Analytica Chimica Acta 645 (2009) 98–104

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Analytica Chimica Acta

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Comparison of chiral separation of basic drugs in capillary

electrophoresis and liquid chromatography using
neutral and negatively charged cyclodextrins
Arkadiusz Kwaterczak, Kazimiera Duszczyk, Anna Bielejewska ∗
Institute of Physical Chemistry PAS, Kasprzaka 44/52, 01-224 Warsaw, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Liquid chromatography (LC) and capillary electrophoresis (CE) are very widely used as chiral separation
Received 23 October 2008 methods. In this publication we try to find if the results obtained in CE and LC with the chiral selector added
Received in revised form 27 February 2009 to the electrolyte and the mobile phase, respectively, can be used as tools for studying weak stereoselective
Accepted 29 April 2009
interactions, and how this information can be useful for optimizing chiral separation processes. The
Available online 5 May 2009
manuscript presents a systematic comparison of chiral discrimination of model compounds in HPLC and
CE using neutral and negatively charged cyclodextrins.
Keywords:
The enantiomeric separation of basic chiral pharmaceuticals such as pheniramine, brompheniramine,
Cyclodextrin complexation
Stability constant
metoxyphenamine, cyclopentolate, doxylamine and ketamine was investigated in capillary electrophore-
Chiral separation sis (CE) and liquid chromatography (HPLC) using negatively charged sulfated-␤-cyclodextrin (S-␤-CD)
Capillary electrophoresis and neutral cyclodextrins (CDs). The apparent stability constants between the model compounds and
Liquid chromatography cyclodextrins were estimated in both techniques. We discuss the influence of the stability constant and
K1 /K2 ratio of the investigated complexes on chiral separation obtained in both techniques.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
The observed chiral separation, in chromatographic or elec-
trophoretic techniques, may be decoupled in two basic steps: chiral
recognition that occurs on the molecular level, and transforma-
tion of the chiral recognition event to the macro-phenomenon of
enatioresolution in both techniques. The phenomenon responsible
for enantioseparation in chromatographic and electrophoretic pro-
cesses is the same; it is the enantioselective interaction between
the enantiomers and a chiral selector. The principal difference
between these two techniques arises from different separation pro-
cess mechanisms and sometimes from different environments of
complexation [1].
The remarkable capacity of cyclodextrin (CD) for enantiosepa-
ration has been used as advantage in many chromatographic and
electrophoretic applications [2–11].
Moreover, chromatographic and electrophoretic methods
together with NMR and molecular modeling were used for
studying weak interactions responsible for molecular recognition
[12–14].
∗ Corresponding author. Tel.: +48 22 3433415.
E-mail address: annab@ichf.edu.pl (A. Bielejewska).
In this study neutral and negatively charged CDs were used as
model selectors to investigate the complexation phenomenon in
both techniques and to compare the mechanism of separation in
CE and HPLC.
The enantiomeric separation of basic chiral pharmaceuti-
cals such as pheniramine, brompheniramine, metoxyphenamine,
cyclopentolate, doxylamine, and ketamine was investigated in cap-
illary electrophoresis (CE) and liquid chromatography (HPLC) using
negatively charged sulfated-␤-cyclodextrin (S-␤-CD) and neutral
cyclodextrins (CDs). To explain the differences between the two
methods, theoretical models presented elsewhere [4,15–21] were
used. The influence of pH conditions and chiral selector concentra-
tions on separation in both techniques was examined. The apparent
stability constants for the model compounds with cyclodextrins
were assessed in CE and HPLC. In particular, the influence of
chiral selector concentration on the time of analysis and enantios-
electivity of studied compounds (with various complex stability
constants) were discussed for both methods.
In this work we tried to answer the question if both meth-
ods can be used as additional tools to study weak stereoselective
interaction, and how the results are compatible. Another question
was whether very good enantioseparation in CE with a negatively
charged cyclodextrin was due only to the mechanism of separa-
tion in this technique, or was the effect of both the mechanism and
the very good enantioselectivity of negatively charged cyclodextrin
with a basic drug on the molecular level.

