Novel chiral ionic liquids stationary phases for the enantiomer separation of chiral acid by high-performance liquid chromatography

Article abstract of DOI:10.1002/chir.22839

Novel chiral ionic liquid stationary phases based on chiral imidazolium were prepared. The ionic liquid chiral selector was synthesized by ring opening of cyclohexene oxide with imidazole or 5,6-dimethylbenzimidazole, and then chemically modified by diffe

Full text of DOI:10.1002/chir.22839

Received: 2 November 2017
DOI: 10.1002/chir.22839
Revised: 8 December 2017
Accepted: 5 January 2018
R E G U L A R A R T I C L E
Novel chiral ionic liquids stationary phases for the
enantiomer separation of chiral acid by high‐performance
liquid chromatography
Shanshan He | Yunchao He | Lingping Cheng | Yaling Wu | Yanxiong Ke
Engineering Research Center of
Abstract
Pharmaceutical Process Chemistry, School
Novel chiral ionic liquid stationary phases based on chiral imidazolium were
prepared. The ionic liquid chiral selector was synthesized by ring opening of
cyclohexene oxide with imidazole or 5,6‐dimethylbenzimidazole, and then
chemically modified by different substitute groups. Chiral stationary phases
were prepared by bonding to the surface of silica sphere through thioene “click”
reaction. Their enantioselective separations of chiral acids were evaluated by
high‐performance liquid chromatography. The retention of acid sample was
related to the counterion concentration and showed a typical ion exchange pro-
cess. The chiral separation abilities of chiral stationary phases were greatly
influenced by the substituent group on the chiral selector as well as the mobile
phase, which indicated that, besides ion exchange, other interactions such as
steric hindrance, π‐π interaction, and hydrogen bonding are important for the
enantioselectivity. In this report, the influence of bulk solvent components,
the effects of varying concentration, and the type of the counterion as well as
the proportion of acid and basic additives were investigated in detail.
of Pharmacy, Ministry of Education, East
China University of Science and
Technology, Shanghai, China
Correspondence
Yanxiong Ke, Engineering Research
Center of Pharmaceutical Process
Chemistry, School of Pharmacy, Ministry
of Education, East China University of
Science and Technology, Meilong Road
130, Shanghai, China.
Email: key@ecust.edu.cn
Funding information
NSF: National Science Foundation, Grant/
Award Number: 21375038
KEYWORDS
5,6‐dimethylbenzimidazole, anion‐exchange, cyclohexene oxide, enantioselectivity, hydrogen
bonding, imidazolium, substituent group, π‐π interaction
1 | INTRODUCTION
then, a large number of CILs have been synthesized by
using in many aspects, such as nuclear magnetic
Ionic liquids (ILs) have been known for many years and
been applied in many ways, such as catalysis,1,2 solvents
in organic synthesis,3 or functional materials in separa-
tion science,4,5 and so on, due to their excellent properties
such as wide liquids ranges, good thermal stabilities, wide
range of viscosities, adjustable miscibility, good solvation
characteristic, and reusability.6-8 Chiral ILs (CILs) are
important due to their potential chiral discrimination
capabilities. The advent of CILs dates back to 1996 when
Herrmann et al synthesized N‐heteocyclic carbenes of
imidazoles.9 A chiral imidazolium chloride IL was
obtained as an intermediate during this reaction. Since
resonance (NMR),10 near‐infrared spectroscopy,11 fluores-
cence spectroscopy,12,13 and so on. But the application in
chromatography is only more used in gas chromatogra-
phy, such as Armstrong and coworkers who have done
pioneering work in introducing highly thermally stable,
uniquely selective, multicationic ILs as gas chromato-
graphic stationary phases.14-16 The application in high‐
performance liquid chromatography (HPLC) is much
smaller than in gas chromatography. Currently, no more
than 10 CIL stationary phases have been reported for
HPLC. For example, Li et al prepared four novel chiral
stationary phases (CSPs) based on β‐cyclodextrin
Chirality. 2018;1–10.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
HE ET AL.
derivatives functionalized by ILs, having a good perfor-
mance on aromatic alcohols and ferrocene derivatives.17
To expand the application of ILs especially for CILs in liq-
uid chromatography, we consider the use of the ion
exchange interaction between the liquid cationic groups
and anion samples.
Enantiopure drugs are always in high demand due to
the different biological and pharmacological activities of
drug enantiomers in the pharmaceutical,18 biological,19
and other industries. The separation of chiral compounds
is particularly important. Especially, the separation of chi-
ral acid is also very vital. In the case of lactic acid,20 2‐
hydroxyglutaric acid,21 and glyceric acid,22 changes in
enantiomeric proportions play a vital role in disease.
Their enantioselective analysis is consequently a major
concern in biomarker studies and clinical analysis. There-
fore, the development of chiral acid separation method is
particularly important.
There are many methods that have been used for the
analysis and preparation of acidic samples, such as super-
critical fluid chromatography,23 gas chromatography,24
liquid chromatography, capillary chromatography,25 and
crystallization.26 Among them, liquid chromatography is
the most widely used. Liquid chromatography is based
on the modified silica gel material comprising either
immobilized small molecules of chiral selectors (CSs; eg,
Cinchona‐based ion exchangers),27 macromolecule CSs
(eg, biopolymers or synthetic polymers),28 or macrocyclic
CSs (cyclodextrin antibiotics).29 The broad family of CSPs
has enabled separation of almost any racemic mixture of
choice, ranging from neutral lipophilic to highly polar
hydrophilic compounds.
In this paper, a novel type of strong anion exchange
CSP was synthesized, in which the imidazole IL was
immobilized onto the modified silica gel material, as
shown in Figure 1. Acidic samples were ionized, with a
FIGURE 1 The synthesis route of chiral stationary phase (CSP) 1 to CSP4: A, Imidazole or 5,6‐dimethylbenzimidazole, 70°C,12 hours;
B, Benzoic anhydride, triethylamine, dichloromethane, room temperature, 4 hours; C, preparative enantiomer separation on S‐Chiral B,
MP:8n‐hexane/2‐ethanol (vol/vol) (overall yield 80%, >99% ee); D, ACN, 70°C, 24H, N2, reflux; E, thiol‐modified silica gel,
azobisisobutyronitrile, methanol, 65°C, N2, reflux; F, EA:H2O = 1:1, 0°C‐rt, 2.5 hours

