Focusing Review

Chromatography, Vol.23 No.1 (2002)

Focusing Review

Method Development of Enantiomer Separations by Affinity
Capillary Electrophoresis, Cyclodextrin Electrokinetic Chromatography
and Capillary Electrophoresis-Mass Spectrometry
Yoshihide Tanaka
Department of Analytical Chemistry, Nippon Boehringer Ingelheim Co. Ltd., 3-10-1, Yato, Kawanishi, Hyogo 666-0193, Japan
Received for review November 27, 2001.

Accepted December 7, 2001.

Abstract
Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic
chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study
in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 µM, without the detection problem. Charged CDs had several advantages for the enantiomer
separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective forthe formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should alsoenhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of
nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.
Keywords: enantiomer separation, capillary electrophoresis, capillary electrophoresis-mass spectrometry, partial filling technique, protein,charged cyclodextrin

Thus, CE has become an attractive analytical tool for the enanti-

1. Introduction
Many of isomeric pharmaceutical drugs frequently exhibit

omer separation during the last decade. Successful examples of ap-

some stereoselectivity for pharmacological activity and/or drug-

plications in our study, which have been obtained by CE and on-protein binding. The separation of enantiomeric mixtures into indi-line CE-mass spectrometry (CE-MS) with several chiral selectors

vidual isomers is one of the most important issues in pharmaceuti-such as proteins and cyclodextrins (CDs), are reviewed. A partial

cal developments. Although high-performance liquid chromatogra-

filling technique was employed to avoid both the deterioration ofphy (HPLC) is the most widespread for this purpose, the method

detection sensitivity of UV photometry caused by the strong UVdevelopments, such as the choice of chiral stationary phases (CSPs)

absorption of chiral selectors and the introduction of nonvolatileand the optimization of separation conditions, are time-consuming.

chiral selectors into the MS instrument.

On the contrary, capillary electrophoresis (CE) has high resolving
2. Theory

power and requires simple operating procedures. It is simply andrapidly performed to choose a chiral selector for the relevant enan-

Concerning the enantiomer separation by CE, Wren and Rowetiomer separation. Only small amounts of the chiral selectors are[1] firstly proposed a simple theoretical model using CD as a chiralrequired for the method developments in comparison to HPLC.

additive:Tel, +81-727-90-2322; fax, +81-727-93-8399;―１
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Chromatography, Vol.23 No.1 (2002)

capillary wall. CDs and their derivatives have been widely accepted as chiral selectors due to their characteristics of the highwater solubility, the weak UV absorption and the excellent chiral
discrimination toward various aromatic enantiomers [2]. However,
all enantiomers are not always separated with native CDs or theirderivatives. In HPLC, various kinds of CSPs have been developed
to separate numerous different enantiomers. Among the CSPs,
protein-based columns have been frequently employed because of
their versatility and broad applicability. In the early 1990s, the usFigure 1. Schematic illustration of the migration behavior in theseparation of a cationic analyte using an anionic CD.

ability of the proteins in CE had attracted the interest of several research groups [2]. We also started the study using some proteinssuch as egg white avidin [3, 4] and ovomucoid [4] in 1993. Be-

A + CD
µs

ACD
µCD

K1 =

[ACD]
[A] [CD]

cause the chiral separations are based on the differential affinity of(1)

the proteins to individual enantiomers, this separation mode is par-

[BCD]
BCD
K2 =
(2)
B + CD
[B]
[CD]
µCD
µs
where A and B are a pair of enantiomers having identical electro-

ticularly named “affinity capillary electrophoresis” (affinity CE). In
this mode, a use of untreated fused-silica capillaries often brought
about the deterioration of peak shapes of analytes due to the ad-phoretic mobilities, µs; and ACD and BCD are diastereomeric in-sorption of the proteins and/or analytes on the inner wall of theclusion complexes having approximately identical apparent electro-

capillary. Therefore, we used a linear polyacrylamide-coated capil-

phoretic mobilities, µCD. The formation constants for inclusion

lary in affinity CE to solve the adsorption problem.

