Complex-precipitation using functionalized chiral ionic liquids with L-proline anion and chromatographic analysis for enantioseparation of racemic amino acids

Article abstract of DOI:10.1002/chir.23011

As one kind of functionalized green medium, chiral ionic liquids (CILs) have been widely applied in fields of asymmetric catalysis, enantioseparation, and so on. In this study, four kinds of amino acid–based CILs were synthesized by using trimethylamine, N-methylpyrrolidine, N-methylimidazole, and tropine as cationic nucleus, respectively. Then their specific optical rotation and solubility in common solvents were determined for further resolution application. The effect of different cations in these CILs was explored on the separation of racemic phenylalanine in complex-precipitation way. Moreover, various factors were systematically investigated for their effects on resolution efficiency, including the type of additive copper salts, the molar ratio of Cu (II) to CIL, pH value, the amount of racemic phenylalanine, and temperature. Under the appropriate conditions, L-phenylalanine mainly existed in solid phase and could be separated from its enantiomers in liquid phase. Furthermore, the mechanism of resolution was studied by thermogravimetric analysis, infrared spectrum, and molecular simulation. The resolution system has characteristics of no organic solvent, fast separation speed, simple resolution process, and easy scale-up.

Full text of DOI:10.1002/chir.23011

Received: 25 May 2018
DOI: 10.1002/chir.23011
Revised: 21 July 2018
Accepted: 31 July 2018
R E G U L A R A R T I C L E
Complex‐precipitation using functionalized chiral ionic
liquids with L‐proline anion and chromatographic analysis
for enantioseparation of racemic amino acids
Huimin Zang
| Shun Yao | Yingjie Luo | Dan Tang | Hang Song
School of Chemical Engineering, Sichuan
University, Chengdu, P. R. China
Abstract
As one kind of functionalized green medium, chiral ionic liquids (CILs) have
been widely applied in fields of asymmetric catalysis, enantioseparation, and
so on. In this study, four kinds of amino acid–based CILs were synthesized
by using trimethylamine, N‐methylpyrrolidine, N‐methylimidazole, and
tropine as cationic nucleus, respectively. Then their specific optical rotation
and solubility in common solvents were determined for further resolution
application. The effect of different cations in these CILs was explored on the
separation of racemic phenylalanine in complex‐precipitation way. Moreover,
various factors were systematically investigated for their effects on resolution
efficiency, including the type of additive copper salts, the molar ratio of Cu
(II) to CIL, pH value, the amount of racemic phenylalanine, and temperature.
Under the appropriate conditions, L‐phenylalanine mainly existed in solid
phase and could be separated from its enantiomers in liquid phase. Further-
more, the mechanism of resolution was studied by thermogravimetric analysis,
infrared spectrum, and molecular simulation. The resolution system has
characteristics of no organic solvent, fast separation speed, simple resolution
process, and easy scale‐up.
Correspondence
Hang Song, School of Chemical
Engineering, Sichuan University,
Chengdu 610065, P. R. China.
Email: hangsong@vip.sina.com
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 81673316
KEYWORDS
characterization, chiral ligand, complex‐precipitation method, ionic liquids, mechanism
1 | INTRODUCTION
and ionic liquids.1,2 They are widely reported in the
application of asymmetric catalysis, enantioseparation,
Functionalized ionic liquids (FILs) are a kind of ionic
liquids with special physicochemical properties, which
are widely used in various fields of science and technol-
ogy in recent years. The design and synthesis of novel
functionalized ionic liquids and their special applications
are at the forefront of current research. As one kind of
functionalized ionic liquids with multifunction, chiral
ionic liquids (CILs) have both the properties of chirality
and so on.3,4 At present, the most widely applied CILs
are imidazole CILs,5-7 and other kinds of CILs include
pyridinium CILs,8-10 thiazole CILs,11 and quaternary
ammonium CILs.12,13
The resolution of racemic amino acids (AAs) is an
important way to obtain AAs with single configuration.
In recent years, the study of their enantioseparation
has been attracting more and more researchers, and
various methods have been emerging that mainly
include chemical resolution,14 membrane resolution,15-17
Huimin Zang and Shun Yao contributed equally to this work.
Chirality. 2018;1–13.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
ZANG ET AL.
enzymatic resolution,18-20 chromatographic resolution,21-
L‐phenylalanine (D,L‐Phe, biological reagent grade,
purity ≥99.5%), and cupric bromide were supplied by
Aladdin Reagent Co, Ltd (Shanghai, China). Tropine
(purity ≥98.9%) was obtained from Huawen Chemicals
Co, Ltd (Zhengzhou, China). All of above chemicals are
at the level of analysis or above.
extraction,26,27 and molecular imprinting technique.28
25
Among them, the solvent extraction method has the
advantages of high separation efficiency, simple equip-
ment, and low energy consumption, but a lot of organic
solvents are used. As green medium, CILs are expected
to replace these conventional solvents. On the other
hand, L‐proline (L‐Pro)‐Cu (II) has been proved as good
ligand in chiral catalysis.29 Inspired by this, Liu et al30
synthesized a series of immidazolium CILs with the
anion of L‐proline and used them in enantioseparation
on the basis of the principle of chiral ligand exchange in
2009. When CILs are used for chiral extraction, it can
be divided into two ways: organic phase extraction and
aqueous two‐phase extraction. Tang et al31 used the reac-
tion between [CnMim][L‐Pro] and Cu2+ to obtain the
complex with the ability of chiral recognition, which
was used to separate D,L‐phenylalanine (D,L‐Phe) from
their ethyl acetate solution and e.e. value of 50% was
achieved in extracted phase. Wu et al32 applied two
kinds of chiral immidazolium ILs and sodium sulphate
to form aqueous two‐phase system (ATPS) in the
enantioseparation of D,L‐Phe. As the result, L‐Phe
was enriched in water phase, and e.e. value was 53%. In
the preliminary studies of our laboratory, Wu et al33
used tropine‐based CILs and inorganic salt solution to
prepare ATPS, and the enantiomeric excess value of
L‐phenylalanine in solid phase was 65%. The formation
of aqueous two‐phase system avoids the use of volatile
organic solvents, but a large amount of inorganic salts
are needed. And there are some difficulties in product
recovery and reuse of the whole system. However,
complex‐precipitation method with CILs in single‐phase
system is rarely studied which has lower requirement
and better reusability, and it is easier to realize fast
resolution and recovery. So this method is explored and
developed in the following study.
