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ORIGINAL
Enantioselective Extraction System Containing Binary Chiral
Selectors and Chromatographic Enantioseparation Method
for Determination of the Absolute Configuration of Enantiomers
of Cyclopentolate
Kamila Szwed •Marcin Go
´recki •Jadwiga Frelek •
Monika Asztemborska
Received: 5 June 2013 / Revised: 1 August 2013 / Accepted: 7 August 2013 / Published online: 23 August 2013
ÓThe Author(s) 2013. This article is published with open access at Springerlink.com
Abstract The distribution coefficients and enantiosepa-
ration of cyclopentolate were studied in an extraction
system containing D-tartaric acid ditertbutyl ester in
organic phase and 2-hydroxypropyl-b-cyclodextrin (HP-b-
CD) in aqueous phase. Various parameters involved in the
enantioseparation such as the type and the concentration of
chiral selectors, pH value and a wide range of organic
solvents were investigated. The maximum enantioselec-
tivity (a=2.13) and optimum distribution coefficients
(K
R
=0.85, K
S
=0.40) were obtained under the following
conditions: 0.10 mol/L HP-b-CD in aqueous phase and
0.20 mol/L D-tartaric acid ditertbutyl ester in decanol as
organic phase. Cyclopentolate is present as a racemic
mixture to the aqueous phase. The potentially different
biological activities of cyclopentolate enantiomers have not
been examined yet. Two chiral liquid chromatography
methods have been developed for the direct separation of
the enantiomers of cyclopentolate. First method was used
for the quantification analysis of cyclopentolate enantio-
mers in aqueous phase. Second method used two chirop-
tical detectors: electronic circular dichroism (ECD) and
optical rotation (OR) for the identification of individual
cyclopentolate enantiomers from the organic phase enri-
ched with (R)-enantiomer. The absolute stereochemistry
was determined by means of the comparison of the
experimental and computed ECD spectra and signs of OR.
The ECD spectra of chiral analytes were measured on-line
using HPLC-ECD technique.
Keywords Column liquid chromatography
Enantioselective extraction Cyclodextrin
Chiral extraction Cyclopentolate
Introduction
Separation of enantiomers is a topic of great interest in
many branches of science such as pharmaceutical, medic-
inal chemistry, agrochemicals and food chemistry [1–3].
The fact that enantiomers usually possess different physical
and pharmacological properties in biological systems [4]
has a strong stimulating influence on the development of
new, more efficient enantioseparation methods.
Asymmetric synthesis and chiral resolution are two
main methods used for obtaining pure enantiomers. Com-
pared to popular resolution methods, asymmetric synthesis
is more expensive and often characterized by low yields
[5]. Chiral resolution methods such as chromatographic
separation, kinetic resolution, and crystallization resolution
also have some disadvantages. Chromatographic methods
can separate racemic mixture on an analytical and pre-
parative level, however, they are rather expensive and
time-consuming [6]. The main disadvantage of kinetic
resolution is the loss of catalytic activity over time [7].
Crystallization is also time-consuming, and the desired
compounds are often obtained as derivatives such as dia-
stereomeric salts [8], diastereoisomeric derivatives [9], or
inclusion complexes [10]. Chiral extraction is cheaper and
easier than other methods. In addition, it can be easily
scaled up to industrial scale [11,12].
K. Szwed (&)M. Asztemborska
Institute of Physical Chemistry, Polish Academy of Sciences,
Kasprzaka 44/52, 01-224 Warsaw, Poland
e-mail: kszwed@ichf.edu.pl
M. Go
´recki J. Frelek
Institute of Organic Chemistry, Polish Academy of Sciences,
Kasprzaka 44/52, 01-224 Warsaw, Poland
123
Chromatographia (2013) 76:1603–1611
DOI 10.1007/s10337-013-2538-z
Chiral selectors play an important role in the efficiency
of enantioseparation.
