Enantioseparation of Racemic Flurbiprofen by Aqueous Two-Phase Extraction With Binary Chiral Selectors of L-dioctyl Tartrate and L-tryptophan

Article abstract of DOI:10.1002/chir.22481

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two-phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two-phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L-dioctyl tartrate and L-tryptophan, which were screened from amino acids, β-cyclodextrin derivatives, and L-tartrate esters. Factors such as the amounts of L-dioctyl tartrate and L-tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L-dioctyl tartrate, 80 mg; L-tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L-dioctyl tartrate and L-tryptophan, which enantioselectively recognized R- and S-enantiomers in top and bottom phases, respectively. Compared to conventional liquid-liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650-657, 2015.

Full text of DOI:10.1002/chir.22481

CHIRALITY 27:650–657 (2015)
Enantioseparation of Racemic Flurbiprofen by Aqueous Two-Phase
Extraction With Binary Chiral Selectors of L-dioctyl Tartrate and
L-tryptophan
1
2
1
1,3
1
1
1
3
ZHI CHEN, WEI ZHANG, * LIPING WANG, HUAJUN FAN, * QIANG WAN, XUEHAO WU, XUNYOU TANG, AND JAMES Z. TANG
1
College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, P.R. China
2
School of Basic Courses, Guangdong Pharmaceutical University, Guangzhou, P.R. China
Faculty of Science and Engineering, University of Wolverhampton, Wolverhampton, UK
3
ABSTRACT
using aqueous two-phase extraction (ATPE) coupled with biphasic recognition chiral extraction
BRCE). An aqueous two-phase system (ATPS) was used as an extracting solvent which was
A novel method for chiral separation of flurbiprofen enantiomers was developed
(
composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in
ATPS for BRCE consideration were L-dioctyl tartrate and L-tryptophan, which were screened
from amino acids, β-cyclodextrin derivatives, and L-tartrate esters. Factors such as the amounts
of L-dioctyl tartrate and L-tryptophan, pH, flurbiprofen concentration, and the operation temper-
ature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum
conditions were as follows: L-dioctyl tartrate, 80 mg; L-tryptophan, 40 mg; pH, 4.0; flurbiprofen
concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for
flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen
enantiomers is discussed and studied. The results showed that synergistic extraction has been
established by L-dioctyl tartrate and L-tryptophan, which enantioselectively recognized R- and
S-enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid
extraction, ATPE coupled with BRCE possessed higher separation efficiency and
enantioselectivity without the use of any other organic solvents. The proposed method is a poten-
tial and powerful alternative to conventional extraction for separation of various enantiomers.
Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: chiral separation; flurbiprofen enantiomers; aqueous two-phase extraction;
biphasic recognition chiral extraction; synergistic extraction
Flurbiprofen, which belongs to the family of 2-arypropionic
of many current chiral selectors is often limited to their sol-
4–27
acid, is one of the well-known nonsteroidal antiinflammatory
ubility for many solvents.2
The enantioselectivity of
1
,2
drugs (NSAID) in clinical use. Its S-enantiomer exhibits
strong antiinflammatory activity by inhibiting the cyclooxy-
genase system, whereas the R-enantiomer has no anti3-
cyclooxygenase activity except gastrointestinal toxicity.
Recently, the R-enantiomer has been found and is being used
monophasic recognition extraction is relatively low with a
chiral selector, whereas biphasic recognition chiral extrac-
tion (BRCE) can greatly improve the enantioselectivity by
the cooperation of both the separation abilities of hydropho-
bic and hydrophilic chiral selectors. The combination of two
chiral selectors enables forming hydrophobic and hydro-
philic complexes with specific enantiomers and then being
3
–6
for the treatment of Alzheimer’s disease
and inhibiting
tumor growth.7 The demand for single enantiomers is grow-
–9
21–27
ing rapidly and draws more and more attention to the chiral
extracted into organic and aqueous phases, respectively.
1
0–13
separation of flurbiprofen.
The key to successful separation is whether chiral selectors
match target enantiomers in the reaction extraction. For
example, tartaric esters and β-cyclodextrin or its derivatives
were used as double chiral selectors in BRCE, and separation
Flurbiprofen is produced in large quantity as a racemic mix-
ture. Various methods such as enantioselective liquid–liquid
extraction (ELLE), crystallization, enzymatic resolution,
kinetic resolution and chromatographic techniques, etc., have
factors α for some aromatic acid enantiomers have been
14–19
21,23,27,28
been exploited for enantioseparation of flurbiprofen.
significantly improved.