complexes, K1 and K2, are defined by the concentration of each spe3.1. Conventional methodcies such as [A], [CD] and [ACD] as given in equations (1) and (2).
A schematic diagram of the migration behavior of an anionic CD

The most common procedure of chiral separations in CE is toand a cationic analyte is shown in Figure 1. In order to obtain thefill a capillary with a separation solution containing a chiral selec-

enantiomer separation, it is required that both pairs of µs and µCD,

tor. The separation conditions are commonly optimized by chang-and of K1 and K2 have different values. When the equilibrium be-

ing the concentration of the chiral selector and/or adding an organic

tween the free and bound forms is quickly established, the apparent

modifier to the separation solution. Although the enantioselectivity

electrophoretic mobility of A, µapp,A is expressed:

should be mostly improved by increasing the protein concentration
as shown in Figure 2, it was not preferable for high sensitive detec-

[ACD]
+
µ
（[A] +[A][ACD]）µ （[A] + [ACD]）µapp,A = µeo +sCDµs + µCDK1 [CD]= µeo +
1 + K1 [CD]tion due to the background UV absorption of the protein solution.
The effective concentration of the protein in HPLC columns was(3)reported at mM level [5], whereas most applications in affinity CE

where µeo is the electroosmotic mobility. The mobility difference

should be performed at the protein concentrations of lower thanbetween the enantiomers, ∆µ, is written as:

100 µM [3, 4]. Only a few successful separations were achieved∆µ =(µs−µCD) (K2−K1) [CD]1 + (K1 + K2) [CD] + K1K2 [CD]2under these low concentrations, suggesting the necessity of solving(4)

the detection problem to increase the number of successful chiralseparations by affinity CE.

From the above equation, the optimum concentration of CD thatgives the maximum ∆µ, [CD]opt, is mathematically obtained:[CD]opt =1(K1K2)1/2

3.2. Partial filling technique(5)

The partial filling technique was firstly introduced byConsequently, the enantiomeric resolution is enhanced by increas-

Valtcheva et al. [6], and we modified the method to be able to per-ing the concentration of chiral selector below the [CD]opt. The

form the continuous and automatic operations using a conventional

above theoretical model has been applied to the other chiral selec-

CE instrument [7]. The technique has been useful to solve thetors, such as proteins and macrocyclic antibiotics.

above-mentioned problem, i.e. the deterioration of detection sensitivity under high protein concentration solutions. The principle isschematically illustrated in Figure 3. After a capillary is washed

3. Affinity capillary electrophoresis using proteins
In CE, most chiral selectors are employed as a solution in awith a running buffer without chiral selectors, a portion of the cap-

background solution unless they are immobilized in a gel or on

illary is filled with a separation solution including a protein by―１
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Chromatography, Vol.23 No.1 (2002)

Yoshihide Tanaka

Figure 2. Effect of the concentration of AVI on the separation of racemic vanilmandelic acid [3]. Instrument, BioFocus 3000; separation
solution, (a) 0 µM, (b) 10 µM, (c) 25 µM, (d) 50 µM AVI dissolved in 50 mM phosphate buffer (pH 4.0); applied voltage, −12
kV; sample injection, 6.9 kPa × 2s (sample concentration, 50 µg/mL); capillary temperature, 25°
C; detection wavelength, 240
nm.

pressurization at 6.9 kPa for 190s. When a coated capillary of 50µm I.D. and 36 cm in total length (31.5 cm to the detector) is em-

(a)

(b)

(c)

(d)

ployed, the zone length of the separation solution in the capillary isapproximately 27 cm. Therefore, the rest portion of the capillary
including the detection window is filled with the running buffer[Figure 3 (a)]. A sample solution is introduced at the end of thecapillary filled with the separation solution [Figure 3 (b)]. Bothends of the capillary are dipped into the running buffer, and a constant voltage is applied for the separation. The partial filling technique has to be performed under the conditions where the protein
does not migrate toward the detection end of the capillary. First,

