Focussing Review

Chromatography, Vol.22 No.3 (2001)

Focussing Review

Enantiomeric Separation by CEC Using Chiral Stationary Phases
Masaru Kato* and Toshimasa Toyo’oka
University of Shizuoka, School of Pharmaceutical Sciences 52-1, Yada, Shizuoka, Shizuoka, 422-8526, Japan
Received for review June１１, 200１.

Accepted September 2５, 200１.

Abstract
Capillary electrochromatography (CEC) is now a promising technique for enantiomeric separation, because of the high separation efficiencyof the technique. In the last few years, many chiral stationary phases (CSPs) were prepared for CEC and acidic, neutral, and basic enantiomers
were separated by these CSPs. These CSPs are classified into three types : (I) open tubular capillary, (II) packed capillary and (III) monolithic
capillary. In this review, we evaluate these types of CSPs and compared separation conditions (inner diameter of capillary and mobile phase)and data (theoretical plate number, separation factor and resolution) by these CSPs.
Keywords: capillary electrochromatography, enantiomeric separation, chiral stationary phasetion modes in detail (10).

1. Introduction
Capillary electrochromatography (CEC) is a hybrid techniqueUsing CSPs is the most popular mode for enantiomeric sepa-of high performance liquid chromatography (HPLC) and capillaryration in HPLC. There are CSPs which are modified with cy-

electrophoresis (CE). CEC shows good separation efficiency be-clodextrins (CDs), modified CDs, Pirkle type, macrocyclic antibi-cause it uses electroosmotic flow (EOF) for pumping the mobile

otics, proteins, cellulose derivatives, etc. These CSPs, well known

phase and can separate both charged and uncharged compounds via

in HPLC, can also be utilized for packing material for CEC. For

electrophoresis and chromatographic separation. These principles

chiral mobile phase additive mode, the solubility of chiral selectorof CEC could be a main driving force for application in many re-in the running buffer and the absorbance of chiral selector at the

search fields, including enantiomeric separation. Reviews havedetection wavelength are critical. CSPs do not need to use a chiralbeen published about CEC and its applications (1-8).

selector in the mobile phase and, thus, have no detection problems.

Separation of enantiomers is very important because some

Another advantage in CEC is the amount of chiral selector for pre-enantiomers show completely different biological activities thanparing a separation column, one mg ; this is about 100 times lesstheir optical isomers (9). For example, thalidomide is a sedative

than that for HPLC. Some chiral selectors are expensive because

drug, but S -(-)-thalidomide is teratogenic. Another example : R -(+)

they are difficult to synthesize. In these cases, enantiomeric separa--limonene smells like orange, but S -(-)-limonene smells liketion is a suitable application field of CEC. Three different ap-

lemon. Thus, separation of enantiomers is required to clarify theproaches are used for preparation of separation column in CEC : (I)biological activity of each isomer. However, it is difficult to sepa-

open tubular capillary, (II) packed capillary and (III) monolithic

rate enantiomers, because they show exactly the same chemical andcapillary.

physical properties except for optical rotation. CEC is a suitable

In this review, we mainly introduce some recent progress in theseparation technique to separate enantiomers, because of its excel-

field of enantiomeric separation using packed and monolithic capil-

lent separation efficiency.

lary, some important papers about open tubular capillary were also

Enatiomeric separation by chromatography, including CEC,

introduced. The kinds of chiral selectors and the parameters affecting

can be performed in three modes : (I) diastereomer formations, (II)the separation on CSPs are summarized on Table 1. We do not dis-

adding chiral selectors to the mobile phase and (III) using chiralcuss molecularly imprinted polymer (MIP) based CSP, because

stationary phases (CSPs). Reviews and books explain these separa-

many reviews have already been published (11-13) on this subject.

*Corresponding author : Tel, +81-54-264-5654 ; fax, +81-54-264-5593 ;e-mail, daikato@u-shizuoka-ken.ac.jp―１５９―

Chromatography, Vol.22 No.3 (2001)

Table 1. Chiral selectors and the separation data by CEC.