But even so, ELLE involves
Among them, chiral extraction is one of the most attractive
techniques due to its simplicity of operation, economic
exploring organic solvent/water systems, which have the dis-
advantages of emulsification and large consumption of toxic,
2
0
efficiency, and ease of scale-up. Currently, ELLE is focused
on the development of chiral selectors. An optimal solvent
extraction system was based on the reactive extraction of a
target enantiomer using one chiral selector or more. Thus,
chiral selectors identified the specific enantiomers from the
racemic mixture and formed diastereomeric complexes,
which could be extracted separately. A variety of chiral selec-
tors such as crown ether, porphyrin, tartaric acid derivatives,
cyclodextrins and their derivatives, etc., were selected as
Contract grant sponsor: State Project for Essential Drug Research and Devel-
opment; Contract grant number: 2011ZX09102-001-31.
Contract grant sponsor: Faculty Development fund Project of Guangdong
Pharmaceutical University; Contract grant number: 52104109.
*Correspondence to: Wei Zhang, School of Basic Courses, Guangdong Phar-
maceutical University, Guangzhou 510006, P.R. China, or Huajun Fan, College
of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, P.R.
China. E-mail: junhuafan@126.com or gzzhangw@163.com
Received for publication 30 March 2015; Accepted 22 May 2015
DOI: 10.1002/chir.22481
Published online 14 July 2015 in Wiley Online Library
(wileyonlinelibrary.com).
1
9–24
chiral selectors in ELLE.
Nevertheless, the application
©
2015 Wiley Periodicals, Inc.

ENANTIOSEPARATION OF RACEMIC FLURBIPROFEN
651
flammable, and volatile organic solvents.21–28 Since the intro-
glutamic acid and glycine were acquired from Sinogel Amino Acid (Anhui,
China); L-tartrate derivatives were synthesized as described in the litera-
ture ; n-hexane and ethanol were of HPLC grade from Merck Darmstadt
(Shanghai, China). All other chemicals were analytical grade from
Guangzhou Chemical Reagent Factory (Guangzhou, China).
duction of aqueous two-phase extraction (ATPE) in chiral
2
0
separation,29 ATPE has been explored in the chiral separation
30–36
of enantiomers.
An aqueous two-phase system (ATPS) is composed of
two or more phase-forming substances dissolved in water
Flurbiprofen stock solution (1.00 mmol/L): Briefly, flurbiprofen
(24.4 mg) was dissolved in ethanol and diluted up to 100 mL in a 100-mL
(
e.g., two polymers, polymer and a salt, two surfactants, or
volumetric flask. It was sealed properly and stored in a refrigerator. Its
standard solution was made from the stock solution when used.
Phosphate buffer solution (PBS) (pH 2.0): A mixture of solution A
(27.5 mL) and solution B (72.5 mL). Solution A was prepared by dissolving
disodium hydrogen phosphate 35.185 g in water and then diluted up to
more surfactants). The phase-forming polymers and surfac-
tants used extensively in the separation system are too viscous
to handle and often form opaque solutions. Recent investiga-
tions shift the paradigm through use of short chain alcohol
and salt. It offers advantages of low viscosity, easy demixing,
solvent recycling, and a more environment-friendly process,
and gains more popularity in extracting active constituents
500 mL and solution B by adding phosphoric acid 8.4 mL to water, mixed
well, and then diluted up to 500 mL.
3
7–43
Methods
from natural products of herb plants.
Thus, it is a green
and potential alternative to conventional ATPS in chiral
separation.
Preparation of ATPS. According to the method described in the liter-
44
ature, ATPS containing ethanol (35.0 %, w/w) and ammonium sulfate
18.0% w/w) was obtained from a mixture of deionized water and ethanol
(
A BRCE combining a L-iso-butyl tartrate and trimethyl-β-
cyclodextrin has the advantage of achieving enantioselective
extraction of flurbiprofen enantiomers with a separation
factor of 1.24, but toxic solvents such as methylene chloride,
showing two-phase separation as a result of adding ammonium sulfate.
Briefly, ammonium sulfate (1.9 g) was dissolved in deionized water
(6 mL) and ethanol (4 mL). The phase ratio (β) was calculated by measuring
the volumes of top (Vtop) and bottom (Vbottom) phases.