［−］

［＋］

［−］

［＋］

the enantiomer separation occurs when the enantiomers migrate
through the separation solution zone [Figure 3 (c)], and thereafter
Figure 3. Schematic illustration of the partial filling technique.
= separation solution,= running buffer,= sample solution,= detection window.

both enantiomers migrate through the running buffer zone at an
identical velocity. The analytes can be detected without interference with the high background UV absorption of the protein solu-―１５―Chromatography, Vol.23 No.1 (2002)

were commercially available in those days. A strong electrostaticinteraction or a large binding constant between charged CDs andanalytes having the opposite charge is expected. In addition, thelarge difference in the electrophoretic mobilities between free analytes and complexed analytes should be advantageous as given inequation (4). We attempted the separations of 50 basic racematesusing anionic CDs as listed in Table 2 [10]. First, each anionic CD
was dissolved in a 50 mM phosphate buffer (pH 5.0) as a separation solution. Owing to the short migration times caused by thestrong EOF, insufficient separations were obtained using an untreated fused-silica capillary. In this study, therefore, a linear
polyacrylamide-coated capillary was used to suppress the EOF.
The coated capillary was also effective in preventing the cationic

Figure 4. Enantiomer separation of racemic vanilmandelic acid by
the partial filling technique with AVI [9]. Runningbuffer, 50 mM phosphate buffer (pH 4.0), separation solution, AVI dissolved in the running buffer (zone injection, 6.9 kPa × 190s); detection wavelength, 210 nm.
Other conditions are as in Figure 2.

analytes from the adsorption on the capillary wall. As given in Table 3, 40 racemates of 50 basic racemates were successfully separated with Rs > 1.2 by choosing a suitable type and concentrationof the anionic CD. The basic analyte is protonated under weakly
acidic conditions, and hence migrates toward the cathode when it isfree from the CD. On the other hand, the anionic CD and the ana-tion [Figure 3 (d)]. As shown in Figure 4, the drift of baseline islyte included by the anionic CD migrate toward the anode becausesignificantly suppressed even if absorbance at 210 nm is employed.

the EOF is almost completely suppressed. The migration direction

A number of enantiomer separations, which may be equivalent toof the analyte depends on the values of µs, µCD and K as given by

those obtained using protein-based CSPs in HPLC, have been ob-equation (3). Therefore, the cationic analyte will migrate toward

tained by the partial filling technique with a high protein concentra-

the anode if it is strongly included into the anionic CD. Figure 6

tions, such as 500 µM, as given in Table 1 [7-9].

shows two typical electropherograms, one is detection at the anodic
side and another is at the cathode side. Most analytes were better
resolved at a low pH and with a low CD concentration because the3.3. Effects of biotin binding to avidin on enantioselectivity
Although egg white avidin (AVI) showed excellent enantiose-

positive charge on the basic analyte decreases with an increase inlectivity towards many acidic racemates, it was not known whether

pH. For the separation of azelastine racemates using β-CD-

the enantioselectivity arose from the interaction between AVI andphosphate sodium salt (β-CD-Phos), however, no resolution was

analytes on the biotin-binding sites. We investigated the effect ofobserved at pH 5.0 and hence the CD was dissolved in a phosphate

the formation of AVI-biotin complex on enantioselectivity [9]. Fig-

buffer at pH 7.0 to obtain the successful separation. It was certain

ure 5 shows the change in the enantiomer separation of racemic 4-that only the non-dissociated forms of the enantiomers interacted

fluoromandelic acid by the addition of D-biotin to the separation

selectively with β-CD-Phos.

solution. Since the modification in the three-dimensional structure

The analytes were classified into three groups according to theof AVI caused by the biotin binding was not well known, it wasconcentration of the CD and the migration behavior as given in Ta-

not concluded that the enantiomers are recognized at the biotin

ble 3: (I) migrate toward the cathode with a low CD concentration;binding sites on AVI from these results. However, it was obvious(II) migrate toward anode with a relatively high CD concentration;that the enantioselectivity was drastically changed. When D-biotin(III) migrate toward the cathode with a high CD concentration. The

was added to the separation solution at the molar ratio of 1:4/AVI:

enantiomers of the group I have relatively large differences be-

D-biotin, not only the enantioselectivity but also the interaction to

tween K1 and K2, and thus easily resolve at low CD concentrations.

the analytes was almost lost [9].