Tartaric acid derivatives are commonly used as chiral
selectors for enantioseparation [13–15]. Nevertheless,
these chiral selectors exhibit a limited ability for enan-
tioseparation that is inadequate in context of industrial
application. Higher enantioselectivity can be obtained by
combination of tartaric acid derivatives with other chiral
selectors [16]. Cyclodextrins (CD) are extensively used in
separation by virtue of their availability and low cost [17–
20]. They are cyclic oligosaccharides that can incorporate
molecules of appropriate size into their hydrophobic cav-
ity, forming the inclusion complexes.
Several papers on the enantioselective extraction system
containing tartaric acid derivatives and CD were also
studied [21–23].
The objective of this study was to investigate tartaric
acid derivatives and b-cyclodextrin derivatives as binary
chiral selectors in extraction of racemic cyclopentolate,
which, as a mydriatic agent, is commonly used during
pediatric eye examinations [24] (Fig. 1). Cyclopentolate is
added as racemic mixture to the aqua phase. Various
parameters involved in enantioseparation of cyclopentolate
were investigated, such as type and concentration of chiral
selectors, pH values, type of organic solvent.
Materials and Methods
Materials
b-Cyclodextrin (b-CD) and trimethyl-b-cyclodextrin (TM-
b-CD) were supplied by Chinoin (Budapest, Hungary).
Hydroxypropyl-b-cyclodextrin (HP-b-CD), dimethyl-b-
cyclodextrin (DM-b-CD) and cyclopentolate (cyclopento-
late) were all obtained from Aldrich Co. (St. Louis, MO,
USA). Sodium 1-octanesulfonate, L-tartaric acid ditertbutyl
ester, D- and L-tartaric acid diethyl ester were bought from
TCI Europe (Zwijndrecht, Belgium). D-tartaric acid diter-
tbutyl ester was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). D-, L-tartaric acid diisobutyl ester
and D-, L-tartaric acid dibutyl ester were synthesised as
described in literature [25].
Analytical Methods
The quantification analysis of cyclopentolate enantiomers
in aqueous phase was performed by HPLC using a Waters
(Milford, MA, USA) Model 515 pump, 717 plus auto-
sampler with 1 lL loop and a Waters UV/VIS detector
Model 2,487 (detection: 220 nm). The mobile phase was
prepared by dissolving 15 mM of b-CD, 1 mM sodium
1-octanesulfonate, 10 mM NaH
2
PO
4
in aqueous ethanolic
solutions (20 % (v/v) EtOH-water) and finally adjusted to
pH 2.0 by addition of phosphoric acid. The column was
Luna 5 lm C18 (2) 100 A 150 mm 91 mm (Phenome-
nex, Torrance, CA, USA). Flow rate of mobile phase was
0.04 mL/min. Chromatographic measurements were per-
formed at the 20 °C.
The identification of cyclopentolate enantiomers in
organic phase was performed using a Jasco analytical
HPLC system equipped with PU-2089 Plus pump with
inner degasser, column oven, a 20 lL sample loop, and
MD-2010 high resolution diode array UV–VIS detector
(600–195 nm). Chiroptical detections were performed
using ECD and OR detectors. An ECD chromatogram
was obtained at a fixed wavelength by setting the flow
cell attachment in the sample chamber of ECD Jasco
J-815 spectrometer, and then introducing an eluant from
HPLC. The optical rotation chromatogram was recorded
using the Jasco OR-2090 detector equipped with 150 W
Hg–Xe-lamp working in the range of white light
(900–350 nm). The signal was processed by ChromNAV
Jasco software. The column was Chiralpak AD (Daicel,
Tokyo, Japan) column (250 mm 94.6 mm, 5 lm). The
mobile phase was
i
PrOH:hexane (90:10, v/v) with a flow
rate of 1 mL/min. The column temperature was set at
20 °C.
