Focusing Review

Chromatography, Vol.26 No.1 (2005)

Focusing Review

Analysis of Chemical Equilibria in Aqueous Solution Related with Separation
Development Using Capillary Zone Electrophoresis
Toshio Takayanagi
The Graduate School of Natural Science and Technology, Okayama University 3−1−1 Tsushimanaka, Okayama 700−8530
Received for review October 30, 2004. Accepted January 18, 2005.

Abstract
Capillary zone electrophoresis has been utilized for the analysis of chemical equilibria in aqueous solution. Electrophoretic mobility of analyte changes along with the addition of interacting reagent in the migrating solution, and precise measurement of the electrophoretic mobility
allows analyzing equilibrium reactions. The capillary electrophoretic method including mathematical analyses was validated through complex
formation reactions, and it was applied to the investigation of ion association reactions. Various types of interactions have been clarified; contributions of hydrophobicity, probability, and aromatic−aromatic interaction were quantitatively evaluated. Ion association reaction in aqueous
solution also elucidated the stepwise reactions of liquid−liquid distribution of ion associates. The technique was further developed to the binding analysis of ions and ion associates to nonionic surfactant micelles.
Keywords: chemical equilibria, intermolecular interaction, capillary zone electrophoresis, electrophoretic mobility, ion association reaction,binding reaction

sure, diffusion coefficients, acid−base, complex−forming, critical

1．Introduction
Capillary zone electrophoresis (CZE) has become a popular

micelle concentration, and binding constants. Capillary zone elec-

technique for the analysis of ionic substances. The separation

trophoresis has its predominant characteristics as mentioned above,mechanism is based on the differences in electrophoretic mobility

and the advantages utilized have been introduced for the equilib-of analyte ions, as well as on the electroosmotic flow. Although therium analysis of acid−base, metal complex, and binding affinity ofseparation efficiency in CZE is amazingly high, various types of

biomolecules [2]. In the CZE analysis of the chemical equilibria,

interacting reagents are often used to control the electrophoretic

the general procedure includes that the electrophoretic mobility ofmobility of analytes, when the resolution among analytes is notanalyte is measured with a series of migrating solutions containing

sufficient. The interacting reagents used are so−called ”modifiers,”

different concentrations of the interacting reagent, and that theand various types of inter−molecular interactions have been util-changes in the apparent electrophoretic mobility is mathematicallyized. Investigation of the modifiers has contributed to establish new

analyzed to give equilibrium constants.

analytical systems. Since the nature of molecular interactions util-

This paper reviews the potential of the CZE techniques forized in the CZE separation is essentially the chemical equilibria inequilibrium analysis in aqueous solution achieved by the present

solution, we can analyze the chemical equilibria by measuring theauthor’s group. The method was validated by the complex forma-change in electrophoretic mobility of analytes, vice versa.

tion analysis of crown ethers, and was further utilized for the analy-

Journal of Chromatography A has issued a special volume en-sis of weak ion−ion interactions in aqueous solution: several inter-titled ”Estimation of Physicochemical Properties by Chroma-

esting factors were elucidated. The ion association reaction in

tographic and Electrophoretic Techniques,” this year (2004) [1]. In

aqueous solution led to understand the stepwise reactions in liquid−that issue, various chromatographic techniques were introduced toliquid distribution of ion−pair extraction. Binding reactions to non-analyze the properties including liquid−liquid partition, vapor pres-ionic surfactant micelle are also interpreted in this review.

Correspondence:
E−mail: takayana@cc.okayama−u.ac.jp―１１―

Chromatography, Vol.26 No.1 (2005)

gen−antibody interaction [6, 7], binding of ligand to protein [8],2. Utilization of capillary zone electrophoresis for equilibrium

binding of carbohydrates to peptides [9], lectin−sugar interaction

analysis in homogeneous solution and its validation[10], and binding of Vancomycin to dipeptides [11] and to precur-

2. 1 Change in electrophoretic mobility of analyte by the interaction with modifiersors [12] have been analyzed. Higher order equilibria, such as ag-

Utilization of modifier additives, including surfactant, hasgregation formation [13] and cyclodextrin binding [14], were suc-been noticed at the early stage of the CZE separations on the con-cessfully analyzed. Theoretical approaches [15] and examination oftrol of electrophoretic mobility of analytes [3, 4]. An interacting re-experimental parameters [16, 17] have been developed to optimize

agent, a modifier, is added in the migrating solution at the concen-

the resolution among chiral compounds and isomers.