However, the K -values of the analytes should be intermediate between those in the group II and group III. The enantiomers of the

4. Capillary electrophoresis using charged cyclodextrinsgroup II have large K -values and the analytes migrate as the com-

4.1. Use of anionic CDs for the separation of basic and neutral

plex toward the anode. Increasing the CD concentration is not ef-

enantiomers

fective to enhance the resolution, because the apparent mobilities

Neutral CDs had been commonly used for the enantiomerof both enantiomers approach the maximum value or the mobility

separations by CE in the early 1990s [2]. Several charged CDsof the CD complexes, and the difference in the mobilities will not―１
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Chromatography, Vol.23 No.1 (2002)

Yoshihide Tanaka

Table 1. Enantiomer separation of racemates by the partial filling technique with proteins [7-9].

increase. On the contrary, decreasing the CD concentration causes

useful for the enantiomeric separations of the group III. The elec-

a decrease in the apparent mobilities, and will increase resolution.

trostatic interaction will enhance the complex formation and a highSome successful separations, e.g. aminoglutethimide and tri-

concentration of the chiral selector can be easily obtained because

hexyphenidyl using β-CD-SBE(IV), were obtained by decreasingof the high solubility of the charged CDs.

the CD concentration. Although we tried to inverse the migration

One limitation of neutral CDs as chiral selectors is that neutral

direction of the analytes to detect at the cathodic side by decreasing

racemates cannot be resolved unless a different discrimination

the CD concentration, the concentration had to be less than 0.05

mechanism such as ionic micellar solubilization is added [11]. The

mM and the enantiomer separations were nearly lost. The enanti-

use of charged CDs offered new possibilities to separate the neutral

omers of the group III have relatively small K -value and the differ-

racemates. These separations were performed using an untreatedence between K1 and K2 is small. The anionic CDs are particularly

capillary and the EOF was used as a driving force for the neutral―１７―Chromatography, Vol.23 No.1 (2002)

Figure 5. Effect of biotin binding to AVI on the separation of racemic 4-fluoromandelic acid [9]. Running buffer, 50 mM phosphate buffer(pH 4.0); separation solution, 100 µM AVI in the running buffer containing (a) 0 µM, (b) 100 µM, (c) 200 µM, (d) 400 µM Dbiotin. Other conditions are as in Figure 4.

Table 2. Description of charged CD derivatives used.

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Chromatography, Vol.23 No.1 (2002)

Yoshihide Tanaka

Table 3. Successful separation of basic enantiomers using anionic CDs [10].

analytes. The migration times and the resolution were affected bystant, K , is obtained from the following equations when it is as-

the change in the EOF velocity. Successful enantiomer separations

sumed that µeo is negligible.

are listed in Table 4.

µs−µapp= K [CD]µapp−µCD(6)4.2. Relationship between the separation conditions and binding

1
1
1
1
=
×
+
µapp−µs (µCD−µs) K [CD] µCD−µs(7)constants
In order to support the three classifications based on the dif-As shown in Figure 7, they were calculated from the slopes of each

ference in binding constants as given in Table 3, the binding con-straight line according to the least-square method. However, four

stants were measured for three racemates, clorprenaline, pri-points should be noted in this experiment: (i) although a single lot

maquine, and sulpiride, chosen from each group. The binding con-of β-CD-Phos was employed, it consisted of a mixture of the CD―１９―Chromatography, Vol.23 No.1 (2002)

Figure 6. Representative electropherograms of the chiral separation of basic enantiomers by CE with an anionic CD derivative [10]. Samples,(a) racemic trimebutine (50 µg/mL), (b) racemic trimipramine (50 µg/mL); sample injection, 6.9 kPa × 2s; instrument, BioFocus
3000; capillary, 36 cm (31.5 cm to the detector) × 50 µm polyacrylamide-coated capillary; separation solution, 5 mM CM-γ-CD in
50 mM phosphate buffer (pH 5.0); migrate direction, (a) toward the cathode, (b) toward the anode; applied voltage, (a) 12 kV, (b)−12 kV; capillary temperature, 20°
C; detection wavelength, 210 nm.