The on-line electronic circular dichroism (ECD) spectra
were recorded between 350 and 200 nm at room temper-
ature. Solutions at maximum ECD absorption were trapped
in the flow cell attachment fixed to the sample chamber of
ECD Jasco J-815 spectrometer. All spectra were recorded
using 100 nm/min scanning speed, a step size of 0.2 nm, a
bandwidth of 2 nm, a response time of 0.5 s, and an
accumulation of five scans. The spectra were background
corrected using mobile phase.
Extraction Experiments
The aqueous phase contained 0.05 mM/L of racemic
cyclopentolate and various concentrations of b-CD deriv-
atives (HP-b-CD, DM-b-CD or TM-b-CD) in 0.10 mol/L
Na
2
HPO
4
/H
3
PO
4
buffer solution. D- and L-tartaric acid
derivatives were used as extractants and dissolved in the
organic phase. 5 mL of organic and 5 mL of aqueous
phases were placed in 25 mL glass-stoppered tube and
HO O
O
N
Fig. 1 Chemical structure of cyclopentolate
1604 K. Szwed et al.
123
shaken sufficiently (10 h) at a constant temperature
(20 °C) to reach equilibrium. After the phase separation,
the concentration of cyclopentolate enantiomers in the
aqueous phase was determined by HPLC. The distribution
coefficients of (S)- and (R)-forms of cyclopentolate
between the organic and aqueous phases (K
S
,K
R
) and
enantioselectivity (a) were evaluated according to the
following equations:
KR¼Concentration of ðRÞcyclopentolate in organic phase
Concentration of ðRÞcyclopentolate in aqueous phase
ð1Þ
KS¼Concentration of SðÞcyclopentolate in organic phase
Concentration of SðÞcyclopentolate in aqueous phase
ð2Þ
a¼KR
KSð3Þ
Computational Details
The conformational search was done using ComputeVOA
[26] program with Merck Molecular Force Field 94
(MMF94) within 5 kcal/mol energy windows, and then all
structures (143) were submitted to the Gaussian 09 pro-
gram [27] for a single-point energy calculation at B3LYP/
6-31G(d) DFT level of theory. In order to improve con-
fidence level for conformers found (17) second optimiza-
tion was performed at B3LYP/aug-cc-pVDZ level in the
3 kcal/mol energy window. Then, finally these 17 struc-
tures were used for simulation of UV/ECD spectra using
B3LYP/aug-cc-pVDZ level. Rotatory strengths were cal-
culated using both length and velocity representations. The
differences between the length and velocity of calculated
values of rotatory strengths were \5 %, and for this rea-
son, only the velocity representations (R
vel
) were taken
into account. The ECD spectra were simulated by over-
lapping Gaussian functions for each transition by means of
the SpecDis program [28]. The final spectra were Boltz-
mann averaged (T=293 K) according to the population
percentages of individual conformers based on the relative
SCF energies. The calculated UV spectra were red-shifted
by 18 nm in relation to the experimental, and conse-
quently ECD spectra were also wavelength corrected.
A Gaussian band-shape was applied with 0.48 eV as a
half-height width. Very similar results were obtained using
B3LYP/6-311??G(2d,2p) and CAM-B3LYP/aug-cc-
pVDZ level of theory.
In order to predict OR sign calculations were made for
the same group of 17 conformers as ECD, in Gaussian 09
package at 589 nm wavelength using the B3LYP/aug-cc-
pVDZ level.
Results and Discussion
The Method for Identification of Absolute
Configuration of Enantiomers of Cyclopentolate
The enantioselective separation of racemic cyclopentolate
was successfully performed using Chiralpak AD column by
application of two chiral detectors: ECD and OR. The first
eluted enantiomer displays a positive ECD and OR values
while second one exhibits opposite signs for both chirop-
tical methods (Fig. 2).
The ECD spectra measured on-line of two elution peaks
are shown in the Fig. 3. The first eluted enantiomer of
cyclopentolate displays two positive ECD bands: a very
weak one at 262 nm and a strong one at 221 nm. The first
band at 262 nm with well-defined vibrational fine structure
is related to the
1
L
b
(p?p*) transition which involves
Fig. 2 UV (upper) and ECD (middle) and OR (lower) chromato-
grams of racemic cyclopentolate on the AD Chiralpak column.
