Focusing Review

Chromatography, Vol.28 No.1 (2007)

Focusing Review

Chiral Separations Using Avidin as a Chiral Selector and Highly Sensitive
Detection Using Thermal Lens Microscopy in Capillary Electrophoresis
Koji Otsuka
Graduate School of Engineering, Kyoto University, Katsura, Nishikyo−ku, Kyoto 615−8510, Japan
Received for review January 31, 2007. Accepted February 2, 2007.

Abstract
A brief review of the author’s studies concerning the fundamental characteristics of micellar electrokinetic chromatography (MEKC) and its
application along with fundamental and application studies on capillary electrophoresis (CE) as well as fundamental studies on microchip
electrophoresis, from the viewpoint of the high performance separation technique, is described. Only limited topics from the authors’ investigations, such as chiral separations by MEKC and affinity capillary electrochromatography and highly sensitive detection schemes including
on−line sample concentration techniques and the use of thermal lens microscopy in CE and MEKC, are presented.
Keywords: capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, microchip electrophoresis,chiral separation, on−line sample concentration, thermal lens microscopy.

the equations describing the relationship between the migration

1. Introduction
During last two decades, capillary electrophoresis (CE) has

time and the retention factor and resolution [6, 7]; (2) theoreticalbecome popular as a high−resolution separation method. It has

and experimental considerations of parameters affecting resolution

been recognized that CE was first introduced by Mikkers et al. [1],and separation performance [6, 7, 9−11]; (3) studies on parameters,

Jorgenson and Lukacs [2], and Hjertén [3], as an automated instru-

such as the structure of the surfactants, pH, and temperature, affect-

mental version of electrophoresis, following the earlier studies [4,ing the selectivity [7, 12]; (4) comparison of the structures of the5]. Generally only ionic or charged solutes could be analyzed bysurfactants in terms of the separation characteristics [7, 13]; (5)CE in principle. However, the development of micellar electroki-measurements of the thermodynamic parameters for the micellar

netic chromatography (MEKC) [6, 7] has successfully overcome

solubilization process [7, 14]; (6) effects of the additives to the mi-such problems. The principle of MEKC is based upon chromatog-

cellar solutions [15−17]; (7) separations of closely related com-

raphy using homogeneous solutions containing an ionic “pseudo-

pounds, e.g., phenylthiohydantoin-amino acids [13], chlorinatedstationary phase” or an ionic micelle, in which both neutral andphenols [18], and aromatic sulfides [19], (8) quantitation in MEKC

charged analytes can be separated electrophoretically. Several[20], (9) chiral separations by CE and EKC [15, 21−25], and bymodes of MEKC-like techniques or electrokinetic chromatography

capillary electrochromatography (CEC) [26−29]. Studies on devel-(EKC) [8] have been available, where some kinds of pseudo-

opments of several on-line sample concentration techniques [30−stationary phases are employed. Among these, however, MEKC is34] and on-line coupling of mass spectrometry (MS) with MEKC

still the most popular technique for the separation of small neutral(MEKC−MS) and CE (CE−MS) [35−40], and the use of thermalmolecules as well as ions by CE.

lens microscopy (TLM) as a high-sensitive detection scheme have

The author and co-workers have continuously made funda-

also been carried out [41]. Recently, the author’s interests havemental and application studies on EKC as well as CE since the in-been extended to the separation and analysis in smaller fields, or

troduction of MEKC in early 1980 s: The key subjects investigatedmicro and nano scale analytical systems, such as microchip electro-

in the early stage, for example, were as follows: (1) derivation ofphoresis (MCE) and microchip sensing device [42−49].

Tel: +81−75−383−2447
Fax: +81−75−383−2450
E−mail: otsuka@mbox.kudpc.kyoto-u.ac.jp―１―Chromatography, Vol.28 No.1 (2007)

This paper briefly overviews recent works of the author’sphy. As well as affinity CE, affinity CEC using proteins as chiralgroup mainly focused on chiral separations by CEC and novel de-selectors has been employed for enantioseparations. In affinity

tection scheme for CE or CE−TLM.

