING TO MITOCHONDRIAL ATPASE
BY
ISOTACHOPHORESIS
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE TECHNISCHE WETENSCHAPPEN AAN DE TECHNISCHE HOGESCHOOL EINDBOVEN,OP GEZAG VAN DE RECTOR MAGNIFICUS, PROF.DR. P.VAN DER LEEDEN, VOOR EEN COMMISSIE AANGEWEZEN DOOR BET COLLEGE VAN DEKANEN IN BET OPENBAAR TE VERDEDIGEN OP
DINSDAG 21 NOVEMBER 1978TE 16.00 UUR
DOOR
JOSEPH PETER MARIE WIELDERS
GEBOREN TE MAASBRACHT
Dit proefschrift is goedgekeurd door de promotoren DP.Ir. C.A.M.G. CramePS
en
We used to tfi'tnk that 'Cf tVe knetiJ one.~ tVe knetiJ two" beaause one and one -z."s t!Jo.
we
are f'tnd'tng that !Je must 'tearn a great deat more.aliout 'and'.CONTENTS
CHAPTER 1 GENERAL INTRODUCTION 1
1.1 Oxidative phosphorylation and beef heart mitoahondrial ATPase
1.2 Maasurement of ADP binding to mi-toabondrial ATPase
Referenaes
CHAPTER 2 STEADY STATE MIXED ZONES IN
ISOTACHOPHO-1
6 9
RESIS . 12
2.1 Introduetion 12
2.2 General aonaepts in eleatrophoresis 12
2.3 Isotaahophoresis 15
2.3.1 General desaription 15
2.3.2 Same examples of
isotaahopho-retia analyses 17
2.4 A model for the transient state 20 2.5 The generation of steady state mixed
zones 25
2.5.1 Generation by the pH of the
Zeading eleatrolyte 26
2.5.2 Generation by aomplex
forma-tion equilibria 32
2.5.3 Generation by the aativity
effe at 33
2.5.4 Generation by the solvent
aomposition 34
2.5.5 Conalusion about the
genera-tion of steady state mixed zones for quantitative analy-sis
2.6 Conaentration profiles in steady state mixed zones.
Referenaes
CHAPTER 3 TRACE ANALYSIS IN COMPLEX MIXTURES BY ISOTACHOPHORESIS 3.1 Introduation 36 36 42 44 44
CHAPTER
CHAPTER
3.2 Minimum deteatable amounts and ana-Lytiaal procedures
3.3 Traae analysis and steady state mixed aones
Referenaes
4 QUANTITATIVE ANALYSIS OF ADP BY STEADY STATE MIXED ZONES
44 52 57 58 4~1 Introduetion 58 4.2 Instrumentation 58
4.3 An operational system for the
quan-tifiaation of ADP 62
4.4 The quantifiaation of ADP, accuracy
and precision 66
4.5 A aomparison ~ith the firefly
luci-ferase assay of ADP
4.6 Quantification of some other aompo-nents
Referenaes
5 PROTEIN.;...LIGAND BINDING STUDIES BY MEANS
OF ULTRAFILTRATION 5.1 Introduetion
5.2 Theoretiaal aspeats of equilibrium
74 76 80 81 81 binding studies 81
5.3 Methode for studying binding
equi-5.4 5.5 libri a 5.3.1 5.3.2 5.3.3
Direct measuring methode Indirect measuring methode The choice of partial ultra-tiltration
The ultrafiltration process A miniature ultrafiltration cell for binding studies
5.6 Disturbing phenomena in
ultrafil-86 86 87 91 91 9"5
tration binding studies 97
5.6.1 Filtration through the
pro-tein gel and the binding
5.6.2
5.6.2.~ 5.6.2.2
Ion e~alusion by electria
potantials over the mem-brane
The 1stirring effeat1
Considerations about the
origin of tbe e~clusion
99 99
effect 108
5.6.2.3 Ion e~clusion by tbe
pro-tein gel Beferences
llO ll2
CHAPTER 6 THE BINDING OF ADP TO BEEF HEART
MITO-APPENDIX
CHONDRIAL ATPASE 115
6.1 Introduetion 115
6.2 Survey of binding studies with
mi-tochondrial ATPase 115
6.3 Binding e~periments 122
6.3.1 Methods~ materials and set
6.3.2
up of the e~periment
E~periments and resuZts
6.4 Discussion and concZusions
Beferences 122 125 132 142 146
ABBREVIATIONS AND SYMBOLS 149
SUMMARY SAMENVATTING CURRICULUM VITAE DANKWOORD 150 152 155 155
CHAPTER 1
GENERAL INTRODUCTION
1.1 Oxidative phosphorylation and beef heart mitochon-dria! ATPase
All living organisme must perferm work in one way or
an-other.in order to stay alive and therefore a continuous energy input is needed. The food that is taken in by higher organisme is breken down in their digestive tract into small molecules, which can be transported to the cells. The main energy yield is obtained in the oxidation of acetyl groups, originating from carbohydrates, fats and proteins, in the Krebs or tricarboxylic acid cycle. This cycle takes place in the mitochondrion, a small
or-ganel (dimensions 1-2 J.1ID) that is situated in the cell
cytoplasm. A schematic representation of this "power plant" of aerobic cells is given Qy figure 1.1. The Krebs cycle produces carbon dioxide and hydragen atoms, which are initially bound by the coenzymes NAD+ and FAD and final-ly react with molecular oxygen after passage through the respiratory chain (or electron transfer chain). The energy that is released stepwise in the passage of hy-drogen atoms (or electrons) from a low to a high redox potentlal along this chain is conserved by the formation
of ATP. The overall reaction is given by (**)
The ATP stock is the major, instant use, energy supply of the organism, and the energy set 'free in the hydralysis of ATP to ADP and Pi is used to drive a large number of endergonic processes. The phosphorylation of ADP, coupled to the oxidation of hydragen atoms, is called oxidative phosphorylation.
The complex system of the electron transfer chain is situated in the mitochondria! inner membrane. Mitochon-dria are ruptured by mild sonication and the most inner membrane fragments reorganise inside out to form
supmi-tochondrial particles, as pictured in figure 1.1. These particles are still capable of oxidative phospho-rylation unless the knob-like projections on the membrane are torn loose by means of further sonication. The iso-lated "knobs" hydrolyse ATP {activity is about 130 pmoles per minute per mg protein) and it was shown by the group of Racker that this ATPase, called coupling factor 1(F1), in its original position attached to the membrane,is the
1 2 3
actual site of the phosporylation of ADP ~ ~ • Research
is being done on the structure and properties of the ele-ments of the ATPsynthase complex (reviewed a.o. by
Senior4 , Pederson6 and Penefsky6~ on the electron
trans-fer chain components (reviewed a.o. by Slater7 and
Skulachev8) and on the coupling of ATP synthesis to the
electron transfer process (review by Boyer et al9 ). Mitochondria! ATPase from beef-heart is a protein com-plex, about 9 nm in diameter, which probably consiste of
a dimer10 with a subunit compositiort a
2
B
2y2öxe 2 • Themo-lecular weight of the complex is calculated to be about 319 kilodaltons of protein material, while the rest of
the total weight of 347 kilodaltons11 is non-protein
ma-teria112.