tration to be much greater than that of the analytes of interest, so
2. 2 Prominent features of the CZE on equilibrium analysis

that the concentration of free modifier is only slightly changed by
the reaction with the analytes. As shown in Figure 1 (a), analytes in

Capillary zone electrophoresis is based on the migration of

the sample solution, A− in this case, interact with the modifier, L,

ionic substances under a constant electric field, and therefore, we

during the electrophoretic separation, and such interactions lead to

can utilize its characteristics with many advantages. It can be no-form the associate LA− accompanying the change in the apparent

ticed at first that the CZE includes electrophoretic separation andmolecular mass, volume and/or charge of the analyte. Apparent

that any pure analytes are not necessary. When a sample solutionchanges in those physicochemical properties are reflected on thecontains several analytes, simultaneous analysis can be performed

apparent electrophoretic mobility of the analyte, µep’. The degree

in a series of the measurements. Coexisting substances, which doof the interaction is different among the analytes; such differences

not affect electrophoretic mobility of analyte and/or equilibrium re-give different apparent electrophoretic mobility values. Conse-

action, are also allowed to exist in the migrating solution. Measure-quently, the resolution among the analytes can be improved.

ment of migration time of analytes from sample injection point to a

Change in the mobility of analyte is due to the interaction with thedetector is an essential piece of information given by the mobility

interacting reagent, and we can analyze the interaction and equilib-

measurement in CZE, and therefore, various types of detection

rium reaction through the measurement of electrophoretic mobility.

methods are available; photometric, fluorometric, and conducto-

A series of different concentrations of modifier give corresponding

metric detections can be chosen depending on the physical proper-

apparent electrophoretic mobility values, as shown in Figure 1 (b),ties of the objective analytes. Other detection methods under devel-

and the set is offered to analyze the equilibrium through mathe-opment are also applicable to the measurement of electrophoretic

matical analysis.

mobility. Depending on the detection system, the CZE method al-

Equilibrium analyses through electrophoretic mobility havelows the concentration of analyte to be reduced to a very low con-succeeded in bio−molecules. Protein−sugar interactions [5], anti-

centration ( ~10−6 M). When the analyte of interest give even tiny

Figure 1. Schematic illustration for mobility change in capillary zone electrophoresis by equilibrium reaction during electrophoretic migration. An anion (A−) is used as analyte ion and neutral substance (L) as an interacting reagent present in the migrating solution. (a)Electrophoretic mobility of anion (µep,A) is apparently reduced to µep,LA by the equilibrium reaction, where K is an equilibrium
constant. (b) Degree of formation of LA by the equilibrium is reflected on the apparent electrophoretic mobility of A− (−µep,A’).

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Chromatography, Vol.26 No.1 (2005)

Toshio Takayanagi

signal, we can use its migration time for the equilibrium analysis.
Low concentration of analytes can exclude the formation of insoluble precipitates, which is often troublesome on general analysis.
2. 3 Validation of the analysis through the complex formationof crown ethers with alkali metal ions
Although the capillary zone electrophoretic method can determine various types of equilibrium constants through measurementsof electrophoretic mobility, as mentioned above, the method should
be validated to establish the reliability. Crown ethers are famous
cyclic polyether and they bind alkali and alkaline earth metal ionsin its ”cavity hole,” depending on the size. The reactivity of 18−
crown−6 with alkali metal ions in various solvents is well established in details [18], and the complex formation reaction was
adopted as a model one to validate the equilibrium analysis by
Figure 2. Changes in electrophoretic mobility of dibenzo−18−
crown−6 by complex formation reaction with alkali
metal ions. M+ : □, Li+ ; △, Na+ ; ○, K+ ; ▽, Rb+ ; ◇,Cs+. Positive electrophoretic mobility is observed foressentially neutral crown ethers by the reaction with alkali metal ion. Non−linear least−squares analysis gavecomplex formation constants in aqueous solution, as
are listed in Table 1. See details in Ref. 20.