2.1.2 | Apparatus
The Shimadzu LC‐20AT high performance liquid chro-
matograph (Kyoto, Japan) consisting of PD‐M20A detec-
tor and Class VP chromatography workstation was used
for the quantitative analysis of the racemic amino acids.
A Welchrom‐C18 column (250 × 4.6 mm, 5 μm) was used
as the HPLC analytical column (Welch Materials,
Austin). A TGL‐16D high speed refrigerated centrifuge
was provided by Baita Xinbao Instrument Factory
(Changzhou, China). The synthesized ionic liquids were
characterized by FT‐IR (KBr disc, Perkin Elmer) and
400 MHz 1HNMR (MeOD, Bruker, Switzerland).
Deionized water used in the experiment was obtained
from the ultra‐pure water system produced by Yufei
Instrument Co, Ltd (Guangzhou, China).
2.2 | Synthesis of the CILs
Triethylamine, N‐methyl pyrrolidine, N‐methylimidazole,
and tropine reacted with 1‐bromobutane to prepare four
kinds of ILs with bromonium anion according to previous
studies.29,31 Then they were loaded on strongly basic
anion‐exchange resin and debromination was achieved.
Finally, the target ionic liquids were obtained after
neutralized with L‐proline. The synthesis procedure was
introduced as followings with pyrrolidium‐type CIL
([BuPyro][L‐Pro]) as an example.
2.2.1 | Synthesis of [BuPyro][Br]
A total of 0.1 mol N‐methylpyrrolidine, 0.1 mol N‐butyl
bromide, and appropriate volume of ethanol were added
in a 100 mL flask. After 24 hours refluxing reaction, the
solvent was removed by reduced pressure distillation.
The residue was washed by ethyl acetate for 2 to 3 times
and dried in a vacuum dryer and target product was
obtained.
2 | MATERIALS AND METHODS
2.1 | Materials and apparatus
2.1.1 | Materials
All the following chemicals were purchased from Kelong
Chemicals Co, Ltd (Chengdu, China): trimethylamine,
N‐methylimidazole, 1‐bromobutane, ethyl acetate, meth-
anol, ethanol, acetonitrile, ammonia, acetic acid, copper
acetate, copper sulfate, copper chloride, copper nitrate
and 201 × 7 type strongly‐alkaline anion‐exchange
resin (pH range: 0‐14, effective particle size: 0.400‐
0.850 mm, expansivity ≤25%, working exchange capacity
≥450 mmol/l). L‐proline (L‐Pro), N‐methylpyrrolidine, D,
2.2.2 | Synthesis of [BuPyro] [OH]
A total of 0.05 mol [BuPyro][Br] was dissolved in an
appropriate amount of methanol in a flask. The solution
was transferred to a pretreated strongly basic anion
exchange resin packed column (2 × 10 cm). The flow rate
of the effluent was controlled at no more than two drops

ZANG ET AL.
3
per second, and methanol was continuously added to the
column to ensure that the resin was immersed below the
liquid level. Effluent solution with pH greater than 8 was
collected, which was [BuPyro][OH] solution.
dd, J = 15.0, 7.5 Hz, ‐CH2 on C‐4 of L‐proline), and 1.04
(3H, t, J = 7.4 Hz, N‐CH3 on L‐proline ring). Other CILs
[BuEt3N][L‐Pro], [BuIm][L‐Pro] and [BuTr][L‐Pro] were
also synthesized in similar way. The synthetic route of
these CILs and their structures are shown in Figure 1.
2.2.3 | Synthesis of [BuPyro] [L‐Pro]
2.3 | Determination of specific optical
The [BuPyro][OH] solution was directly dropped in
methanol solution containing 0.06 mol L‐proline and
then stirred for 8 hours at room temperature (20°C). Then
the solvent was removed under vacuum, and frozen
acetonitrile was added into the remaining liquid. As a
result, redundant L‐proline was precipitated and removed
by filtering, and then viscous yellowish liquid was
obtained after concentration, which was the target
product of [BuPyro][L‐Pro] and preserved in a cold
drying environment. Its structure was proved by spectral
data, and FT‐IR (KBr disc) showed absorbance at 3351,
3296 cm−1 (νO‐H); 3045, 3012 cm−1 (νas C‐H and νs C‐H of
N+CH3); 2952, 2909 cm−1 (νas C‐H, νs C‐H of N+CH2, N
+CH); 2881 cm−1 (ν C‐H); 1491, 1473, 1442, 1405 cm−1
(δas C‐H, δs C‐H); 1577, 1431 cm−1 (νas C‐O, νs C‐O);
1533 cm−1 (δ N‐H) and 869 cm−1 (ωC‐H); meanwhile 1H
NMR spectrum (400 MHz, MeOD) exhibited proton
signals at 3.83 ppm (1H, dd, J = 8.6, 6.2 Hz, ‐OH),
3.78~3.27 (8H, m, ‐CH2 × 4 on pyrrolidine ring),
3.13~3.04 (4H, m, ‐CH2 × 2 on L‐proline ring), 2.24 (5H,
m, ‐CH2 and ‐CH3 on alkyl chain), 1.90 (2H, dd, J = 10.4,
4.3 Hz, ‐CH2 on alkyl chain), 1.81 (1H, s, ‐NH), 1.45 (2H,
rotation and solubility of CILs
For chiral compounds, the specific optical rotation of a
given substance is a constant which is equal to the optical
rotation when the concentration is 1 g/mL. In this study,
0.2 g CILs was dissolved in 20 mL distilled water, and
then the CILs solutions with the concentration of
0.01 g/mL were prepared. The specific optical rotation
degree was measured in a 100 mm polarimeter tube at
25°C and 589 nm by using Autopol I polarimeter
(Rudolph Research Analytical). Moreover, in the investi-
gation for dissolution behavior of CILs, a certain amount
of CILs was placed in a dry glass jacketed reaction bottle;
the temperature of jacket was controlled at 25 0.05°C
by thermostatic water bath, and the solvent was slowly
added into the jacketed reaction bottle until all the CILs
was thoroughly dissolved. Then the solubility of the
CILs in the solvent can be observed by weighing the mass
of the solvent added accurately. In this study, the solubil-
ity properties of four kinds of CILs in 12 common
organic solvents (methanol, ethanol, acetone, dichloro-
methane, acetonitrile, ethyl acetate, toluene, chloroform,
FIGURE 1 Synthesis route of chiral
ionic liquids and their structures

4
ZANG ET AL.