2
2
1
,2-dichloroethane, etc., were used. In this work, we report
VTop
β ¼
a novel method of chiral separation of flurbiprofen enantio-
mers using BRCE-coupled ATPE. In order to separate
enantiomers from a racemic mixture, chiral selectors were
introduced into the hydrophobic and hydrophilic phases of
ATPS and chiral separation by ATPS was therefore achieved.
Chiral selectors, for this purpose, were screened from amino
acids, β-cyclodextrin derivatives, and L-tartrate esters. Fac-
tors such as the amounts of chiral selectors, pH, flurbiprofen
concentration, and the operation temperature were investi-
gated in terms of chiral separation of flurbiprofen enantio-
mers. The mechanism of chiral separation of flurbiprofen
enantiomers was discussed for further investigation. Without
any toxic solvent, enantioselectivity of the proposed method
for flurbiprofen was greatly improved when using L-dioctyl
tartrate and L-tryptophan as chiral selectors (as shown in
Fig. 1). BRCE-coupled ATPE was proved to be a green, effec-
tive, and promising alternative for chiral separation of
flurbiprofen enantiomers.
VBottom
Extraction experiments. The extraction experiments were performed
in a graduated tube. Two mL of racemic flurbiprofen (0.10 mmol/L),
L-tryptophan (40 mg), and L-dioctyl tartrate (150 mg) were added in the
graduated tube and then mixed well with ATPS (10 mL). The mixture
was adjusted to a pH of 4.0 using PBS solution (pH = 2.0) and mixed thor-
oughly before being placed on a water bath oscillator and shaken for 3 h
at 25 °C. After demixing, volumes of top (Vtop) and bottom (Vbottom
)
phases were measured for the calculation of the phase ratio β. Concentra-
tions of R-flurbiprofen and S-flurbiprofen of samples collected from the
top phase were analyzed by HPLC and those from the bottom phase were
calculated using a subtraction method. The partition coefficient K and the
separation factor α were obtained according to the following equations:
CT;R
CB;R
KR ¼
Ks ¼
(1)
(2)
(3)
CT;S
CB;S
MATERIALS AND METHODS
KR
KS
KS
KR
α ¼
and K
ðIf KR > KSÞor α ¼
ðIf KR < KSÞ
Materials
Racemic flurbiprofen (purity >98%) was purchased from Tokyo Chem-
ical Industry (Tokyo, Japan); R-flurbiprofen (purity >99%) was purchased
from American Alpha Reagent (Shanghai, China). Carboxymethyl-β-
cyclodextrin (CM-β-CD) was provided by Sichuan Antibiosis (Chengdu,
China); β-cyclodextrin (β-CD, purity ≥99%) was purchased from Shanghai
Bio Science Technology (Shanghai, China); methyl-β-cyclodextrin (Me-β-
CD) and hydroxypropyl-β- cyclodextrin (HP-β-CD) were obtained
from Shandong Binzhou Zhiyuan Bio-Technology (Binzhou, China);
L-tryptophan was bought from Aladdin Industrial (Shanghai, China);
Where K
S-flurbiprofen, respectively;
R
S
are partition coefficients of R-flurbiprofen and
B,R are concentrations of
C
T,R and C
R-flurbiprofen in top and bottom phases, respectively; CT,S and CB,S are
concentrations of S-flurbiprofen in top and bottom phases, respectively.
HPLC analysis. The quantification of flurbiprofen enantiomers in the
top phase was performed on an Agilent 1200 Infinity chromatograph
(Agilent Technologies, Palo Alto, CA) at 25 °C with a UV detector at
4
5
254 nm. The chromatographic conditions were as follows: the column
Fig. 1. The chemical structure of flurbiprofen (a), L-dioctyl tartrate (b), and L-tryptophan (c).
Chirality DOI 10.1002/chir

6
52
CHEN ET AL.
was Phenomenon Lux 5u Cellulose-3 (250 × 4.6 mm, 5 μm). The mobile
phase was methanol: 0.1% formic acid (85:15, v/v). The injection volume
was 20 μL. The flow rate was 1.0 mL/min. It was observed that the R-
and S-flurbiprofen enantiomers were completely separated from each
other, with an indication that the R-enantiomer was eluted first against
the S-enantiomer. The detector responses were linear from 2.38 μg/mL
to 238.00 μg/mL of both R-flurbiprofen (A = 45802C+61.350,r = 0.9996)
and S-flurbiprofen (A = 45518C+52.100,r = 0.9991). The detection limit
was 0.0600 μg/mL for R-flurbiprofen and 0.0621 μg/mL for S-flurbiprofen.
