Focusing Review

Chromatography, Vol.26 No.2 (2005)

Focusing Review

Capillary electrophoresis using chemical and physicochemical reactions
Atsushi Taga
Faculty of Pharmaceutical Sciences, Kinki University, 3−4−1, Kowakae, Higashi−osaka, Japan
Received for review November 26, 2005. Revised manuscript received January 23, 2006. Accepted January 30, 2006.

Abstract
Chemical and physicochemical reactions can be carried out in a capillary tube on capillary electrophoresis. And utilizing in−capillary reactions on capillary electrophoresis allows quick determination of reaction rate constants and association constants between biological components with small samples. This paper summarizes “in−capillary derivatization” and “affinity capillary electrophoresis” as capillary electrophoretic analyses using chemical and physicochemical reactions.
Key words: Capillary electrophoresis, In−capillary derivatization, Affinity capillary electrophoresis, o−phthalaldehyde, carbohydrate

1. Introduction
Recently capillary electrophoresis is coming more popular toanalyze not only general small compounds but also large moleculesof biological samples. This separation tool requires just small
amounts of samples and running buffers. Various modes of separation can be carried out only with filling a capillary tube with running buffers. We noticed this advantage of capillary electrophoresis
what allows ultra−micro scale kinetic studies. In this paper chemical and physicochemical reactions carried out in a capillary tube on
capillary electrophoresis were investigated multilaterally.
2. In−capillary derivatization
Derivatization in a capillary is an excellent technique to reduce a sample amount for conversion to a derivative amenable tothe detection system installed in the apparatus. It can be carried out

Figure 1. Electropherograms of selected amino acids derivatizedwith OPA by at−inlet (a) and pre−capillary (b) derivatization. Capillary, untreated fused silica (72 cm, 50 µm i.
d.); running buffer, 50 mM borate buffer (pH 10.0); applied voltage, 30 kV; detection, UV absorption at 230nm; reaction conditions, R (3 s)−S (1 s)−R (3 s)−B (5 s)were introduced in this order for (a), and 30 µL of R
was added to 5 µL of S for (b), standing time for reaction was 20 min (S, sample solution, R, reagent solution, B, running buffer).

by three formats (at− inlet, zone−passing, and throughout−capillary) depending on the place where reaction takes. It should bestressed that all these formats allow rapid, automatic derivatization,by using only small amounts of samples (sample amount can be reduced to a thousandths of that in pre−capillary derivatization).
2. 1. At−inlet technique [1]In−capillary derivatization at the capillary inlet named “at−in-Correspondence: Atsushi Taga, Faculty of Pharmaceutical Sciences, Kinki University, 3−4−1, Kowakae, Higashi−osaka, Japan (Postalcode 577−8502)
E−mail: punk@phar.kindai.ac.jp
Phone: +81−6−6721−2332 (EXT: 3856)
Fax: +81−6−6721−2353―１３―

Chromatography, Vol.27 No.1 (2006)

let technique” is a similar format to pre−capillary derivatizationand the simplest to analyze data among the three techniques of in−capillary derivatization. Solutions of a reagent and a sample are introduced successively into the capillary inlet, and stood for appropriate time followed by application of voltage for electrophoretic
separation. Two introduction modes of each solution are available;
tandem and sandwich modes. These two introduction modes withvarious introduction times were investigated on mixing and reaction efficiency. In this investigation, a mixture of selected aminoacids was used as a sample solution and an o−phthalaldehyde(OPA) solution was used as a derivatization reagent. As a result theFigure 3. Schematic illustration of the zone−passing technique ofin−capillary derivatization.

sandwich mode by reagent (3 sec)−sample (1 sec)−reagent (3 sec)−running buffer (5 sec) in this order gave the highest efficiency. Thisefficiency is similar to pre−capillary derivatization as shown inFigure 1. This technique of in−capillary derivatization has a specialof fast reactions. Figure 4 shows the influence of the reagent intro-feature, that various solutions can be used as electrophoretic solu-duction time on derivatization yields. The phenylalanine derivative

tions. According to the advantage of this feature, fifteen of seven-gave a peak having half height compared with the peak of cinnamic

teen amino acids included in H−type amino acids could be wellacid added as an internal standard by introduction time of 10 sec. A

separated by using a phosphate buffer containing neutral detergent

similar and a higher peak height were given by 40 sec and 120 sec,

as shown in Figure 2.

respectively.
Reaction time on each electropherogram in Figure 4 can be es-2. 2. Zone−passing technique [2]Figure 2. Separation of amino acids derivatized with OPA by theat−inlet technique of in−capillary derivatization. Running buffer, 50 mM phosphate buffer (pH 6.8) containing CHAPS at the concentration of 3.2 mM. The otherconditions are as in Figure 1 a.