petroleum ether, hexane, ether, and water) were studied,
and related grading standard was according to Chinese
pharmacopoeia (2015 edition).
excess values (e.e., %) of D‐ and L‐Phe in the solid or
liquid phase were analyzed.
The percentages of D,L‐Phe in different phases were
obtained under above conditions. The e.e. value and yield
of enantiomers were calculated according to Equations (1)
to (3) as following:
2.4 | Quantitative analysis of amino acid
enantiomers by HPLC
e:e:L−PheðD−PheÞ ¼ ∣ωL−
−ωD− he in solid ðliquidÞ∣=
Chiral HPLC method was used for qualitative and
P
P
he in solid ðliquidÞ
quantitative analysis of D,L‐Phe.
A Welchrom‐C18
∣ωL−Phe in solid ðliquidÞ þ ωD−Phe in solid ðliquidÞ∣×100%;
column (5 μm, 250 × 4.6 mm) was used as the HPLC
analytical column (Welch Materials, Austin). The chiral
mobile phase was composed of L‐Pro (12 × 10−4 mol/L),
copper acetate (6.0 × 10−4 mol/L) and the volume ratio
1:4 of methanol to water which was adjusted to pH 5 with
acetic acid. Other HPLC conditions were as follows: flow
rate of 1 mL/min, injection volume of 20 μL, column
temperature of 25°C, and detection wavelength
of 212 nm.34 The HPLC determination of D,L‐Phe was
mainly based on the comparison with retention time of
standard compounds. In detail, the retention time of
D‐Phe was 8.17 minutes and the retention time of L‐Phe
was 11.25 minutes; D‐Phe had shorter retention time
than L‐Phe. The mechanism is that D‐type amino acid
is more difficult to form complex with L‐Pro and Cu2+
in the chiral mobile phase compared to L‐type amino acid
and would be eluted from the column with priority. On
the contrary, L‐type amino acid is eluted later and has
longer retention time.35 The chromatogram is shown in
Figure S1.
(1)
À
Á
YL−Phe ¼ mL−Phe in solid= mL−Phe in solid þ mL−Phe in liquid ×100%;
(2)
À
Á
YD−Phe ¼ mD−Phe in liquid= mD−Phe in solid þ mD−Phe in liquid ×100;
(3)
where ω and m are the mass fraction and mass of L‐Phe
and D‐Phe in solid or liquid phase, respectively.
On the other hand, the evaluation of racemic
compounds resolution is mainly based on the enantiomer
excess value, that is, the e.e. value, but it can only be used
as the optical purity parameter of the target product and
cannot reflect the actual production capacity of the
resolution system and the yield of the target enantiomers.
Therefore, the concept of enantioseparation effectiveness
(α) is proposed according to Schuur et al,36 which is
calculated according to Equation (4):
α ¼ e:e:×Y;
(4)
2.5 | Resolution of amino acid
enantiomers with CIL by complex‐
precipitation method
where Y represents the yield of L‐Phe in the solid phase,
0 ≤ Y ≤ 1; e.e. is the excess value of enantiomer in the
solid phase, 0 ≤ e.e. ≤ 1. D‐Phe is the target product in
the liquid phase and also has α value corresponding to
the solid phase. The resolution effect can be evaluated
through the combination of above indexes, which can
reflect the application value of the resolution system
comprehensively.
The whole resolution method consisted of several pro-
cesses is shown in Figure S2. A total of 6.0 × 10−4 mol
CIL and 7.2 × 10−4 mol copper salt was mixed in a
5 mL centrifugal tube, and then 1 mL deionized water
was added into the mixture. After that, hydrochloric acid
or sodium hydroxide was used to adjust the water solu-
tion to the specific pH value. The CIL and copper salt
could be thoroughly dissolved to form a blue clear trans-
parent solution through the assistance of ultrasound
wave for 2 minutes. Finally, 10 mg racemic mixture of
D,L‐Phe was added in the blue solution and dissolved
completely by oscillating. After kept in constant tempera-
ture water bath for 30 minutes, the biphase formation
completed. The solid phase was obtained through high
speed centrifuge for 5 minutes (6000 r/min) and then
diluted with deionized water to 25 mL. A total of 20 μL
of this solution was injected into the HPLC system for
quantitative analysis of related enantiomers in order to
evaluate resolution result. Finally, the enantiomeric
2.6 | Resolution mechanism research
The mechanism of complex‐precipitation of racemic
amino acids was analyzed by the following methods.
The solid phase were washed with deionized water, then
dried and the dark blue powders were obtained. The
product was firstly analyzed by FT‐IR analysis (KBr disc)
in the wavenumber range of 400 to 4000 cm−1; then
thermogravimetric analysis (TG) was performed on STA
449 F3 simultaneous thermal analyzer (NETZSCH, Selb,
Germany), and heating rate was set at 10°C/min in the
temperature range of 30°C to 800°C and under nitrogen

ZANG ET AL.
