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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 In this study neutral and negatively charged CDs were used as
model selectors to investigate the complexation phenomenon in
The observed chiral separation, in chromatographic or elec- both techniques and to compare the mechanism of separation in
trophoretic techniques, may be decoupled in two basic steps: chiral CE and HPLC.
recognition that occurs on the molecular level, and transforma- The enantiomeric separation of basic chiral pharmaceuti-
tion of the chiral recognition event to the macro-phenomenon of cals such as pheniramine, brompheniramine, metoxyphenamine,
enatioresolution in both techniques. The phenomenon responsible cyclopentolate, doxylamine, and ketamine was investigated in cap-
for enantioseparation in chromatographic and electrophoretic pro- illary electrophoresis (CE) and liquid chromatography (HPLC) using
cesses is the same; it is the enantioselective interaction between negatively charged sulfated--cyclodextrin (S--CD) and neutral
the enantiomers and a chiral selector. The principal difference cyclodextrins (CDs). To explain the differences between the two
between these two techniques arises from different separation pro- methods, theoretical models presented elsewhere [4,15–21] were
cess mechanisms and sometimes from different environments of used. The influence of pH conditions and chiral selector concentra-
complexation [1]. tions on separation in both techniques was examined. The apparent
The remarkable capacity of cyclodextrin (CD) for enantiosepa- stability constants for the model compounds with cyclodextrins
ration has been used as advantage in many chromatographic and were assessed in CE and HPLC. In particular, the influence of
electrophoretic applications [2–11]. chiral selector concentration on the time of analysis and enantios-
Moreover, chromatographic and electrophoretic methods electivity of studied compounds (with various complex stability
together with NMR and molecular modeling were used for constants) were discussed for both methods.
studying weak interactions responsible for molecular recognition In this work we tried to answer the question if both meth-
[12–14]. 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
∗ Corresponding author. Tel.: +48 22 3433415. the very good enantioselectivity of negatively charged cyclodextrin
E-mail address: annab@ichf.edu.pl (A. Bielejewska). 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
Brompheniramine
2.2. Apparatus and procedure
Maleate salt
Table 2
Stability constants and separation parameters obtained by HPLC experiments.
Conditions Compounds
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- cyclopentolate, for which the stability constant with -CD is of
crepancy between the estimated stability constants obtained for the same order as for S--CD). For compounds with one nitro-
cyclopentolate in both methods but the K1 /K2 ratio is the same. The gen atom there are no differences between pH 7 and 2.5, while
differences in stability constant estimation can arise from the sol- for pheniramines and doxylamine the stability constants in pH
vent’s influence on complexation (the addition of 20% of ethanol to 2.5 are one order of magnitude higher than for pH 7. At pH 7 for
the mobile phase in the HPLC experiment). all the studied compounds, discrimination between enantiomers
As CE is the more efficient method, chiral recognition is bet- was visible. Rs = 3.0; 0.9; 1.3; 2.1; 4.3, 1.1 for brompheniramine,
ter visible in this method than in HPLC. For -CD, in CE ˛ > 1 was pheniramine, doxylamine, cyclopentolate, metoxyphenamine and
obtained for five compounds and in HPLC only for two compounds ketamine, respectively (see Table 3).
(see Tables 2 and 3). In HPLC, estimating the stability constant was possible only
As CE is a much cheaper and faster method, the stability con- in pH 7. The estimated stability constants were about ten times
stants for various cyclodextrins were estimated. For the studied lower (for cyclopentolate and ketamine only five times lower) than
compounds, TM--CD forms very weak complexes. The stability those obtained in CE. Chiral recognition in HPLC is visible only for
constant for DM--CD is very similar to that obtained for -CD. brompheniramine and cyclopentolate (see Table 2).
Among the neutral CDs the best chiral selectors for the studied From both techniques it can be said that the strongest S--CD
compounds are -CD and HP--CD. complexe was formed with brompheniramine with K1 /K2 = 1.44.
For the studied compounds the best recognition between enan- In both CE and HPLC (similarly as for -CD) the (+)enantiomer of
tiomers was obtained for cyclopentolate (K1 /K2 = 1.32, K1 /K2 = 1.45 bropheniramine is complexed by S--CD stronger than that of the
and K1 /K2 = 1.26 for -CD, for HP--CD and TM--CD, respectively) (−) one. Similar results were obtained earlier by Chankvetadze et
(see Table 3). al. [22,23].
3.2. Negatively charged cyclodextrin 3.3. The influence of selector concentration on enantiomer
retention and migration
In the CE measurements it was possible to estimate the sta-
bility constant for three pH values. For pH 10 where the model According to Eq. (1), in a chromatographic system with CDs
compounds are weakly protonated, the order of magnitude for added to the mobile phase, retention depends directly on the
the stability constant is similar as for neutral CD. For pH 7 and retention in the chromatographic system without CD. It also
2.5 the stability constants are much higher (one exception is 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
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 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
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
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