Figure 4. Electropherograms of phenylalanine derivatized with
OPA by the zone−passing technique of in−capillary
derivatization. Capillary, untreated fused silica (70 cm,50 µm i. d.); running buffer, 50 mM borate buffer (pH10.0); applied voltage, 5 kV; detection, UV absorption
at 230 nm. Introduction of sample and reagent solutions; S (10 s)−B (10 s)−R (10 s) (a), S (10 s)−B (10 s)−
R (40 s) (b) or S (10 s)−B (10 s)−R (120 s) (c) by thehydrostatic method (10 cm), where S, ES and R aresample solution, electrophoretic solution and reagent
solution, respectively.

Amino acids will generally migrate more slowly than OPA reagent in an alkaline solution in the presence of electroosmotic flow
because amino acids have negative charge. When a sample solutionof amino acids is introduced followed by a reagent solution ofOPA, the OPA zone will pass the sample zone. While these zones
overlap, the derivatization reaction proceeds as shown in Figure 3.

timated from half bandwidth of OPA and migration velocity dis-Reaction time can be easily controlled by changing introduction

tance between OPA and phenylalanie. Remaining phenylalanine

time of a reagent solution, hence, zone−passing technique of in−concentration can be calculated from each peak response of pheny-capillary derivatization is a convenient technique for kinetic studies

lalanine−OPA derivative using a calibration curve. A natural loga-―１４―Chromatography, Vol.27 No.1 (2006)

Atsushi Tagarithm plot of remaining phenylalanine concentration to the reaction
time gave a straight line with good linearity, consequently reaction
rate constant can be obtained from the slope of this straight line.
2. 3. Throughout−capillary technique [3]Throughout−capillary technique of in−capillary derivatizationis the simplest technique. It can be performed by carried out electrophoretic analysis in a running buffer containing a reagent for thetarget samples. Figure 5 shows separation of selected amino acids
derivatized by throughout−capillary derivatization. Each aminoacid gave relatively broad peaks in comparison with at−inlet andpre−capillary derivatizations. The peak widening was likely to becaused by the detection time difference between derivatives reacted
at the early stage and the late stage.
Figure 6 shows a magnification of a peak of phenylalanine

Figure 6. Analysis of phenylalanine as the OPA derivative by thethroughout−capillary technique of in−capillary derivatization. The analytical conditions are as in Figure 5.
gave a straight line with good linearity, and from this straight line areaction rate constant could be obtained with just one electrophoretic analysis.
3. Affinity capillary electrophoresis
Recently human gene mapping has been completed, hence
studies of biological components having complex physical functions are becoming more important. Interactions of biological samples can be observed by capillary electrophoretic analyses calledaffinity capillary electrophoresis. Affinity capillary electrophoresis
was investigated using interactions between proteins and carbohydrates as model systems. This technique has two systems. The firstis “normal system” which uses proteins as samples and ionic carbohydrates as additives to running buffers. The second is “reversesystem” which uses carbohydrate derivatives as samples and pro-Figure 5. Analysis of a mixture of selected amino acids as OPA
derivatives by the throughout−capillary technique of in−capillary derivatization. Capillary, untreated fused silica(70 cm, 50 µm i. d.); running buffer (reagent solution),
50 mM borate buffer (pH 10.0) containing OPA (50mM); applied voltage, 20 kV; detection, UV absorption
at 230 nm.

teins as additives.
3. 1. Normal system [4,5]In the normal system of affinity capillary electrophoresis,
small molecules of carbohydrates are used as an additive to a running buffer. Neutral carbohydrates are unable to change the velocity of a protein having a large molecular weight, because they haveanalyzed by throughout−capillary derivatization. It gave a fronting

no charge and also their small size. Accordingly, lactobionic acidpeak, because the derivative has stronger negative charge, andhaving both a carboxylic group and a galactose residue was used asmoves more slowly than the unreacted phenylalanine. From a dataan additive to a running buffer. Recinus communis agglutinin

in Figure 6, amount of phenylalanine derivatized before the reac-(RCA60) which is galactose specific lectin was used as a sample.

tion time (t) can be calculated from the part peak area, marked ∆A.