CEC, mainly following three modes are used: (1) packed CEC (P−CEC) [55,56], (2) monolithic CEC (M−CEC) [29, 57], and (3)2. Chiral separations

open tubular CEC (OT−CEC) [28, 58]. In P−CEC, preparing a frit2.1. Chiral separation by MEKC

in the capillary to dam the packing materials is sometimes trouble-

Chiral separation is one of major objectives of CE as well as

some, whereas in M−CEC the preparation of capillaries is time−

MCE and a number of successful studies has been reported [50−consuming for 2−4 days. In OT−CEC, however, the preparation of54]. As for MEKC, mainly following two modes are employed in

the stationary phase of proteins onto the inner wall of the capillarychiral separations: (1) MEKC using chiral micelles and (2) cy-

can be achieved by simple modification procedures. Although aclodextrin (CD) modified MEKC (CD−MEKC).

low phase ratio in OT−CEC, relatively shorter analysis time com-

In the former system, an ionic chiral micelle is used as a chiralpared to P−CEC and M−CEC is expected and thus, higher separa-

pseudostationary phase. When a pair of enantiomers is injected totion efficiency can be achieved in chiral separations. A short condi-

the MEKC system, each enantiomer is incorporated into the chiraltioning time, reproducible column preparation, and easy operation

micelle. However, the equilibrium constant for each enantiomer isare also advantageous in OT−CEC. The immobilization of proteins

expected to be different more or less among the enantiomeric pair;

onto the capillary surface for OT−CEC has been attained by twothat is, the degree of solubilization of each enantiomer into themethods: physical adsorption (dynamic coating) and covalent bind-chiral micelle would be different each other and hence, the differ-

ing methods. Generally, in the former method the stability of theence in the retention factor would be obtained, resulting a different

prepared capillary is not good due to a significant loss of the sta-

migration time.

tionary phase, while in the latter better stability can be expected.

While in the latter system or CD−MEKC, an ionic achiral mi-

Only a few groups, however, have reported the use of the proteinscelle, e.g., sodium dodecyl sulfate (SDS), and a neutral CD are

chemically immobilized capillaries for the chiral affinity CEC

typically used as a pseudostationary phase and a chiral selector, re-analysis.

spectively. At the time of the introduction of a pair of enantiomers
2.2.1. OT−CEC chiral separation

into the system, two major distribution equilibria are considered forthe enantiomers, namely (1) the equilibrium between the aqueous

To obtain a stable stationary phase with easy manipulations,

phase and the micelle, i.e., the micellar solubilization and (2) theavidin, a basic protein, was covalently fixed on the inside wall ofequilibrium between the aqueous phase and CD, i.e., the inclusion

the capillary by Schiff base formation reaction [59, 60] and the pre-complex formation. Each enantiomer may have a different equilib-

pared capillary was applied to the OT−CEC enantioseparation [61].

rium constant for the inclusion complex formation among the enan-

Under the optimized condition, the successful chiral separation oftiomeric pair due to the enantioselectivity of the CD. Consequently

abscisic acid was attained within 10 min, as shown in Figure 1

each enantiomer exists in the aqueous phase at a different time[61]. Effects of pH and organic additives on the OT−CEC separa-

among the enantiomeric pair, so that the time spent in the micelletion efficiency revealed that both the electrostatic and the hydro-

would be varied. In some cases, additionally, an ionic chiral mi-

phobic interactions between the analytes and avidin play an impor-celle, e.g., a bile salt, is also used as a chiral pseudostationary

tant roll in the chiral separation of these racemic acidic compounds.

phase with a CD.

The longer lifetime of the capillaries, for 50 days with over 100
chromatography

runs, in comparison with the physical adsorption method shows(CDEKC), where a CD derivative having an ionizable group isthat the loss of avidin suppressed by the immobilization throughused as a chiral pseudostationary phase, has become popular in re-

the covalent bond. The prepared capillary was useful for the MS

cent since several commercially available ionic CD derivatives

detection because of the stable immobilization of protein or lesshave appeared. Although the CDEKC technique is actually beyond

desorption from the capillary surface. The modification techniquethe field of MEKC, it is important method for enantiomer separa-can be applied to a microchannel on a quartz chip to achieve a hightion by CE.

speed electrochromatographic enantioseparation.