CZE; the reaction and its equilibrium constants are presented in
Eqs. (1) and (2).
M+ + L
KML =

ML+

(1)

[ML+]
[M+] [L](2)

where M+, L, and ML+ are metal ion, crown ether and the complex,
respectively, and KML is an equilibrium constant. Crown ethers,

mobility is more convenient than with the migration time; i.e., mi-such as dibenzo−18−crown−6 (DB 18 C 6) and benzo−18−crown−

gration time includes experimental parameters such as capillary

6 (B 18 C 6), are essentially neutral, and they become cationiclength and applied voltage. The migration time also contains thethrough the incorporation of alkali and alkaline earth metal ionscontribution of electroosmotic flow. Several linear analyses have

[19, 20]. The crown ethers were used as analytes, and some alkali

been proposed and have succeeded on various types of equilibrium

metal salt as interactive reagent is present in the migrating solution.

analysis [21, 22]. The linear analyses, however, are applicable to

Along with the increase in the concentration of alkali metal ion, thesuch reactions as to give sufficient change in the mobility; somecrown ether becomes a cationic complex and the apparent electro-

experimentally obtained data is used as constants, and in such

phoretic mobility of the crown ether increases as is shown in Fig-

cases, they must be obtained very correctly. Thus, such linear

ure 2. The complex formation constants are determined through theanalyses possess their unavoidable limitations, as long as one han-mobility change by analyzing it mathematically.

dles experimental data as constants. Non−linear analysis methods

While some mathematical approaches have been proposed toare more reliable for handling small changes in electrophoretic mo-analyze the equilibrium reactions, handling with the electrophoretic

bility and weak interactions. Schwarz et al. used a fitted function

Table 1. Complex formation constants (log KML) of crown ethers
M+

Crown ether(L)
DB 18 C 61)

Li+

Na+

K+

Rb+

Cs+

(−0.3 ± 0.2)

1.18 ± 0.02

1.67 ± 0.02

1.14 ± 0.03

0.96 ± 0.06

2)

DB 18 C 6

0

1.16

1.67

1.08

0.83

DB 18 C 63)

−

4.4

5.0

4.2

−

4)

18 C 6

−

0.8

2.03

1.56

0.99

B 18 C 65)

−

1.38

1.744

1.15

(0.88)

1) Determined through CZE. Error: 3 σ 2) Reported values determined by spectrophotometry. 3) Values in methanol asa medium. 4) Reported values for 18−crown−6. 5) Reported values for Benzo−18−crown−6.

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Chromatography, Vol.26 No.1 (2005)

for the analysis of aggregate formation [13], and we adopted a non

CZE besides the mobility change, and applied the analysis to an−linear least−squares analysis [23]. Havel and Janos developed aunstable substance in an aqueous solution.

Fortran 77−based CELET program to determine equilibrium con-

Phenolphthalein is one of the most famous acid−base indica-

stants [24]. Prominent features in those non−linear analyses are: alltors, but its property has not been known in details, even textbooks

experimental data are handled equivalently, and the electrophoretic

still contain confused data. It is because Phenolphthalein is unsta-

mobility of species can be also optimized through the analysis.

ble in aqueous alkaline media and gradually decomposes [28, 29].

The complex formation constants determined by using non−Compared with the classical photometric method, the CZE method

linear least−squares analysis are summarized in Table 1 [20, 25];

has a prominent advantage that gradually decomposing species isthey agreed very well with the reported values. In addition to theseparated from equilibrium species of interest during electropho-

reliability of the analysis, the CZE method has offered its superior-retic separation (Figure 3). Thus, the two steps of acid dissociation−5

ity. Solubility of DB 18 C 6 in water is as low as 1.3 x 10 M, andreaction of phenolphthalein were firstly clarified in detail; values

it was sometimes troublesome on precise equilibrium analysis. The

for pKa1 and pKa2 are 8.84 and 9.40, respectively [30]. Comparison

CZE analysis, however, the concentration of DB 18 C 6 is allowedof the results with other pH indicators validated the values.

to be less than 10−5 M, and we can get rid of the solubility problem
owing to the photometric detection sensitivity.

3. Equilibrium analysis of ion association reaction in aqueous

When less UV−absorptive 18−crown−6 was subjected, thesolutioncrown ether was added in the migrating solution as a modifier, and3. 1 Capillary zone electrophoresis as a powerful tool to inves-the electrophoretic mobility of alkali metal ions was monitored us-

tigate ion association reactions

ing indirect photometric reagent [26]. Fair results were obtainedInteractions between organic ions have widely been used inthrough the mobility analysis, and the equilibrium constants ob-

the research field of analytical chemistry by coupling it with distri-tained agreed well with the reported values.

bution to hydrophobic media, including ion−pair solvent extraction, ion−pair reversed−phase high performance liquid chromatography, etc. [31]. The interaction between organic ions in aqueous

