Enantioselective liquid-liquid extraction of valine enantiomers in the aqueous two-phase system formed by the cholinium amino acid ionic liquid copper complexes and salt

Article abstract of DOI:10.1016/j.molliq.2019.111599

It was widely reported that the heterocyclic ionic liquids (ILs) had relatively high biotoxicity and the cholinium amino acid ionic liquids (ChAAILs) were much environmentally friendly. Therefore, the ChAAILs/salt aqueous two-phase systems (ATPSs) were developed for the chiral resolution of valine enantiomers in this study. The copper complexes were prepared by ChAAILs and Cu(Ac)₂, which can be used as both the phase-forming components and the chiral selectors. A series of factors affecting the enantioseparation result were investigated, including type and concentration of ChAAILs, molar ratio of ChAAIL and Cu(Ac)₂, type and concentration of salt, concentration of valine, and operation temperature. The valine enantiomers were preferentially recognized in the ChAAIL-rich phase (top phase) and the maximum e.e. value of 55.6% was obtained in the top phase. This ATPS used for the enantioseparation of valine enantiomers has the advantages of environmental friendliness, high enantioseparation efficiency and simple operation by one step extraction.

Full text of DOI:10.1016/j.molliq.2019.111599

Journal of Molecular Liquids 294 (2019) 111599
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Enantioselective liquid-liquid extraction of valine enantiomers in the
aqueous two-phase system formed by the cholinium amino acid ionic
liquid copper complexes and salt
a
a
a,
a
b,
Lianglei Liu , Danyu Sun , Fenfang Li ⁎, Shaoping Ma , Zhijian Tan ⁎⁎
a
College of Chemistry and Chemical Engineering, Central South University, Changsha, China
Institute of Bast Fiber Crops and Center of Southern Economic Crops, Chinese Academy of Agricultural Sciences, Changsha, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2019
Received in revised form 19 August 2019
Accepted 20 August 2019
Available online 21 August 2019
It was widely reported that the heterocyclic ionic liquids (ILs) had relatively high biotoxicity and the cholinium
amino acid ionic liquids (ChAAILs) were much environmentally friendly. Therefore, the ChAAILs/salt aqueous
two-phase systems (ATPSs) were developed for the chiral resolution of valine enantiomers in this study. The cop-
per complexes were prepared by ChAAILs and Cu(Ac)
nents and the chiral selectors. A series of factors affecting the enantioseparation result were investigated,
including type and concentration of ChAAILs, molar ratio of ChAAIL and Cu(Ac) , type and concentration of
2
, which can be used as both the phase-forming compo-
2
Keywords:
salt, concentration of valine, and operation temperature. The valine enantiomers were preferentially recognized
in the ChAAIL-rich phase (top phase) and the maximum e.e. value of 55.6% was obtained in the top phase. This
ATPS used for the enantioseparation of valine enantiomers has the advantages of environmental friendliness,
high enantioseparation efficiency and simple operation by one step extraction.
Aqueous two-phase system
Cholinium amino acid ionic liquids
Copper complexes
Valine enantiomers
Enantioseparation
©
2018 Elsevier B.V. All rights reserved.
1
. Introduction
As a commonly used liquid-liquid extraction technology, aqueous
two-phase system (ATPS) was first developed by Albertson [14]. In
the last few decades, various ATPSs including the PEG/dextran [15],
PEG/salt [16], low molecular weight alcohol/salt [17], anionic/cationic
surfactant [18], ionic liquid (IL)/salt [19], and deep-eutectic solvent
(DES)/salt systems [20] had broad application in the extraction field. Re-
cently, ATPSs were also developed in the ELLE of various enantiomers
[21–26]. For example, we ever developed a PEG/salt ATPS for the
enantioseparation of α-cyclohexyl-mandelic-acid enantiomers in our
previous work [27]. However, the high viscosity and cost of PEG re-
stricted its application, moreover, additional chiral selector of β-
cyclodextrin derivate should be added. Similarly, additional chiral selec-
tors should be added in majority studies about the enantioseparation of
enantiomers in different ATPSs [21,22,24,26]. Pan and co-workers de-
veloped an IL/salt ATPS for the chiral resolution of amino acids enantio-
mers [28], the imidazolium-based chiral ILs was used as both the phase-
forming component and chiral selector. Although ILs were considered as
a class of “green solvents” in recent years, more and more studies
proved that the imidazolium-based ILs had high biotoxicity and low bio-
degradability [29–31]. Therefore, to choose the more environmentally
friendly ILs used in the ATPS is of great significance. Cholinium amino
acid ionic liquids (ChAAILs) are considered as one kind of much greener
solvents among the commonly used ILs due to the materials for prepa-
ration of them are natural sources [32–34].