2.2. Chiral separation by affinity CEC

2.2.2. P−CEC chiral separation

Moreover,cyclodextrin

electrokinetic

CEC, the hybrid method of CE and HPLC, is also a useful

The use of magnetic polymer particles (MPs) was investigatedtechnique for the chiral separation owing to the high efficiency of

to fabricate a capillary for the P−CEC analysis [62]. The MPs

capillary electromigration and the high selectivity of chromatogra-

could be retained with the magnetic field in the capillary even ap-―２―

Chromatography, Vol.28 No.1 (2007)

Koji Otsuka

plying high voltage for the CE separation, so that the fritless capillary for P−CEC can be easily prepared and the length of the packed
MPs zone also be easily adjusted by changing the magnet length.
By employing the fritless avidin coated MPs packed capillary, thechiral separation of ketoprofen was successfully attained with thepacking length of 10 cm, as shown in Figure 2 [62]. Effects of themodification condition of avidin, pH of the separation solution, andthe packing length on the enantioseparation were investigated. Under the optimal condition the repeatability for the retention time of
ketoprofen was better than 1.5% in the relative standard deviationand the capillary-to-capillary reproducibility was also acceptable in
the prepared fritless capillaries.
3. Highly sensitive detection schemes
3.1. On−line sample concentration
The low concentration sensitivity in CE is an inherent defect,
which is caused by the limited volume of sample that can be injected into the capillary and the narrow optical pathlength for on
Figure 1. CEC separation of abscisic acid enantiomers using capillaries prepared with avidin concentrations of 2.0 mg/
mL. Capillary, 50 cm (effective length, 41.5 cm) × 50
µm I.D.; BGS, 10 mM phosphate buffer (pH 5.0); sample, 0.1 mg/mL abscisic acid; injection, 50 mbar, 3 s;
separation, −20 kV; detection, 230 nm; temperature, 25°C.

capillary detection in case of the absorbance detection. By using
sample stacking technique, this problem can be successfully reduced in CZE. The sample stacking phenomenon is caused by thedifferent mobilities of the target ions across a boundary, which
separates regions of high and low electric fields. Sample stacking
in MEKC, however, is not as straightforward as in CZE, since aneutral analyte is not affected by the enhanced electric field. We
have achieved several methods for on-line sample concentrations
for neutral analytes in MEKC using low conductivity solutions
containing no micelles to prepare sample solutions: (1) the hydrodynamic sample injection mode, including normal stacking mode
(NSM) [63], reversed electrode polarity stacking mode (REPSM)[64], stacking with reversed migrating micelles (SRMM) [65], andstacking using reverse migrating micelles and a water plug (SRW)[30]; (2) the electrokinetic sample injection mode, including fieldenhanced sample injection (FESI) [66] and field enhanced sample
injection with reverse migrating micelles (FESI−RMM) [67]. It has
been found that sample stacking of neutral analytes under acidic
conditions, i.e., SRMM, SRW, and FESI−RMM, offers several advantages over those in neutral conditions, i.e., NSM, REPSM, and
FESI. By using these concentration techniques, high sensitivity enhancement factors, e.g., 1000-fold increase in detection sensitivity,
are easily obtained and optimization schemes are simply encoun-

Figure 2. Enantioseparation of ketoprofen by affinity P−CEC
with avidin immobilized MPs: Capillary, 52 cm×50 µm
I.D. (30 cm to the detector); packed length, 10.0 cm;
BGS, 10 mM acetate buffer (pH 5.0); avidin concentration for immobilization, 5.0 mg/mL; detection wavelength, 200 nm; applied voltage, −10 kV; sample, 2.5µg/mL ketoprofen.

tered.
The development of sweeping has successfully introduced aremarkable enhancement of the detection sensitivity, typically as
5000 to 100000-fold increase in detectability [68, 69]. Fundamental and application studies on sweeping have been extensively reported. The combination of sweeping with stacking or sweepingwith the dynamic pH junction technique has been also investigated―３―