2. 4 Analysis of acid−base property of PhenolphthaleinAcid−base reaction is a popular equilibrium to analyze it bysolution has been discussed through the hydrophobicity of ions, as

CZE. We only change the pH of the migrating solution, and thewell as the electrostatic interactions. While electrostatic interaction

mobility measurement gives acid dissociation constants (pKa).

works strongly between small ions in the solvents with low dielec-

Many studies have been reported on acid dissociation reaction bytric constants, the hydrophobic one works with relatively large ionsusing CZE analysis [27]. We noticed the separation features inin the solvents with high dielectric constants such as water. It goes
without saying that electrostatic interaction has been establishedthrough conductivity measurements. However, hydrophobic (organic) ion associates easily precipitate in aqueous solution, and we
did not have available tool to utilize the reaction in aqueous phaseand to analyze the equilibrium, until the potential of capillary zone
electrophoresis was thoroughly noticed.
Capillary zone electrophoresis has prominent feature that theelectrophoretic separation is performed in homogeneous solution.
Terabe and Isemura used cationic polyelectrolytes to resolve anionic analytes by utilizing ion−ion interactions [32, 33]. Quaternaryammonium ions have also been used to improve the resolution
among isomers by utilizing the ion association reaction in aqueous
solution [34]. Important questions may arise how the selectivity
emerges and what kind of interactions work on ion−ion interactions
in aqueous media. Ion−pair solvent extraction could give the ion
association constant in aqueous solution, but the reliability was too

Figure 3. Electropherograms for Phenolphthalein and Methyl Orange at different pH conditions. pH: a) 8.64; b) 9.42; c)
9.81; d) 11.60. Signals: 1, Phenolphthalein; 2, Methyl
Orange (mobility standard); S, ethanol (EOF marker).
See detail conditions in Ref. 30.

inferior [35]. Capillary zone electrophoresis can handle the concentration of ion associate as low as 10−5 M level or less, and ion association reactions in an aqueous solution can be directly analyzed
through the change in electrophoretic mobility of analytes. Figure 4―１
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Chromatography, Vol.26 No.1 (2005)

Toshio Takayanagi

Figure 4. Electropherograms for aromatic anions in the absence and presence of ion association reagent. In the presence of
TBA+, the resolutions among isomers are developed. See details in Ref. 23.

C+ + A−
Kass =

C+A−[C+A−][C+] [A−](3)(4)

where C+, A−, and C+A− are pairing cation, pairing anion, and 1:1
ion associate, and Kass is ion association constant.
3. 2 Factors contributing to the ion associability in aqueous solution
Electrostatic interaction in solution is a classical one, and theinteraction is well known and established in details. Smaller ionsattract with each other, and the interaction is weakened by solvation. The electrostatic interaction works weakly in high dielectric−
constant media such as water, because those solvents solvate andFigure 5. Changes in electrophoretic mobility of aromatic anionswith increasing concentrations in TBA+. See details in
Ref. 23.

their inter−ionic distance is lengthened to be than their crystal ionicradii, as seen in Stokes’ radii. Electrostatic interaction between aromatic anions and primary to quaternary ammonium ions was examined by capillary zone electrophoresis [37]. An evident trend in ion

shows a typical separation improvement for 11 aromatic anions by+using tetrabutylammonium ion (TBA ) as an ion association re-associability was observed: from electrostatic interactions with pri-agent [23]. Electrophoretic mobility of the analyte anions de-mary ammonium ions to hydrophobic interactions with quaternary+creased with increasing concentration of TBA in the migrating so-ammonium ions. In most aromatic anions, minimum ion associa-

lution (Figure 5). We first determined the ion association constants

tion constants were obtained with secondary ammonium ions. Re-

using linear analysis [36], and the reliability and the accuracy wereversal of migration order among anionic isomers was also ob-much developed by using non−linear least−squares analysis [23].

served, along with the change in ion associability. The phenome-

The interactions discovered are summarized in the following sec-

non is very useful for designing separation systems; we can choosetion. The equilibrium reaction and its constant are represented in

the interacting reagent and its interactions for developing separa-

Eqs. (3) and (4), respectively, unless noticed.

tion selectivity.