Table 4. Successful separation of neutral enantiomers with anionic CDs [10].

K2 = 2.37 × 102 M−1

derivatives having different degrees of substitution; (ii) although

α = 1.0[CD] was not equal to the added CD concentration, but it was notwhere α is K2/K1. The (α-value for the enantiomers of sulpiride wascorrected; (iii) the EOF was not completely suppressed and hencecalculated to be 1.004 with the binding constants.

the absolute mobility could not be obtained. (iv) viscosity changes
with increasing [CD] were not taken into account. The rough bind-

4.3 Comparison between anionic CDs and neutral CDs

ing constants were given as follow:
Clorprenaline (Group I)

In order to compare the anionic CDs with neutral CDs, we−12

K1 = 2.4 × 10 M

K2 = 4.4 × 102 M−1Primaquine

10 mM β-CD and 7 M urea, 10-40 mM heptakis(2,6-di-O -methyl)-−13β-CD (DM-β-CD), 10-20 mM heptakis(2,3,6-tri-O -methyl)-β-CD(Group II) K1 = 1.7 × 10 M

K2 = 2.1 × 103 M−1
Sulpiride

tried chiral separations of basic racemates using five neutral CDs:
α = 1.82(TM-β-CD), 10-40 mM hydroxypropyl-β-CD and 10-40 mM γ-CD

α = 1.2−1[10]. Most racemates that were successfully separated using an an-(Group III) K1 = 2.36 × 10 M

―２０―

Chromatography, Vol.23 No.1 (2002)

Yoshihide Tanaka

ionic CD were also successfully separated using a 50 mM phos-mM DM-β-CD. Although relatively high concentrations as high asphate buffer (pH 5.0) containing one of the neutral CDs. However,

5 mM were necessary for the separation of most above mentioned

eleven racemates, acebutolol, bisoprolol, bunitrolol, chlor-

racemates, they were all successfully resolved using charged CDs.

mezanone, meclizine, metoprolol, nebracetam, oxyphencyclimine,

However, these concentrations were much lower than those em-

sulconazole, sulpiride and trimipramine, could not be resolved atployed for neutral CD derivatives. The use of anionic CDs was

all in our experiments with the neutral CDs. Furthermore, the sepa-

considerably effective for the enantiomer separation of basic race-

ration of eight racemates, atenolol, azelastine, ketamine, metaneph-mates.

rine, nicardipine, pindolol, salbutamol and thioridazine were insuf4.4. Use of cationic and amphoteric CDs for the separation officient even with 40 mM DM-β-CD. The complete separation ofacidic enantiomers

salbutamol racemate was reported by Rogan et al. [12] using 112

Because it was expected that cationic CDs showed high enan−1

tioselectivity to anionic enantiomers as the anionic CDs did to ba-−1

Figure 7. Plot of (µapp−µs) versus [CD] for the calculation of
the binding constants with β-CD-Phos [10]. Samples, (a)clorprenaline, (b) primaquine, (c) sulpiride. Conditions,see Table 3.

sic racemates, we performed several enantiomer separations ofacidic racemates using a commercial cationic β-CD derivative having quaternary ammonium groups (QA-β-CD). QA-β-CD is positively charged over the wide range of pH in comparison with thecationic CDs having amino groups, and is practically useful for theenantiomer separations by CE. Successful enantiomer separations
are listed in Table 5. The enantioselectivity of QA-β-CD was compared with several neutral CDs. As the results, three racemates
which were not resolved with neutral CDs, i.e., racemic (cis, trans)-abscisic acid, racemic 4-bromomandelic acid and racemic
chrysanthemum-monocarboxylic acid, were separated completely
with QA-β-CD [13].
An amphoteric β-CD derivative (AM-β-CD) having both quaternary ammonium groups and carboxyl groups was also commercially available. Because the degrees of the substitutions are equal
between cationic and anionic substituents, the amphoteric CD has

Table 5. Successful separation of acidic enantiomers using cationic and amphoteric CDs [13].