Chromatography, Vol.28 No.1 (2007)

to achieve further enhanced sensitivity [70−72].
3.2. Thermal lens microscopy
As one of powerful schemes improving the detection sensitivity in CE, the use of TLM has been investigated as a universal ultra−high−sensitive detector. In the TLM detection scheme, the key toachieve a higher sensitivity is to match the excitation and emission
volumes to the separation system, which will provide a low scattering and luminescence background. As well as a confocal microscopic fluorometric detection, the focusing of laser beams on thecapillary can facially align in the TLM detection. Although a simple way to make a CE−TLM system possible is the use of the oncapillary method, optical configurations of TLM become complicated since the capillary has a curved surface. The reduce of boththe sensitivity and reproducibility will be caused by the complicated refraction, reflection, and scattering of the two laser beams
on the capillary surface. To solve the problems, an interface chip
(IFChip), which has a flat surface and is connected to the separation capillary for the CE separation, has been introduced [73, 74].
The IFChip was used as a detection window for TLM, so that asufficient separation efficiency and 100-times higher detection sensitivity than that obtained with CE−UV analysis were achievedsince the complicated optical configuration by curved surface ofthe capillary was improved by using the microchannel with a semiFigure 3. Schematic illustration of the CE−IFChip system for
TLM detection.

circular shape. An extended study on the CE−IFChip−TLM system
has been carried out to make highly sensitive detection scheme
possible for the separation of nonfluorescent and neutral analytes
by the MEKC mode, in combination with sweeping to obtain further improvement of the concentration sensitivity [41]. The schematic illustration of the CE−IFChip−TLM system is shown in Figure 3, and a typical electrokinetic chromatogram of Sudan R obtained by a sweeping−MEKC condition with the IFChip−TLM system is shown in Figure 4, where dilute sample was introduced withan injection length of 258 mm under an acidic condition (pH 3.0).
The value of sensitive enhancement factor (SEFheight) was calculated by comparing the peak height obtained in the sweeping condition with that in the conventional MEKC taking into account thedilution factor regardless of the injection volume of the sample solution. The obtained SEFheight was ca. 3.9×106 under the acidic condition. Even though the sample amount injected was considered,
the concentration efficiency was estimated to be as low as 18000

Figure 4. Sweeping−MEKC−IFChip−TLM detection of Sudan R(SdR) at the concentration of 1.8×10−12M under theacidic condition. Data acquisition was started after
sample injection. BGS, 1.5×10−2M SDS (pH 3.0); sample matrix, no SDS (pH 3.0) with a conductivity similar
to that of the BGS; applied voltage, −20 kV; injection,hydrodynamically 15 cm for 1100 s (injected length,258 mm).

and hence, an ultra sensitive detection of nonfluorescent compounds was successfully achieved by sweeping−MEKC with the
IFChip−TLM system, due to both high spatial resolution of TLM(~µm) and high retention factor of neutral and hydrophobic azo
dyes. Further investigations toward the ultrahigh-sensitive detection by using TLM in both CE and MCE are still proceeded in
combination with novel separation media and detection fields.

―４―Chromatography, Vol.28 No.1 (2007)

Koji Otsuka

leagues of his studies, especially to Professors Nobuo Tanaka

4. Conclusion
The author has been studying on fundamentals and applica-(Kyoto Institute of Technology), Richard N. Zare (Stanford Uni-

tions of MEKC since its invention and thinks that MEKC has obvi-

versity) , and Takehiko Kitamori (The University of Tokyo) for

ously become one of major modes in CE. As a proof of this fact,

their intimate supports. He also thanks Dr. Fumihiko Kitagawa

Zare found all those articles having the words “Micellar Electroki-(Kyoto University) for his many efforts to the recent research sub-netic Chromatography” or “MEKC” in its title or abstract since

jects at Kyoto University.