During the last decades saveralhypotheses have been
proposed13~14~15
for the mechanism of oxidative phospho-rylation. Energy transduetion can be split up into three interacting events: the redox processes, the storage of energy liberated in these processes and the use of this intermediate energy in ATP synthesis. It is well accepted now that the redox processes build up a proton gradient over the mitochondria! inner membrane, the matrix space having a lower proton concentratien then the inter mem-brane space. This gradient can be regarded as a
high-energy intermediate. It has been demonstrated that an ar-tificial pH gradient is able to drive ATP synthesis by
ATPases embedded in the membrane16~17~18• On the other
hand these ATPases are able to build up a membrane
po-tential upon hydralysis of ATP19• In analogy with the
Na+K+- and ca 2+-ATPases the coupling ATPase of oxidative phosphorylation can be called a (reversible) proton trans-locating ATPase. outer membrane inner membrane submltoehondrial particles intermembrane space -+t-H--matrix spaee SONICATION SONICATION =
=
outer membrane fragmentsFigure 1.1 Scheme of the mitochondrion and the preparation
of mitochondrial ATPase fF
1).
Considering the actual ATP synthesis, we can distin-guish two hypotheses, which are nat mutually exclusive but may even be partially supplementary.
agree-ment with the model of phosphorylation over pentacoordinat-ed intermpentacoordinat-ediates. It has been discusepentacoordinat-ed by Korman and
McLick21•22 that ATP synthesis and hydrolysis and the cha-racteristic exchange reactions catalyzed by mitochondria! ATPase could be explained by a pseudorotatien mechanism with trigonal bipyramidal intermediates. Phosphate is
pro-tonated directly20 by protons passing through a proton
"well" into the catalytic site of the enzyme complex. In the trigonal bipyramidal complex, that is formed with ADP, the oxonium group would be a good leaving group and so ATP synthesis beoomes complete.
While Mitchell focuses largely on the molecular events in
the catalytic center, Slater23 and Boyer24 point to the
apparent need of protein conformational changes in ATP
synthesis. It has been shown25•26 that F1 containes
"tight-ly bound" ATP and ADP, which are not removed by mild pro-cedures like ammoniumsulfata precipitation or gel fil-tration, but are lost after dissociation of the protein in subunits. So, apparently, streng binding is related to the quaternary structure of F1 • The ratio ATP/ADP, tightly bound on the enzyme (2 ATP and 1 ADP in recent measurements
e.g.lZ) and the indications of a conformational change
du-ring electron transfer coupled ATP synthesis27• 28 • 29 led
Slater23 to the following hypothesis. Electron transfer
energy is used for changing the binding affinities of sub-strates and products via conformational changes. The major
energy input is needed for the release of tightly bound ATP and not for. the formation of it. After binding of ADP
and P1 the enzyme relaxes into a "closed" state, where
ATP is formed spontaneously and is tightly bound. Energy is needed for the transformation back to the "open" state, where ATP is bound weakly and dissociates from the enzyme. In recent formulations this hypothesis was extended to a me-chanism involving two interacting sites.
Independently,Boyer24 came toa similar mechanism as
18
an explanation of his studies on 0 exchange reactions.
The insights gained in ATP synthesis, were recently bundled in a multi-author review9 •
Figure 1.2 shows a model for ATP synthesis, which is simplified for the sake of clearness for just one site. This may be incomplete but it wil! serve as an illustration of the starting point of this thesis. Reaction I is the bin-ding of ADP and Pi by the enzyme. Reaction II is a conforma-tional change that alters the binding site or transports the
ADP ADP
'f-/E~""'
y
I ll"""*
ADPE\
v
•IEP;
ATP-\ IV*
E·ATP E-ATPFigure 1.2 SimpZified saheme
of ATP synthesis. The asterisk indiaates a different
aonfor-mat~on of the enz.yme .. aompared with the initiat aonformation.
reactants into the catalytic centre. Several authors21.o30- 32
have argued that the catalytic site should be hydrophobic or water free. Reaction III is the actual synthesis, that might follow a pathway over pentacoordinated intermediates. Reactants, intermediatea and products are stabilized by
amino acid side groups and Mg2+ bas a task in shielding
the negative charge of the ADP and/or Pi oxygens. The for-mation of an oxonium group does not obligatory need a di-rect interaction between protons from the proton well, but
may pro~eed via pronated amino acid groups. Reaction IV
again is a conformational change, which brings the products back to the aqueous phase, where the ATP dissociates in
re-action V. This scheme is equivalent to the Slater/Boyer hypothesis, extended with some suggestions about the way how the conformational changes lead to the actual
phos-phorylation. Extension of this sche~e to an alternating,
double site model or even taking into account the
speci-fic ATP and ADP binding properties of the a and
B
sub-10 .
units (Slater ) increases the complexity, but does not essentially change it.
However, it is important to recognize the simultanecue presence of several molecules of bound ATP and ADP on the enzyme complex. In the model of figure 1.2 the tightly bound nucleotidescan be involved,in the reactions II, III and IV. The Ka (see paragraph 5.2) for the tightly bound
nucle-otidesis less than 0.1 llM25, which is much lower than any
reported values for the ~of ATP hydralysis by F1 (KmATP =
0.79 - 1.25 mM4) or the Ki for its competitive inhibition
by ADP (KiADP = 30- 100 llM4 ).
Hence, in addition to tight binding also weak binding must take place, either as a first step in ATP synthesis or hydralysis (scheme 1.2 reaction I, or V backwards) preced-ing the tight bindpreced-ing, or as a separate process leavpreced-ing the tightly bound nucleotides out of the direct reaction path.
In any case, as part of the study of ATP synthesi~the
binding affinities and the number of sites for ATP and ADP (and Pi) were in need of further characterisation. Since the reaction sequence of ATP hydralysis by F1 apparently in-volves the same reaction steps as ATP hydralysis or its op-posite,ATP synthesis, by the membrane bound enzyme complex, such binding studies have been performed with the isolated enzyme in first instance.