5
atmosphere. Finally, and a manual docking was carried
out among three ligands in the obtained complexes, and
the binding energy of the two kinds of complexes with
different configuration was calculated.
influence the solubility of CILs in different solvents.
Related investigation for above solubility behaviors can
lay the foundation for their application and recovery.
3.2 | Resolution of racemic amino acids
3.2.1 | Screening on CILs
3 | RESULTS AND DISCUSSION
3.1 | Specific optical rotation and
Four kinds of amino acid anionic ionic liquids with
different cationic structure are compared in equal
mole, including straight chain‐type ([BuEt3N]+), cyclic
alkane‐type ([BuPyro]+), cyclic aromatic type ([BuIm]
+), and polycyclic type ([BuTr]+). Actually, the formation
process of coordination unit between metal ions and
other ligands besides water in aqueous solution should
be considered as the process that the water molecules
around the metal ions of Cu2+ are substituted by other
ligands. As long as the alkyl chain on the cations of ionic
liquids reaches a certain length, the resulting hydropho-
bic region will be strong enough around the ligands
which can hinder the formation of hydrated copper ions.
It is beneficial to reduce the competition between water
molecules and ionic liquids and improve the stability of
ternary complex. In the ternary complex (L‐Pro‐Cu
(II)‐D/L‐Phe), copper ion containing space orbitals play
the role of central atom, CIL and phenylalanine with
coordination atoms (oxygen atom and nitrogen atom)
can provide the lone pair electrons as ligands. So the
butyl‐substituted chain was finally selected on four kinds
of cations through preliminary experiments. As a result,
the e.e. values of D‐ and L‐Phe in solid and liquid
solubility of CILs
20
The measurable specific optical rotation ([α]D ILexp) of
four ionic liquids is −27.75 ([BuEt3N][L‐Pro]), −25.27
([BuPyro][L‐Pro]), −27.06 ([BuIm][L‐Pro]), and − 30.18
([BuTr][L‐Pro]), respectively. The results indicate the
specific optical rotation values of amino acid based ionic
liquids with different cations are relatively close. So it
can be concluded that the optical activity mainly
originates from the anion of L‐proline. Moreover, the
solubility properties of four kinds of CILs in 12 common
organic solvents are listed in Table 1. Four kinds of amino
acid ionic liquids are all soluble in polar and intermediate
polar solvents and insoluble in weak polar solvents. It
has been proved that these ionic liquids possess high
polarity, and all of them can be dissolved in the listed
solvents with the polarity higher than or equal to
that of acetone. They have the same side chain length
and L‐proline anion, so their difference in solubility is
mainly determined by the cationic parent structure.
Triethylamine, N‐methylpyrrolidine, N‐methylimidazole,
and tropine as cationic nucleuses have different polarity
and interaction with solvent molecules, which directly
TABLE 1 Solubility properties of four ILs in common solvents at room temperaturea
Solvent
[BuEt3N][L‐Pro]
[BuPyro][L‐Pro]
[BuIm][L‐Pro]
[BuTr][L‐Pro]
Water
Very easily soluble
Very easily soluble
Easily soluble
Easily soluble
Soluble
Very easily soluble
Very easily soluble
Freely soluble
Freely soluble
Soluble
Easily soluble
Easily soluble
Easily soluble
Easily soluble
Soluble
Very easily soluble
Very easily soluble
Very soluble
Soluble
Ethanol
Methanol
Acetonitrile
Acetone
Soluble
Chloroform
Ethyl acetate
Dichloromethane
Ether
Soluble
Soluble
Insoluble
Soluble
Sparingly soluble
Sparingly soluble
Slightly soluble
Very slightly soluble
Insoluble
Sparingly soluble
Slightly soluble
Slightly soluble
Very slightly soluble
Insoluble
Insoluble
Slightly soluble
Slightly soluble
Slightly soluble
Very slightly soluble
Insoluble
Insoluble
Insoluble
Toluene
Insoluble
Hexane
Insoluble
Petroleum
Insoluble
Insoluble
Insoluble
Insoluble
aAccording to Chinese pharmacopoeia, basic solubility of substance is defined as followings: very easily soluble: 1 g solute can be dissolved in less than 1 mL
solvent, easily soluble: 1 g solute can be dissolved in 1 to 10 mL solvent, soluble: 1 g solute can be dissolved in 10 to 30 mL solvent, sparingly soluble: 1 g solute
can be dissolved in 30 to 100 mL solvent, slightly soluble: 1 g solute can be dissolved in 100 to 1000 mL solvent, poor soluble: 1 g solute can be dissolved in 1000
to 10 000 mL solvent, insoluble: 1 g solute cannot be dissolved in 10 000 mL solvent.

6
ZANG ET AL.
FIGURE 2 Comparison of various CILs on e.e. value (%) and yield
phases in resolution of phenylalanine by four CILs are
shown in Figure 2A. Related experimental conditions
were as follows: 6.0 × 10−4 mol ionic liquid,7.2 × 10−4 mol
Cu (Ac)2, 1 mL deionized water, 10 mg D,L‐Phe and
constant temperature at 5°C. It can be seen from
Figure 2A that e.e. value of enantiomer in solid phase
is higher than that in liquid. The precipitated particles
could be regularly arranged as blue needle crystals, and
the generated solid phase is more ordered than the liquid
phase, so the entropy is reduced after precipitation.
Consequently, the isomer which can form the more
stable complex is preferred to be precipitated. On the
other hand, there is an obvious difference using different
cationic CILs to resolute racemic amino acid, the result
shows [BuPyro][L‐Pro] has the best resolution result, so
the following scale‐up and recovery experiment selected
[BuPyro][L‐Pro] as object to carry out our research.