0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2009.04.049
 A. Kwaterczak et al. / Analytica Chimica Acta 645 (2009) 98–104 99

2. Experimental Table 1
The chemical formulas of the studied compounds.

2.1. Materials

␤-Cyclodextrin (␤-CD), heptakis (2,3,6-tri-O-methyl)

␤-cyclodextrin (TM-␤-CD), heptakis (2,6-di-O-methyl) ␤-
cyclodextrin (DM-␤-CD), hydroxypropyl ␤-cyclodextrin (HP-␤-CD)
were supplied by Chinoin (Budapest, Hungary), sulfated-␤-
cyclodextrin (S-␤-CD) was supplied by Aldrich (Steinheim, Pheniramine
Germany). According to specification the substitution of S-␤-CD Maleate salt
was 7–11 molecules mol−1 ␤-CD, for the calculation we used
average substitution (9), molecular mass 2062 g mol−1 .
The compounds for the study: bropheniramine racemate, (+)
brompheniramine, pheniramine, metoxyphenamine, doxylamine,
cyclopentolate, ketamine as salts (see Table 1) were supplied by
Sigma (Steinheim, Germany). All other reagents and solvents were
of analytical grade and were used as received.

Brompheniramine
2.2. Apparatus and procedure
Maleate salt

Chromatographic experiments were performed using a Waters

(Vienna, Austria) Model 590 pump, a Rheodyne type injector with
5 ␮L loop and a Waters UV–vis detector Model 490 (detection:
220 nm). The mobile phases were phosphate buffer solutions (pH
2.5 and 7, estimation of the binding constant was possible only for
pH 7, for lower pH the retention of the compounds on the reversed
phase column was too short) with ethanol modifier (20%, v/v) and Doxylamine
Succinate salt
appropriate concentration of CDs (for stability constant estimation,
five solutions in the concentration range 0–15 mg mL−1 were used).
The column used was: Luna 5 ␮m C8 (2) 100A 150 mm × 1 mm Phe-
nomenex (Le Peck Cedex, France).
Flow rate was 0.04 mL min−1 . All chromatographic measure-
ments were performed at the ambient temperature of the
air-conditioned room (about 23–25 ◦ C).
Electrophoretic separations were performed on a Spectrophore- Cyclopentolate
sis Ultra (Thermo Separation Products, San Jose, California, USA) Hydrochloride salt
using a 75 ␮m i.d. fused-silica capillary with total length of 63 cm
and effective length of 58 cm to the detector window. The capil-
lary was conditioned by passing a 0.1 M NaOH solution for 20 min
and then washed with distilled water for 10 min, and finally equili-
brated with an appropriate running buffer for 2 min. Between runs
the capillary was washed with the buffer for 2 min.
The electrolyte solution was prepared using an 18 mM Ketamine
tris(hydroxymethyl)aminomethane (Tris) solution adjusted to the Hydrochloride salt
appropriate pH with H3 PO4 or NaOH, for the complexation study
solutions contain the appropriate amount of cyclodextrin (for sta-
bility constant estimation three different pH: pH 2.5, 7, and 10 were
studied, for each pH five solutions in the concentration range of
0–15 mg mL−1 were used).
The samples were introduced by applying hydrodynamic pres- Metoxyphenamine
sure (0.8 psi) for 2 s and the separation temperature was 25 ◦ C. The Hydrochloride salt
applied voltage was 25 or −25 kV. The electro-osmotic flow (EOF)
was determined using DMSO as the neutral marker.
For both methods the elution order was checked only for
brompheniramine by injecting the scalemic mixture (a mixture data were analyzed on the basis of the model described earlier
containing one enantiomer in excess). [15,16,13].
The solute retention factor k1 in the chromatographic system
2.3. Binding constant estimation described by the model can be defined by the equation