1.2 Maasurement of ADP binding to mitochondria! ATPase The experiments described in this thesis were restricted
to the study of ADP binding. An indirect measuring method,
invalving the use of ultrafiltratien for separating the
reactants, has been selected for this purpose, as will be
discussed in chapter 5. The calculation of binding con-stante requires a rather sensitive and accurate analytica! methad for the determination of the equilibrium concen-tratien of free ligand. As a rule of thumb, estimating the Specificatiens of such analytica! method, ene should maas-ure as a minimum,equilibrium concentrations corresponding
with 20-80% saturation.33 of the enzyme in a simple one
site system. Taking a KiADP of 30 llM as an estimation of
concentra-tion of 10 ~M that the free ADP concentratien ranges from about 10 to 120 pM. Since more than one weak site is ex-pected, the analytica! method should be able to determine concentrations down to about 1 pM. In addition the
avai-labl.e sample volume is rather low: 30 40 pl (paragraph
6.3.1). In table 1.1 the methods which seem suitable for this purpose are compared.
Especially the radiometria methods are very sensitive. Liquid scintillation methode have been used in several binding studies with mitochondria! and related ATPases (survey in paragraph 6.2). However, the use of labelled ligands has the severe disadvantage that binding data are difficult to interpret because of the occurrence of ex-change reactions between nucleotides which are tightly
bound, weakly bound or present i~ the medium (see
para-graph 6.2).
Table 1.1 Comparison of analytical methods suitable for
sensitive ADP and ATP determ~nation.
Method LoWèr concentrati-on Absolute amount Usual sample Reference limit(pM) needed (pxnoles) volume{pl)
Bioluminesoenc:e 0.1-1 20 5 - 50 34, 35, 36
Liqu.id sc:intillation about 0.02 0.1 100 5 - 1000 371 38, 42 (direct or after sample preparation) Hiqh performance liquid ohromatography 1 - 5 10 l - 20 ... q. 39 Isotachophoresis 1 - 5 10 1 - 5 41 wi th steady state mixed zones
Binding studies should preferably be done with the na-tura! ligand. In addition, an analytica! method which also verifies the identity of the component being quantified, is
undoubtedly desirable • Hence the firefly luciferase as-say or high performance liquid chromatography could be
used. Isotachophoresis40, performed with "pure" zones,
cannot be used to determine mieromelar nucleotides in a centimolar buffer solution (chapter 3). Still it was con-sidered advantageous that the whole spectrum of
consti-tuents of the filtrate sample could be measured in the same analysis.
In chapter 2, 3 and 4 theoretica! and practical aspects
are described of a new isotachophoretic method~ This
im-plies the use of steady state mixed zones and gives a solution for trace analysis of specifically detectable components like nucleotides. The sensitiv·ity of this new method is comparable with that of high performance liquid chromatography for nucleotide analysis and is close to that of the luciferase assay, which measures specifically ATP
(or ADP in a coupled analysis).
Since the number of components, measured by isotacho-phoretic analysis of the filtrate (all anions), exceeds the number which can be foliowed by liquid chromatography,
isotachophoretic analysis wit~ steady state mixed zones
was selected for this binding study with mitochondria! APTase. The advantage of the quantification of all anions
in the isotachophoretic analysis is demonstratea by the discovery of disturbing effects inthe ultrafiltratien procedure (chapter 5).
From the fitting of binding roodels to the data obtain-ed from the binding experiments (chapter 6),it is con-cluded that the binding of ADP can be described bes·t by the presence of 1.2 to 1.4 binding sites per enzyme
mole-cule {Kd
= 1.2- 1.3 ].lM). However, a model with one
rela-tively strong and about 1.5 weak binding sites cannot be ruled out.
REFERENCES (Chapter 1)
1 Pullman M.E., Penefsky H.S., Datta A. and Racker E.,
J.Biol.C~em. 235 (1960) 3322.
2 Fessenden J.M. and Racker E., J.Biol.Chem. 241 (1966) 2483.
3 Racker E. and Horstman L.L., J.Biol.Chem. ~ (1967)
2547.
4 Senior A.E., Biochim.Biophys.Acta 301 (1973) 249.
5 Pedereen P.L., Bioenergetics §. (1975) 243.
6 Penefsky H.S. in The Enzymes, Vol X (Boyer P.D. et al
eds.), Acad.Press, New York (1974) 375.
7 Slater E.C. in Electron Transfer Chains and Oxidative Phosphorylation (Quagliariello E. et al eds.), North-Holland Publ.Co., AmsterdamfOxford (1975) 3.
8 Skulachev V.P •. in Dynamica of Energy-Transducing Mem-branes (Ernster L. et al eds.), Elsevier Publ.Co., Amsterdam/Oxford/New York (1974) 243.
9 Boyer P.D., Chance B., Ernater L., Mitchell P., Racker E. and Slater E.C., Ann.Rev.Biochemistry 46
(1977) 966.
10 Slater E.C., Symposium on Structure and Functions of Biomembranes, Nagoya (1977).
11 Knowles A.F. and Penefsky H.S., J.Biol.Chem.
a!Z
(1972) 6617.
12 Muller J.L.M., Rosing J. and Slater E.C., Biochim.
Biophys.Acta ~ (1977) 422.
13 Slater E.C., Nature~ (1953) 975.
14 Mitchell P., Nature 191 (1961) 144.
15 Boyer P.D. in Oxidases and Related Redox Systems (King T.E. et al eds.), Vol 2, Wiley, New York (1965) 994.
16 Jagenderf A.T. and Uribe E., Proc.Nat.Acad.Sci. USA 55 (1966) 170.
17 Racker E. and Stoeckenius
w.,
J.Biol.Chem. ~ (1974)18 Thayer
w.s.
and. Hinkle P.C., J .Biol.Chem. 250 .. (1975) 5330.19 Drachev L.A., Jasaitis A.A., Mikelsaar H., Nemeeek I.B.,
Semenov A.Y., Semenova E.G., Severina l.I. and
Skulachev V.P., J.Biol.Chem. l§! (1976) 7077.
20 Mitchell P., Biochem.Soc.Trans.! (1976) 399.
21 Korman E.F. and McLick J., Bioorg.Chem. ~ (1973) 179.
22 Young J.H., Korman E.F. and McLick J., Bioorg.Chem
1
(1974) 1.
23 Slater E.C., ibidem as reference 8, 1.
24 Boyer P.D., ibidem as reference 8, 289.
25 Harris D.A., Rosing J., van de Stadt R.J. and Slater E.c.,
Biochim.Biophys.Acta 314 (1973) 149.
26 Rosing J., Harris D.A., Kemp A. Jr. and Slater E.C.,
Biochim.Biophys.Acta 376 r1975) 13.
27 van de Stadt R.J., de Boer B.L. and van Dam K.,
Bio-chim.Biophys.Acta 292 (1973) 338.
28 Bertina R.M., Schrier P.I. and Slater E.c., Biochim.
Biophys.Acta 305 (1973) 503.
29. Chang T. and Penefsky H.S., J .Biol.Chem. ill_ (1974)
1090.