―２１―Chromatography, Vol.23 No.1 (2002)

no net charge in a buffer solution unless the pH is extremely low ormass. In addition, some analytes having no strong UV absorption

high. AM-β-CD showed the different enantioselectivity as compare

can be detected by MS detection. Concerning the enantiomer sepa-

with the other CDs, e.g. racemic 4-chloromandelic acid was sepa-

rations by CE-MS, nonvolatile components, such as chiral addi-

rated completely only with AM-β-CD [13].

tives and nonvolatile electrolytes, contaminated the ESI nozzle of
the CE-MS interface and/or the orifice plate during the run. They
may also cause a decrease in ionization efficiency of the analytes at

5. Enantiomer separation by capillary electrophoresis-mass

the ESI interface. Furthermore, the nonvolatile components arespectrometry
We have performed several application studies by CE-MS,

blown into the orifice plate when a capillary is washed with a sepa-that is, composition analysis of charged CDs [14] and ionic surfac-

ration solution prior to the run. As an electrolyte, either ammonium

tants [15] by capillary zone electrophoresis-MS (CZE-MS), a si-

formate or ammonium acetate buffer is usually employed. In ordermultaneous determination of charged and neutral analytes by CZE-

to avoid the introduction of the chiral selector into the interface, we

MS, separations and detections by EKC-MS using sodium dodecyl

employed the partial filling technique described as Section 3.2. The

sulfate, β-CD-SBE(IV) and 4-sulfonated calix[6]arene as pseudo-

separation conditions as listed in Table 1 were employed for CE-

stationary phases [16], and enantiomer separations by CE-MS [17,MS by changing the buffer electrolytes from sodium phosphate to18]. The electrospray ionization (ESI) interface is employed in theammonium acetate, and enantiomer separations were successfully

above mentioned experiments. This review article is focused on theobtained as shown in Figure 8. The optical purity test is one of theenantiomer separations by CE-MS. Coupling of CE with MS detec-

important items in chiral separation. The determination of opticaltion offers several advantages over the UV detection method. The

purity of commercial camphorsulfonic acid enantiomers was per-MS detector has relatively high sensitivity as compared to the con-formed. The enantiomeric separation was achieved using DM-β-ventional UV detector, and structural information of the analytes is

CD as a chiral selector. From 1% to 2% of the enantiomeric impu-

also obtained simultaneously on the separation. Even if some inter-rity was easily detected as shown in Figure 9.

fering compounds are co-eluted, target analytes are separately de6. Conclusion

tected with high sensitivity according to the specific molecularDuring the last decade, CE has become a useful analytical toolfor chiral separations through the numerous efforts by many researchers. We believe that our investigations have contributed tothe progress of chiral separations by CE. In this review article,
many successful enantiomer separations are demonstrated. The excellent results suggest the wide application range for chiral separa-

Figure 8. Enantiomer separation of (a) racemic ibuprofen, (b) racemic ketoprofen and (c) racemic warfarin by affinity CEMS with AVI [17]. Capillary, 50 µm i.d. × 80 cm
polyacrylamide-coated capillary; running buffer, (a), (b)40 mM ammonium acetate buffer (pH 6.0) containing
10% (v/v) 2-propanol, (c) 40 mM ammonium acetate
buffer (pH 6.0) containing 10% (v/v) ethanol; separation
solution, 100 µM AVI in the running buffer; sheath liquid, a mixture of the running buffer and methanol (1:1),
5 µL/min; voltage, −30−(−4.5) kV; detection, (a) m/z
205.0, (b) m/z 253.0 and (c) m/z 306.6 (negative ionmode).