2.3.1. Liquid chromatography k

n  
0
Our experimental set-up consists of a C8 column with a buffer- k1 = (1)
ethanol mobile phase and appropriate concentration of different 1+ i=1
K
i i
· [CD]i
cyclodextrins.
In order to establish the stoichiometry and stability of the where k1 and k0 are the retention factors observed in the systems
CD complexes, changes of the retention factor of the analyte with or without CD, Ki is the stability constant for the formed com-
with concentration of individual CDs have been followed. The plexes, and [CD] is the concentration of CD in the mobile phase.
100 A. Kwaterczak et al. / Analytica Chimica Acta 645 (2009) 98–104

The stability constants can be fitted by non-linear least-square

procedures according to the model using Eq. (2)

1 1 K1 [CD] K1 K2 [CD]2 K1 K2 K3 [CD]3

= + + + + ··· (2)
k1 k0 k0 k0 k0
The shape of the reciprocal of k versus CD concentrations pro-
vides information on the stoichiometry and stability constants of
the complex, in the case of 1:1 stoichiometry, the reciprocal of
k is a linear function of [CD] while for higher stoichiometry the
relation between 1/k and [CD] becomes parabolic. For the studied
compounds the reciprocal of k was a linear function of CD con-
centrations, therefore linear regression was used to calculate the
stability constants. Moreover, the elution order of the enantiomers
can provide information which enantiomer forms a more stable
complex. According to the model, the one forming more stable com-
plexes with a complexing agent present in the mobile phase should
be eluted faster from the column.

2.3.2. Capillary electrophoresis

The electrophoretic mobility of the analyte was calculated from
the observed migration times according to Eq. (3)
 
LD Lt 1 1
ep = obs − eof = − , (3)
V tm teof

where ep is the electrophoretic mobility of the analyte, obs is the

observed (apparent) mobility of the analyte, eof is the mobility of
the neutral marker—DMSO, Lt is the total length of the capillary,
LD is the length of the capillary to the detector window, V is the
applied voltage, tm is the migration time of the analyte, teof is the
migration time of the neutral marker—DMSO which moves with
the electro-osmotic flow (EOF).
The model used for calculation was described earlier [14,17,18].
Following the changes of ep as a function of CD concentra-
tion, the binding constants K and the mobility of the complex
comp were fitted by non-linear least-square procedures (the
Levenberg–Marquardt algorithm) according to the 1:1 stoichiome-
try model using Eq. (4)
free + comp K[CD]
ep = (4)
1 + K[CD]
where free is the electrophoretic mobility of the free analyte, comp
is the electrophoretic mobility of the complex and K is the stability
constant of the complex. The free of the analyte was measured in
single experiments without CD in the solution. The stability con-
stants obtained in linear regression for the HPLC experiment were
used as the starting K, and the mobility of the cyclodextrin alone
was used as the starting comp .
According to the assumed model, for normal polarity (+25 kV),
from two enantiomers of positively charged compounds the one
forming more stable complexes with CD (the neutral one moving
with electro-osmotic flow toward the negative electrode, or the
negatively charged one moving to the positive electrode) should be
eluted second from the capillary. For reversed polarity with S-␤-CD
(−25 kV, pH 2.5), the enantiomers forming more stable complexes
should be eluted first.
2.4. Resolution
The quality of separation evaluated by the separation factor Rs
was defined as:
t2 − t1
RS = , (5)
(1/2)(w1 + w2 )
where t is the retention time for the first and second eluted enan-
tiomer respectively and w is the peak width at the base.
Fig. 1. The influence of pH on the electrophoretic mobility of the studied com-
pounds.
3. Results and discussion
In the preliminary study the electrophoretic mobility of the
studied compound in various pH was estimated using Eq. (3) (see
Fig. 1).
The studied compounds can be divided into two groups: the
first group is pheniramine, brompheniramine and doxylamine, for
which two nitrogen atoms can be protonated, and the second group
comprises the rest of the compounds (compounds with one nitro-
gen atom), for which electrophoretic mobility does not change
between pH 2.5 and 7. To study the complexation phenomena in
electrophoresis, on the basis of the assumed model and the elec-
trophoretic mobility of the studied compounds, pH 2.5 was chosen
for neutral CD, while three different pH conditions, 2.5, 7 and 10
were chosen for negatively charged S-␤-CD. pH 2.5 was chosen for
neutral CD to maximize the difference between the mobility of the
compounds and selectors. For this pH all the studied compounds
were positively charged and moved at their highest mobility (see
Fig. 1). This differed from the mobility of neutral cyclodextrin mov-
ing with the mobility of EOF, which in this pH is rather slow. Because
S-␤-CD has its own mobility, it was interesting to consider different
pH.
For the chromatographic studies, estimating the stability con-
stant was possible only for pH 7 (for lower pH the retention of the
compounds on the reversed phase column was too short).
The stability constants fitted by the described models on the
basis of chromatographic and electrophoretic experiments are pre-
sented in Tables 2 and 3
3.1. Neutral cyclodextrin
Both methods seem to be complementary for the study of com-
plexation phenomena. Although for ␤-CD the stability constants
were estimated in different pH (for CE pH 2.5, for HPLC pH 7),
it can be seen that brompheniramine forms stronger complexes
with ␤-CD than pheniramine and doxylamine do. Complexation of
pheniramine and doxylamine by ␤-CD is very similar. ␤-CD forms
 A. Kwaterczak et al. / Analytica Chimica Acta 645 (2009) 98–104 101