It can indicate that this kind of ionic liquid has the
strongest complexation with copper ion and L‐Phe, so
the hydrophobic ternary complex is easy to form. If the
Van der Waals volume without solvent sphere of
interacting groups is large or there is great steric
hindrance near the coordination atoms on multidentate
ligand, they can interfere with the chelating formation,
or/and reduce the stability of ternary complex, thus
affecting the separation performance. L‐proline is the
anion of the CILs, and it is also the origin of the chirality
of ionic liquids. The coordination atoms in ligands
directly provide the lone pair electrons to form the
coordination bonds with the central atom (copper ion),
which are oxygen atom of carbonyl‐group and nitrogen
atom of amino‐group in the proline‐based anion of CILs.
Furthermore, the steric hindrance of the cation moiety
influences the interaction of coordination atoms and
copper ions, and then results in the different resolution
performance of CILs. Besides that, the cation moiety
can also interact with these chelating groups on the
anion. If their interaction is stronger, it is less conducive
to the chelation between the anion and other complex
parts. In order to explore the interaction between
the anion and cation, Gaussian 09 (Gaussian, Inc.,
Wallingford CT) was used to obtain the energy‐optimized
structures of four CILs, which are shown in Figure 2B.
The method selected was Ground State at the level
of HF/6‐31G(d). As a result, the calculated distance
between the anion and cation center is accorded
with the order of [BuPyro][L‐Pro]
> [BuEt3N][L‐
Pro] > [BuTr][L‐Pro] > [BuIm][L‐Pro], which can prove
above effect resulting from the cationic structure.
3.2.2 | Effects of various conditions on
enantioseparation results
In the process of using CIL [BuPyro][L‐Pro] to separate
target racemic phenylalanine, the involved experimental
conditions were investigated systematically, which
included the types of metal salts, the molar ratio of
Cu (II) to CILs, pH value, the amount of racemic
phenylalanine, and temperature. The detailed influence
of different factors on the e.e. value and yield are shown
in Figure 3A‐D.
Types of metal salts
According to our preliminary experiment and literature,37
the combination of L‐proline as the chiral ligand and
Cu (II) as the central atom has strong enantiomeric
recognition ability. Hence, a series of copper salts were
firstly selected for further study. In order to make
comprehensive comparison and then obtain the ideal
resolution effect, other transition metals containing space
orbitals such as Fe3+/Co2+/Cr6+/Zn2+ were also consid-
ered. As the result, eight kinds of salts including Cu
(Ac)2, CuSO4, CuCl2, CuBr2, Zn (Ac)2, CoCl2, CrCl6, and
FeCl3 were studied in this experiment under the following

ZANG ET AL.
7
FIGURE 3 Effects of various factors on the enantioseparation results
conditions: 6.0 × 10−4 mol [BuPyro][L‐Pro], metal ion‐to‐
IL molar ratio = 1.2, 10 mg D,L‐Phe, 1 mL deionized water,
5°C. It can be seen from Table 2 that only metal salts of Cu
(Ac)2, Zn (Ac)2, CoCl2, and CrCl6 can precipitate com-
plexes solid in the same conditions. Among them, the
solid complex precipitated by salt Cu (Ac)2 has the highest
e.e. value. The less hydrophilic anion will promote the
precipitation of ternary complex to a certain extent. On
the contrary, the anion with strong hydrophilicity cannot
only form ion pairs with copper ions but also form
hydrogen bonds with the water molecules, so the solubility
of the complexes in water will increase, which is not
beneficial for the improvement of their yield.
Cu (II)‐to‐IL molar ratio
Effect of Cu (II)‐to‐IL molar ratio was explored under the
following conditions: 6.0 × 10−4 mol [BuPyro][L‐Pro],
10 mg D,L‐Phe, 1 mL deionized water, 5°C. The results
of investigating the ratio of copper salt to ionic liquid
[BuPyro][L‐Pro] are illustrated in Figure 3A. When
the molar ratio of Cu (Ac)2 to CIL is 1, the e.e. value of
L‐Phe in solid phase reaches its maximum (80.99%).
Meanwhile, when the molar ratio of Cu (Ac)2 to ILs is
0.6, the yield of L‐Phe peaks at 60.59%. According to pre-
vious studies, the activity coefficient of ions will decrease
with the increase of ionic strength within a certain range.
TABLE 2 Effect of salt types on the separation results
If There is
e.e.% of L‐Phe
Salts
Any Complex
in Solid Phase
38
The resolution mechanism in this paper is due to the
Cu (Ac)2
CuSO4
CuCl2
Y
N
N
N
Y
Y
Y
N
75.3%
different stability of the ternary complex formed by race-
mic amino acids in water. When the Cu (II)‐to‐IL molar
ratio changes, the varied ionic strength of the solution
will affect the activity coefficient of all ions participating
in the coordination reaction, thus affecting the stability
of complexes with different configuration. When the ionic
strength is low, the solubility of complex formed by L‐Phe
is lower than that formed by D‐Phe, and the solubility of
both the two complexes will decreases with the increase
‐
‐
CuBr2
Zn (Ac)2
CoCl2
‐
3.4%
10.2%
10.9%
‐
CrCl6
FeCl3

8
ZANG ET AL.
Amino acid amount
of ionic strength in a certain range. According to the
experimental results, it is found: (1) When the molar ratio
of Cu (Ac)2 to CIL is increased from 0.2 to 1, the e.e. value
of L‐AA complex in solid phase will become higher, and
the solubility of L‐AA complex will decrease with the
increase of ionic strength. In this range of molar ratio, the
L‐AA complex can become more and more stable in solu-
tion, which is also more stable than D‐AA complex; (2)
when the molar ratio is increased from 0.2 to 0.6, the e.e.
value of L‐Phe in the solid phase will rise and the yield is
increased. If the molar ratio is further increased from 0.6
to 1.4, the ligand of ionic liquid will be used up. So L‐Phe
has to combine with the large amount of free Cu2+ ions
to form water‐soluble complex, which can make the
precipitation amount less and reduce the separation effi-
ciency. As the result, the e.e. value of L‐Phe in the liquid
phase will decrease with the increase of Cu (II)‐to‐IL molar
ratio, while the percentage of D‐Phe in the solid phase is
also increased, resulting in the rapid decrease of e.e. value
in solid phase under high salt concentration.