Figure 9. Optical purity test of camphorsulfonic acid enantiomers[17]. Conditions: sample solution, (a) (R) -(+)camphorsulfonic acid enantiomer and (b) (S) -(−)camphorsulfonic acid enantiomer in water (100 µg/mL);
running buffer, 40 mM ammonium formate buffer (pH4.0); separation solution, 50 mM DM-β-CD in the running buffer; detection, m/z 230.8 (negative ion mode).
Other conditions are as in Figure 8.

―２２―Chromatography, Vol.23 No.1 (2002)

Yoshihide Tanaka[2]

tions by CE as well as conventional HPLC method. The method

Terabe, S.; Otsuka, K.; Nishi, H. J. Chromatogr. A 1994,
666, 295.

development for separation conditions can be also performed easily[3]

in a short-time.

Tanaka, Y.; Matsubara, N.; Terabe, S. Electrophoresis 1994,
15, 848.

Coupling of CE with MS detection offers several advantages[4]over the UV detection method. However, nonvolatile chiral addi-

Terabe, S.; Ozaki, H.; Tanaka, Y. J. Chin. Chem. Soc. 1994,
41, 251.

tives such as proteins and CDs are incompatible with either ESI in[5]terface or MS instrument in CE-MS. The partial filling technique

Ahmed, A.; Ibrahim, H.; Pastoré, F.; Lloyd, D. K. Anal.
Chem. 1996, 68, 3270.

has been practically useful to solve the problem. Although several[6]

applications are introduced in this paper, the type of chiral selectors

Valtcheva, L.; Mohammad, J.; Pettersson, G.; Hjertén, S. J.
Chromatogr. 1993, 638, 263.

does not restrict the MS detection by the partial filling technique. A
number of applications of enantiomer separations have been al-[7]

Tanaka, Y.; Terabe, S. J. Chromatogr. A 1995, 694, 277.

ready reported using UV detection, and the separation conditions[8]

Tanaka, Y.; Terabe, S. Chromatographia 1997, 44, 119.

should be transferred to CE-MS by changing the buffer electrolyte[9]

Tanaka, Y.; Terabe, S. Chromatographia 1999, 49, 489.

from the nonvolatile species to volatile one. CE-MS combined with

[10] Tanaka, Y.; Yanagawa, M.; Terabe, S. J. High Resoult.
Chromatogr. 1996, 19, 421.

the partial filling technique can be applied to a wide range of the[11] Terabe, S.; Ozaki, H.; Otsuka, K.; Ando, T. J. Chromatogr.

separations.

1985, 332, 211.
[12] Rogan, M. M.; Altria, K. D.; Goodall, D. M. Electrophoresis

Acknowledgement

1994, 15, 808.

Most of these studies were performed at Faculty of Science,
Himeji Institute of Technology, and the author is very grateful to[13] Tanaka, Y; Terabe, S. J. Chromatogr. A 1997, 781, 151.

Professor S. Terabe, Dr. K. Otsuka and the members of the Ter-

[14] Tanaka, Y.; Kishimoto, Y.; Terabe, S. Anal. Sci. 1998, 14,383.

abe’s laboratory including the graduates. The author is also grateful[15] Tanaka, Y.; Kishimoto, Y.; Otsuka, K.; Terabe, S. Bun-for many useful suggestions to Professor T. Nakagawa in Graduate

sekikagaku 1998, 47, 563.

School of Pharmaceutical Sciences, Kyoto University. Some pro-[16] Tanaka, Y.; Kishimoto, Y.; Otsuka, K.; Terabe, S. J. Chro-tenis and racemic samples were kindly gifted by Dr. Y. Ishihama

matogr. A 1998, 817, 49.

and Eisai Co., Dr. H. Nishi and Tanabe Seiyaku Co., Dr. H. Wada[17] Tanaka, Y.; Kishimoto, Y.; Terabe, S. J. Chromatogr. A

and Shinwa Chemical Co., and Professor J. Stobaugh.

1998, 802, 83.
[18] Tanaka, Y.; Otsuka, K.; Terabe, S. J. Chromatogr. A 2000,

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