This combination was used as a model interaction system for theThe reaction time (t) can be estimated from migration times of de-basic study of the normal system. Figure 7 shows electrophero-rivative and unreacted phenylalanine. A natural logarithm plot ofgrams of RCA60 in the absence and presence of lactobionic acid inremaining unreacted phenylalanine concentration to reaction timeneutral phosphate buffers. In the absence of lactobionic acid, this―１５―

Chromatography, Vol.27 No.1 (2006)

an acidic carbohydrate can be determined as mentioned above. In
this determination the carbohydrate as ligand must have an electriccharge. A convenient method was investigated for conversion ofneutral carbohydrates having no electric charge to a derivative having strong negative charge by dithioacetalation with 2−mercaptoethanesufonic acid sodium salt as shown in Figure 8. The yield of
this derivatization was investigated for maltose series oligosacchaFigure 7. Concentration−dependent retardation of the RCA60 peakin running buffers containing lactobionic acid. Capillary, untreated fused silica (85 cm, 50 µm i. d.); runningbuffer, 50 mM phosphate buffer (pH 6.8) containing lactobionic acid at the concentrations of (a) 0, (b) 0.2, (c)0.3, (d) 0.4, (e) 0.5, (f) 1.0, (g) 5.0 and (h) 10.0 mM; applied voltage, 20 kV; detection, UV absorption at 220nm; sample solution, 50 mM phosphate buffer (pH 6.8)
containing RCA60 and mesityl oxide to concentrations of1 mg/ml and 1 mM, respectively. Peaks: 1= mesityl oxide (neutral marker); 2= RCA60.

rides. Maltose, maltotriose and maltopentaose gave the yields of
94.7%, 96.2% and 96.0%, respectively. These derivatives could be
conveniently used as the negatively charged additives to runningbuffers for determination of their Ka values to a Lentil lectin(LCA) of glucose recognizing protein. The Ka values increased
with their polymerization degree.
3. 2. Reverse system [6,7]
It is difficult to separate carbohydrate derivatives having thesame size by capillary zone electrophoresis. By adding a protein

lectin gave a peak almost superimposed on that of a neutral marker.

recognizing carbohydrate structures to a running buffer, oligosac-

When the lactobionic acid was added to the running buffer, RCA60

charide derivatives can be separated from each other. This separa-was separated completely from the neutral marker by forming ation technique, named “reverse system”, must be one of the separa-complex with the negative charged ligand. A plot of migration timetion modes to separate the compounds having micro heterogenei-of RCA60 to concentration of the lactobionic acid gave a curve hav-ties. Separation of five kinds of disaccharides derivatized with 1−ing a plateau at the concentration of more than 5 mM. Theoreticaltreatment to estimate an association constant is mentioned briefly
below. The association constant (Ka) can be written as [P−C] / [P][C] (P, protein; C, carbohydrate; P−C, complex). The migration
time of the protein changes depending on the carbohydrate concentration in the running buffer. A plot of reciprocal migration time
shift to reciprocal carbohydrate concentration gives a straight line.
Ka can be estimated from the slope (A) and the Y−intercept (B) of
this straight line and the migration time of the protein in the absence of the carbohydrate (t1) by the equation Ka = A−1 t1−1 (Bt1 + 1).
The double reciprocal plot for RCA60 and lactobionic acid gave astraight line, and Ka could be estimated as a value of 3.3 x 103(M−1). The reproducibility of the determination of Ka was 3.6% asa relative standard deviation for five repetitions of a series of binding experiments on five successive days.
The association constant of interaction between a protein andFigure 9. Separation of several disaccharides derivatized withPMP by capillary zone electrophoresis (a) and affinity
capillary electrophoresis in the presence of LCA (b) or
RCA60 (c). Capillary, linier polyacrylamide−coatedfused silica (50 cm, 50 µm i. d.); running buffer, 50mM phosphate buffer (pH 6.8) (a), 50 mM phosphatebuffer (pH 6.8) containing LCA at a concentration of
10 mg/ml (b), 50 mM phosphate buffer (pH 6.8) containing RCA60 at a concentration of 5 mg/ml (c); applied voltage, −15 kV (a, b) or −18 kV (c); detection,UV absorption at 245 nm.