Chromatographic conditions are described in experimental section
Enantioselective Extraction System Containing Binary Chiral Selectors 1605
123
orbitals of chirally perturbed benzene ring. The second one
at 221 nm is an admixture of the
1
L
a
(p?p*) transition
from aromatic ring and of the ester n?p* transition.
The absolute configuration (AC) of eluted enantiomers
were determined on the basis of the comparison of the
experimental and computed ECD spectrum and sign of
OR. The calculations were performed using the Time
Dependent Density Functional Theory (TDDFT) method
for an arbitrarily chosen (R)-enantiomer. This combined
analysis has already proven efficient and reliable for the
assignment of the AC of various chiral organic molecules
[29–31].
In order to assure a reliable AC assignment, first a
thorough conformational analysis by molecular mechanics
(MMF94 force field) was carried out to find the lowest-
energy conformers. Then, all of the conformers were
optimized at B3LYP/6-31G(d) level of theory. In order to
refine the data, the reoptimization was performed at a
higher level (B3LYP/aug-cc-pVDZ). Finally, 17*con-
formers within the range of 3 kcal/mol were selected for
ECD and OR calculations. The OR calculations were car-
ried out at the wavelength of 589 nm. As can be seen in
Fig. 4, the Boltzmann averaged ECD spectrum of the (R)-
enantiomer is in an excellent agreement with the experi-
mental spectrum of the first eluted peak.
An additional proof of this AC assignment comes from
calculated value of OR at 589 nm. For the same set of
conformers, as for ECD spectra, the simulated Boltzmann
average value of OR is positive.
In conclusion, this combined experimental and theoret-
ical analysis of ECD and OR data allowed us to confidently
assign the AC to be (R) for the first eluted peak and (S) for
the second one in accordance with experimental data. This
method was used only to determine which enantiomer is
enriched in the organic phase. It was found that in the
organic phase (R)-cyclopentolate was present in excess.
Fig. 3 ECD spectra measured on-line of two elution peaks at 10.9
and 12.0 min, respectively
Fig. 4 Top Computed ECD
spectrum at the B3LYP/aug-cc-
pVDZ level of theory obtained
as a population-weighted sum at
293 K of individual conformers
of (R)-cyclopentolate compared
to measured on-line ECD
spectrum of the first peak eluted
at 10.9 min. Bottom Optimized
structures of the three lowest-
energy conformers
1606 K. Szwed et al.
123
Effect of Organic Solvents on Chiral Extraction
Table 1shows the influence of various organic solvents on
distribution coefficients and enantioselectivity. The aque-
ous phase contains 0.05 mmol/L racemic cyclopentolate,
0.10 mol/L Na
2
HPO
4
/H
3
PO
4
buffer and 0.10 mol/L HP-b-
CD. There is no extractant in organic phase. The large
influence of organic solvent on the extraction efficiency has
also been investigated in previous works [32,33].
Table 1illustrates, extraction performance of different
kinds of organic solvents. When 1,2-dichloroethane,
dichlorobenzene and cyclohexane were used as solvent,
very low distribution coefficients were obtained. This may
be caused by the fact that the cyclopentolate is poorly
extracted into the organic phase.
When alcohol groups were used as solvents, relatively
higher distribution coefficients were obtained. The distri-
bution coefficients and enantioselectivity increase with the
elongation of length of alkyl chain of alcohol.
The organic solvents can penetrate into the aqueous
phase and can form complexes of different stabilities with
b-HP-CD [34]. In addition, the more hydrophilic solvents
more easily penetrate into aqueous phase. The organic
solvent in the aqueous phase can cause a displacement of
the analyte from the CD cavity. The result is a decrease in
enantioselectivity.
When cyclohexane was used, high enantioselectivity
was obtained but low distribution coefficients were found.