It's well known that the enantiomers in chiral compounds usually
have different physical and chemical properties because of the tinny dif-
ferences in their structures. In particular, chiral drugs with different
pharmacological activities are being paid more and more attention,
and the demand for optically pure compounds is sharply increased.
Nowadays, the chiral separation of enantiomers is still the most popular
method to obtain the single enantiomer, including liquid chromatogra-
phy [1], gas chromatography [2], capillary electrophoresis [3], mem-
brane resolution [4], biocatalytic resolution [5], and enantioselective
liquid-liquid extraction (ELLE) [6]. Compared with the other
enantioseparation methods, ELLE is drawn more and more attention
owing to its simple operation, low cost, mild conditions, and easy for
scale-up production [7–9]. Therefore, ELLE was widely applied in the
chiral separation of various enantiomers, such as mandelic acid [10],
1
,2- amino alcohols [11], 4-chlorophenylglycine [12], phenylalanine an-
alogues [13], and so on.
⁎
Corresponding author.
⁎
⁎ Correspondence to: Z. Tan, Institute of Bast Fiber Crops and Center of Southern
Economic Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China.
E-mail addresses: lfflqq@csu.edu.cn (F. Li), tanzhijian@caas.cn,
tanzhijiantgzy@csu.edu.cn (Z. Tan).
https://doi.org/10.1016/j.molliq.2019.111599
0167-7322/© 2018 Elsevier B.V. All rights reserved.

2
L. Liu et al. / Journal of Molecular Liquids 294 (2019) 111599
The objective of this work is to develop a much greener ChAAIL/salt
make it clear again. The mass of each component added to the tube was
recorded. And the above procedures were repeated to obtain sufficient
data for constructing the phase diagrams.
ATPS for the chiral separation of valine enantiomers. L-valine is an es-
sential amino acid for vertebrates, it can also be used in the cosmetics,
pharmaceuticals, or as precursor for herbicides synthesis [35,36]. D-
valine has been used for the synthesis of drugs and agricultural pesti-
cides [37]. Therefore, racemic valine was chosen as the target compound
used for the chiral separation in this study. In order to achieve this goal,
a series of ChAAILs were synthesized. Then the copper complexes were
formed by ChAAILs and Cu2 , which can be used as both the phase-
forming components and chiral selectors in ATPS. The major factors
influencing the enantioseparation result were optimized in this process.
2.4. Octanol-water partition coefficient (Pow) of amino acids
The Pow is an important parameter for the hydrophobicity [43],
which was measured for five amino acids according to the reported pro-
cedures in this study [44–46]. An amino acid standard solution of
2.0 mg/mL was prepared with a 50 mmol/L octanol-saturated phos-
phate buffer (pH 7.0). The amino acid solution was mixed with octanol
+
(
saturated with phosphate buffer) at an equal volume. The mixture was
2
. Materials and methods
stirred for 30 min, then centrifuged at 10000 rpm for 5 min. The amino
acid concentrations in two phases were respectively determined by a
UV–Vis spectrophotometry. The Eq. (1) was used to calculate the
octanol-water partition coefficient (Pow).
2
.1. Materials and reagents
ChAAILs were synthesized by the following reagents, including cho-
line chloride (ChCl, 98%), L-proline (L-Pro, 99%), L-methionine (L-Met,
9%), L-alanine (L-Ala, 99%), L-cysteine (L-Cys, 99%), and L-histidine
L-His, 99%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai,
Coctanol
Cwater
Pow ¼
ð1Þ
9
(
where Coctanol was the amino acids concentration in octanol phase,
while Cwater was the amino acids concentration in water phase.