For comparison, HPLC analysis of L-dioctyl tartrate in the top phase was
also carried out at 25 °C with a UV detector at 220 nm and 254 nm as well.
The enantioselectivity of L-tartaric esters was lost when the
number of carbons in the ester chain is smaller than 5. The
hydrophilic amino acids also show interesting results, which
is very useful in ATPE. In this group, L-tryptophan has a
separation factor of 1.26, much better than the best of
β-cyclodextrin derivatives. Therefore, L-tryptophan was chosen
as a chiral selector in parallel with L-dioctyl tartrate, which is
more hydrophobic in nature.
Interaction of Flurbiprofen With L-Dioctyl Tartrate and
L-Tryptophan at pH 4
UV–vis analysis. The interaction of flurbiprofen with L-dioctyl tartrate
The chemical structures of flurbiprofen, L-dioctyl tartrate,
and L-tryptophan are illustrated in Figure 1. In terms of hy-
drophilicity, it could be on the order of L-tryptophan >
flurbiprofen > L-dioctyl tartrate. L-tryptophan is slightly solu-
ble in water, so the hydrophilicity was largely achieved
through pH 4 ionization. The interaction of flurbiprofen with
L-dioctyl tartrate and L-tryptophan in the top phase of APTS
was investigated with UV–vis analysis. Figure 2 shows the
UV–vis spectra of the mixture of flurbiprofen, L-dioctyl tar-
trate, and/or L-tryptophan in the top phase after ATPE. It
reflected the hydrophobicity of flurbiprofen (Log P = 4.16)
with a broad absorption at 248 nm. Adding L-dioctyl tartrate
significantly enhanced this absorbance, while adding L-
tryptophan reduced this absorbance in terms of broadness
and strength. The combination of both L-dioctyl tartrate and
L-tryptophan was logically in the middle and provided consid-
eration in exploiting BRCE-coupled ATPE.
and L-tryptophan was evaluated under UV Vis analysis. UV spectra from
2
00 nm to 400 nm were obtained by scanning the top phase of the ATPS,
which was coupled with BRCE. Briefly, L-dioctyl tartrate (80 mg) and L-
tryptophan (40 mg) were added to the flurbiprofen solution separately.
Then the mixture was added to ATPS (10 mL), vortex-mixed, and pH
adjusted to 4 using PBS (pH 2.0). The top phase was collected after oscil-
lation for 3 h at 25 °C and analyzed using a Shimadzu 2550 UV–vis Spec-
trophotometer (Shimadzu Scientific Instrument, Japan).
RESULTS AND DISCUSSION
Screening of Chiral Selectors
ATPS of ethanol/(NH ) SO was selected as the extraction
4
2
4
solvent. It was screened from seven ATPS systems in our re-
40
cent practices. There are three types of chiral selectors,
namely, amino acids, β-cyclodextrin derivatives, and L-tartaric
esters for chiral separation of flurbiprofen enantiomers.
Screening of chiral selectors was carried out using ATPS of
ethanol/(NH ) SO at a phase ratio β of 2. Enantioselectivity
4
2
4
Strategic Design of an ATPS With L-Dioctyl Tartrate and
L-Tryptophan Chiral Selectors
of the extraction was assessed by separation factor α, which
was calculated by measuring partition coefficients of R-
enantiomer and S-enantiomers. The obtained results as
shown in Table 1 indicate that there was a significant differ-
ence in the enantioselective extraction in ATPS with these
three types of chiral selectors and their chiral recognition ca-
pacities were assessed using separation factor α in this study.
Among β-cyclodextrin derivatives, β-cyclodextrin has a higher
separation factor α of 1.12 and enantioselectivity than the
other three, such as methyl-β-cyclodextrin, hydroxypropyl-
β-cyclodextrin, and carboxymethyl-β-cyclodextrin, whose separa-
tion factors were ~1.0. Compared with β-cyclodextrin derivatives,
L-tartaric esters had a much higher α value when a longer
ester chain of tartrate was used. L-dioctyl tartrate has a separa-
tion factor of 1.81, considered the strongest chiral selector in
the group, L-dipentyl tartrate of 1.37, considered the second.