Distribution coefficients for cyclopentolate enantiomers
were higher with alcohol groups. According to these
observations, decanol was chosen as a suitable organic
solvent giving the highest distribution coefficients with
satisfied enantioselectivity.
Effect of b-Cyclodextrin Derivatives
The CD are capable of forming inclusion complexes with
compounds having a size, shape and polarity compatible
with the dimensions of their cavity. Cyclopentolate can
form inclusion complexes with the following derivatives of
b-CD: HP-b-CD, DM-b-CD, TM-b-CD. Stability constants
of complexes b-CD derivatives (HP-b-CD, DM-b-CD,
TM-b-CD) with cyclopentolate were determined by capil-
lary electrophoresis technique [35] Cyclopentolate forms
the most stable complexes with DM-b-CD (1,769.2 L/mol)
and the least stable with the TM-b-CD (7.93 L/mol). Sta-
bility constant complex of cyclopentolate with HP-b-CD
(590.1 L/mol) has a value in between.
The distribution coefficients and enantioselectivity were
determined by chiral extraction (0.10 mol/L b-CD deriva-
tives in aqueous phase and no addition of tartaric acid
derivatives to organic phase).
In Table 2, the highest enantioselectivity and distribu-
tion coefficients of (R)-enantiomer were achieved when
HP-b-CD was used, but with lowest distribution coeffi-
cients of (S)-enantiomer. However, when DM-b-CD and
TM-b-CD were used significantly lower enantioseparation
was achieved.
It can also be found in Table 2that K
R
are always higher
than K
S
, which indicates that HP-b-CD preferentially
interacts with (S)-cyclopentolate and this complex is
retained in the aqueous phase.
Among the three b-CD derivatives, HP-b-CD has the
highest enantioselectivity and was chosen as a chiral
selector in aqueous phase. It was reported that HP-b-CD
was also previously used for enantioselective extraction,
among others with binary selector systems [22,36].
Effect of Tartaric Acid Derivatives
The distribution coefficients and enantioselectivity were
also determined in chiral extraction containing 0.10 mol/L
HP-b-CD in aqueous phase and 0.20 mol/L-tartaric acid
derivatives in organic phase.
Table 3shows that the distribution coefficients and
enantioselectivity increased when tartaric acid derivatives
were added to the organic phase. In this case the enantio-
selective extraction system has stronger chiral separation
ability than the monophasic extraction.
The use of D-tartaric acid derivatives as an additive led
to the higher K
R
value than K
S
value observed when the
L-tartaric acid derivatives were used. This result indicates
Table 1 Effect of organic solvent on Kand a
Organic solvent K
R
SD K
S
SD aSD
1,2-Dichloroethane 0.050 0.037 0.041 0.035 1.220 0.053
Dichlorobenzen 0.035 0.022 0.020 0.025 1.750 0.076
Cyclohexane 0.087 0.020 0.011 0.018 7.909 0.118
n-Hexanol 0.365 0.058 0.258 0.051 1.411 0.064
n-Heptanol 0.396 0.054 0.272 0.053 1.465 0.091
n-Octanol 0.461 0.060 0.264 0.065 1.745 0.097
n-Decanol 0.487 0.063 0.276 0.059 1.831 0.086
Aqueous phase: [HP-b-CD] =0.10 mol/L, [cyclopentolate] =
0.05 mM/L, pH 6.0
Table 2 Effect of b-CD derivatives
b-CD derivatives K
R
SD K
S
SD aSD
HP-b-CD 0.52 0.074 0.27 0.051 1.96 0.131
DM-b-CD 0.48 0.090 0.35 0.076 1.33 0.068
TM-b-CD 0.45 0.076 0.30 0.050 1.49 0.095
Aqueous phase: [b-CD derivatives] =0.10 mol/L, [cyclopento-
late] =0.05 mmol/L, pH 6.0
Enantioselective Extraction System Containing Binary Chiral Selectors 1607
123
that the D-tartraic acid derivatives have stronger recogni-
tion ability for (R)-cyclopentolate than for (S)-
cyclopentolate.
