Extraction of phenylalanine enantiomers by aqueous two phase systems containing combinatorial chiral selector

Article abstract of DOI:10.1002/cjoc.201100224

In order to obtain a better enantioselectivity of phenylalanine enantiomers and establish the optimal chiral extraction conditions, the distribution behavior was investigated in aqueous two-phase systems which were composed of polyethylene glycol and ammonium sulfate containing combinatorial chiral selector: β-cyclodextrin and HP-β-cyclodextrin. The influence of the molar concentration ratio of combinatorial chiral selectors, the total molar concentration of combinatorial chiral selectors, pH value, buffer type and its concentration were thoroughly studied, respectively. The results show that the enantioselectivity reaches 1.53 under the optimal chiral extraction conditions. This extraction is a potential economical and effective way for chiral resolution. Copyright

Full text of DOI:10.1002/cjoc.201100224

FULL PAPER
DOI: 10.1002/cjoc.201100224
Extraction of Phenylalanine Enantiomers by Aqueous Two
Phase Systems Containing Combinatorial Chiral Selector
Chen, Xiaoqing(陈晓青)
Liu, Li(刘莉)
Jiao, Feipeng*(焦飞鹏)
Wang, Zhen(王珍)
School of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China
In order to obtain a better enantioselectivity of phenylalanine enantiomers and establish the optimal chiral ex-
traction conditions, the distribution behavior was investigated in aqueous two-phase systems which were composed
of polyethylene glycol and ammonium sulfate containing combinatorial chiral selector: β-cyclodextrin and
HP-β-cyclodextrin. The influence of the molar concentration ratio of combinatorial chiral selectors, the total molar
concentration of combinatorial chiral selectors, pH value, buffer type and its concentration were thoroughly studied,
respectively. The results show that the enantioselectivity reaches 1.53 under the optimal chiral extraction conditions.
This extraction is a potential economical and effective way for chiral resolution.
Keywords aqueous two phase extraction, combinatorial chiral selectors, chiral resolution, enantioselectivity,
phenylalanine enantiomers
Introduction
resolution of racemic terbutaline and mandelic
acid.[11,12]
An aqueous two-phase system (ATPS) is a new
separation technique and shows unique advantage of
separation and purification in biology substance, natural
production, antibiotics, etc.[1] These systems are formed
when aqueous solutions of two mutually incompatible
components are separated into two phases of different
densities under the force of gravity. It has been reported
that aqueous two phase extraction shows significant
advantages including less process time, small interfacial
tension, non-residual solvents, innocuity, facile opera-
tion conditions, and easy polymer recycling.[2-8] Seller-
gren and Ekberg proposed the use of aqueous two-phase
system by a few counter-current extractions for semi-
preparative chiral separation at 1988.[9] However, it does
not achieve any important result from then on in the
field of chiral separation.
Based on the previous results, the distribution be-
havior of phenylalanine enantiomers (D,L-Phe, Figure
1a) was investigated in ATPS composed of polyethylene
glycol (PEG) and ammonium sulfate [(NH4)2SO4] con-
taining combinatorial chiral selectors: β-cyclodextrin
(β-CD, Figure 1b) and hydroxypropyl-β-cyclodextrin
(HP-β-CD, Figure 1c). The influence of the molar
concentration ratio of combinatorial chiral selectors, the
total molar concentration of combinatorial chiral selec-
tors, pH value, buffer type and its concentration were
studied, respectively. This study provides an environ-
mental friendly, effective and economical chiral separa-
tion method and is important for the development of
aqueous two phase extraction (ATPE) technique.
O
HO
Combinatorial methods have more and more impor-
tant effect on screening medication lead compound and
a widely application in chemistry research. Vries et al.[10]
first proposed the application of combinatorial methods
in chemistry resolution, which is a significant break-
through in resolution methodology. They took the com-
bination of diaryl formyl derivant to separate chiral
amine by crystallization. The results showed that the
combination improved resolution efficiency greatly,
accelerated crystallization and increased enantioselec-
tivity. The combinatorial resolution was also applied in
OH
O
NH2
OH
*
a
O
*
OH
7
b
OR
O
CH3
CH2 CH
*
RO
*
O H
R =
n = 1,2, ...
OR
O
n
*
7
c
Figure 1 Molecular structures of (a) D,L-Phe, (b) β-CD and (c)
HP-β-CD.
*
E-mail: jiaofp@163.com
Received August 24, 2011; accepted October 20, 2011; published online March 7, 2012.
Chin. J. Chem. 2012, 30, 965—969
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Chen et al.