Table 2
Stability constants and separation parameters obtained by HPLC experiments.

Conditions Compounds

Brompheniramine Pheniramine Doxylamine Cyclopentolate Metoxyphenamine Ketamine

S-␤-CD
pH 7, 20% EtOH K1 [L mol−1 ] 351 ± 37 289 ± 46 175 ± 21 315 ± 70 65 ± 6 118 ± 5
K2 /K1 1.44 1.00 1.00 1.07 1.00 1.00
k2 8.5 1.5 2.4 4.7 1.2 2.2
˛ = k2 /k1 1.32 1.00 1.00 1.09 1.00 1.00
RS 0.9 0 0 0.4 0

pH 2, 20% EtOH
k2 – – – 2.4 0.7 0.5
˛ = k2 /k1 – – – 1.10 1.00 1.00
RS – – – 0.3 0 0

pH 2, 10% EtOH
k2 – – – 6.1 2.2 2.0
˛ = k2 /k1 – – – 1.17 1.00 1.00
RS – – – 1.13 0 0

␤-CD
pH 7, 20% EtOH K1 [L mol−1 ] 183 ± 9 59 ± 2 52 ± 13 202 ± 7 15 ± 1 29 ± 0.2
K2 /K1 1.05 1.00 1.00 1.33 1.00 1.00
k2 6.5 2.3 2.7 2.8 1.7 2.9
˛ = k2 /k1 1.06 1.00 1.00 1.26 1.00 1.00
RS 0.4 0 0 0.8 0 0

pH 7, 10% EtOH
k2 – – – 9.8 6.8 –
˛ = k2 /k1 – – – 1.28 1.00 –
RS – – – 0.7 0 –

pH 2, 20% EtOH
k2 0.7 – – 1.3 0.7 0.4
˛ = k2 /k1 1.00 – – 1.08 1.00 1.00
RS 0 – – 0.3 0 0

pH 2, 10% EtOH
k2 3.3 – – 4.0 2.4 1.8
˛ = k2 /k1 1.00 – – 1.29 1.00 1.14
RS 0 – – 0.6 0 0.2

k2 retention factor for the second eluted enantiomer, data k2 /k1 for 15 mg mL−1 for native CDs and S-␤-CD concentration.