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2. Type of capillary

which reduces the free silanol groups. The treatment reduces not

2.1 Open tubular capillary

only interactions between silanol and analyte, but also the EOF,

Open tubular capillary is coated by the chiral selectors physi-

which means separation time is extended. Some specially designed

cally or chemically onto the internal capillary wall and there is no

packing particles based on ion-exchange phase, organic polymer,

packing material inside the capillary. The first report of enanti-

sol-gel etc. have been developed to promote the EOF (16). A seri-

omeric separation using open tubular CEC (o-CEC) was describedous drawback of p-CEC is the difficulty of frit fabrication (frits

by Mayer and Schurig in 1992 (14). They coated the capillary wall

prevent packing particles from flowing away). Frit fabrication with

with permethyl-β-CD. The main drawback of o-CEC is low sample

a good repeatability, permeability and durability is technically dif-loading capacity, due to the low surface area of o-CEC (inner di-

ficult, because the inner diameter of capillary is narrow (100-50

ameter of an o-CEC capillary is less than 50 µm and the thicknessµm) and the frit length is very short (1-2 mm). Furthermore, the fritof coating is less than 1 µm). By etching the internal wall of theitself disturbs separation efficiency by encouraging bubble forma-

capillary, the surface area increased up to 1,000- fold and reducetion or by disturbing the flow profile of the mobile phase at the in-the loading problem (15).

terface between the packed section and the frit (17-19). Bubbles
lead to increase of baseline noise, and sometimes current breaks
down which stop EOF. To prevent bubble formation, the separation

2.2 Packed capillary
In packed CEC (p-CEC), the capillary is filled with chiralcan be performed under a pressurized condition, in which both inlet

modified particles, many of which are also used as packing parti-

and outlet vials are pressurized by gas. The pressurizing systemcles for HPLC columns. p-CEC is the most common CEC mode

also helps to compensate for the slow EOF (20). Although the sys-and numerous numbers of commercially available LC packing ma-

tem is useful, the frit still deteriorates the separation efficiency. To

terials with different selectivities are applicable. Packing particles

overcome these frit problems, another mode of CEC, that is, m-for HPLC columns, however, suffer an end-capping treatment,

CEC was developed.

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having six, seven and eight glucopyranose units, respectively.

2.3 Monolithic capillary
m-CEC consists of a single piece of porous solid packing ma-

Guttman et al. reported the first enantiomeric separation using p-

terial, without a frit. Gusev (21) defines this “monolithic stationary

CEC with immobilized CDs in 1988 (28). In this study, poly-phases” as “a continuous unitary porous structure prepared by inacrylamide gel and β- or γ-CD were packed inside the capillary.

situ polymerization or consolidation inside the column tubing and,

These discharged compounds does not cause fast EOF, hence

if necessary, the surface is functionalized to convert it into a sorb-

acidic analytes migrate toward the anode by electrophoresis withent with the desired chromatographic binding properties.” Theforming complexes with β- or γ-CD. Dansyl-Ser enantiomers weremonolithic structure is fixed to a capillary by chemical or physicalseparated using this technique (α = 1.12, N = 100,000/m).

interaction, which prevents it from being pushed out from the cap-

Some o-CECs were prepared using CDs and modified CDs.

illary by EOF or electrophoretic forces. There are two main meth-

These reports compared CEC with other separation methods (GC,ods of m-CEC preparation as follows :HPLC, OTLC, SFC) using the same capillary (29-34). Li and(a) The monolithic structure is prepared by co-polymerization

Lloyd reported enantiomeric separations of neutral drugs andof a homogeneous mixture of chiral selector and monomer

amino acid derivatives using p-CEC packed with β-CD modified(acrylamide or methacrylate). After polymerization, chiral recogni-

particles (35). The separation efficiency is better than that of β-CDtion is achieved either through (1) molecular recognition of ana-

additives in the mobile phase. Leliévre et al. used hydroxypropyl-

lytes by the chiral selector, or (2) physical recognition of analytes

β-CD as a chiral selector (36). Separation efficiency of p-CEC

in the cavities remaining throughout the monolithic. The latter,

packed with the chiral selector was compared with that of ODS

which is the basis for MIP separations, takes advantage of highly

packed p-CEC with the chiral selector additive in the mobile phase.

selective spatial recognition properties of the cavities, which simi-

The latter method achieved higher selectivity and resolution in alar to that of antibodies or receptors.

shorter analysis time. Zhang et al. also used hydroxypropyl-β-CD
as a chiral selector for p-CEC (37). They packed particles with sul-(b) The monolithic structure is prepared from slurry solution

fate and separated acidic compounds within a short time.