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Experimental
Chemicals
Racemic Phe was purchased from Alfa Aesar, a
Johnson Matthey Company (American). β-CD and
HP-β-CD were purchased from Chemical Reagent Si-
nopharm Group Co. Ltd (Shanghai, China). (NH4)2SO4
was obtained from the third chemical factory (Jiaozuo,
China). PEG with molecular weight 2000 was supplied
by Chemical Reagent Sinopharm Group Co. Ltd
(Shanghai, China). Perchloric acid was purchased from
Guangfu Institute of Fine Chemicals (Tianjin, China).
Disodium hydrogen phosphate was purchased from
Xin’an Science and Technology Co. Ltd (Jiaozuo,
China). Acetic acid and phosphoric acid were purchased
from Chemical Reagent Factory of Hunan Normal Uni-
versity (Changsha, China). All the chemicals are of
analytical-reagent grade.
Quantification
The quantification of Phe enantiomers in the bottom
phase was performed by the liquid chromatograph
(Shimadzu, Japan) with a Crownpak CR(＋) column
(150 mm×4.0 mm i.d.) (Daicel, Japan) and coupled
with an SPD-10 UV/VIS spectrophotometer detector
(Shimadzu, Japan) and an AT-130 temperature controller
(Autoscience, Tianjin, China).
The mobile phase for Phe enantiomers was perchlo-
ric acid aqueous solution with pH 2.0 at a flow of 0.8
mL/min. The UV spectrometer was operated at 200 nm,
while the column temperature was set at 25 ℃. The
retention time of (D)-enantiomer is less than that of
(L)-enantiomer (Figure 2).
Figure 2 (a) Chromatogram of D,L-Phe in the lower phase.
Initial aqueous concentration of D,L-Phe was 400 mmol/L, pHeq
5.50; (b) separation of Phe enantiomers after extraction with
combinatorial chiral selectors β-CD and HP-β-CD.
which is expressed in term of separation factor (α) and
enantiomeric excess (ee), are important parameters to
estimate aqueous two-phase chiral-extraction perform-
ance of extractant. They can be calculated by the fol-
lowing formulas:
KD C /C
(1)
(2)
(3)
＝
t,D b,D
K
C /C
t,L b,L
＝
L
ATPE
α
K /K
D L
＝
For each extraction experiment, an ATPS was pre-
pared by mixing 5 mL of a given concentration of chiral
selectors aqueous solution with Phe enantiomers and 20
mL of a given quantity PEG2000 solution and
(NH4)2SO4. The pH value was adjusted with 5 mL of
buffer solution and measured by a pH meter. The con-
tents were mixed thoroughly using a magnetic stirrer at
the rotation speed of 800 r/min. Subsequently the mix-
ture was separated for about 2 h in a thermostat water
bath with fixed temperature. The phase separation was
completed by putting the contents in a vibrating centri-
fuge at the speed of 3000—3500 r/min, operated for
about 6—8 min. After clear separation of the two phases,
the volumes of the top and bottom phase were noted.
The concentration of D,L-Phe enantiomer in the bottom
phase was analyzed by HPLC. The concentration of
D,L-Phe enantiomer in the top phase was calculated by
subtractive method. Each experiment was repeated
twice at least and the standard deviation between two
should be less than ±2%.
Top phase:
C
t,D |Ct,L
ee＝
×100%
×100%
(4)
Ct,D＋Ct,L
Bottom phase:
C
b,L |Cb,D
ee＝
(5)
Cb,L＋Cb,D
among which Ct,D and Cb,D represent the concentration
of D-Phe enantiomer in the top phase and bottom phase,
respectively. Similarly, Ct,L and Cb,L represent the con-
centrations of L-Phe enantiomer, respectively. KD and
KL represent the distribution coefficient of D-Phe enan-
tiomer and L-Phe enantiomer, respectively.
In the process of ATPE, chiral extraction is carried
out by the formation of two diastereomeric inclusion
complexes between chiral selector and D,L-enantiomer
due to potential molecular interactions, hydrogen bond,
Theory of ATPE
Distribution coefficient (K) and enantioselectivity
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Chin. J. Chem. 2012, 30, 965—969

Extraction of Phenylalanine Enantiomers by Aqueous Two Phase Systems
polarization, induction, or electrostatics existing. These
two diastereomeric inclusion complexes, with different
physical and chemical properties, have different interac-
tion with PEG of the top phase, which leads to different
distribution behavior of Phe enantiomer in ATPS.
similar to distribution trend reported previous study,[20]
which shows combinatorial chiral selectors own better
separation effect.