the weakest complexes with metoxyphenamine. There is some dis-
crepancy between the estimated stability constants obtained for
cyclopentolate in both methods but the K1 /K2 ratio is the same. The
differences in stability constant estimation can arise from the sol-
vent’s influence on complexation (the addition of 20% of ethanol to
the mobile phase in the HPLC experiment).
As CE is the more efficient method, chiral recognition is bet-
ter visible in this method than in HPLC. For ␤-CD, in CE ˛ > 1 was
obtained for five compounds and in HPLC only for two compounds
(see Tables 2 and 3).
As CE is a much cheaper and faster method, the stability con-
stants for various cyclodextrins were estimated. For the studied
compounds, TM-␤-CD forms very weak complexes. The stability
constant for DM-␤-CD is very similar to that obtained for ␤-CD.
Among the neutral CDs the best chiral selectors for the studied
compounds are ␤-CD and HP-␤-CD.
For the studied compounds the best recognition between enan-
tiomers was obtained for cyclopentolate (K1 /K2 = 1.32, K1 /K2 = 1.45
and K1 /K2 = 1.26 for ␤-CD, for HP-␤-CD and TM-␤-CD, respectively)
(see Table 3).
cyclopentolate, for which the stability constant with ␤-CD is of
the same order as for S-␤-CD). For compounds with one nitro-
gen atom there are no differences between pH 7 and 2.5, while
for pheniramines and doxylamine the stability constants in pH
2.5 are one order of magnitude higher than for pH 7. At pH 7 for
all the studied compounds, discrimination between enantiomers
was visible. Rs = 3.0; 0.9; 1.3; 2.1; 4.3, 1.1 for brompheniramine,
pheniramine, doxylamine, cyclopentolate, metoxyphenamine and
ketamine, respectively (see Table 3).
In HPLC, estimating the stability constant was possible only
in pH 7. The estimated stability constants were about ten times
lower (for cyclopentolate and ketamine only five times lower) than
those obtained in CE. Chiral recognition in HPLC is visible only for
brompheniramine and cyclopentolate (see Table 2).
From both techniques it can be said that the strongest S-␤-CD
complexe was formed with brompheniramine with K1 /K2 = 1.44.
In both CE and HPLC (similarly as for ␤-CD) the (+)enantiomer of
bropheniramine is complexed by S-␤-CD stronger than that of the
(−) one. Similar results were obtained earlier by Chankvetadze et
al. [22,23].

3.2. Negatively charged cyclodextrin
In the CE measurements it was possible to estimate the sta-
bility constant for three pH values. For pH 10 where the model
compounds are weakly protonated, the order of magnitude for
the stability constant is similar as for neutral CD. For pH 7 and
2.5 the stability constants are much higher (one exception is
3.3. The influence of selector concentration on enantiomer
retention and migration
According to Eq. (1), in a chromatographic system with CDs
added to the mobile phase, retention depends directly on the
retention in the chromatographic system without CD. It also
depends indirectly on the stability constants of the complex and
102 A. Kwaterczak et al. / Analytica Chimica Acta 645 (2009) 98–104

Table 3
Stability constants and separation parameters obtained by CE experiments.

Conditions Compounds

Brompheniramine Pheniramine Doxylamine Cyclopentolate Metoxyphenamine Ketamine

S-␤-CD
pH 10 K1 [L mol−1 ] 24 ± 0.52 150 ± 5 90 ± 3 16 ± 0.5 172 ± 10 47 ± 0.5
K2 /K1 1.07 1.16 1.20 1.11 1.25 1.00
1 /2 1.05 1.14 1.18 1.12 1.20 1.00
RS 0.2 0.5 0.6 0.3 0.6 0

pH 7 K1 [L mol−1 ] 3462 ± 143 2323 ± 37 1218 ± 69 1640 ± 161 753 ± 15 619 ± 5

K2 /K1 1.44 1.07 1.09 1.32 1.16 1.10
1 /2 1.10 1.02 1.07 1.12 1.44 1.12
RS 2.96 0.9 1.26 2.14 4.26 1.09

pH 2.5 K2 [L mol−1 ] 18,397 ± 771 56,454 ± 1184 18,421 ± 10 1320 ± 3272 960 ± 68 681 ± 5
K1 /K2 1.01 1.01 1.00 1.38 1.79 1.11
2 /1 1.01 1.01 1.00 1.08 1.29 1.08
RS 0.6 0.6 0 2.5 10.7 2.2

pH 2.5, neutral cyclodextrins

␤-CD K1 [L mol−1 ] 137 ± 0.5 18 ± 0.5 20 ± 0.5 1036 ± 22 14 ± 0.5 87 ± 0.5
K2 /K1 1.07 1.00 1.09 1.32 1.08 1.07
1 /2 1.02 1.00 1.02 1.04 1.01 1.02
RS 0.6 0 1.8 0.5 0.8 0.6