1990, as shown in Table 1 [75]. Zare also said, “this selection does

Financial supports from the governmental foundations, includ-

not gauge the full impact of MEKC, but it does serve as an interest-

ing the Ministry of Education, Culture, Sports, Science and Tech-

ing measure”. Continuous studies on high performance microscalenology, the Japan Society for the Promotion of Science (JSPS), theseparation methods not only by MEKC but also by CE, CEC, andJapan Science and Technology Agency (JST), the Ministry of

MCE have been carried out in the author’s research groups. In thisEconomy, Trade and Industry, as well as from private foundations

focusing review, however, only his recent typical studies on CE, i.

and companies are greatly acknowledged.

e., chiral separations by affinity CEC techniques using avidin as a

Finally, the author is grateful to the Society for Chroma-chiral selector and highly sensitive detection methods for CE em-

tographic Sciences for presenting him with the 2006 Award of theploying on-line sample concentration techniques and TLM detec-

Society for Chromatographic Sciences.

tion, are briefly described.
References

At the present stage, many papers and review articles on[1]

MEKC as well as CE and MCE are available, where fundamental

Mikkers, F. E. P.; Everaerts, F. M.; Verheggen, Th. P. E. M.
J. Chromatogr. 1979, 169, 11−20.

characteristics, theoretical treatments, and applications are de[2]scribed from various points of view, so that one can easily refer toJorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298
−1302.

those references when detailed information is required.
Acknowledgments[3]

Hjertén, S. J. Chromatogr. 1983, 270, 1−6.

[4]

Hjertén, S. Chromatogr. Rev. 1967, 9, 122−219.

[5]

The author would like to express his gratitude to Professor

Virtanen, R.; Kivalo, P. Suomen Kemistilehti B 1969, 42,
182−184.

Shigeru Terabe (Graduate School of Engineering, Kyoto Univer[6]sity; Professor Emeritus, University of Hyogo) for his continuous

Terabe, S.; Otsuka, K.; Ichikawa, K.; Tsuchiya, A.; Ando, T.
Anal. Chem. 1984, 56, 111−113.

supervision and invaluable suggestions and advice. The author
greatly acknowledges to all the collaborators, coworkers, and col-[7]

Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1985, 57, 834

Table 1. Number of articles on MEKC published since 1990 ineach period a[8]Terabe, S. Trends Anal. Chem. 1989, 8, 129−134.

−841.

PeriodNumber of Articles

1990−1991
1990−1992
1990−1993
1990−1994
1990−1995
1990−1996
1990−1997
1990−1998
1990−1999
1990−2000
1990−2001
1990−2002
1990−2003
1990−2004
1990−2005

58
102
179
298
438
591
827
1022
1240
1461
1699
1957
2245
2466
2633

[9]

Terabe, S.; Utsumi, H.; Otsuka, K.; Ando, T.; Inomata, T.;
Kuze, S.; Hanaoka, Y. HRC CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1986, 9, 666−670.

[10] Terabe, S.; Otsuka, K.; Ando, T. Anal. Chem. 1989, 61, 251−260.
[11] Otsuka, K.; Terabe, S. J. Chromatogr. 1989, 480, 91−94.
[12] Otsuka, K.; Terabe, S. J. Microcol. Sep. 1989, 1, 150−154.
[13] Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 332,
219−226.
[14] Terabe, S.; Katsura, T.; Okada, Y.; Ishihama, Y.; Otsuka, K.
J. Microcol. Sep. 1993, 5, 23−33.
[15] Otsuka, K.; Terabe, S. Electrophoresis 1990, 11, 982−984.
[16] Terabe, S.; Ishihama, Y.; Nishi, H.; Fukuyama, T.; Otsuka,
K. J. Chromatogr. 1991, 545, 359−368.
[17] Otsuka, K.; Higashimori, M.; Koike, R.; Karuhaka, K.;
Okada, Y.; Terabe, S. Electrophoresis 1994, 15, 1280−
1283.

a Listed by Zare [75].

[18] Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1985, 348,―５―

Chromatography, Vol.28 No.1 (2007)

resis 2001, 22, 3509−3513.

39−47.

[43] Tachibana, Y.; Otsuka, K.; Terabe, S.; Arai, A.; Suzuki, K.;[19] Otsuka, K.; Terabe, S.; Ando, T. Bull. Chem. Soc. Jpn.