China). The racemic D, L-valine (HPLC purity N98%), inorganic salts of
potassium phosphate and monopotassium phosphate (AR purity
N98%), derivatization reagents of O-phthalaldehyde (OPA) and N-
acetyl-L-cysteine (NAC) were also purchased from Aladdin Reagent
Co., Ltd. (Shanghai, China). Octanol (99%) was purchased from Macklin
Reagent Co., Ltd. (Shanghai, China). Three dyes of 2,6-dichloro-4-(2,4,6-
2.5. Determination of Kamlet-Taft polarity parameters
The hydrogen-bond donating ability (α), hydrogen-bond accepting
ability (β), and dipole/polarizability (π*) are the Kamlet-Taft polarity
parameters [47]. The dyes of RD, DENA, and NA were mixed with the
target ChAAILs. The DENA and NA concentrations were both 5.0
T
triphenyl-N-pyridino)-phenolate [E (33)], 4-nitroaniline, and N, N-
diethyl-4-nitroaniline were bought from Molbase (Shanghai) Biotech-
nology Co., Ltd. (Shanghai, China). Acetonitrile (HPLC grade) was
bought from TEDIA Company, Inc. (Fairfield, OH, USA).
−
5
−4
×
10 mol/L, the RD concentration was in the range of 2.2 × 10
–
−3
8
.6 × 10 mol/L. A UV–Vis spectrophotometer was used to measure
the maximum absorption wavelength of the dyes. The polarity parame-
ters were calculated according to the Eqs. (2)–(6) [48,49].
2
.2. Synthesis of ChAAILs and copper complexes
A two-step synthesis method was used to prepare the ChAAILs [38],
104
λðnmÞ
À
Á
namely choline-L-proline ionic liquid ([Cho][Pro]), choline-L-histidine
ionic liquid ([Cho][His]), choline-L-alanine ionic liquid ([Cho][Ala]),
choline-L-methionine ionic liquid ([Cho][Met]), and choline-L-cysteine
ionic liquid ([Cho][Cys]). Firstly, to a round-bottom flask, potassium hy-
droxide: choline chloride = 1.3:1 and methanol were added. This mix-
ture was blended at room temperature for 12 h, choline hydroxide
[Cho][OH]) was obtained after removing the potassium chloride by fil-
tration. Then, [Cho][OH] was mixed with the amino acids at a ratio of
:1.1, the mixture was blended at a low temperature for 12 h. Lastly,
−1
vmax cm
¼
ð2Þ
ð3Þ
28592
λmax
ETð33Þ ¼
ðRDÞ
πꢀ ¼ 0:314½27:52−vmaxðDENAÞꢁ
ð4Þ
ð5Þ
(
α ¼ 0:0649ETð33Þ−2:03−ð0:72πꢀÞ
1
ChAAILs were obtained after filtration, purification and vacuum drying.
1
The synthesized ChAAILs were characterized by FT-IR and HNMR, and
½1:035vmaxðDENAÞ þ 2:64−vmaxðNAÞꢁ
β ¼
ð6Þ
the results were in accordance with that in our previous work and other
similar studies [32,39,40].
The copper complexes were prepared by the following procedures.
To a tube, copper acetate and ChAAIL at a mole ratio of 1:2 were
added. The mixture was stirred for 10 min to make copper acetate
completely dissolve in ChAAIL. Then the mixture was centrifugated for
2:8
where vmax was the maximum experimental wave number, E
the solvatochromic parameter (kcal/mol), λ was absorption wave-
length, and λmax was the maximum absorption wavelength.
T
(33) was
2
.6. Enantioseparation of valine enantiomers
5
min at 10000 rpm to remove the redundant copper acetate. And the
obtained supernate was the complexes. The FT-IR spectra for the com-
plexes were provided in the Supplementary materials (Figs. S1–S5).
All the data in FT-IR spectra showed that the characteristic peaks around
The copper complexes, salt, racemic valine and water were added
into a tube. The mixture was vortexed and then centrifuged for com-
plete phase separation. The concentrations of valine enantiomers in
both the top and bottom phases were determined by HPLC. To assess
the chiral selectivity of this ATPS, the enantiomeric excess (e.e.) value
of valine in the top (ChAAIL-rich phase) or bottom (salt-rich phase)
phase was calculated according to the Eq. (7):
−
1
6
50 cm
belonged to the metal-ligand bands (Cu\\O and Cu\\N
2+
bonds) [41]. The UV spectra of Cu , [Cho][Pro], Cu([Cho][Pro])
2
com-
plexes were shown in Fig. S6, the results can also prove that the com-
plexes were successfully prepared.