HE ET AL.
3
negative charge, under the mobile phase of acetonitrile
(ACN)/50‐mmol acetic acid (HAc)/50‐mmol triethylamine
(TEA) (vol/vol/vol), taking ion interaction with the posi-
tive stationary phase. In the separation process of acidic
samples on this type of CSP, except the ion exchange, there
were π‐π interaction and hydrogen bonding interaction.
In addition, in this study, influences of the mobile
phase composition concerning acid‐to‐base ratio as a sub-
stitute for proton activity, protic‐to‐aprotic solvent ratio,
and counterion variation on the overall chromatographic
performance were investigated to reveal the ion exchange
process as the main mechanism for retention and to map
the conditions under which this new material can be suc-
cessfully operated. These aspects will be discussed in
detail in the following sections.
elementar vario EL III (Germany). The CSP of preparation
of monomeric compounds was purchased from Acchrom
Technologies (China). Other chemicals and equipment
are described in the Supporting Information.
2.2 | Chromatographic condition and
sample preparation
For evaluation, each CSP 1 to CSP 4 (2.5 g) was slurried in
methanol (20 mL) and packed into a 150 × 4.6‐mm HPLC
column with methanol (100 mL) as propulsion solvent
under a pressure of 50 MPa. All the chromatographic sep-
arations were performed on Waters HPLC system
consisting of a 515 HPLC pump, 2489 UV/Vis detector,
7725i manual injector, and model 1500 column heater
(Waters, USA).
All the analytes (AR1‐AR10), as shown in Figure 2,
used for chiral separation evaluation were dissolved in
HPLC‐grade ethanol to form about 1‐mg/mL concentra-
tion. Dead time t0 was measured with 1,3,5‐tri‐t‐
butylbenzene as the void volume marker by using the elu-
ent of methanol. The flow rate was set at 1 mL/min, and
column temperature was held constantly at 20°C. Ultravi-
olet detection wavelength was set at 254 or 220 nm, and
the injection volume was 1 μL.
2 | MATERIALS AND METHODS
2.1 | Instrumentation
High‐performance liquid chromatography‐grade spherical
silica gel (5‐μm particle size; 10‐nm pore size; 300‐m2/g
surface area) was purchased from Acchrom Technologies
1
(China). H‐NMR and 13C‐NMR identifications were car-
ried out on a Bruker AVANCE 400 (Germany) at a temper-
ature of 25°C. Elemental analysis was measured on an
FIGURE 2 The chemical structure of AR 1 to AR 10