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Chromatography, Vol.26 No.1 (2005)

Ion association constants (Kass) of aromatic anions were determined with different sizes of quaternary ammonium ions to establish the concept on hydrophobic interactions [23, 38, 39]. A linear
free energy relationship (LFER) was examined with the increase in
the size of quaternary ammonium ions; the carbon number of alkyl
chains in quaternary ammonium ion was used as an index of hydrophobicity. Logarithmic value of Kass increased linearly with increasing bulkiness/carbon number of quaternary ammonium ion(Figure 6). The mean slope, 0.06, represents the contribution of hydrophobicity per one carbon atom of quaternary ammonium ions inaqueous solution. The contribution, 0.06 in logarithmic unit, isfound to be about one−tenth of that obtained in liquid − liquid distribution system of ion associates, 0.6 [31].

Figure 7. Linear free energy relationship between ion association
constants and carbon number of ion associates formed
between aromatic cations and anions. The mean slope,
0.14, contains the contribution of hydrophobicity, andthe contribution of aromatic − aromatic interaction is
deduced to be 0.08 for log Kass (Ref. 42).
Aromatic−aromatic interaction also plays an important role inion associability in aqueous solution [42]. Ion association constants
between aromatic ions are determined and plotted against the number of carbon atom on the ion associate, as is shown in Figure 7. A
typical interaction was observed with anthracene ring − pyrene
ring; the ion association constant between 1−propylbenzo[f]quinolinium ion and 1−pyrenecarboxylate ion reached to 102.74, while the
Figure 6. Linear free energy relationship between ion association
constants and the carbon number of quaternary ammonium ions (Q+) as an index of hydrophobic parameter.
Symbols correspond to individual aromatic anions. The
mean slope, 0.06, is attributed to the contribution of hydrophobicity in ion association reaction in an aqueous
medium (Ref. 23).

ion associate became composed of a monovalent cation and an anion. Higher ion associability provides that low concentration of ion
association reagent can realize sufficient resolution among analytes. Such operating conditions reduce the electric current andJoule heat, and stabilize the migration time. The linear free energy
relationship provided the increase in Kass values by 100.14 per one
aromatic carbon. Considering that the hydrophobicity of carbon

The Kass values are compared based on the number of charge.

atom is contributing to the ion associability by 100.06, one can esti-

Divalent anions are found to have large Kass values than monova-

mate the contribution of aromatic−aromatic interaction to ion asso-

lent ones by the order of about 0.3 in logarithmic units, which

ciability to be 100.08. Aromatic−aromatic interaction was also re-means that divalent anions are twice as associable as monovalentvealed with divalent viologen cations; hydrophobicity, multipoint

anions (log 2 = 0.3) [40].

interaction, and aromatic−aromatic interaction are synergistically

Divalent quaternary ammonium ions are also designed to util-

utilized with the viologen cations [43].

ize multipoint interactions, and the distance between two ammo-

Factors clarified for ion association reactions in aqueous solu-

nium sites is controlled with the chain length of methylene grouption are summarized in Table 2. The present author reviewed the[41]. Ion association constants were determined and ion associabil-

investigation of ion association reaction through CZE in detail [44,ity was discussed on the basis of intramolecular ion−ion distance.

45].

Ion associability was proved to be contributed from fitted distance
between intramolecular ionic sites and hydrophobicity of the methylene group.

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Toshio Takayanagi

Table 2. Intermolecular ion−ion interactions found to work in an aqueous solution

3. 3 Stepwise aspects of ion association reaction in aqueous so-where C+, A−, and C+A− are pairing cation, pairing anion, and 1:1

lution on liquid−liquid extraction and its CZE analysis

ion associate, and Kex, Kass, and KD,IP are extraction constant, ion

Ion−pair extraction has widely been used in analytical andassociation constant, and distribution coefficient of ion associate

separation sciences. The stepwise reaction of ion−pair extraction is

between aqueous and organic phases. Subscript (o) denotes that theconveniently expressed with formation of electrically neutral ion−

species exists in the organic phase. It can be easily understood fromassociate in aqueous solution and distribution of the ion−associate

Eq. (5) that the extraction constant simply consists from the prod-

to water−immiscible organic phase, as is illustrated in Figure 8.

uct of Kass and KD,IP. Once the ion association reaction in aqueous

The extraction reaction is represented by Eq. (5):solution is clarified by mobility measurements in CZE, the extraction constant can easily be divided into two stepwise reactions of[C+A−] (o)Kex = += Kass x KD,IP[C ] [A-]Kass and KD,IP. We have determined stepwise reactions of Kass and(5)