Effect of amino acid amount was explored under the fol-
lowing conditions: 6.0 × 10−4 mol [BuPyro][L‐Pro],
6.0 × 10−4 mol Cu (Ac)2, 1 mL deionized water (pH = 4),
5°C. As can be seen from Figure 3C, if the concentration
of amino acids is low, the resulting complex is less likely
to be agglomerated and the resolution is less selective. As
the amount of amino acid increases, the e.e. value in the
solid phase rises rapidly from the lower level and then
decreases. When the amount of racemic amino acid is
10 mg, the e.e. value reaches its maximum (81.49%). In
summary, the dosage of D,L‐Phe is closely related with
the concentration of ternary complexs and then has an
obvious effect on solid precipitation and separation
selectivity. The increase of amino acid is beneficial for
more complex and higher e.e. value in a certain degree,
but its limited solubility should be fully considered
for the possible precipitation together with the complex
during centrifuge.
Resolution temperature
Effect of temperature was explored under the following
pH value
conditions: 6.0
×
10−4 mol [BuPyro][L‐Pro],
The change of hydrogen ion concentration in the solution
often affects the coordination equilibrium through the
protonation with AAs or their ions. Here sodium acetate
and acetic acid were used to adjust pH value in the
following system at 5°C: 6.0 × 10−4 mol [BuPyro][L‐pro],
Cu (II)‐to‐IL molar ratio = 1, 10 mg D,L‐Phe, 1 mL
deionized water with different pH values. Because the
ligand in ternary complex is amino acidic ion rather than
strongly acidic ion, H+can combine with it to regenerate
amino acid when the concentration of hydrogen ions is
increased in aqueous solution or the value of pH becomes
lower. According to above mechanism, the concentration
of hydrogen ions in solution can affect the coordination
equilibrium and the stability of complex. As can be seen
from Figure 3B, when the pH value is varied from 3 to 7,
the e.e. value of the solid phase is less affected; and when
the pH value is 4, the e.e. value reaches its maximum
(81.08%). It can be concluded that strong acidic or alkaline
environment is not conducive to the separation of racemic
amino acids. N atom in phenylalanine can bind with
proton in strong acidic conditions to form quaternary
ammonium ions and its lone electron pair will be
occupied,39 which cannot make it coordinate with Cu2+
ion to form a complex. When the pH value reaches 8, it
can be observed that the complex amount is more than that
obtained in acidic condition, but the e.e. value decreases.
More importantly, there is the blue cotton‐like sediment
appearing in the solution. It is because the copper ions
can be hydrolyzed to become copper hydroxide precipita-
tion when the pH value is 8. Therefore, the alkaline
environment should be avoided for efficient resolution.
6.0 × 10−4 mol Cu (Ac)2, 1 mL deionized water, 10 mg
D,L‐Phe. According to the previous experiments, it was
found that the solid precipitation of the ternary complex
was very rapid, so the investigation in this section was
carried out by controlling the temperature of adding
deionized water. As shown in Figure 3D, the e.e. value
of L‐Phe in the solid phase increases continuously with
the increase of temperature. When the temperature rises
to 10°C, the e.e. value reaches its maximum as same as
the yield of L‐Phe. If the resolution temperature is further
increased, the thermal motion of molecules will be
accelerated and the racemization of the amino acids can
be enhanced, so the e.e. value begins to decrease.
Excessive temperature is also not beneficial for the yield
of L‐Phe, because it will improve the solubility of the
ternary complex in water and hinder the precipitation
process. On the other hand, it was found the temperature
below 10°C could accelerate precipitation of solid
phase significantly. Under these circumstances, the
effect of adsorption and occlusion was more obvious, so
the coprecipitation of different enantiomers occurred
more easily and then e.e. value would decrease.
The whole precipitation process did not need too low
temperature, which was also not beneficial for efficient
resolution.
As a result, the highest e.e. value 85.72% of L‐Phe in
solid phase was finally obtained in single separation
when the resolution system was composed of
6.0 × 10−4 mol CIL [BuPyro][L‐Pro], 6.0 × 10−4 mol Cu
(Ac)2, 1 mL deionized water and 10 mg D,L‐Phe,

ZANG ET AL.
9
meanwhile 10°C was chosen as the resolution tempera-
ture and the pH value was 4.
anionic exchange resin and then the alkaline solution
containing Na+ and [Bupyro]+ was obtained. Then the
solution was neutralized with L‐proline ethanol solution
after concentration, and sodium salt was filtered and the
solvent was removed by vacuum distillation to acquire
the CIL. As for the solid phase resulting from complex‐
precipitation, 0.1 M HCl aqueous solution was used to
dissolve it and adjust the pH to 7, and L‐proline anions
and phenylalanine were transformed into free amino
acids which could be precipitated and removed by
10 000 r/min centrifuge for 5 minutes. Based on the
solubility difference of L‐phenylalanine and L‐proline,
the former was obtained by washing with water for three
times.
In order to further investigate the reliability and
practicability of above separation conditions, a scale‐up
experiment for separating the D‐ and L‐Phe using ionic
liquid [BuPyro][L‐Pro] was conducted. Both of the
volume of [BuPyro][L‐Pro] and Cu (Ac)2 aqueous
solution and D,L‐phenylalanine amount were magnified
tenfold. It was found the e.e. value of solid phase of
L‐Phe in the amplified experiment was 80.5% in single
resolution, which was slightly reduced compared to the
e.e. value (85.7%) of the solid phase in the small‐scale
test before. However, there is no obvious change of the
e.e. value of liquid phase. The overall resolution effect
is basically satisfied, and it was helpful to obtain the
higher e.e. value of solid phase by reducing the amount
of amino acid in the scale‐up system. L‐phenylalanine
could be recovered in the solid phase according to
above posttreatment procedures, and the supernatant
(containing [BuPyro]+ and Cl−) could be directly used
as the raw material and react with L‐pro through the
anion exchange resin to reacquire the CIL of [BuPyro]
[L‐Pro].