30. Williams R.J.P., ibidem as reference 7, 417.
31. Banks B.E.C. and Vernon C.A., J,Theor.Biol. 29 (1970)
301.
32. McGuinness E.T. and Zuleski F.R., Bioinorg.Chem. 5
(1975) 73.
33 Deranleau D.A., J.Am.Chem.Soc.
2!
(1969) 4044.34 Strehler B.L. in Methods of Enzymatic Analysis
(Bergmeyer H.U. ed.), Acad.Press, New York (1974)
2112.
35 Kimmich G.A., Randles J. and Brand J.S., Anal.Biochem •
.22.
(1975) 187.36 Stanley P.E. and Williams S.G., Anal.Biochem. 29
(1969) 381.
37 Cooney D.A., Jayaram H.N., Homan E.R. and Motley C.F.,
38 Cheung e.P. and Marcus A., Anal.Biochem. 69 (1975) 131.
39 Andersen F.S. and Murphy R.c., J.Chromatogr. ~
(1976) 251.
40 Everaerts F.M., Beekers J.L. and Verheggen Th.P.E.M.,
Isotachophoresis; Theory, Instrumentation and Appli-cations, J.Chromatogr.Library Vol VI, Elsevier Publ.
Co., Amsterdam/New York (1976).
41 Wielders J.P.M. and Everaerts F.M. in Electrofocusing
and Isotachophoresis (Radola B.J. and Graesslin D. eds.), Walter de Gruyter, Berlin (1977) 527.
42 Dyer A., An Introduetion to Liquid Scintillation
CHAPTER 2
STEADY STATE MIXED ZONES IN ISOTACHOPHORESIS
2.1 Introduetion
Isotachophoresis is a recently developed analytica! technique, halonging to the group of electrophoretical methods. This chapter wil! briefly describe its principles and will further deal with theoretica! aspects of steady state mixed zones in isotachophoretic analyses.
2.2 General concepts in electrophoresis
Electrophoresis is the movement of a charged particle, e.g. an ion in an electrolyte solution, under the influence of an external electric field. The relation between the
electrophoretic velocity v (cm.s-1) and the field strength E
(V .cm -1 ) is represented by
V = m. E (2 .1)
where m is called the effective mobility, that is the ave-rage velocity per unit field strength of a certain component
under the loc al condi tions • The dimens i ons of m are cm 2 • v -1 •. s -1
and generally its value lies between 10-3and 10-4~ For
anions the m is defined to be negative.
The specific conductance of an electrolyte solution (K,
w-~cm-1) is given by
(2.2) where F is the Faraday and ei and zi are respectively the concentratien (mol.cm-1 and the valency of ionic species i. As a raferenee value for the effective mobility is adopted
the mobility at infinite dilution (m
0) where no mutual
in-fluence of the ions exists. This m
0 is,only determinedby the
solvent and the temperature. On the assumption that Stokes' law can be applied to the migration of small ions in a
(2. 3)
6 'Jf. r •
n
This formula expresses the relationship between the m0 and the charge, the solvated ion radius (r) and the absolute
viscosity (n) of the solvent, but it must be considered
only as a rough description of the m0•
However, the movement of a particular ion is not
inde-pendent of th~ presence of other ions. Due to the cloud of
opposite charged ions around a particular ion, we have to take into account a relaxation effect and an
electrophore-tic effect on the central ion's mobility. An expression
for this is given by the well-known Debye-HÜckel-Onsager equation, dealt with in detail in raferences 1 and 2.
-3. -2 -1
For concentrations higher than about 10 to 10 greq .1 ,
where ion pair formation becomes more and more important, an activity coefficient has to be used to relate the theo-retica! data to the experimental effective mobilities. Up till here the ion is considered as an inert charged par-ticle, but in practice it may be a reactant in a chemica!
(or even physical) process. The most commonlcase for such a chemica! process is that of protolysis for weak
electro-lytes. Tiselius 3 wrote for this case for the effective
mo-bility, neglecting all other factors:
(2.4)
wherein ai is the dissociation degree (ci/ctotal) for sub-species i of a certain component. Of· course other equilibria, e.g. complex formation equilibria, will also partly dater-mine the effective mobility.
Concluding, the effective mobility of a component is very complex and it would be extremely dif.ficult to derive a formula which correctly describes all contributing factors to the effective mobility. For the practice of
electropho-retic analysis the following expression for the effective mobility is useful:
m =
L
a i • B. • y i • f i • m0i l. i
(2. 5)
wherein ai represents the effect of equilibria other than protolysis, yi is a correction for the relaxation and electrophoretic effect and fi beoomes important in elec-trophoresis in supporting media and corrects for e.g. friction or pore tortuosity. It is obvious that the tempa-rature influences most of the constituent parameters
(in-cluding the m0) of equation 2.5 and will be very important
for the effective mobility.
For the achievement of the electrophoretic separation of sample components, it will often be necessary to in-fluence their mobilities. The pH of the separation medium is the operational parameter, which is most frequently used for this aim in the case of weak electrolytes. In isotacho-phoresis, analyses are usually performed between pH 3 and 10, see paragraph 2.3.2. Complex formation equilibria have also been used 4 ,S as wel! as the electrolyte
concentrat-ion 2 in order to achieve an electrophoretic separation.
But for practical reasens isotachophoretic analyses have
-2 -3 -1
to be performed at a 10 - 10 greg.l ·concentration level
(paragraph 2.3.2) which greatly limits the use of the acti-vity factor in equation 2.5 for the effectuation of a shift in the effective mobility.
Last but not least are the possibilities effered by the solvent (or solvent mixtures) for influencing the effective mobilitiesi a change in the dipole moment and the dielee-trio constant will change the ion's solvation and also a change in viscosity will reflect in the m
0l. . (equation 2.3),
in addition the ai, the ai and the yi will be changed upon a change in solvent composition.
2.3 Isc>tachophoresis 2.3.1 General description
Isot.achophoresis is an electrophoretic method by which ionic components are mainly separated on the basis of dif-ferent effective mobilities. These components beoome ordered in general according to decreasing effective mobilities into consecutive zones with sharp boundaries.
The principles are illustrated by figure 2.1. Three dif-ferent electrolytes are used in discrete compartments. For anion analyses the leading electrolyte contains one
anion (L) with a higher effective mobility than any sample
anion, whereas the terminating electrolyte contains one anion (T) with a lower effective mobility than any sample
anion. The common cation (Q) of the leading and
termina-ting electrolytes is selected on.account of its buffering capaoity around the pH ohosen for the analysis and this
counter ion Q should preferably have a low effeotive mobi··
lity.