HP-␤-CD K1 [L mol−1 ] 41 ± 0.5 9±1 10 ± 1 590 ± 10 12 ± 1 27 ± 3

K2 /K1 1.06 1.00 1.07 1.45 1.08 1.10
1 /2 1.02 1.00 1.01 1.04 1.01 1.01
RS 0.6 0 0.8 1.2 1.0 0.2

DM-␤-CD K1 [L mol−1 ] 253 ± 5 26 ± 1 28.5 ± 0.5 1770 ± 15 30 ± 1 106 ± 2

K2 /K1 1.00 1.00 1.05 1.00 1.07 1.01
1 /2 1.00 1.00 1.01 1.00 1.02 1.02
RS 0 0 1.1 0 1.1 0.8

TM-␤-CD K1 [L mol−1 ] 13 ± 0.5 2 ± 0.5 3 ± 0.5 8 ± 0.5 4 ± 0.5 4 ± 0.5

K2 /K1 1.00 1.00 1.00 1.26 1.00 1.00
1 /2 1.00 1.00 1.00 1.03 1.00 1.00
RS 0 0 0 1.2 0 0

1 , 2 : mobility for the first and second eluted enantiomer respectively; data 1 /2 for 15 mg mL−1 for native CDs and 10 mg mL−1 S-␤-CD concentration.

decreases with the increase in CD concentration. For example, illustrated by the electropherograms for brompheniramine and
the retention factor of brompheniramine changes from 23.0 with- metoxyphenamine presented in Fig. 3. The much higher stability
out CD to 12.5 and 8.5 (data for the second eluted enantiomer) constants for brompheniramine cause longer analysis in the case of
for the same concentration (0.005 M) for ␤- and S-␤-CD, respec- normal polarity (25 kV) in pH 7 and very short analysis in the case
tively. For cyclopentolate the retention factor changes from 12.3 of reverse polarity in pH 2.5.
to 5.8 and 4.7, respectively. The higher the stability constant, the
higher the reduction of retention and the shorter the time of
analysis.
In CE the mobility of positively charged analytes decelerates
with the increase of the concentration of non-charged CD. In this
case, for normal polarity, the time of analysis becomes longer
but not longer than the time of migration of neutral compounds.
With a negatively charged selector the migration is reduced and
even inverted (see Fig. 2). The influence of the values of sta-
bility constants with S-␤-CD on the time of analysis is well

Fig. 3. Electropherograms of brompheniramine and metoxyphenamine enan-

Fig. 2. The influence of concentration of various CDs on the electrophoretic mobility tiomers. Conditions: pH 2.5 (−25 kV) or pH 7 (25 kV), S-␤-CD concentration
of cyclopentolate (first enantiomer). 10 mg mL−1 (for the reset see Section 2).
 A. Kwaterczak et al. / Analytica Chimica Acta 645 (2009) 98–104 103

Fig. 5. Enantioselectivity ˛ for cyclopentolate obtained in the CE experiment with

the increase in concentration of ␤-CD, HP-␤-CD and TM–␤-CD.
Fig. 4. Enantioselectivity ˛ for brompheniramine and cyclopentolate obtained in
the HPLC experiment with the increase in S-␤-CD concentration.

3.4. Enantioselectivity as a function of chiral selector

concentration

3.4.1. Neutral cyclodextrin

3.4.1.1. Liquid chromatography. By definition, the chromatographic
enantioseparation factor (˛) can be written as:
kII
˛= (6)
kI
where II and I refer to the enantiomers eluted from the column as
the second and first ones, respectively.
The combination of Eq. (1) for 1:1 stoichiometry and Eq. (6)
produces
1 + KI [CD]
˛= (7)
1 + KII [CD]
˛ changes asymptotically with the CD concentration from 1.0
([CD] = 0) to the constant value of KI /KII .
Fig. 4 represents typical data obtained experimentally.