3.2.3 | Comparison with the separation
results of nonaromatic AAs
In order to find more rules in separation of racemic
amino acids with the developed method, D,L‐serine and
valine are selected as the representatives of nonaromatic
AAs and their resolution results are shown in Table 3.
It shows that the resolution of D,L‐valine was better than
that of D,L‐serine, because the water solubility and
stability of the complexes formed by different amino
acids and chiral ligands are different. However, the
enantiosepararion result of D,L‐Phe is better than theirs,
and it is obvious the difference of the coordination
between D/L‐AA and Cu (II) will be more significant if
there is a group with larger Van der Waals volume in
the structure of AA, which can result in enough steric
hindrance for effective chiral recognization.40
3.3 | Scale‐up and posttreatment research
for the resolution of amino acids
In order to further investigate the multiplication perfor-
mance of above method for enantioseparation of amino
acids, it was designed about a tenfold scale‐up experiment
based on the screened experimental conditions (0.6 mol/L
[BuPyro][L‐Pro] and Cu (Ac)2 aqueous solution, 0.1 g D,
L‐Phe, 10°C). Furthermore, in the previous studies of
similar resolution, the posttreatment of obtaining a single
configuration of the target amino acids from complexes
was rarely mentioned, so it is difficult to be applied in
the actual preparation and the recovery of ionic liquids
has not been effectively realized, which will cause the
waste of expensive CILs and increase the operation cost.
It also cannot meet the requirements of green chemistry.
In this section, an ionic liquid recovery strategy and
actual procedure to obtain target amino acids with single
configuration are proposed as following. After the resolu-
tion, the system was centrifugally filtrated to remove solid
phase, and the liquid phase flowed through 201 × 7 type
3.4 | Characterization of ternary complex
and resolution mechanism
In aqueous solution, the copper ions can be coordinated
with adjacent water molecules as ligand to form hydrated
ions. So it is known that the so‐called free metal ions
in water solution are actually hydrated metal ions.
Therefore, the formation process of coordination unit
between metal ions and other ligands besides water in
aqueous solution should be considered as the process that
the water molecules around the metal ions of Cu2+ are
substituted by other ligands.
TABLE 3 The properties of nonaromatic AAs and their
enantioseparation resultsa
e.e. value of
S/g
(100 g−1
L‐Phe in solid
phase
AAs
pI
)
M (mg)
According to ligand‐field theory, Cu2+ belongs to the
3d9 electronic configuration. In the electrostatic field of
asymmetric ligand, the energy improvement of the five
D,L‐serine
D,L‐valine
5.68
5.68
38
30
20
55.55%
63.50%
4.50
2
2
degenerated orbits (dxy, dxz, dyz, dz , dx ‐y2) is different,
aS is the solubility of the amino acids in water at 25°C; M is the amount of
the amino acid.
and the splitting of energy levels will occur which leads

10
ZANG ET AL.
to the generation of new orbits. During the coordination
process, the six ligands are distributed along six directions
of x, y and z axes and approach towards d orbital of Cu2+
ions to form octahedron. As the result, the electric field
2
2 2
forces vary in different orbits, dz and dx ‐y are affected
apparently by electrostatic repulsion and their energy rise
remarkably, then the double‐fold degenerate orbit of
dγ or eg forms. On the other hand, dxy, dxz, and dyz
result in threefold degenerate orbit of dε or t2g, which
have lower energy. Among them, t2g and eg orbits will
split into low‐energy and high‐energy π orbitals when
coordinate bonds form; the energy improvement of
the former is less, the distance between Cu2+ and
ligand is longer and interaction is weaker; the latter
is completely opposite. As for the coordination process
involving CIL with L‐proline anion, D,L‐Phe and
Cu2+, the former two have O and N atoms as ligand
teeth in their structures which can result in stronger
coordination than CH3COO− and H2O. The ligands
surrounding Cu2+ ions are substituted by nucleophilic
reaction. In the formation of complexes, ligand
exchange can be used to explain the separation mecha-
nism (see Equation (5)). The stability constant of the
coordination ions is the same as all other equilibrium
constants, which can be very different under external
conditions. In the formation of complex, all the factors
mentioned in Section 3.2 will affect the coordination
equilibrium.
Cu2þð½BuPyroꢀ½L−ProꢀÞxðH2OÞyðAcÞz þ L−Phe→Cu2þ
ð½BuPyroꢀ½L−ProꢀÞxðL−PheÞyðAcÞz↓ þ H2O
Cu2þð½BuPyroꢀ½L−ProꢀÞxðH2OÞyðAcÞz þ D−Phe→Cu2þ
(5)
ð½BuPyroꢀ½L−ProꢀÞxðD−PheÞyðAcÞz þ H2O:
As mentioned above, the amino acid ions (both pro-
line and phenylalanine) have two coordination atoms of
O and N, which often combine with the central atom to
form chelating individual. They are located on the same
plane as copper (II) and the same chemical atoms are in
the para‐position; thus, the system has the lowest energy
and is stable. Consequently, α‐ and β‐amino acids used to
form the five‐ and six‐membered chelating ring, respec-
tively; and the coordination ratio is usually 1:1. The TG
diagram of precipitated complex is shown in Figure 4A.
In the temperature range of 30 to 160°C, the first stage
of weightlessness (1.95%) occurs for the loss of bound
water in the complex. During 160 to 250°C, the second
stage of weightlessness (53.98%) is mainly related with
the decomposition of the ionic liquid and phenylalanine
in the precipitates. During 280 to 320°C, the third
stage of weightlessness (11.78%) accords with the decom-
position of the acetate radical according to the following
FIGURE 4 A, Thermogravimetric analysis and B, FT‐IR of
precipitated complex together with C, molecular modeling of
ternary complex
equation:2Cu(CH3COO)2
→
Cu2O
+
C2H6
+
CH4 + CO2 + 4CO + H2O. Finally, the residue of Cu2O
accounted for 35.33% of the total mass. Based on these
thermogravimetric data and the contents of H, C, and N
element measured by EA3000 elemental analyzer
(Euro vector S.P.A, USA), it can be calculated and proved
that CIL to L‐Phe to Cu (Ac)2 approximates to 1:1:1 in the

ZANG ET AL.