KD,IP for ion associates of tetrabutylammonium ion − phenolate andbenzoate ions by using Kex values determined by solvent extraction
and Kass values determined by CZE [40, 46, 47]. The equilibrium
constants are summarized in Table 3. Both Kass and KD,IP values increase with increasing molecular volume, and consequently the extraction constant, Kex, increases. However, the magnitude in the increase is larger in KD,IP than Kass, and the distribution process isfound to contribute effectively. The results well agree to the factthat contribution of hydrophobicity in Kass is 1/10 of that in Kex.
Alkali metal ions can be extracted into organic phase by using
dibenzo−18−crown−6 (DB 18 C 6) and picrate ion (Pic−), as illustrated in Figure 9. The entire extraction reaction (Kex) involves four

Figure 8. Ion−pair extraction of simple 1:1 ion associate into organic phase. The extraction constant, Kex, comes fromthe product of Kass and KD,IP.

stepwise reactions (KD,L, KML, KMLX, and KD,MLX). The extraction
constant is expressed as in Eq. (6).

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Table 3. Equilibrium constants concerning liquid−liquid distribution of ion associates between an aqueous and a chloroform phase*Kex =KML x KMLX x KD,MLX
KD,L

(6)

Complexation analysis provides the KML value, and values ofAnion

log Kasslog KD,IP

log Kex**Kex and KD,L can be determined through solvent extraction study.

Phenolate ionsPhenolate

0.40

−0.70

−0.30

perimentally. We have succeeded in the direct analysis of ion−pairo−Nitrophenolate

0.64

1.53

2.17

m− Nitrophenolate
p− Nitrophenolate

0.60
0.80

1.09
1.72

1.69
2.52

2, 4− Dinitrophenolate
2, 6− Dinitrophenolate

0.80
0.68

3.21
3.30

4.01
3.98

method, the successive equilibrium constants have been determined

2, 4, 6−Trinitrophenolate

0.67

4.86

5.53

selectivity for potassium ion against sodium ion was also evaluated

0.69

−0.01

0.68

p−Nitrobenzoate

0.55

1.12

1.67

Benzenesulfonate ions
Benzenesulfonate

0.69

1.03

1.72

0.64

2.38

3.02

1.19
1.13

1.02
2.12

2.21
3.25

However, it was impossible to resolve KMLX and KD,MLX values ex-

Benzoate ionsBenzoate

formation reaction between alkali metal−crown ether complexes
and pairing anions in aqueous solution (KMLX) using the CZE
for the first time [20]; they are summarized in Table 4. Extraction
by comparing the stepwise reactions. It can be seen in Table 4 thatp−Nitrobenzenesulfonate
Naphthalene−2−carboxylate (2−NC)
Naphthalene−2−sulfonate (2−NS)

distribution of ion associate, M−DB 18 C 6+ − Pic−, between aqueous and benzene phases is predominant on extraction selectivity.
The selectivity study was further carried out with benzo−18−crown−6 [48] and 18−crown−6 [49].
4． Binding reaction to nonionic surfactant micelle
4. 1 Binding analysis of aromatic anions to nonionic surfactant* Tetrabutylammonium ion was used as a pairing cation.
** Reference values.

micelleSince the development of Micellar Electrokinetic Chromatography by Terabe et al. [3,4], surfactant micelles have been utilizedin capillary electrophoresis as a pseudo−stationary phase. Non−ionic surfactant micelles were found to modify the resolution ofionic substances by Matsubara et al. [50]. We performed binding
analysis of aromatic anions to the nonionic surfactant micelle [51];the binding reaction is illustrated in Figure 10. The electrophoretic
mobility of aromatic anions decrease with increasing concentrationof nonionic surfactant, resolution among positional isomers wasdeveloped. Binding constants to nonionic surfactant micelle were
determined using the mobility change and aggregation number of

Figure 9. Ion−pair extraction of alkali metal ions (M+) with DB 18
C 6 (L) and hydrophobic anion (X−). The overall extraction constant, Kex, consists of KD,L, KML, KMLX, and
KD,MLX (Ref. 20).

the surfactant. The binding constants were validated throughfluorometry. Because the binding reaction includes distribution tohydrophobic media, less charged ions are more likely to be boundto the micelle. Different types of surfactant micelles are examinedbased on hydrophilicity − lipophilicity balance (HLB) and aggrega-

Table 4. Equilibrium constants related to ion−association extraction of alkali metal ions
Crown ether