The new method of chiral separation of flurbiprofen enan-
tiomers was designed based on ATPS with the incorporation
of BRCE. Figure 3 shows an illustration of this modification.
ATPS has a top phase of ethanol and a bottom phase of
(NH ) SO aqueous solution. L-dioctyl tartrate (L-OT), char-
4 2 4
acterized by two long hydrocarbon chains and hydroxyl
groups, was largely dissolved in the top phase, while L-
tryptophan (L-T), with a pKa of 5.88, was ionized at pH 4
and preferably dissolved in the bottom phase. Flurbiprofen
(HA), with a pKa of ~4.24, acquired perfect equilibrium at
pH 4.0. The separation efficiency relies on the chiral resolu-
tion in both phases.
TABLE 1. Screening of chiral selectors in ATPS of ethanol/
(
4 2 4
NH ) SO under excess of chiral selectors (CFlurbiprofen =
0
.10 mmol/L, pH 2.0 and temperature 25 °C)
Chiral selector
K
R
K
S
α
β-cyclodextrin
0.25
0.47
0.97
0.86
0.67
0.79
1.09
1.36
0.84
0.74
1.47
1.79
0.28
0.46
0.96
0.86
0.67
0.78
0.80
0.75
0.82
0.79
1.68
1.42
1.12
1.02
1.01
1.00
1.00
1.02
1.37
1.81
1.02
1.08
1.14
1.26
Methyl-β-cyclodextrin
Hydroxypropyl-β-cyclodextrin
Carboxymethyl-β-cyclodextrin
L-di-iso-butyl tartrate
L-dibutyl tartrate
L-dipentyl tartrate
L-dioctyl tartrate
L-phenylalanine
L-glycine
L-glutamic acid
L-tryptophan
Fig. 2. UV Vis spectra of interaction of flurbiprofen with L-dioctyl tartrate
and/or L-tryptophan in the top phase after APTE.
Chirality DOI 10.1002/chir

ENANTIOSEPARATION OF RACEMIC FLURBIPROFEN
653
tartrate and from 10 mg to 80 mg for L-tryptophan. Figure 4
shows that the chiral selection of the BRCE-coupled ATPE
in terms of K , K , and α for flurbiprofen enantiomers, pro-
R
S
viding that other factors were kept under L-tryptophan
40 mg), pH 2.0, flurbiprofen (0.10 mmol/L), and temperature
(
at 25 °C. The results present a clear rise of K with L-dioctyl
R
tartrate of 80 mg. There was no change of K in line with the
S
addition of L-dioctyl tartrate, indicating the preference of the
R-enantiomer with L-dioctyl tartrate. The optimized separa-
tion factor α was also obtained when L-dioctyl tartrate of
8
0 mg was used. Figure 5 shows that the chiral selection of
the BRCE-coupled ATPE in terms of K , K , and α for
R
S
flurbiprofen enantiomers, providing that other factors were
kept under L-dioctyl tartrate (80 mg), pH 2.0, flurbiprofen
(
0.10 mmol/L), and temperature at 25 °C. The results
Fig. 3. Schematic illustration of the biphasic recognition in aqueous two-
phase extraction. L-dioctyl tartrate (L-OT), L-tryptophan (L-T), and
showed a simultaneous rise of both K and K values with
R
S
4 2 4
flurbiprofen (HA) in ATPS of ethanol/(NH ) SO aqueous solution.
L-tryptophan of 60 mg, indicating no significant preference
of either the R-enantiomer or S- enantiomer. The optimized
separation factor α was obtained when L-tryptophan of
Optimization of the Formulation of Chiral Selective ATPS
and Other Key Parameters in Extraction
4
0 mg was used, but it was not as significant as that when
A chiral selective ATPS was proposed with two chiral selec-
tors, namely, L-dioctyl tartrate and L-tryptophan and an ATPS
of ethanol/(NH ) SO aqueous solution. Although the pH
was set at 4, it was not optimized for chiral separation of
flurbiprofen enantiomers. In this study, we optimized the for-
mulation in terms of the amounts of chiral selectors and the
concentration of flurbiprofen, the system pH value, and the
operation temperature.
L-dioctyl tartrate was used.