2
.3. Construction of the phase diagrams
e:e:% ¼ │
CL−CD│
│CL þ CD│
ꢂ 100%
ð7Þ
The cloud point method was used to construct the phase diagrams
for ChAAIL/salt ATPS [42]. ChAAILs was added to a 10 mL centrifuge
tube. The salt solution was added dropwise into the tube to make mix-
ture become cloudy. After that a certain amount of water was added to
where C
L D
and C were the concentration of L-Val and D-Val enantio-
mers, respectively.

L. Liu et al. / Journal of Molecular Liquids 294 (2019) 111599
3
2
.7. Derivatization of valine
Table 1
The logPow values of five amino acids.
The precolumn derivatization has been widely used to analyze the
His
Pro
Ala
Met
Cys
amino acids enantiomers, and the derivatization of valine was per-
formed by the reported procedures [50,51]. Equal amount of NAC and
OPA were dissolved in a mixed solution of absolute ethanol and sodium
borate buffer at a volume ratio of 1:5 to obtain a solution concentration
of 2.0 mg/mL for both NAC and OPA. The derivatization reagent was pre-
pared and stored at 4 °C for use. The derivatization reagent and valine
sample were mixed at a volume ratio of 1:1 for 3 min. The sample was
injected to HPLC after filtration by 0.22 μm millipore filter.
logPow
−1.01
−0.81
−1.3
−1.04
−0.95
It was also reported that the phase-forming ability of ChAAILs de-
creased with the increasing of hydrogen bond forming ability [52]. The
greater the hydrogen bond ability of ChAAILs, the easier to form hydro-
gen bonds with water molecules, which resulted in the decreasing of
phase separation ability. The results of Kamlet-Taft polarity parameters
were shown in Table 2. It was reported in previous studies that the α × β
value can reflect the hydrogen bond ability of ILs [53]. The results in
Table 2 indicated that the hydrogen bond ability of ChAAILs followed
the order [Cho][Pro] b [Cho][His] b [Cho][Cys] b [Cho][Ala] b [Cho]
2
.8. HPLC conditions
A Dionex UltiMate 3000 HPLC system and a Kromasil C18 chromato-
graphic column (250 × 4.6 mm i.d., 5 μm) were used to analyze the sam-
ples. Acetonitrile and sodium dihydrogen phosphate aqueous solution
[
Met], which roughly accorded with the inverse order of the phase-
forming ability. These results proved that the hydrophobicity/hydrophi-
licity and hydrogen bond forming ability can jointly affect the phase-
forming ability of ChAAILs.
(
20 mmol/L, pH = 6.8) at a volume ratio of 18:82 were used as the mo-
bile phase. The flow rate was 0.8 mL/min with isocratic elution; the
oven temperature was 30 °C; the detection wavelength was 350 nm;
and the sample injection volume was 20 μL.
3
.2. Screening the optimal ChAAIL
To screen the optimal ChAAIL, five ChAAILs were chosen to form the
complexes with Cu2 . The results were shown in Fig. 2(a), it can be seen
+
3
. Results and discussion
2
that the Cu([Cho][Pro]) /salt ATPS had the relatively higher
3
.1. Phase behavior of ChAAILs/salt ATPSs
enantioseparation ability in both the top and bottom phases among
the five ATPSs. Overall, the chiral resolution ability of ChAAILs was
ranked as follows: [Cho][Pro] N [Cho][Ala] N [Cho][Cys] N [Cho][Met] N
[Cho][His]. This can be attributed to the stability of the complexes
Because the enantioseparation was performed in the ATPS, and thus
the phase behavior must be considered. The phase diagrams can pro-
vide some information whether the components can form the ATPS or
what amount of each component needed for constructing the ATPS. Be-
cause the introduction of Cu2 can interfere the observation of the cloud
point, so only the phase diagrams for ChAAILs/salt ATPSs were studied.
The phase diagrams for different ChAAILs/K PO ATPSs were shown in
3 4
Fig. 1. The results in Fig. 1 showed that the phase-forming ability of
ChAAILs was in the order of [Cho][Pro] N [Cho][Cys] N [Cho][Ala] N
formed by ChAAILs, Cu(Ac)
sults were proved in other studies [41,54]. Therefore, [Cho][Pro] was
chosen for further studies. Moreover, the Cu([Cho][Pro]) complexes
2
and valine enantiomers, and the similar re-
+
2
concentration should also be studied, and the results were shown in
Fig. 2(b). It showed that the maximum e.e. value was obtained at
26 wt% complexes concentration. The lower complexes concentration
resulted in insufficient chiral resolution, while higher complexes con-
centration will extract more valine into the ChAAIL-rich phase resulting
in the decreasing of recognition ability for enantiomers.