of CSP and monomer. After polymerization, CSP is encapsulated

Schurig and co-workers used permethyl-β-CD as a chiral se-by porous polymer and is fixed within capillary.
These structural features do not cause frit problems. Further-

lector for enantiomeric separation of mephobarbital on both p-CEC

more, m-CEC is prepared in one step using in situ polymerizationand m-CEC (20, 38, 39). m-CEC obtained better efficiency than p-

inside the capillary ; hence, preparation of m-CEC is easier than p-

CEC. In another example of m-CEC, Végvári et al. prepared m-

CEC. A serious problem in m-CEC is the limited choices of mono-

CEC from acrylamide gel containing 2-hydroxy-3-allyloxy-propyl-

mer. The monolithic structure needs to be porous, robust and stableβ-CD as a chiral selector (40). They separated acidic, neutral andand it also needs to be charged for EOF. Despite this problem, m-

basic drugs using m-CEC. Koide and Ueno prepared m-CEC com-

CEC is a popular trend in CEC.

posed of acrylamide gel and allyl carbamoylated β-CD (41-45). Al-

There are several new trends in CEC research. One is the use

lyl carbamoylated β-CD was trapped inside the gel by chemical orof non-aqueous mobile phases. Many CSPs in HPLC obtained suc-

physical interactions. Acidic, neutral and basic drugs were sepa-

cessful enantiomeric separation in non-aqueous mobile phases,

rated enantiomerically by m-CEC and mephobarbital showed asuch as ethanol or mixtures of hexane and iso-propanol (22, 23).

good enantiomeric separation (α = 1.03, N = 224,000/m).

Non-aqueous mobile phases have also been used in CEC. This
3.2 Small molecules (amino acid derivatives and Pirkle type)

method is attractive when solubility or stability problems exist inaqueous buffers, but run-to-run reproducibility problems still needSome amino acid derivatives or drug derivatives are used as

to be solved (24). The other trends are hyphenation technology ofchiral selectors in HPLC. These chiral selectors make it easy to re-

CEC with mass spectrometry or CEC separation system on a chip.

verse the elution order of enantiomers by changing a configuration

The details of these new trends have been reported in some papersof the chiral selectors. Wolf et al. modified 3 µm silica particles

including this special issue (25-27).

with (S )-naproxen and (3 R , 4 S )-Whelk-O for p-CEC, and separated more than 30 neutral compounds (46, 47). Enantiomeric sepa-

3. Chiral selectors

rationof

N - [ 1- ( 4-bromophenyl ) ] -2,2-dimethylpropionamide

3.1 CDs and modified CDs

showed good efficiency (Rs = 30.95, N = 200,000/m).

CDs and modified CDs are the most widely used compounds

Fluorescently derivatized amino acids and non-protein amino

as chiral selectors for enantiomeric separation in LC, GC and CE.

acids were separated by a p-CEC packed with 5 µm aminopropyl

CDs are cyclic oligosaccharides with truncated cylindrical molecu-

silica-gel

lar shapes, and have particular names, α-, β- and γ-CD for thosenaphthylglycine (48). Amino acids and non-protein amino acids

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Masaru Kato and Toshimasa Toyo’oka

3.4 Proteins

were derivatized with the fluorogenic reagent, 4-fluoro-7-nitro-2, 1,
3-benzoxadiazole (NBD-F). Resolution ranged from 1.21 to 8.29,

Proteins are also used as chiral selectors in HPLC. But com-with a plate height from 8.7 to 39 µm. NBD-amino acids were also

position of applicable mobile phase is restricted, because the three-separated by m-CECs packed with 5 µm aminopropyl silica-gel

dimensional structures of proteins are dramatically changed by pH

modified with (S )-N -3,5-dinitrobenzoyl-1-naphthylglycine and (S )or organic modifiers in the mobile phase. Bovine serum albumin-N -3,5-dinitrophenylaminocarbonyl-valine (49). These modified

and cellobiohydrolase I was used as chiral selectors for m-CEC byparticles were fixed within a capillary by porous monolithic struc-