Nakamura, S. J. Chromatogr. A 2003, 1011, 181−192.

1986, 950−955.

[44] Liu, B.−F.; Sera, Y.; Matsubara, N.; Otsuka, K.; Terabe, S.

[20] Otsuka, K.; Terabe, S.; Ando, T. J. Chromatogr. 1987, 396,

Electrophoresis 2003, 24, 3260−3265.

350−354.

[45] Tachibana, Y.; Otsuka, K.; Terabe, S.; Arai, A.; Suzuki, K.;[21] Otsuka, K.; Terabe, S. J. Chromatogr., 1990, 515, 221−226.

Nakamura, S. J. Chromatogr. A 2004, 1025, 287−296.

[22] Otsuka, K.; Kawahara, J.; Tatekawa, K.; Terabe, S. J. Chro-[46] Kitagawa, F.; Aizawa, S.; Otsuka, K. Anal. Sci. 2005, 21, 61

matogr. 1991, 559, 209−214.

−65.

[23] Otsuka, K.; Terabe, S. J. Liq. Chromatogr. 1993, 16, 945−[47] Kitagawa, F.; Tsuneka, T.; Akimoto, Y.; Mizuno, J.; Shoji,953.

S.; Otsuka, K. In Micro Total Analysis Systems 2005;[24] Wu, H.−L.; Otsuka, K.; Terabe, S. J. Liq. Chromatogr. Re-

Jensen, K. F.; Han, J.; Harrison, D. J.; Voldman, J., Eds.;

lat. Technol. 1996, 19, 1567−1577.

Proceedings of µTAS 2005; TRF: San Diego, CA, USA,[25] Yamaguchi, T.; Matsubara, N.; Tanaka, Y.; Otsuka, K.; Ter-

2005; pp. 331−333.

abe, S. Chromatography 1998, 19, 15−18.

[48] Kitagawa, F.; Kubota, K.; Sueyoshi, K.; Otsuka, K. Sci.

[26] Otsuka, K.; Mikami, C.; Terabe, S. J. Chromatogr. A 2000,

Tech. Adv. Mater. 2006, 7, 558−565.

887, 457−463.

[49] Sueyoshi, K.; Nagai, H.; Wakida, S.; Nishii, J.; Kitagawa,[27] Kawamura, K.; Otsuka, K.; Terabe, S. J. Chromatogr. A

F.; Otsuka, K. Meas. Sci. Technol. 2006, 17, 3154−3161.

2001, 924, 251−257.
[28] Liu, Z.; Otsuka, K.; Terabe, S. J. Sep. Sci. 2001, 24, 17−26.

[50] Otsuka, K.; Terabe, S. In Capillary Electrophoresis Tech-[29] Liu, Z.; Otsuka, K.; Terabe, S.; Motokawa, M.; Tanaka, N.

nology; Guzman, N. A., Ed.; Chromatographic Science Series Vol. 64; Marcel Dekker: New York, 1993; Chapter 20.

Electrophoresis 2002, 23, 2973−2981.

[51] Otsuka, K.; Terabe, S. Trends Anal. Chem. 1993, 12, 125−[30] Quirino, J.P.; Otsuka, K.; Terabe, S. J. Chromatogr. B

130.

1998, 714, 29−38.

[52] Terabe, S.; Otsuka, K.; Nishi, H. J. Chromatogr. A 1994,[31] Quirino, J.P.; Terabe, S.; Otsuka, K.; Vincent, J.B.; Vigh, G.

666, 295−319.

J. Chromatogr. A 1999, 838, 3−10.

[53] Otsuka, K.; Terabe, S. J. Chromatogr. A 2000, 875, 163−[32] Otsuka, K.; Hayashibara, H.; Yamauchi, S.; Quirino, J.P.;

178.

Terabe, S. J. Chromatogr. A 1999, 853, 413−420.

[54] Otsuka, K.; Terabe, S. In Chiral Separations − Methods and[33] Kim, J.−B.; Otsuka, K.; Terabe, S. J. Chromatogr. A 2001,

Protocols; Guebitz, G.; Schmid, M. G., Eds.; Humana: To-912, 343−352.

towa, NJ, 2004; Chapter 20.