Influence of different molar concentration ratio of
combinatorial chiral selectors
The effect of various molar concentration ratios of
combinatorial chiral selectors for the distribution be-
havior of racemic Phe was investigated under the same
conditions as above except that the concentration ratio
of β-CD and HP-β-CD was variational though the total
concentration was 0.01 mol/L fixedly. Table 2 shows
that, α and ee reached maximum when the molar con-
centration ratio of β-CD to HP-β-CD was 2∶3. The
possible reason is that different ratio of β-CD to
HP-β-CD has different chiral recognition ability and
enantioselectivity separation efficiency for racemic Phe.
HP-β-CD has better chiral recognition ability while
β-CD has higher separation efficiency. Therefore, the
optimal concentration ratio of β-CD to HP-β-CD is 2∶3
in combinatorial chiral selectors.
Results and Discussion
Influence of different chiral selectors
The composition of ATPE system should be a pa-
rameter under control. The molecular weight of PEG
and mass fraction of (NH4)2SO4 were decided after
thoroughly literature survey. Molyneux et al.[13] sup-
ported that small molecular weight PEG can be dis-
solved in the water while high molecular weight PEG is
less hydrosoluble. Thus, some researchers adopted small
molecular weight PEG, such as PEG400 PEG425
PEG600 and PEG725, to separate amino enantiomers in
aqueous phase.[14,15] Other researcher groups suggested
to use high molecular weight PEG, from PEG3350,
PEG6000 to PEG 8000, to separate hydrophobic amino
acids.[16,17] However the viscosity of ATPE system in-
creases dramatically by increasing the molecular weight
or concentration of PEG.[18] Pedro et al.[17] proposed that
the ATPE systems with a mass fraction of PEG between
10% to 50% showed better separation in the PEG/salt
system. Moreover, Gao et al.[19] proved that, the mass
fractions of PEG and ammonium sulfate should be 10%
—30% and less than 20%, respectively. In our work,
PEG2000 was selected with a mass fraction of 30%
whilst the quality fraction of (NH4)2SO4 is 20%. Larger
molecular weight PEG may increase the viscosity of
ATPE system and reduce the separation efficiency.
Table 2 The influence of ratio of combinatorial chiral selectors
on K, α and ee
C(β-CD)
C(HP-β-CD)
4∶1
∶
ee/% (lower
ee/% (higher
KL
KD
α
phase)
phase)
3.12 2.17 1.44
3.30 2.21 1.49
3.25 2.15 1.51
3.30 2.33 1.53
3.44 2.36 1.46
7.11
8.65
9.81
10.27
7.86
10.44
11.59
12.83
13.31
11.23
3∶2
1∶1
2∶3
1∶4
The concentration of Phe was 3.00 mmol/L. the
concentration of chiral selector was 0.01 mol/L (in
combinatorial selectors, C(β-CD)∶C(HP-β-CD)＝1∶
1). The pH of Na2HPO4-H3PO4 buffer solution was 5.5
and the temperature was 25 ℃. The influence of dif-
ferent chiral selectors on the distribution behavior of
racemic Phe was studied. As shown in Table 1, ATPS
shows extractive effect but no resolution ability without
chiral selector. Moreover, the parameters of α and ee
were improved using combinatorial selectors composed
of β-CD and HP-β-CD instead of single selector such as
β-CD or HP-β-CD, though K was not noticeably
improved. The distribution characteristics of Phe were
Influence of different total concentration of combi-
natorial chiral selectors
The effect of various total concentrations of combi-
natorial chiral selectors for the distribution behavior of
racemic Phe was investigated under the same conditions
as the section of Influence of different chiral selectors,
except that the molar concentration ratio of β-CD to
HP-β-CD is 2∶3 in combinatorial chiral selectors. As
shown in Figure 3, α and ee rapidly increase with the
increasing total concentration of combinatorial chiral
selectors within the range of 5—10 mmol/L, and then
the increases slow down, finally they get stable when
the concentration exceeds 10 mmol/L. This conversion
of changing rate permits concentration variation in a
wide range with a stable resolution. Hence, the experi-
mental results reappear easily. On the contrary, the use
of single chiral selector usually has an optimal concen-
tration. The slight change of concentration nearby the
optimal concentration could cause noticeable variation,
which causes a difficult control of separation conditions
and has difficulty in repeating results. Stephen reported
a model for the separation of pairs of enantiomeric
molecules in capillary electrophoresis.[21] Once the con-
Table 1 Relationship between chiral selectors and resolution
results
ee/% (lower ee/% (higher
KL KD
α
phase)
0.02
phase)
0.05
None
β-CD
1.98 1.92 1.03
3.04 2.10 1.45
3.49 2.37 1.47
7.08
10.40
11.03
HP-β-CD
β-CD and HP-
7.62
β-CD as combina- 3.25 2.15 1.53
9.81
12.83
torial selector
Chin. J. Chem. 2012, 30, 965—969
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Chen et al.