3.4.1.2. Electrophoresis. For electrophoresis, the equation for ␣ can

be written as:
epI
˛= (8)
epII
where II and I refer to the enantiomers eluted from the capillary as
the second and first ones, respectively (for normal polarity +25 kV,
for the positively charged compounds ˛ > 1).
The combination of Eqs. (8) and (4) for 1:1 stoichiometry pro-
duces
Fig. 6. Enantioselectivity ˛ for brompheniramine and cyclopentolate obtained in the
(free + comp KI [CD])(1 + KII [CD]) CE experiment with the increase in S-␤-CD concentration for pH 2.5 (−25 kV) and 7,
˛= (9)
(free + comp KII [CD])(1 + KI [CD]) 10 (25 kV). (For pH 2.5, ˛ < 1 because of the reverse elution order of the enantiomers.)

If the complex mobility is zero, the relationship is the same as for

the chromatographic system and ˛ changes asymptotically with CD
concentration from 1.0 ([CD] = 0) to the constant value to KII /KI . But
even if the mobility of the positively charged compound with neu-
tral cyclodextrin is very low, the plot of ˛ = f ([CD]) has a maximum
for optimum concentration of [CD].
The experimental enantioselectivity values for cyclopentolate
obtained for various CDs are presented in Fig. 5.
If we assume similar mobility for a cyclopentolate complex with
various CDs, the optimal concentration depends only on the stabil-
ity constants. For a high stability constant with ␤-CD, the maximum
enantioselectivity is obtained for low concentration of the selector,
for TM-␤-CD, for which the stability constant with cyclopentolate
is very low, optimal concentration was not achieved in the experi-
mentally tested concentration range.
tors. The only difference may occur on the molecular recognition
level.
The role of counter-current mobility of chiral selector and
selectand for separation has been discussed for the first time
in 1994 [24]. For electrophoresis studies, if the mobility of the
selector is opposite to the free analyte, the complex mobility
may have the opposite direction to the free analyte. In such a
case the theoretical plot of ˛ = f ([CD]) has discontinuity (infi-
nite selectivity) [19,25]. The selectivity obtained in our study is
demonstrated in Fig. 6. In the experimentally studied range of
concentrations, selectivity decreased with the increase in concen-
tration.
4. Conclusions

3.4.2. Negatively charged cyclodextrin Both methods may be complementary for studying complexa-
For a chromatographic model with a chiral selector in the mobile tion phenomena. Due to higher efficiency of CE, chiral recognition
phase there is no difference between charged and neutral selec- is better visible in CE. Knowledge on complexation phenomena and
104 A. Kwaterczak et al. / Analytica Chimica Acta 645 (2009) 98–104
the mechanism of separation for both techniques is very instructive
for designing and optimizing chiral separation.
For model compounds, if the retention of the analyte on the
stationary phase in a chromatographic system is sufficient, better
selectivity and a shorter time of analysis are obtained for higher
concentration of CD added to the mobile phase. According to the
mechanism, enantioselectivity cannot be higher than the K1 /K2
ratio. In chromatography there is no difference in the separation
mechanism process between charged and uncharged cyclodex-
trin, and the only differences may arise from molecular interaction
between CD and analyte. From the obtained data, not much better
stereoselectivity is observed for negatively charged S-␤-CD than for
neutral CD.
For the investigated compounds in CE, better enantioselectivity
was obtained for S-␤-CD than for neutral cyclodextrin. This is due
rather to the separation mechanism than complexation phenom-
ena, because S-␤-CD in a chromatographic system does not exhibit
better separation ability than ␤-CD. When the mobility of the chiral
selector is opposite to the mobility of the analyte, even a small dif-
ference in complexation between enantiomers can produce good
enantioselectivity. For the model compounds in the investigated
concentration range the selectivity decreased with the increase in
CD concentration. Only for TM-␤-CD, for which the stability con-
stant is very low, selectivity increased with the increase in CD
concentration.
Acknowledgment
The authors acknowledge the financial support of the Polish
Ministry of Science and Higher Education (grant No. N204 044
32/1046).
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