Befere the isotaohophoretic analysis is started, the se-paration oompartment (usually a teflon capillary) and the anode oompartment are filled with the leading eleotrolyte. The oathode oompartment is filled with the terminating eleotrolyte. The sample, whioh is oonsidered as the third electrolyte, is introduced in between the leading and ter-minating electrolytes. This arrangement is pictured in fi-gure 2.1a, the sample containing the anions A and B. When an electrio current is passing through this system, all ionic components will migrate according to equation 2.1. In this
example mA is greater than ~· hénce A will gradually
mi-grate ahead of the mixed zone AB while B will stay behind (figure 2.1b and 2.1c). The separation phase, when the zone boundaries are moving at different velocities, is called the transient state. The diminishing mixed zone AB is cal-led a transient state mixed zone. In the steady state (fi-gure 2.ld) all zone boundaries have the same velocity (the
a
b
c
Ld
ae
®f
- L L 0 a -L a A a L a A B Q I A* B*e
a• fH Q**I : I
~ ~1:.[
e
I ' ~~ VL,A1 VA.AB r••e
B T T* T**e
Q Q Q• a** a••I
~
A*I
r• e•a• o•
T**e
T T* T**e
a a• o••Figure 2.1 Saheme of the sepaPation pPoaess foP anion
analysis in isotaahophoresis. In figuPes a up to and inclu-ding d the sample anions A and B ape being sepaPated due to a suffiaient diffePence in theiP effeative mobilities. In figuPe e and f pheiP effective mobilities aPe equal~ no sepaPation takes place and a steady state mixed zone is obtained. FuPtheP explanation in the text.
Whenever two components have the same effective mobility, no resolution takes place in general and a steady state mixed zone will be obtained. This is pictured in figures
2.le and 2.lf. According to equation 2.1 an equal migration of all components in their respective zones (L,A,B and T)
can only be achieved by a stepwise increase of the field strength in these zones, in the direction fram leading electrolyte towards terminating electrolyte, to compensate for the decreasing effective mobilities. As a result
thereof, the effect of diffusion is effectively counter-acted and very sharp transitions between the zones are ob-tained. The same principles are applicable for cation ana-lysis.
The conditions of the sample component zone in the steady
state {e.g. the constituent~ concentration) are strictly
regulated and are determined by the leading electrolyte's nature and concentration and by the properties of the con-stituents within that particular zone (see paragraph 2.4).
The basic arrangement of electrolytes, needed for the
performance of iso:tachophoretic ~nalyses (figure 2.1a),
can be extended for special purposes. An example is given by raferenee 6.
2.3.2 Same examples of isotachophoretic analyses Isotachophoretic analyses are usually performed at electrolyte concentrations of 10-2 to 10-3
gramequiva-lente per liter. The use of a lower concentration is
in favour of decreasing the minimal detectable amount but is limited by the increasing resistance of the electroly-te and the stability of the constant current power supply at the very low driving currents, needed under. this
con-dition. The pH at which the analysis can be done is
usuallyin between pH 3 and pH 10 for 10-2 greq. 1-1 • This range is also concentration _dependent and decreases at lower concentrations since the mobilities of H+ and OH- differ an order of magnitude fram those of all other ions. At the extremities- of this pH range the current will be more and more carried by the H+ or OH- ions and finally the isotachophoretic separation will be disturbed. The upper limit of the useful pH scale is also partly set by the presence of carbon dioxide in the solvent (usually
water). Details about the selection of an operational sys-tem for isotachophoretic analysis are presented by
2
Everaerts et al • A current stabilised power supply is
used since a constant velocity of the zones along a detec-tor simplifies the quantitative analysis.
As an illustration of the method and in order to demon-strate how analytica! data are obtained, figure 2.2 shows some isotachopherograms of a mixture before and after en-zymatic converslons have taken place. In figure 2.2a only hexokinase (HK) was added while in figure 2.2b hexokinase as well as glucose-6-phosphate dehydrogenase (G6P-DH) was
used 7 •
Since the concentration of a sample component in its own zone is regulated by theKohlrauschfunction (paragraph 2.4) the length of its zone is linearly proportional to the amount that has been injected. At constant injection volume the length of a zone in the isotachopherogram reflects the concentratien in the sample. In figure 2.2a the length of the ATP zone after the c.:onversion (L2) is only half the lengthof that before the conversion (L1). This is in
agree-ment with the full conversion of the 3 mM glucose at 6 mM
ATP in the original mixture. Sample concentrations are ge-nerally obtained from a calibration graph.
Figure 2.2 (opposite page) Isotaahopherograms of a
mix-ture before (Zeft side} and after (right sidel the indiaa-ted enzymatia aonversions have taken pLaae. Phe mixture aontained 3 mM gtuaose, 6 mM NADP, 12 mM Mgct 2, buffered
with Tris at pH B.O. Leading eZeatroZyte 5.10-3 M CZ-/6-aminoaaproia aaid (pH 4.01, terminating eZeatroZyte 5.10-3 M gtutamate. Phe anions in the zones are indiaated by their respeative resistanae Zever. R
=
resistanae, T=
time, A=
absorption at 254 nm (100% absorption indiaated).@
-Lt I l Olut- G6P- N ADP- NADPH- 6PO- ATP-_100% HK ATP + G - G-6-P + ADP. Mg2+ Glut- 06P- N ADP- NADPH- 6PG-_fOO% + HK M Z+
ATP + 0 + NADP g ADP + 6-PG + NADPH + H+
Qualitative information about the constituent of a
par-ticular zone can be obtained from the recording
Of
thesignal of a universa! detector (e.g. resistance measuring) or/and from the use of a specific dateetion like uv ab-sorption measurements. The isotachopherograms of figure 2.2 show the recording of the resistance R, which increases stepwise from the leading electrolyte zone towards the zone of the terminating electrolyte (glutamate). To facilitate the determination of the zone lengths, the first deriva-tive of the reaiatanee signal has also been recorded. The lower part of the isotachopherogram is the recording of the UV absorption at 254 nm of the zones, the rectan-gular profiles clearly demonstrata the constancy of the concentrations in the zones.
The (reduced) step height HNÁDP' that is the difference in the resistance of the NADP zone and the leading elec-trolyte zone, is characteristic for NADP under the condi-tions of this particular operational system. To correct for variations in the instrument settings, relativa step heights( e.g. HNADP/Hreference> are used for identifica-tion purposes. The overall conversion of the figure 2.2b reaction can be fellewed directly by maasurement of the absorbance at 340 nm. It is possible to fellow all the individual ionic reactantsof conversiena a andb in one assay by the use of isotachophoresis. The total time of analysis for such experiments is about 15 minutes and the reproducibility is better than 2% for both quantitative and qualitative aspects (stepheightreproducibility). 2.4 A model for the transient state
In this paragraph the processas that occur during an isotachophoretic analysis will be considered in more de-tail. Extensive treatises of the relations between the concentrations,charges and mobilities of the sample ions in their respective zones in the steady state, have been given for streng and weak electrolytese.g. 2, 8 • The theory
9
electrophore-sis" is also applicable to the transient and steady stat~ of isotachophoretic analysis.