Chiral extraction performance is related to the structure
of chiral selector. It is very important to investigate the
influence of different branched alkyl side chains of tartaric
acid derivatives on the distribution coefficients and
Table 3 Effect of tartaric acid derivatives
Tartaric acid derivatives K
R
SD K
S
SD aSD
L-Tartaric acid diethyl
ester
0.67 0.064 0.34 0.068 1.97 0.043
D-Tartaric acid diethyl
ester
0.75 0.079 0.38 0.070 1.98 0.056
L-Tartaric acid dibutyl
ester
0.66 0.120 0.34 0.108 1.94 0.099
D-Tartaric acid dibutyl
ester
0.68 0.118 0.35 0.087 1.95 0.127
L-Tartaric acid
diisobutyl ester
0.82 0.096 0.40 0.093 2.02 0.069
D-Tartaric acid
diisobutyl ester
0.85 0.073 0.42 0.062 2.06 0.091
L-Tartaric acid
ditertbutyl ester
0.82 0.086 0.39 0.071 2.09 0.068
D-Tartaric acid
ditertbutyl ester
0.85 0.098 0.40 0.065 2.13 0.082
Organic phase: (tartaric acid derivatives) =0.2 mol/L, aqueous
phase: [HP-b-CD] =0.10 mol/L, [cyclopentolate] =0.05 mmol/L,
pH 6.0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.00 0.05 0.10 0.15 0.20 0.25 0.30
K
[HP-β-CD] (mol/L)
1.5
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
0.00 0.05 0.10 0.15 0.20 0.25 0.30
α
[HP-β-CD] (mol/L)
Fig. 5 Effect of HP-b-CD concentration on Kand a. Organic phase:
[D-tartaric acid ditertbutyl ester] =0.20 mol/L
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
K
[D-tartaric acid ditertbutyl ester] (mol/L)
1.6
1.7
1.8
1.9
2
2.1
2.2
2.3
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
α
[D-tartaric acid ditertbut
y
l ester] (mol/L)
Fig. 6 Effect of D-tartaric acid ditertbutyl ester concentration on
Kand a. Aqueous phase: [HP-b-CD] =0.10 mol/L, [cyclopento-
late] =0.05 mM/L, pH 6.0
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
α
pH
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
K
p
H
Fig. 7 Effect of pH on K and a. Aqueous phase: [HP-b-
CD] =0.10 mol/L, [cyclopentolate] =0.05 mM/L. Organic phase:
[D-tartaric acid ditertbutyl ester] =0.20 mol/L
1608 K. Szwed et al.
123
enantioselectivity. The distribution coefficients and enanti-
oselectivity increase slightly with the degree of alkyl chain
branching. Because the bulky alcohols might allow for a
more stereospecific and stronger interaction with cyclo-
pentolate, which increases the stabilization of complexes of
chiral selector with cyclopentolate molecules. Consistent
with observations by Prelog et al. [37] the highest enanti-
oselectivity is provided by extensive tartaric acid deriva-
tives. However, the differences in the values of the
distribution coefficients and enantioselectivity are too small
for any discussion structural effects on the chiral recognition
mechanism. Therefore, D-tartaric acid ditertbutyl ester was
chosen as the chiral selector in the organic phase.
Effect of Chiral Selector Concentrations
Figures 5and 6both summarize the effect of concentration
of HP-b-CD in the aqueous phase and D-tartaric acid dit-
ertbutyl ester in the organic phase on the distribution
coefficients and enantioselectivity. With the increase of
HP-b-CD concentration, the distribution coefficients for
cyclopentolate enantiomers decrease, which can be
explained by the higher amount of complexes formed in the
aqueous phase. The enantioselectivity increases steadily,
up to the concentration of HP-b-CD at 0.10 mol/L. When
the concentration of HP-b-CD is over 0.10 mol/L the
enantioselectivity decreases subtly. The behavior of
enantioselectivity can be explained by the results of the
cooperation of HP-b-CD in the aqueous phase and D-tar-
taric acid ditertbutyl ester in the organic phase, which is in
accordance with below results.