Pairing anion

(L)

(X−)

DB 18 C 6
DB 18 C 6
DB 18 C 6
DB 18 C 6
DB 18 C 6

picratepicratepicrateM+

Constant

log Kex 1)
log KD,L 3)
log KML 4)log KMLX 4)
log KD,MLX 5)

Li+

Na+

K+

Rb+

Cs+

−

2.21

4.65

3.75

3.07
2.9

2)

−0.3
−
−

1.24 ± 0.02
2.23 ± 0.29
1.7

1.67 ± 0.02
1.95 ± 0.10
3.9

1.07 ± 0.02−−

0.94 ± 0.02−−

1) Ion−association extraction constants with picrate ion to benzene. 2) Estimated values. 3) Reported value. 4) Determined through CZE.
Error: 3 σ 5) Distribution coefficient of ion associate, ML+X−, calculated by KD,MLX = (Kex x KD,L) / (KML x KMLX).

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Chromatography, Vol.26 No.1 (2005)

Toshio Takayanagi

ing constant of ion associates (KB,IA).
Since the charge of aromatic anion is reduced by ion association reaction, KB,IA as ion associate gave larger values than KB foreach aromatic anion. The results can be explained from distribution
phenomena that less charged species are more likely distributed tohydrophobic media. In the comparison among aromatic anions, the
KB,IA values for such ion associates as are formed from monovalentanions and pairing cations are larger than those formed from divalent anions and pairing cations. The results also agree with the distribution phenomena that electrically neutral or less charged species are more likely bound to hydrophobic media than morecharged ones, while divalent anions are more associable with pairing cations than monovalent anions. The ion−association − micelle
Figure 10. Binding reaction of anionic species to nonionic surfactant micelle in connection with the change in electrophoretic mobility of the analyte anions (Ref. 51).

distribution CZE is also proved to be useful to develop separation
systems.
4.3 Binding analysis of phenols to nonionic surfactant micelletion number. More hydrophobic micelles bind aromatic anions

When binding of nonionic substances, such as phenol, to non-strongly.

ionic surfactant micelle is subjected to analyze, direct measurementof the binding reaction is impossible by the electrophoretic method.

4.2 Binding analysis of ion associates to nonionic surfactant
micelleIt is because both charges of the neutral analyte and the bounded

Binding of ion associates to nonionic surfactant micelle (Fig-

species are zero and we cannot observe any change in electropho-

ure 11) was also explored [52]. As can be seen in the scheme, theretic mobility in a usual measurement. In such case, change in acidreaction is realized in the presence of both ion association reagent

dissociation constant is utilized for the equilibrium analysis, as isand nonionic surfactant micelle, and three equilibria take part in theshown in Figure 12. Apparent acid dissociation constant, Ka’,entire system. Ion association reaction (Kass) and binding reaction

should become larger in the presence of NIS micelles, because neu-of anion (KB) were separately examined in the presence of either

tral species would be more easily bound to the micelle than the an-

ion association reagent or nonionic surfactant micelle, and the equi-ionic species [53]. Binding constant of phenols, KB,HA, has the rela-librium constants obtained were used for the determination of bind-tionship with Ka in aqueous solution, Ka’ in the presence of micelle, and binding constant of anions (KB,A), as is written in Eq. (7).
KB,HA =

Figure 11. Schematic representations for ion−association nonionic
micellar electrokinetic chromatography. Ion association reaction in aqueous solution, binding of anion tononionic surfactant micelle, and binding of ion associate to nonionic surfactant micelle work cooperatively(Ref. 52).

Ka
( 1 + KB,A) − 1
Ka’

(7)

Figure 12. Schematic representations for binding of phenol derivatives to nonionic surfactant micelles (Ref. 53).

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Chromatography, Vol.26 No.1 (2005)

834−841.