The formation of the steady state configuration of "pure" zones and mixed zones wil! be presented in a re-latively simple model. It will be shown that this model is suitable to explain the use of steady state mixed zones in isotachophoretic analysis.
In this model the following assumptions are made:
- All constituents are completely dissociated, but these
ions can be multivalent~
- The mobilities are not influenced by e.g. temperature
and activity effects~
- Conveetien and diffusion are negligible.
Considering the. effect on the electrolyte composition by the passage of an electrio current through it, i t will be
clear that the input of e~ectric charge in a small volume
element of the electrolyte must be equal to the output (at constant current density), independent of the kind of ion that is responsabie for this charge transport. From this
starting point the following expression was derived by
Kohlrausch10
constant (2.6)
: -3
where ·Cn is the concentratien (greq.cm ) of component n. Hence, the concentrations in any part of the system are regulated by the electrolyte, whi.ch was originally present in that part. In figure 2.la three different electrolytes can bedistinguished, consequently three different
regulat-ing functions exist(w1 , w8, wt) for respectively the
lea-ding, sample and terminating electrolyte compartment. Application of the principle of electroneutrality puts equation 2.6 in the form
1:
constant (2. 7)where concentrations and mobilities of anions are given a negative sign. The use of the relative mobility ri
leads to
. r. - r
0
w = LJ.. ei (rJ. r ) =
i . Q
LC·R·
i J. J.Dole11 extended the Kohlrausch theory and derived
(2. 8)
(2. 9)
(2 .10)
The V a of this equation is the displacement of the
boun-a,...,
dary between two electrolyte phases a and B, expressed in cm3• Faraday -l ;Fora stationary boundary, like I and II of figure 2.1, he deduced for a component present on both sides of that boundary
(2 .11)
and for a moving boundary (V . a,., a # O)
(2 .12) We can now write down the relations between the concentra-tions in any of the zones of figure 2.1 from the equaconcentra-tions
2.9, 2.11 and 2.12. In the following the notation CA ,m
indicates the concentration of A in the mixed zone, while
*
or**
indicates that the sample, respectively thetermi-nating electrolyte oompartment is conoerned. For the transient state mixed zone holds
Replacing the ratio of the components B and A in the sample by
it will be clear from equation 2.11 that
Combination of equation 2.15 and 2.13 leads to
c
A,m =Equation 2.11 can be rewritten for component A at boundary I
(2.14)
(2.15)
( 2 .16)
(2.17)
This equation represents the factor by which A as well as
B is concentrated or diluted upon the passage of boundary I,
either in a transiènt state mixed zone (figure 2.1b) or in a steády state mixed zone (figure 2.le). For the case of N
anionic components in the sample, of which A and B will
farm a steady state mixed zone {in formulas abbreviated to SSMZ) the concentratien of A in the steady state mixed zone will be given by CA,SSMZ c RA + PBA~ + + PNA~ = A,m RA + PBA~ N CL~
1:
Ri piA*
i=A=
cA,m· ws.
RA+ PBA~ {2 .18)A in the complete sample, adapted to the leading electro-lyte conditions.
Once the concentrations in the zones are known, the veloeities of the moving boundaries can be calculated and from this the time is obtained that is needed for
reaching the steady state (t5) .
The general form of the moving boundary equation can
12
be written as
(2.19) For the velocity of a separation boundary, where a com-ponent is present on just one side, equation 2.19 is sim-plified. For vA,AB' the velocity (cm.s -l)·of the boundary of the pure zone A and the mixed zone of A and B(figure 2.lb), is obtained
(2.20) and for the velocity vL,A
( 2. 21) In the example of figure 2.la-d the steady state will be reached as soon as zone A has its maximal length. Hence for the resolution time tr can be written
(2.22)
wherein qA is the amount of A in the sample(greq) and
e(cm2) is the area of a cross-section of the separation
compartment. Insartion of equation 2.2 and E
=
I/K,I being the current density (A.cm- 2), in equation 2.20 results in
Ill:BI
(2.23)
After a similar transcription of equation 2.21 and insert-ing this tagether withequations 2.8, 2.16 and 2.23 in 2.22 we obtain
rA- rQ + PBA(rB- rQ) rA - rB
( 2. 24)
where J means current(A). A similar treatment for the
re-. 13
salution time is presented by M~kkers et al • This
equa-tion shows that tr wil! beoome irifinite for rA
=
rB andno resolution wil! be achieved. However, for this case a steady state will be reached as soon as the sample has
ad-justed its concentratien to the w1 • In figure 2.lf the
steady state mixed zone of A and B has a fixed length and migrates at the isotachophoretic velocity. The time
need-ed for the achievement of this steady state (tst> is
giv-en by
(qA + qB) F (rA - rQ)
J rA
Whenever rB is negligible , compared with rA and r
0,
equation 2.24 reduces to
=
qA FJ r·
·A
2.5. The generation of steady state mixed zones
(2~25)
(2.26)
Up til! this study isotachophoresis has mainly been dis-cussed in terms of separating ionic species for analytica!
e.g.2 i e.g.14
or preparat ve purposes. However, werking in
com-ponents, can be very benificial. It wiÎl be explained in detail in chapter 3, that steady state mixed zones offer a valuable tool for trace analysis in isotachophoresis, if the trace component can be specifically detected.
Therefore attention has to be paid to the selection ofan electrolyte system that does nat induce different effec-tive rnahilities but works the opposite way. Isotachopho-retic analysis with steady state mixed zones needs the careful matching of the effective rnahilities of two com-ponents; the carrier component, which carries the major part of the electric current in the mixed zone, and the trace component which has to be quantified.
sa, the factors whichcontribute to the effective mobility (equation 2.5) have to be considered once again.
2.5.1 Generation by the pH of the leading electrolyte Searching for equal rnahilities as a function of the pH of the system is the opposite of what one has to do for selecting a system, in which the components are "separated on pKa. values". Inthefarmer case,the intersections of the m versus pH plots are interesting. These plots (fi-gure 2.3) show sections where the m is nat ar slightly influenced by smal! pH fluctuations, while near the pKa values a big shift will occur. If the conditlans within a zone were nat partially determined by the properties of that particular sample component (pKa values and mobi-lities), then every intersectien of two m versus pH plots would lead to a steady state mixed zone for this pair of.components.
It will be demonstrated that the carrier component should determine the pH of the steady state mixed zone
and that the trace component should nat possess a pK a
value within 1-1.5 pH unit of the pH of the steady state mixed zone. The best conditlans arise whenever the m versus pH plots of the couple overlap over a wide pH ran-ge, especially when bath components migrate at a pH, which differs more than about 1.5 pH unit from the com-ponents' pKa values.