[Cho][His] N [Cho][Met]. The hydrophobicity/hydrophilicity may be an
important factor influencing the phase-forming ability of ChAAILs. The
ChAAILs used in this study have the same cation and different amino
acids anions, and the amino acids can retain their properties in ChAAILs
3.3. Effect of molar ratio of [Cho][Pro] and Cu(Ac)
2
[25], so the logPow values of five amino acids were determined. The re-
sults were shown in Table 1, and they indicated that the hydrophobicity
of amino acids was in the order of Pro N Cys N His N Met N Ala, which
roughly accorded with the phase-forming ability of ChAAILs.
Copper ion is a key and most common ion for forming the chiral
complexes with amino acids. It is located at the center of the complex
and links the other two molecules together to form a chiral ternary com-
plex [41,54]. Therefore, the molar ratio of [Cho][Pro] and Cu(Ac)
key factor affecting the enantioseparation ability. The molar ratios of
:1–1:1 for n([Cho][Pro]): n(Cu(Ac) ) were investigated, as can be
2
is a
25
20
15
10
5
0
8
2
[
[
[
[
[
Cho][L-Ala]
Cho][L-Pro]
Cho][L-His]
Cho][L-Cys]
Cho][L-Met]
seen in Fig. 3 that the maximum e.e. value was obtained at the molar
2+
ratio of 2:1. In general, two [Cho][Pro] and one Cu could form a com-
2+
2
plex (2[Cho][Pro] + Cu → Cu([Cho][Pro]) ). During the chiral resolu-
tion of the ternary complex, a [Cho][Pro] was replaced by the target
amino acid to form two complexes of Cu[Ch][Pro][D-Val] and Cu[Cho]
[Pro][L-Val]. Due to the different stability of the two complexes, the dis-
tribution of the complexes in two phases is different, which resulted in
the chiral resolution of the valine enantiomers. When the Cu2+ concen-
tration was low, the ternary complexes can't be formed or the
Table 2
The Kamlet-Taft polarity parameters for ChAAILs.
⁎
ChAAILs
E
T
(33)
π
α
β
α × β
0
10 20 30 40 50 60 70 80 90
[
[
Cho][Ala]
Cho][Met]
51.80
51.06
56.28
53.05
54.05
0.61
0.46
2.40
0.81
1.66
1.29
1.25
1.45
1.35
1.36
1.49
1.66
1.18
0.86
1.22
1.92
2.08
1.71
1.17
1.66
AAILs concentration (wt%)
[Cho][Cys]
[
[
Cho][Pro]
Cho][His]
3 4
Fig. 1. The phase diagrams for different ChAAILs/K PO ATPSs.

4
L. Liu et al. / Journal of Molecular Liquids 294 (2019) 111599
(
a) 50
60
Salt-rich phase
ChAAIL-rich phase
ChAAIL-rich phase
Salt-rich phase
40
30
20
10
0
50
40
30
20
10
8
:1
4:1
2:1 1.5:1
1:1
n ([Ch][Pro]): n (Cu(Ac)2)
Fig. 3. Effect of the molar ratio of [Cho][Pro] and Cu(Ac)
valine enantiomers, 26 wt% [Cho][Pro], 30 wt%
experiments were performed at room temperature and without adjusting the system pH.
2
on the chiral resolution of the
K PO , 10 mg/mL valine. The
3 4
(
b)
ChAAIL-rich phase
Salt-rich phase
6
5
4
3
2
1
0
0
0
0
0
0
0
Cu2+ than the molecular valine [55]. K
than K HPO , so the chiral resolution ability of Cu([Cho][Pro])
PO
3 4
has more stronger alkalinity
/K PO
PO ATPS
2
4
2
3
4
ATPS is also much stronger. Therefore, Cu([Cho][Pro])
was chosen for the further studies.