On the contrary, with increase of D-tartaric acid diter-
tbutyl ester concentration, the distribution coefficients for
cyclopentolate enantiomers increase and the enantioselec-
tivity increases up to the concentration of D-tartaric acid
ditertbutyl ester at 0.20 mol/L. When the concentration of
D-tartaric acid ditertbutyl ester exceeds 0.20 mol/L, the
enantioselectivity begins to decrease.
This is because a large amount of complexes for
cyclopentolate enantiomers were formed in the organic
phase which led to an increase of the distribution coeffi-
cients and the enantioselectivities were the results of the
cooperation of HP-b-CD in the aqueous phase and D-tar-
taric acid ditertbutyl ester in the organic phase.
Time (min)
Time (min)
(a)
(b)
D-tartaric acid ditertbutyl ester
(S) - Cyclopentolate
(R) - Cyclopentolate
0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00
0.00 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 40.00
Fig. 8 Chromatograms of
cyclopentolate enantiomers;
abefore enantioselective
extraction bafter
enantioselective extraction
under optimal conditions
(aqueous phase: [HP-b-
CD] =0.10 mol/L,
[cyclopentolate] =0.05 mM/L,
pH 6.0. Organic phase
(decanol): [D-tartaric acid
ditertbutyl ester] =0.20 mol/L)
Enantioselective Extraction System Containing Binary Chiral Selectors 1609
123
As seen in Figs. 2and 3, the enantioselectivity reaches a
maximum at the ratio 2:1 in the molar concentration of D-
tartaric acid ditertbutyl ester to HP-b-CD. A possible
explanation of this behavior is that HP-b-CD can form
inclusion complexes with cyclopentolate enantiomers, and
the complexation ability of HP-b-CD toward (S)-cyclo-
pentolate is higher than toward (R)-cyclopentolate. This
statement is backed by experimental data presented in
Table 2(K
R
=0.52, K
S
=0.27).
Effect of pH
Figure 7shows the effect of pH on the distribution coef-
ficients and enantioselectivity of cyclopentolate enantio-
mers. As seen in Fig. 2, distribution coefficients decrease
with increase of pH. On the other hand, the enantioselec-
tivity increases with increase of pH.
The possible reasons may be that the ratio between
protonated and unprotonated cyclopentolate decreases
with the rise of pH value. D-tartaric acid ditertbutyl ester
has the chiral recognition ability limited mainly for the
neutral molecule of cyclopentolate. Ionic cyclopentolate
only exists in aqueous phase. In search for the opti-
mum combination of the highest distribution coefficients
and favorable enantioselectivity, the pH 6.0 was an
appropriate choice for extraction of cyclopentolate
enantiomers.
Conclusions
Liquid–liquid extraction proved to be a powerful tool to
achieve chiral separation.
In order to obtain sufficient enantioselectivity, it is
necessary to optimize conditions for the chiral extraction.
The type and concentration of chiral selectors, the pH
value, the choice of organic solvent were clearly identified
as important factors influencing enantioselectivity.
As seen in Fig. 8, high enantioselectivity and distribu-
tion coefficients for cyclopentolate were obtained using
0.10 mol/L HP-b-CD and 0.20 mol/L D-tartaric acid dit-
ertbutyl ester in decanol as organic phase. Optimum pH
was about six for the chiral separation of cyclopentolate.
The enantiomeric excess is 61.13 % for the aqueous phase
under the optimal condition.
Acknowledgments The research was partially supported by the
European Union within European Regional Development Fund,
through grant Innovative Economy (POIG.01.01.02-14-102/09). The
author (MG) acknowledges financial supports of the National Science
Centre, Poland, grant UMO-2011/03/N/ST4/02426 and a grant No
G34-15 for computational time at the Interdisciplinary Centre for
Mathematical and Computational Modelling (ICM) of University of
Warsaw, Poland.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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