Nilsson and coworkers (58-60). They used glutaraldehyde to entrap

ture, which was prepared by sol-gel reaction. The m-CEC preparedthese proteins inside the capillary. Six β-adrenergic antagonistsby (S )-N -3,5-dinitrobenzoyl-1-naphthylglycine showed better

were separated by m-CEC. Li and Lloyd reported enantiomericenantiomeriseparation

separations of 10 enantiomers using p-CEC packed with 5 µm par-

than

that

by

( S ) -N -3,5-

dinitrophenylaminocarbonyl-valine. The other m-CEC was pre-ticles modified with α1-acid glycoprotein (61). The theoretical plate

pared by co-polymerization of 2-hydroxyethyl methacrylate (N -L-

number of benzoin was low (5,800), because the velocity of mass

valine-3, 5-dimethylanilide) carbamate and acrylamide (50). N -(3,transfer is slow between protein and enantiomers. They also pre-5-dinitrobenzoyl) leucine diallylamide enantiomers were separated

pared a p-CEC packed with human serum albumin to separate 3

by the m-CEC with a resolution of 2, and theoretical plate number

drugs (62). The theoretical plate numbers of the first eluted enanti-

for the first eluted enantiomer of 61,000/m. Schmid et al. preparedomer of temazepam and the separation factor of it were 7,000/m

m-CEC composed of polyacrylamide gel containing N -(2-hydroxy

and 2.4, respectively. Bovine serum albumin was used as chiral se--3-allyloxypropyl)-L-4-hydroxyproline (51). They added copper in

lector of o-CEC (63). Hofstertter et al. bound bovine serum albu-

the mobile phase and separated amino acids and biological com-min onto the capillary wall chemically and separated dinitrophenyl

pounds by ion-exchange mode (α-methyl-dopamine : α = 2.65).

(DNP)-amino acids and 3-hydroxy-1,4-benzodiazepines. Ly-

Liu et al. used amino acid (Lys) or peptides (Lys-Tyr and Lys-Ser-

sozyme, cytochrome c and avidin were also used as chiral selectors

Tyr) for chiral selectors for o-CEC (52). These chiral selectors haveof o-CEC (64, 65). These proteins were adsorbed onto the capillary

positively charged and adsorbed onto the capillary wall by flushingwall through two kinds of force (electrostatic attraction and hydro-a solution containing the chiral selectors into the capillary. These

phobic interaction). Lysozyme, cytochrome c and avidin column

columns separated Phe, Tyr and fenoprofen with the resolution of

separated 5,5 and 16 enantiomers, respectively. Column-to-column

1.3 to 2.0.

reproducibility of the avidin column (1.1%) was superior to run-torun reproducibility of it (2.2%) (65).

3.3 Macrocyclic antibiotics
3.5 Quinine-base anion-exchange

Macrocyclic antibiotics are popular for chiral selectors in
HPLC. These selectors have both a binding site (hydrogen bond-Lindner and co-workers used anion exchange type CSPs

ing, π-π interaction, etc.) and a cavity for forming the host-guest

modified with t-butyl-carbamoyl quinine as a chiral selector (66-complex. Dermaux used vancomycin as a chiral selector for p-CEC

70). Quinine has five chiral centers and two basic amino groups :(53). Owens and co-workers also used vancomycin as a chiral se-

the tertiary quinuclidine group and the aromatic quinoline group.

lector (54, 55). They separated drugs (β-blocker and nonsteroidal

These amino groups have a positive charge at low pH ; thus EOF

anti-inflammatory drugs) using polar organic and reversed-phasemoves towards the anodes. Anions migrated toward the anode (themode. The resolution and theoretical plate number for thalidomide

same direction of the EOF), hence the anions eluted within a short

was 13.8 and 190,000, respectively. Another macrocyclic antibi-

time. Glutethimide was separated ; its resolution was 4.32 andotic, teicoplanin, was also used as a chiral selector for p-CEC.

theoretical plate number of the first eluted enantiomer was

Carter-Finch and Smith separated tryptophan enantiomers ; the120,000/m with t-butyl-carbamoyl quinine as a chiral selector.

HETP of the first eluted enantiomer was 56 µm and the resolution

They also prepared m-CEC by co-polymerizing O -[2-(methacry-

was 1.17 (56). About 30 enantiomers (β-blocker, nonsteroidal anti-

loxyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine

inflammatory drugs etc.) were separated by the group of Owens

acrylate (69, 70). N -Derivatized aminoacids were separated by m-(57). Resolution of tryptophan and alprenolol were 1.74 and 3.27,

CEC and resolution of DNP-Val and theoretical plate number ofrespectively, and theoretical plate numbers were 29,000/m andthe first eluted enantiomer were 6.28 and 242,000/m, respectively.

and

meth-

136,000/m.
3.6 Cellulose derivatives
Cellulose derivatives, amylose-tris (3,5-dimethylphenylcarbamate), cellulose-tris (3,5-dimethylphenylcarbamate) and cellulose

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Chromatography, Vol.22 No.3 (2001)

tris (4-methylbenzoate), are some of the most popular chiral selec-

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