Optimization of pH. The window opened for optimization was
from pH 2.0 to 6.0, which considered the acidity of
flurbiprofen and L-tryptophan. Figure 6 shows the effect of
4
2
4
pH on KR, K , and α, providing that other factors were kept un-
S
der L-dioctyl tartrate (80mg), L-tryptophan (40mg), flurbiprofen
(0.10 mmol/L), and temperature at 25 °C. The results indicate
that K was affected more than K and both decreased when
S
R
Optimized amounts of chiral selectors. The windows opened
for optimization were from 20 mg to 100 mg for L-dioctyl
the pH was more than 3 until reaching bottom values at
pH 4.0. There were no massive increase in either K and K
R
S
Fig. 4. The effect of L-dioctyl tartrate concentration on K (a) and α (b) for flurbiprofen enantiomers. mL-tryptophan = 40 mg, CFlurbiprofen = 0.10 mmol/L, pH 2.0, and
temperature 25 °C.
Fig. 5. The effect of L-tryptophan concentration on K (a) and α (b) for flurbiprofen enantiomers. mL-dioctyl tartrate = 80 mg, CFlurbiprofen = 0.10 mmol/L, pH 2.0, and
temperature 25 °C.
Chirality DOI 10.1002/chir

6
54
CHEN ET AL.
Fig. 6. The effect of pH on K (a) and α (b) for flurbiprofen enantiomers. mL-dioctyl tartrate = 80 mg, mL-tryptophan = 40 mg, CFlurbiprofen = 0.10 mmol/L, and temperature
5 °C.
2
values from pH 4.0 to 6.0. It appeared that at pH 4.0 the BRCE-
coupled ATPE achieved the best enantioselectivity, which was
agreeable with the acidity value of flurbiprofen (pKa = 4.24),
when a one-to-one ratio of the dissociation equilibrium of two
the K value increased more rapidly above the 0.10 mmol/L,
therefore nonselective partitioning prevailed. It appeared that
the flurbiprofen concentration might be limited by the
amounts of chiral selectors based on enantioselective
complexation in the presence of L-dioctyl tartrate and
L-tryptophan.
ꢀ
chemical moieties (HA and A ) was reached.
Optimization of flurbiprofen concentration. The window opened
for optimization was from 0.05 to 0.25 mmol/L in consider-
ation of that 0.10 mmol/L of flurbiprofen was used above.
Figure 7 shows the effect of flurbiprofen concentration on
K , K , and α, providing that other factors were kept under
Optimization of the operation temperature. The window opened
for optimization was from 15 to 35 °C when BRCE-coupled
ATPE was exploited. Figure 8 shows the effect of the opera-
tion temperature on K , K , and α, providing that other fac-
R ,
S
R ,
S
L-dioctyl tartrate (80 mg), L-tryptophan (40 mg), pH 4.0, and
tors were kept under L-dioctyl tartrate (80 mg), L-tryptophan
temperature at 25 °C. The results indicated that both KR
(40 mg), pH 4.0, and flurbiprofen (0.10 mmol/L). The results
and K values increase with flurbiprofen concentration, while
indicate that the operation temperature only affected K and
S
R
the α value started decreasing after 0.10 mol/L. Noticing that
α values at 25 °C, with a relatively unchanging K value. This
S
Fig. 7. The effect of flurbiprofen concentration on K (a) and α (b) for flurbiprofen enantiomers. mL-dioctyl tartrate = 80 mg, mL-tryptophan = 40 mg, pH 4.0, and temper-
ature 25 °C.
Fig. 8. The effect of temperature on K (a) and α (b) for flurbiprofen enantiomers. mL-dioctyl tartrate = 80 mg, mL-tryptophan = 40 mg, CFlurbiprofen = 0.10 mmol/L, and
pH 4.0.
Chirality DOI 10.1002/chir

ENANTIOSEPARATION OF RACEMIC FLURBIPROFEN
655
Fig. 9. HPLC Chromatograms of R-flurbiprofen and S-flurbiprofen enantiomers in the top phase before (a) and after extraction (b).
was unusual in conventional extraction, which was acceler-
ated by temperature rise. The phenomenon could be ex-
plained by the fact that the enantioselective complexation
between the L-dioctyl tartrate and R-enantiomer was achieved
at 25 °C and an optimized α value of 2.34 was obtained. Com-
paratively, the effect of temperature on chiral separation of
flurbiprofen enantiomers was quite different from other bi-
Chiral Separation of BRCE-Coupled ATPE in Terms of
HPLC Analysis
Figure 9 shows the HPLC chromatograms of R-flurbiprofen
and S-flurbiprofen in the top phase before (a) and after
extraction (b). The concentration ratio of R-flurbiprofen and
S-flurbiprofen were 1.0 and 1.92 before and after extraction,
respectively. E.E.% (enantiomer excess percentage) of R-
flurbiprofen in the top phase reached 43.9% according to
equation (4). BRCE-coupled ATPE enriched the collection
of R-flurbiprofen enantiomer with about a 50% increase in R-
enantiomer in the top phase.