FULL PAPER
Figure 4 Influence of the pH values on the distribution behav-
ior of racemic phenylalanine.
Figure 3 Influence of the total molar concentration of combi-
natorial chiral selector on the distribution behavior of racemic
phenylalanine.
vestigated under the same conditions as the section of
Influence of different chiral selectors, except that the
molar concentration ratio of β-CD to HP-β-CD is 2∶3
in combinatorial chiral selectors. The buffer solution in
this study contains H3PO4-Na2HPO4, citric acid-sodium
citriate (Cit-NaCit), HAc-NaAc, H3PO4-Na2B4O7,
Cit-TEOA and Cit-Tris. Table 3 indicates that, the sepa-
ration effect of H3PO4-Na2HPO4 buffer solution is the
best in all the experimental buffer solution. The possible
reason is that TEOA competes for the cavity of CD with
object. The competition could reduce the affinity be-
tween the object and the cavity of CD and weaken the
action existing in sample molecule and chiral selector.
The other four buffer solutions showed less enantiose-
lectivity ability based on the experimental data (Table 3).
The reasons for less recognition ability could be ex-
plored in the further study.
centration of chiral selector is lower or higher than the
optimum concentration, the separation rate decreased
dramatically with the decreasing of mobility difference.
The mobility difference decreased over 50% when the
concentration of chiral selector was 20 mmol higher
than optimum condition. That is the reason why the
popularization and application of the single chiral se-
lector have been restricted.
Influence of pH
The effect of pH of Na2HPO4-H3PO4 buffer for the
distribution behavior of racemic Phe was investigated
under the same conditions as the section of Influence of
different chiral selectors, except that the molar concen-
tration ratio of β-CD and HP-β-CD is 2∶3 in combina-
torial chiral selectors. Figure 4 shows that the pH has
obvious effect on the distribution behavior of racemic
Phe. The items of α and ee achieve the maximum at the
same time when pH is 5.5. The possible reason is that
Phe reaches its isoelectric point when pH is 5.5 in this
case, Phe exists in form of molecule. Its ionization
process increases in other pH, which leads to ionic Phe
more than molecular Phe. However, HP-β-CD has little
recognition effects for ionic Phe. Therefore, K, α and ee
decrease when the pH is higher or lower than the
isoelectric point. The extraction system of pH 5.5 has
been selected in this experiment.
Table 3 Influence of different buffer on the distribution behav-
ior of racemic phenylalanine
Item
KL
H3PO4/Na2HPO4Cit/NaCitHAc/NaAcCit/TEOACit/Tris
3.30
2.16
1.53
2.56
1.80
1.42
3.12
2.11
1.48
2.12
1.70
1.25
1.86
1.58
1.18
KD
α
ee/%(lower
phase)
ee/%(higher
phase)
10.27
13.31
7.01
8.65
4.92
7.81
4.02
6.98
10.32
11.68
Influence of different kinds and concentration of
buffer solution
Figure 5 indicates that the appropriate concentration
of buffer solution is 0.02 mol/L in the experiment. The
concentration of buffer solution has the similar influ-
ence on separation effect. The mutual restraint between
The effect of type and concentration of buffer solu-
tion for the distributive action of racemic Phe was in
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Chin. J. Chem. 2012, 30, 965—969

Extraction of Phenylalanine Enantiomers by Aqueous Two Phase Systems
ions reinforces with the increase of buffer solution con-
centration. It minishes the chance that anion and cation
combine into molecule from electrolyte dissociation,
subsequently decreases the concentration of weak elec-
trolyte molecule and increases ionic concentration as
well as degree of dissociation. Thus, the separation ef-
fect was weakened.
conditions can be more easily using combinatorial chiral
selectors instead of single chiral selector. At the same
time, separation efficiency has also been improved.
Therefore, we conclude the method we proposed is an
economical and effective way for chiral resolution.
Acknowledgements
The authors would like to thank National Natural
Science Foundation of China (Nos. 20805058 and
201176262) and Provincial Natural Science Foundation
of Hunan (No. 09JJ3026) for the financial supports of
this work.
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The chiral separation behavior of racemic Phe was
investigated in aqueous two-phase systems composed of
polyethylene glycol and ammonium sulfate containing
combinatorial chiral selector: β-CD and HP-β-CD. The
experimental results show that a better enantiosepara-
tion efficiency can be obtained using combinatorial
chiral selectors. The optimized concentration ratio of
β-CD to HP-β-CD is 2∶3 in combinatorial chiral se-
lectors. The total concentration of combinatorial chiral
selectors is 0.01 mol/L and pH of Na2HPO4-H3PO4
buffer solution is 5.5. The research indicates that re-
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