Figure 8. Procedure for introduction of 2−mercaptoethanesulfonate (MerES) tags to a carbohydrate.

―１６―Chromatography, Vol.27 No.1 (2006)

Atsushi Taga

phenyl−3−methyl−5−pyrazolone (PMP) by capillary zone electro-constants of each derivative could be determined simultaneously. It

phoresis is shown in Figure 9 a. It gave group−separation of gluco-

is notable that the small difference in Ka values was well differenti-

bioses and galactosyl glucoses. Addition of LCA having glucose

ated and increasing tendency of Ka with degree of polymerizationand mannose specificity resulted in the splitting of these group−in these homologous series of oligosaccharides was clearly indi-separated peaks into four peaks in total (Figure 9b). Addition ofcated.

RCA60 having galactose specificity gave a quite different migration
4. Conclusions

profile (Figure 9c). Since migration time delay of melibiose andlactose by addition of RCA60 were great, simultaneous determina-

Various capillary electrophoretic analyses with chemical andtion of association constants of melibiose and lactose to RCA60 was

physicochemical reactions were observed from our previous pa-

examined by the same way as in the normal system. The plots ofpers. Usefulness of in−capillary derivatization in the first half andreciprocal migration time shifts of melibiose and lactose derivat-

affinity capillary electrophoresis in the latter half were mentioned

ized with PMP to reciprocal concentration of RCA60 gave straightin this review. The noticeable point is the convenience of carrying

lines, and determined Ka values of melibiose and lactose were 8.9

out various modes of analyses simply by filling appropriate run-x 103 (M−1) and 1.1 x 104 (M−1), respectively. Based on this result aning buffers and reagents into an open tubular fused silica capil-multiple solute system containing a larger number of ligands werelary. This feature of capillary electrophoresis will allow more reli-

examined. Figure 10 shows the electropherograms of 8−aminon-able ultra−micro scale analyses for complex functional studies.

aphthalene−1,3,6−trisulfonate (ANTS) derivatives of isomaltooliAcknowledgement

gosaccharides having various degrees of polymerization in the absence (Figure 10a) and presence (Figure 10b) of LCA. Figure 10

The author acknowledges to Emeritus Professor Susumu

indicated that the ANTS derivatives having any degree of polym-

Honda, Kinki University, for his kind help. The author also ex-erization were retarded in the presence of LCA, and associationpresses his gratitude to all collaborators in this work. The present
author is very grateful to the Japan Society for Chromatographic
Sciences who selected him as the recipient of the Encouragement
Award in 2005 and gave him an opportunity to publish this review
paper. This study was partly supported by the Sasagawa Scientific
Research Grant from the Japan Science Society.
References[1]

Taga A.; Honda S. J. Chromatogr. A 1996, 742, 243−250.

[2]

Taga A.; Sugimura M.; Honda S. J. Chromatogr. A 1998,802, 243−248.

[3]Taga A.; Nishino A.; Honda S. J. Chromatogr. A 1998, 822,271−279.

[4]

Honda S.; Taga A.; Suzuki K.; Suzuki S.; Kakehi K. J.
Chromatogr. 1992, 597, 377−382.

[5]

Figure 10. Electropherograms of isomaltooligosaccharides derivatized with ANTS in the absence and presence ofLCA. Capillary, linier polyacrylamide−coated fusedsilica (50 cm, 50 µm i. d.); running buffer, 50 mM
phosphate buffer (pH 6.8) (a) or the same buffer containing LCA at a concentration of 0.6 mg/ml (b); applied voltage, −15 kV; detection, UV absorption at
280 nm.

Taga A.; Mochizuki M.; Itoh H.; Suzuki S.; Honda S. J.
Chromatogr. A 1999, 839, 157−166.

[6]

Taga A.; Yabusako Y.; Kitano A.; Honda S. Electrophoresis
1998, 192, 2645−2649.

[7]

Taga A.; Uegaki K.; Yabusako Y.; Kitano A.; Honda S. J.
Chromatogr. A 1999, 837, 221−229.

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