11
TABLE 4 The calculated energy results of complex (D,L‐Phe‐Cu
(II)‐CILs)
composition of blue complex, which can prove that Cu
(II) is coordinated with six ligands and octahedral geo-
metric configuration in the complex.
Configuration
For further structural analysis, the infrared spectrum
of the ternary complex is shown in Figure 4B. The
main IR data of the ternary complex are as follows:
IR (KBr, cm−1): 3435, 3295.27, 3249, 3180, 2966.07,
1615.51, 1496.09, 1448.24, 1387.86, 1366.92, 1316.3, 1396,
1198.29, 1110.75, 1088.34, 935.94, 889.09, 851.92, 827.04,
745.5, 704.22, 647.31, 600, and 553.29. In which, the peak
at 3435 cm−1 accords with the absorbance of residual
water, two peaks at 3295 and 3249 cm−1 belong to νas
and νs of primary amine group in L‐Phe. The absorbance
of secondary amine group in the anion of ionic liquid
is weak which is covered by the adjacent peaks. The sig-
nal at 3180 cm−1 is related with νas and νs of N+CH3
and Ar‐H, and the peak slightly below 3000 cm−1 can
be ascribed to the absorbance of stretching vibration of
–CH3 and –CH2–. Because of the oxygen atom of carbonyl
group interacts with copper ion to form coordination
bond, which results in redshift of absorbance peaks.
So 1615.51 cm−1 is lower than the wavenumber of
of Complex
△H (kcal/mol)
△G (kcal/mol)
D‐Phe‐Cu (II)‐CILs
L‐Phe‐Cu (II)‐CILs
−64.48
−64.89
−53.12
−54.25
and the interaction difference between D,L‐AA com-
plexes. The results are shown in Figure 4C and
Table 4, which prove that the enthalpy is negative. So
the coordination is a spontaneous process, and the
binding energy of L‐Phe‐Cu (II)‐CILs is less than that
of D‐Phe‐Cu (II)‐CILs, indicating the L‐complex is
more stable, consistent with the experimental results.
The results of energy optimization and spatial structure
of D,L‐complexes also prove that the main part of ionic
liquids participating in the formation of complexes is
the anion of L‐proline. When N atom in the cationic
nucleus becomes the quaternary ammonium form, it
will also form an atypical hydrogen bond with the coor-
dination center of Cu (II).
νas
in uncoordinated CIL. Meanwhile, the
C=O(COOH)
absorbance at 1387 cm−1 belongs to νs C=O(COOH) of CIL
anion in the ternary complex. Moreover, because of the
nitrogen atom of amino acid can also coordinate with
copper ion, the C‐N vibration is red‐shifted to 1400 to
1350 cm−1. The peaks in the range of 1200 to 1000 cm−1
accords with the absorbance of stretching vibration of
C‐O and C‐C, and the signals at 745 and 704 cm−1
can be ascribed to the out‐of‐plane bending of Ar‐H
in L‐Phe.
In the following calculation research, molecular struc-
tures were firstly developed in Gauss‐View 5.0, and then
they were optimized at the level of HF/6‐31G(d) (Method:
Ground State) with Guassian 09 software (Gaussian, Inc,
Wallingford Connecticut) to obtain the configurations
with lowest energy. In the quantitative calculation for
coordination, the part of L‐Pro‐Cu (II)‐D/L‐Phe was
selected as host (main body) and [BuPyro]+ was treated
as guest. The binding energy (△G, kcal/mol) can be
obtained according to Equation (6).
4 | CONCLUSION
In this study, a series of CILs with L‐proline anion were
synthesized by using triethylamine, N‐methyl pyrrolidine,
N‐methylimidazole, and tropine as cationic nuclear,
and their specific optical rotation and solubility in different
common solvents were determined after structural identi-
fication. Moreover, the properties of CILs separating D,L‐
Phe by complex‐precipitation method were investigated.
The relationship between cationic structure of CILs and
their performance was discussed, and the effects of
main factors on the separation results of racemic
phenylalanine using these ionic liquids were studied in
detail. The suitable conditions for resolution D,L‐Phe in
this experiment are 6.0 × 10−4 mol CIL [BuPyro][L‐pro],
6.0 × 10−4 mol Cu (Ac)2, 1 mL deionized water (pH = 7),
10 mg D,L‐Phe and constant temperature at 10°C. As the
result, 85.7% e.e. value of L‐phenylalanine in solid phase
could be achieved. After tenfold scale‐up of the resolution
system, it still showed an ideal resolution performance.
Finally, the study of resolution mechanism indicated
that the electron pairs of CILs anion and spatial configura-
tion of CILs played important role in the resolution
process.
À
Á
△Gbind ¼ Gcomplex− Ghost þ Gguest
;
(6)
where Gcomplex is Gibbs free energy of the complex,
Ghost is Gibbs free energy of L‐Pro‐Cu (II)‐D/L‐Phe,
Gguest is Gibbs free energy of [BuPyro]+. If the value
of binding energy of the complex is negative, it indicates
that the coordination compound can be formed between
the host and guest, and the lower the binding energy,
the easier to form a stable complex. The Gauss‐View
software was also used to depict the spatial structure
ACKNOWLEDGMENT
Preparation of this paper was supported by the National
Natural Science Foundation of China (No. 81673316).

12
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16. Afonso CAM, Crespo JG. Recent advances in chiral resolution
through membrane‐based approaches. Angew Chemie ‐ Int Ed.
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ORCID
Huimin Zang http://orcid.org/0000-0001-9583-3141
17. Xie R, Chu L‐Y, Deng J‐G. Membranes and membrane processes
for chiral resolution. Chem Soc Rev. 2008;37(6):1243‐1263.
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