2
/K
3
4
3 4
The concentration of K PO should also be optimized. The results in
Fig. 4(b) show that the e.e. values increased with the increasing of salt
concentration from 15 to 25 wt%, then decreased with further increas-
ing of salt concentration. The volume of salt-rich phase increased with
the increasing of salt due to more free water was drawn into the salt-
rich phase in ATPS [56]. The polarity of water is strong due to the elec-
tronegativity of oxygen and hydrogen-oxygen bond [25]. Therefore,
the polarity and hydrogen-bond interaction decreased in ChAAIL-rich
phase, maybe which is more suitable for the enantioseparation. Accord-
ingly, the polarity and hydrogen-bond interaction increased in salt-rich
phase, but the enantioseparation ability was slight influenced by that.
However, excessive water could affect the stability of the complexes
in salt-rich phase, hence affected the enantioselectivity of the com-
plexes. The further increasing of salt will pull more water from the
ChAAIL-rich phase to salt-rich phase, which will destroy the stability
of complexes and result in the decreasing of enantioselectivity. To sum
up the above viewpoints, 25 wt% salt concentration was chosen in this
study.
21
23
26
30
36
2+
[
Cho][Pro]-Cu concentration (wt%)
Fig. 2. (a). The e.e. values for valine enantiomers in different ChAAILs/K
Cho][Pro], n([Cho][Pro]): n(Cu(Ac) ) = 2:1, 30 wt% K PO , 10 mg/mL valine. (b). Effect of
Cu([Cho][Pro]) complexes concentration on the enantioseparation result, n([Cho][Pro]):
n(Cu(Ac) ) = 2:1, 30 wt% K PO , and 10 mg/mL valine. The experiments were performed
3 4
PO ATPSs, 24 wt%
[
2
3
4
2
2
3
4
at room temperature and without adjusting the system pH.
complexes concentration was low, which resulted in the poor
3.5. Effect of valine concentration
2+
enantioseparation result. However, when the concentration of Cu
was too high, part of the Cu2 precipitated due to salting-out effect.
Moreover, the precipitate of valine was also observed, which resulted
in the decrease of enantioseparation ability. Therefore, the n([Cho]
+
The valine concentration at the range of 5–35 mg/mL was optimized.
The results in Fig. 5 showed that the maximum e.e. value in both two
phases were obtained at the valine concentration of 15 mg/mL. The
[
Pro]): n(Cu(Ac)
.4. Effect of salt type and concentration
The salt type and concentration are also important factors affecting
2
) = 2:1 was chosen for further studies.
ligand-exchange reaction Cu([Cho][Pro])
2
+ D, L-Val → Cu[Cho][Pro]
[D-Val] + Cu[Cho][Pro][L-Val] occurred in the extraction process [41].
3
The reaction can be continuously promoted at the valine concentration
below 15 mg/mL, and the e.e. values increased. When the valine concen-
tration was 15 mg/mL, the molar ratio of valine was basically equal to Cu
the chiral resolution ability in ATPS. Eleven common salts were consid-
ered as the phase-forming components in this study. It was found that
2
([Cho][Pro]) , and the complexes concentration of Cu[Cho][Pro][D/L-
Val] was maximized, resulting in the optimal chiral selectivity. When
the valine concentration was larger than 15 mg/mL, further reaction
for Cu[Cho][Pro][D-Val] and Cu[Cho][Pro][L-Val] was promoted, namely
the phase-forming ability of [Cho][Pro] was reduced after forming the
complexes with Cu2 , only K
+
PO
and K
HPO
can form ATPSs with
3
4
2
4
the complexes. The enantioseparation results were shown in Fig. 4(a),
it can be seen that the Cu([Cho][Pro]) /K PO ATPS and Cu([Cho][Pro])
/K HPO ATPS had better chiral resolution ability. Since the two basic
salts can result in the solution pH N 7, valine exists at ionic state in
this ATPS, and the ionic valine is more likely to form complexes with
Cu[Cho][Pro][D-Val] + Cu[Cho][Pro][L-Val] + 2D, L-Val → Cu[L-Val]
2
+
2
3
4
Cu[L-Val][D-Val] + Cu[D-Val] + 2[Cho][Pro]. However, the new ter-
2
2
2
4
nary complexes no longer had chiral resolution ability, so the e.e. values
gradually decreased with increasing of valine concentration. Therefore,
15 mg/mL valine concentration was chosen in this study.