1
9–21
phasic recognition extraction systems.
CT;R ꢀ C
T;S
E:E:ð%Þ ¼
ꢁ100%
(4)
CT;R þ C
T;S
Mechanism of Chiral Resolution of Flurbiprofen
Enantiomers
The strategic design of ATPS with specific chiral
selectors was evaluated and supported with about a 50% in-
crease of R-flurbiprofen enantiomer enriched in the top
phase. From the chromatograms in Figure 10, flurbiprofen
enantiomers in the top phase were detected at 254 nm for
maximum adsorption, while L-dioctyl tartrate without a
chromophoric group was not observed at 254 nm but at
2
20 nm. The enrichment might be due to the enantioselective
Fig. 10. HPLC chromatograms of L-dioctyl tartrate in the top phase before
complexation between the L-dioctyl tartrate and R-enantiomer
was achieved at 25 °C, and resulted in the reduction of L-
and after enantiomeric complexation at 220 nm in comparison with that at
2
54 nm.
Fig. 11. The process of enantioselective extraction of flurbiprofen with L-dioctyl tartrate and L-tryptophan by ATPE.
Chirality DOI 10.1002/chir

6
56
CHEN ET AL.
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dioctyl tartrate after ATPE, shown in Figure 10. The quantity
change of L-dioctyl tartrate before and after ATPE also
revealed the interaction of flurbiprofen enantiomers with chiral
selectors. In this study, a cutoff concentration of flurbiprofen
1
7
. Grösch S, Schilling K, Janssen A, Maier TJ, Niederberger E, Geisslinger
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(
0.10 mmol/L) provided a separation factor of 2.34, which indi-
cated that chiral separation occurred at a concentration of
flurbiprofen that chiral selectors limited. Figure 11 illustrates
this enantioselective complexation between chiral selectors
and R- and S-enantiomers based on phenomena observed.
First, the R-enantiomer and S-enantiomer were ionized at
pH 4.0, and although flurbiprofen is hydrophobic in nature,
partition into the ATPS aqueous bottom phase was driven by
ionization. Second, L-dioctyl tartrate was added and spontane-
ously dissolved in ethanol due to the least ionization occur-
ring, whereas L-tryptophan was added and more easily
dissolved in the aqueous bottom phase due to ionization.
Third, L-dioctyl tartrate had preference in enantioselective
complexation with the R-enantiomer, providing an opportunity
of L-tryptophan forming enantioselective complexation more
with the ionized S- enantiomer. As a result, L-dioctyl tartrate
and L-tryptophan had synergistic effects on extraction of
flurbiprofen enantiomers in binary chiral recognition. This
was agreeable with our UV Vis spectral analysis in Figure 2.
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CONCLUSION
A novel method of chiral separation of flurbiprofen enantio-
mers, namely, BRCE-coupled ATPE, was developed based on
chiral selectors enhanced biphasic recognition in both top
and bottom phases of the ATPS used. The chiral selectors were
L-dioctyl tartrate in the top phase and L-tryptophan mainly in
the bottom phase of the ethanol / ammonium sulfate system.
It was confirmed that L-dioctyl tartrate enriched the top
phase and had preferential complexation with flurbiprofen
R-enantiomer, and L-tryptophan enriched the bottom phase
and attracted the ionized flurbiprofen S-enantiomer. The new
1
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1
1
1
2
2
5
0% increase in extrac-
BRCE-coupled ATPE enabled about a
tion of flurbiprofen R-enantiomer in the top phase in compari-
son with that extracted purely with ATPE. A certain cutoff
concentration of flurbiprofen was exploited in BRCE-coupled
ATPE to avoid the occurrence of nonchiral selective ATPE.
Compared to conventional enantioselective extraction, BRCE-
coupled ATPE showed a higher separation efficiency. More-
over, it is a simple, cost-effective method, which is easy to scale
up and friendly to the environment without the addition of any
organic solvent. This method could be further developed as a
potential and powerful alternative to conventional extraction
for separations of various enantiomers.
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