When Ka’ value is determined experimentally at certain condi[5]Honda, S.; Taga, A.; Suzuki, K.; Suzuki, S.; Kakehi, K. J.

rium constants, KB,HA values are larger than the corresponding KB,A

[6]

Shimura, K.; Karger, B. L. Anal. Chem., 1994, 66, 9−15.

values in the order of 0.22 − 0.96 in logarithmic units. The hydro-[7]

Mammen, M.; Gomez, F. A.; Whitesides, G. M. Anal.

tions, we can determine KB,HA values by using Eq. (7) and theChromatogr., 1992, 597, 377−382.

known values of Ka and KB,A. From the comparison of the equilib-

Chem., 1995, 67, 3526−3535.

phobic concept of micelle is also applied in this case: less charged[8]

neutral species is more likely bound to the micelle. It can be also

Gomez, F. A.; Avila, L. Z.; Chu, Y.−H.; Whitesides, G.
Anal. Chem., 1994, 66, 1785−1791.

noticed from the comparison that KB,HA values are slightly larger[9]than KB,IA values, which means that ion associates are not com-

Heegaard, N. H. H.; Robey, F. A. Anal. Chem., 1992, 64,
2479−2482.

pletely neutral as in the protonated species, but are in the state[10] Kuhn, R.; Frei, R.; Christen, M. Anal. Biochem., 1994, 218,

where the positive and negative charges remain in the ion associ-131−135.

ates.

[11] Carpenter, J. L.; Camilleri, P.; Dhanak, D.; Goodall, D. J.
Chem. Soc., Chem. Commun., 1992, 804−806.

5. Conclusion[12] Liu, J.; Volk, K. J.; Lee, M. S.; Pucci, M.; Handwerger, S.

The fundamental criterion in affinity capillary electrophoresis

Anal. Chem., 1994, 66, 2412.

is the control of electrophoretic mobility of analytes by using inter-[13] Schwarz, M. A.; Neubert, R. H. H.; Ruttinger H. H. J. Chro-acting reagent in the migrating solution. We have performed vari-matogr. A, 1996, 745, 135−143.

ous types of equilibrium analysis through the electrophoretic mo-[14] Bowser, M. T.; Chen, D. D. Y. Anal. Chem., 1998, 70, 3261

bility in capillary zone electrophoresis by utilizing its prominent−3270.

features. Various types of molecular interactions have been clari-[15] Wren, S. A. C.; Rowe, R. C. J. Chromatogr., 1992, 603,

fied in ion association reactions, in addition to the classical electro-

235−241.

static interaction. Exploration of ion association reaction in aqueous solution allowed us to investigate the stepwise reactions in two[16] Nielen, M. W. F. J. Chromatogr., 1991, 542, 173−183.

−phase distribution phenomena including liquid−liquid distribution[17] Ingelse, B. A.; Claessens, H. C.; Wal, S.; Duchateau, A. L.

of ion associates. Nonionic surfactant micelle as hydrophobic ma-

L.; Everaerts, F. M. J. Chromatogr. A, 1996, 745, 61−71.

trix was elucidated through binding analysis of anions, ion associ-[18] Izatt, R. M.; Bradshaw, J. S.; Nielsen, S. A.; Lamb, J. D.;
Christensen, J. J.; Sen, D. Chem. Rev., 1985, 85, 271−339.

ates, and nonionic substances, as well as its utilization as separation
modifier. When one is aware that molecular interactions are widely[19] Okada, T. J. Chromatogr. A, 1995, 695, 309−317.

used in separations and separation instruments, the equilibrium can[20] Takayanagi, T.; Iwachido, T.; Motomizu, S. Bull. Chem.
Soc. Jpn., 1998, 71, 1373−1379.

further be analyzed through physicochemical properties easily.

[21] Rundlett, K. L.; Armstrong, D. W. J. Chromatogr. A, 1996,
721, 173−186.

Acknowledgements[22] Rundlett, K. L.; Armstrong, D. W. Electrophoresis, 2001,

The author acknowledges to Prof. Motomizu, Okayama Uni-

22, 1419−1427.

versity, for his fruitful advises. The present author is very grateful[23] Takayanagi, T.; Wada, E.; Motomizu, S. Analyst 1997, 122,

to the Japan Society for Chromatographic Sciences who selected57−62.

him as a recipient of the Encouragement Award in 2004 and gave
him an opportunity to publish this review paper. This study was[24] Havel, J.; Janos, P. J. Chromatogr. A, 1997, 786, 321−331.

partly supported in part by a Grant−in−Aid for Scientific Research[25] Manege, L. C.; Takayanagi, T.; Oshima, M.; Iwachido, T.;
Motomizu, S. Bull. Chem. Soc. Jpn., 1999, 72, 1301−1306.

(No.15750068) from Japan Society for Promotion of Science.

[26] Takayanagi, T.; Motomizu, S. Chem. Lett., 1999, 523−524.
[27] Bruzzoniti, M. C.; Mentasti, E.; Sarzanini, C. J. Chroma-

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