Calculations concerning the stability of a steady state mixed zone, generated by the pH of the leading
electroly-t~, have to show whether there will be a difference
be-tween the effective mobilities of the pair of components under the conditions predicted by the regulating function
and the concentr~tion ratio of the components. If we
con-sider the case of ratios carrier/trace larger than about 20, which is often found when the determination of traces:· by steady state mixed zone analysis is useful,then we can
-
salicylale®
pH 0 5 9 11 13 '10 Meff 50 30®
10 0 11Figure 2.3 Effeative mobiZity aa function of the pH of
the aoZution aaZcuZated from Ziterature data of phoapha-te,. acetate and saZieyZate (figure a} and of ATP,. ADP,. pimelate and aeetate (figure b}. The aurvea for ATP and ADP are estimated as described in the te~t.
obtain valuable information from the oonditions oaloulat-ed for the "pure" zones of the steady state mixoaloulat-ed zone oomponents in the same operational system.
This modifioation of isotaohophoretio analysis was initiated by the need for the determination of traces ADP
(and ATP). However, no accurate data of the mobilities and pKa values of these nuoleotides are available (see also page 31). Therefore the possibility of the genera-tion of steady state mixed zones by the pH of the leading eleotrolyte was studied by oaloulations on two model
sys-tems: phosphate/aoetate and phosphate/salioylate1
s.
Figure 2.3a shows that the theoretica! plots of the ef-feotive mobility versus the pH of the salution for phos-phate and aoetate, have two interseotions: at pH 5.36
and at pH 6.93. Henoe in a salution at one.of these pH
values the effeotive mobilities of aoetate and phosphate will be equal. Using the same pKa and mobility data as in figure 2.3 (data were obtained from the literature16,l7,lB)
2 8
and the computer program X3 developed by Beokers 1
, the
plots shown in figure 2.4 were obtained. It is olear from figure 2.4a and b, that at the pH of the leading
eleotro-lyte (= 5.14) where the effeotive mobilities are fully
equal for aoetate and phosphate, being in their respeo-tive zones, the pH of their respeorespeo-tive zones is different. The pH of a fiotitious mixed zone in this operational
sys-tem is partially determined by the concentratien ratio
phosphate/aoetate. At a lew ratio,the pHmixed zone ap-proaches the pHacetate• Although this value is higher than
the pHphosphate' there will be hardly any difference in
the phosphate effective mobility in the fictitious mixed zone and that in a "pure" phosphate zone since the m ver-sus pH plot for phosphate is rather flat in this region. This implies that a mixed zone of low phosphate/acetate ratio will be stable at this pH of the leading electro-lyte and thus a steady state mixed zone can be expected. The reverse, a high ratio phosphate/aoetate causes a decrease of the acetate effective mobility of about 5%
42 35 40 33
@
38 31 5.0 5.2 5.4 6.5 6.7 6.9 7.1/.
PHzone ..-;;Y
/
.~
5.5 5.3 6.9 6.6 5.0 5.2 5.4 6.5 6.7 6.9Figure 2.4 (a+b) Calaulated plots for the effeative
mo-bility in the zone and the pH of the zone as funation of the pH of the leading eleatrolyte ( 10-2 M Cl-/area-tinine) for phosphate ( .& ) and aaetate ( • ) .
Figure 2.4 (c+d) Calaulated plots for the effeative
mo-bility in the zone and the pH of the zone as funation of the pH of the leading eleatrolyte (10-2 M Cl-/histidi-nel for phosphate ( .& ) and aaetate ( e).
for the acetate in the ·fictitious mixed zone as eeropar-ed with the acetate in a "pure" zone. Acetate will accu-roulate at the rear of the mixed zone and gradually leave this zone. No steady state mixed zone will be possible.
A similar discussion can be had for the ether inter-sectien of the effective mobility versus pH curves of acetate and phosphate (at pH 6.93). From the plots
this case only a phosphate-rich mixed zone can exist as a steady state mixed zone.
Figure 2.5 shows the plots of the effective mobility versus the pH of the leading electrolyte and the pHzone versus the pH of the leading electrolyte for phosphate
30
3.7 4.1 4.5 4.9 5.3 5.7
6.1
3.7
3.7 4.1 4.9 5.7
Figure 2.5 Calaulatea plots for the effeative mobility
in the zone and the pH of the zone as function of the pH of the leading eleatrolyte (10- 2 M Cl-/8-aminocaproia acid) for phosphate (A) and saUcylate ( e).
and salicylate. For these two components the resulting pHposphate and pHsalicylate are equal over a large pH of the leading electrolyte region, while the mobilities are almost equal. This implies that over a large pH range of the leading electrolyte a steady state mixed zone will be possible for both high and low ratios
phos-2
phate/salicylate. Experiments showed that already when
the pH of the leading electrolyte is ca. 4.2 a steady st~te
in-accuracies in the data used for the calculations and to temperature and activity effects, for which no correction has been made in these calculations.
so, i t will be clear that a mixed zone is a steady sta- · te mixed zone under one of the followinq conditions at a pH of the leadinq electrolyte where the components mi-qrate at equal mobilities in their "pure" zones.
Tzye I: Neither comJ?onent has a pKa within about 1.5 pH
unitsof,the pH of the mixed zone. This is the most stable steady state mixed zone under all concentratien ratios and is very suitable for quantitative analysis by means of steady state mixed zones.
For example: phosphate/salicylate at a pH of the leadinq electrolyte 4 to 6 and several systems presented in chapter 4.
Type II: One of the components of a COUJ?le A and B (e.q.A) has a pKa value near the pHmixed zone· A steady state mix-ed zone can be formmix-ed only at a high ratio A/B and this type is much less suitable for quantification purposes. Examples are presented by the acetatefphosphate systems.
TyPe III: Both components have a pKa near the pHmixed zone" Except wh:en the ·PKa and mo .va lues are identical, the pH zone versus the pH of the leadinq electrolyte plots do not over-lap. The possibilities for a steady state mixed zone are dependent of the concentratien ratio in a more complex way then for the type II steady state mixed zone. An ex-ample of this rare type is presented by the ATP/phosphate system in fiqure 2.8.
For the determination of traces ADP by means of a type I steady state mixed zone,we must have reliable pKa data. Analyses in an operational system at pH 7.8 or higher are
suqqested by the values qiven by Perrin16, pKal and
pKa2 < 2, pKa3
=
3.95 and pK 4=
6.26 at 25°C in 0.15 M NaCl.a 16
A pKa3
=
4.2 has also been reported • Besides thedepen-denee of the pKa upon the ionic strength, also the
presen-. + + 2+ 2+
strongly influence the pKa values for adenine nucleotides
19, 20, 21• In the absence of these kinds of cations and by
the use of the formula presented by Phillips et a119, a
pKa4 of 6.98-7.00 was calculated at 25°C for an ionic strength 0.8 to 1.0 x 10-2 , which is normal in an isotacho-phoretic analysis at a 10-2 N leading ion concentration. Tris is an inert cation as fas as complexation with adenine
nucleotides is concerned22 and can be used in an
operatio-nal system for isotachophoresis in the pH region 7.6-8.6 (pKa of Trisis 8.12).