[34] Kim, J. −B.; Quirino, J. P.; Otsuka, K.; Terabe, S. J. Chro-[55] Li, S.; Lloyd, D. K. Anal. Chem. 1993, 65, 3684−3690.

matogr. A 2001, 916, 123−130.

[56] Lloyd, D.K.; Li, S.; Ryan, P. J. Chromatogr. A 1995, 694,[35] Muijselaar, P. G.; Otsuka, K.; Terabe, S. J. Chromatogr. A

285−296.

1998, 802, 3−15.

[57] Kato, M.; Sakai−Kato, K.; Matsumoto, N.; Toyo’oka, T.

[36] Tanaka, Y.; Kishimoto, Y.; Otsuka, K.; Terabe, S. J. Chro-

Anal. Chem. 2002, 74, 1915−1921.

matogr. A 1998, 817, 49−57.

[58] Liu, Z.; Zou, H.; Ye, M.; Ni, J.; Zhang, Y. Electrophoresis[37] Otsuka, K.; Smith, C. J.; Grainger, J.; Barr, J. R.; Patterson,

1999, 20, 2891−2897.

Jr., D. G.; Tanaka, N.; Terabe, S. J. Chromatogr. A 1998,[59] Walt, D.R.; Agayn, V.I. Trends Anal. Chem. 1994, 13, 425−

817, 75−81.

430.

[38] Tanaka, Y.; Kishimoto, Y.; Otsuka, K.; Terabe, S. Bunseki[60] Higashiyama, S.; Noda, M.; Kawase, M.; Yagi, K. J. Bio-

Kagaku 1998, 47, 563−569.

med. Mater. Res. 2003, 64 A , 475−482.

[39] Isoo, K.; Otsuka, K.; Terabe, S. Electrophoresis 2001, 22,[61] Kitagawa, F.; Inoue, K.; Hasegawa, T.; Kamiya, M.;

3426−3432.

Okamoto, Y.; Kawase, M.; Otsuka, K. J. Chromatogr. A[40] Tanaka, Y.; Otsuka, K.; Terabe, S. J. Pharm. Biomed. Anal.

2006, 1130, 219−226.

2003, 30, 1889−1895.

[62] Okamoto, Y.; Ikawa, Y.; Kitagawa, F.; Otsuka, K. J. Chro-[41] Kitagawa, F.; Tsuneka, T.; Akimoto, Y.; Sueyoshi, K.;matogr. A 2007, in press.

Uchiyama, K.; Hattori, A.; Otsuka, K. J. Chromatogr. A[63] Quirino, J. P.; Terabe, S. J. Chromatogr., A 1997, 781, 1192006, 1106, 36−42.

−128.

[42] Sera, Y.; Matsubara, N.; Otsuka, K.; Terabe, S. Electropho-―６―

Chromatography, Vol.28 No.1 (2007)

Koji Otsuka[71] Kim, J.−B.; Otsuka, K.; Terabe, S. J. Chromatogr. A 2001,[64] Quirino, J. P.; Terabe, S. J. Chromatogr., A 1997, 791, 255−

932, 129−137.

267.

[72] Britz−McKibbin, P.; Otsuka, K.; Terabe, S. Anal. Chem.

[65] Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 149−157.

2002, 74, 3736−3743.

[66] Quirino, J. P.; Terabe, S. J. Chromatogr., A 1998, 798, 251−[73] Uchiyama, K.; Hibara, A.; Sato, K.; Hisamoto, H.; Tokeshi,

257.

M.; Kitamori, T. Electrophoresis 2003, 24, 179−184.

[67] Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 1893−1901.

[74] Uchiyama, K.; Tokeshi, M.; Kikutani, Y.; Hattori, A.;[68] Quirino, J. P.; Terabe, S. Science 1998, 282, 465−468.
[69] Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638−1644.

Kitamori, T. Anal. Sci. 2005, 21, 49−52.

[70] Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023−1030.

[75] Zare, R. N. J. Chromatogr. A 2006, 1106, 4.

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