L. Liu et al. / Journal of Molecular Liquids 294 (2019) 111599
5
(a)
70
ChAAIL-rich phase
Salt-rich phase
50
40
30
20
10
0
Salt-rich phase
60
50
ChAAIL-rich phase
40
30
20
10
0
5
10 15 20 25 30 35
Valine concentration (mg/mL)
K HPO
4
K PO
2
3
4
(
b)
Fig. 5. Effect of valine concentration on the chiral extraction of valine enantiomers. The
extraction conditions were as follow: n ([Ch][Pro]): n (Cu(Ac) ) = 2:1, 25 wt% K PO
The experiments were performed at room temperature and without adjusting the
system pH.
60
50
40
30
20
10
2
3
4
.
ChAAIL-rich phase
Salt-rich phase
with other chromatographic methods, this ELLE can obtain higher e.e.
value than many reported method used for the chiral separation of va-
line enantiomers [58–60]. Moreover, this enantioseparation can be
achieved by one single step extraction, so it has great application poten-
tiality in chiral separation field.
4. Conclusions
In this work, ChAAILs/salt ATPSs were constructed and used for the
chiral resolution of valine enantiomers. The complexes formed by
ChAAILs and Cu2 were used as both the phase-forming component
and the chiral selector in the enantioseparation process. It was found
+
15
20
25
30
35
that the Cu([Cho][Pro])
under the following conditions of n ([Ch][Pro]): n (Cu(Ac)
5 wt% K PO , 15 mg/mL valine, and 25 °C operation temperature. It
2
/K
3
PO
4
ATPS had the best enantioselectivity
K PO concentration (wt%)
3
4
2
) = 2:1,
2
3
4
Fig. 4. (a) Effect of salt type on the enantioseparation result, 26 wt% [Cho][Pro], n([Cho]
Pro]): n(Cu(Ac) ) = 2:1, 30 wt% salt, 10 mg/mL valine. (b) Effect of K PO
concentration on the enantioseparation result, 26 wt% [Cho][Pro], n([Cho][Pro]): n(Cu
Ac) ) = 2:1, 10 mg/mL valine. The experiments were performed at room temperature
and without adjusting the system pH.
was also demonstrated that the valine enantiomers were preferentially
recognized in the ChAAIL-rich phase. The maximum e.e. values of 55.6%
[
2
3
4
(
2
7
6
5
4
3
0
0
0
0
0
ChAAIL-rich phase
Salt-rich phase
3
.6. Effect of operation temperature
The extraction temperature is one of the most important factor af-
fecting chiral resolution ability. It was reported that the amino acid
began to denature at the temperature higher than 50 °C [57], and thus
the effect of different temperature from 4 to 50 °C on the chiral resolu-
tion was studied. As shown in the Fig. 6, it can be seen that the e.e. values
first increased and then decreased with the increasing of temperature.
The maximum e.e. value was obtained at 25 °C. Since the temperature
can affect the active state of ions in the solution, as the rising of temper-
ature, more and more ions in the solution absorbed energy from the
normal state to an active state at which chemical reaction was prone
to occur, so the e.e. value increased. As the continue rising of tempera-
ture, the structure of the complexes in the solution was destroyed,
resulting in the decreasing in chiral resolution. Therefore, 25 °C was
chosen.
20
10
0
5 10 15 20 25 30 35 40 45 50
Temperature (°C)
In this study, the maximum e.e. values of 55.6% and 35.3% were ob-
tained in the ChAAIL-rich phase and the salt-rich phase, respectively
under the optimal conditions. To compare the enantioseparation results
Fig. 6. Effect of temperature on the chiral extraction of valine enantiomers. The extraction
conditions were as follow: n ([Ch][Pro]): n (Cu(Ac) ) = 2:1, 25 wt% K PO , 15 mg/mL
valine. The experiments were performed without adjusting the system pH.
2
3
4

6
L. Liu et al. / Journal of Molecular Liquids 294 (2019) 111599
20] W.W. Deng, Y. Zong, Y.X. Xiao, Hexafluoroisopropanol-based deep eutectic solvent/
[
and 35.3% were obtained in the ChAAIL-rich phase and the salt-rich
phase, respectively. This ATPS used for the enantioseparation of valine
enantiomers has the advantages of simple and rapid operation within
a single step extraction, high enantioselectivity, and good environmen-
tal friendliness, which is potentially used in the chiral separation of
other enantiomers.
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Acknowledgement
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17–631.
This work was financially supported by the National Natural Science
Foundation of China (No. 21878326), the Training Program for Excellent
Young Innovators of Changsha (No. kq1802018), and the Hunan Provin-
cial Natural Science Foundation (No. 2019JJ50713).
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