A pKa3 of 4.0-4.2 and a pKa4 of 7.0 for ADP point to the pH region near 5.6 or to a pH equal to or above 8.5 where a steady state mixed zone with ADP could be formed. This is illustrated by figure 2.3b. To construct this
fi-3- 2- 4-
3-gure the mobilities of ADP 1 ADP 1 ATP and ATP were
estimated by using literature data on components that could farm a steady state mixed zone with these nucleotides at a certain pH. It is clear that the best conditions for a steady state mixed zone,containing ADP or ATP,can be ex-pected at the high pH region. Experiments at the narrow pH range near 5.6 for ADP containing mixed zones have been performed (see paragraph 2.5.3) but the results were not satisfactorily for the determination of traces of ADP.
Accepting that the generation of a steady state mixed zone-should take place at a pH that is minimally one pH unit apart from the pKa values of the components leaves,open the question what parameter would be most suitable for the final balancing of a latent couple that could farm such a mixed zone.
2.5.2 Generation by complex formation equilibria
In principle the effective mobility of either the car-rier or the trace component could be adjusted to that of
the trace or the carrier,respectively1 by the use of
com-plex formation reactions, taking place during the migra-tion process. The counter ion, or an addimigra-tional ion in the leading electrolyte with the same sign as the counter ion, could be the complexing agent. However, in order to obtain
a desired adjustment, the concentratien of this complex-ing agent in the steady state mixed zone must have a fix-ed ratio to that of the other reactant in the complex for-mation reaction. Hence, this approach to the generation of such mixed zone for analytica! purpose is not suitable when different ratios of carrier and trace component are expected in the samples to be analysed. Each ratio car-rierjtrace would need a different complexing agent con-centration in the operational system.
2.5.3 Generation by the activity effect
It can be estimated by means of the Debye-Hückel-On-Onsager equation that the effective mobility of a
diva--5 2 -1 -1
lent anion (m0 = -50.10 cm V s ) decreases from
-47.6 to -39.5 as aresult of an increase in concentra-tien from 5.10- 4 to 10-2 M in thè presence of a
mono-valent counter ion (m0 = 20.10- 5). Fora monovalent anion
with the same m0 the effective mobility would decrease
from -49.0 to -45.7. It is clear that matching effective mobilities by.optimalisation of the concentratien of the operational system could be useful if the latent mixed zone components, possessing nearly equal effective mobi-lities, have different valencies.
For example it was experimentally found that ADP had a slightly higher effective mobility than
2-hydroxybuty--5
ric acid (m0
= -35.10
and pKa=
3.76) in an operationalsystem with leading electrolyte 5 mM Cl-/histidine (pH 5.7)1
whereas with leading electrolyte 20 mM Cl-/histidine
(pH 5.7) ADP migrated as a separate zone behind the 2-hy-droxybutyric acid zone. Optimalis.ation of this system led
to a steady state mixed zone at 10.5 mM Cl- leading ion
concentratien (figure 2.6).
However, this particular system was judged to be un-suitable for the determination of ADP in trace analysis since the distribution of ADP over the mixed zone varies largely as function of the ratio carrier/trace. This is a reflection of the influence,at the pH of the zone,of the pKa values of ADP on i ts effect! ve mobili ty.
The opposite, i.e. using the activity effect to achieve a difference in effective mobilities, has been
demonstrat-ed by Everaerts et al2 in the separation of chloride and
4-Fe (CN6) , \ .---rO% A I Figure 2.6 Isotaahopherogram of a
steady state mixed zone of ADP and 2-hydroxybutyrate. The operational system ~as:Zeading eZeatroZyte 10.5 mM CZ-/histidine (pH 5.70)~ termina-ting eZeatrolyte ± 10 mM glutamate~
further as in table 4.1. The sample aontained 2 nanomates ADP and 20 na-nomotes· 2_-hydroxybutyrate.
R resistanae~ T =time~ A =·ab-sorption at 254 nm~ 1 = chloride, 2 = ADP, 3 2-hydroxybutyrate,
50% 4 = gZutamate.
2.5.4 Generation by the solvent composition
Conductances in solvent mixtures have since long been measured because they give an insight in the properties of solvent mixtures and reflect solute - solvent inter-actions. In isotachophoresis only recently composed solvents have been used to influence electrophoretic
mobilities15•23 although the use of solvents other than
water had proven for some time to be quite useful for particular problems. The change in a constituent's effec-tive mobility upon the addition of other solvents to an aqueous electrolyte is very complex and cannot be explain-ed as a result of a simple change in viscosity as prexplain-edict- predict-ed by equation 2.3 ar by the so-callpredict-ed Walden's rule that is derived from this equation. The influence of the ion
on the structure of the solvent in its vicinity is dis-cussed by a.o. Kay and Evans 24 for aqueous solutions and
by Kay25 for aqueous mixtures of water and alcohols. The
change in structure and properties of aqueous solutions after the addition of a small volume percentage (lessthan
10%) ·Of alcohols such as me.thanol . , ethanol and propanol
is elaborately dealt with in the raferences 26, 27 and 28. A decrease in the effective mobilities after the addition of a small volume percentage of alcohol to an aqueous electrolyte is expected from bath the decrease in the dielectric constant and the increase in the structure
of~the solvent26~21cawarent
increasing viscosity). Theo-retica! values for the constantsin the Debye-JIÜckel-· · Onsager formula and for the acitivity coefficient constant
are presented by Shedlovsky29 fo~ a number of water/
methanol ratios.
Finally the influence of the solvent additives on the pKa values of.the ionic solutes needs to be considered. For example the pKa of acetic acid in water/methanol and
water/ethanol has been studled by several authorsa•0•29 - 32
In general it seems that the change in the pKa values can be neglected at very low percentages alcohol, e.g. less than 5 vol% methanol. Shedlovsky and Kay30 found an
in-crease of 0.13 pH units for the pKa of acetic acid (25°c) in the presence of 10 vol% methanol. Bates et a1 33 re-port a shift in the pKa of Tris from 8.12 (100% water) to 8.09 (8.1 wt% methanol) at 25°C. In trace analysis with steady state mixed zones such small changes in the pKa value of the mixed zone couple will be not important since the pH of the zone is minimally 1-l.S pH units apart from these pKa values.
Although the effect.of solvent additives on the effec-tive mobilities is hard to describe exactly, in practice it proved to be the most suitable tool for the final ba-lancing of the mobilities of a latent couple for a steady state mixed zone.