4
HE ET AL.
Performance of the separation system was monitored
in resolution (Rs), selectivity factor (α), and retention time
(k1,k2), according to the usual formulae:
interaction, and hydrogen bond interaction of different
derivation groups on chiral separation. The structures of
the four stationary phases and the chromatographic
results were shown in Figure 1 and Table 1, respectively.
Chiral stationary phase 1 and CSP 2 were prepared by
ring opening of cyclohexene oxide with imidazole or 5,6‐
dimethylbenzimidazole, respectively, and then substituted
by benzoate group. The only difference of two CSPs was the
part of imidazolium moiety. This design was to investigate
the effect of cationic moieties on chromatographic behavior.
The enantio separations of the samples on CSP 1 are poor.
Only AR 4 can be partially separated with selectivity factor
value of 1.05. Compared with CSP 1, CSP 2 had better enantio
separation result, on which AR1 to AR3 beside AR 4 were
separated under the mobile phase of 6H/4 isopropanol (I)/
0.2 trifluoroacetic acid (vol/vol/vol) on CSP 2. As shown in
the Figure 3A, AR 1 cannot be resolved on CSP 1. It was
completely resolved into enantiomers on CSP 2 (α = 1.15,
Rs = 1.77). More steric hindrance and π‐π interaction pro-
vided by the cationic imidazolium moiety are the possible
reason for the improvement of enantioselectivity.
2ðt2−t1Þ
w1 þ w2
k2
; α ¼ ; k1 ¼
k1
t1−t0
t2−t0
Rs ¼
; k2 ¼
t0
t0
where t1 and t2 are the retention times and w1 and w2 are
the extrapolated peak widths at the baseline.
2.3 | Preparation of chiral stationary
phases
The structures and the synthesis route of CSP 1 to CSP 4
were shown in Figure 1, and the details were shown in
the Supporting Information.
3 | RESULTS AND DISCUSSION
3.1 | Preparation of CSP 1 to CSP 4
Strong anion exchangers CSPs 1 to 4 were prepared as
outlined in Figure 1, relying on established methodolo-
gies. At first, cyclohexene oxide 1 was considered as
“spring‐loaded” rings for nucleophilic opening reaction
with imidazole or 5,6‐dimethylbenzimidazole under sol-
vent‐free conditions to get the racemic imidazolyl or 5,6‐
dimethylbenzimidazolyl alcohols 2.30 Derivation of 2 with
different functional group provided the target compounds
3.31,32 The racemic mixture 3 was resolved chromato-
graphically on a 250 * 20‐mm ID column packed with cel-
lulose tris(3,5‐dimethylphenylcarbamate) coated silica
spheres (S‐Chiral B). Typically, on analytical scale, the
resolution of racemic 3A to 3D using a normal mobile
phase provided excellent selectivity (α = 2.03, Rs = 5.68)
and the transfer of these conditions to a preparative level
allowed high sample loading of 100‐mg neat racemate per
injection while baseline separation was still maintained.
Thus, the collected fractions were virtually enantiopure
(data were shown in the Supporting Information). Then,
compound 3 was reacted with 4 to afford the target IL 5
and then covalently bonded to thiol‐functionalized silica
gel through thioene “click” reaction. The loading of CSs
corresponding to CSP 1, CSP 2, CSP 3, and CSP 4 were
0.21, 0.16, 0.15, and 0.15 mmol/g according to elemental
analysis, respectively (see in the Supporting Information).
According to the data of element analysis, it can be seen
that the bulkiness of the CSP affects the loading amount.
The effect of substitute group on oxygen atom on the
enantioselectivity was also investigated. Chiral stationary
phase 3 has the substitution of (3,5‐dimethylphenyl)
carbamoyl group on oxygen atom and the same
imidazolium moiety as CSP 2. These CSPs have much dif-
ferent enantioselectivity from CSP 2. AR4‐AR10 that can-
not be separated on CSP 2 were resolved a little on CSP 3
under the mobile phase was 8H/2I/50‐mmol HAc/50‐
mmol TEA, as shown in Figure 4A. The retention time
of AR1 to AR3 was very long under this mobile phase. Dif-
ferent steric hindrance and the additional side of hydro-
gen donor site on carbamoyl group may respond for the
change of the enantioselectivity.
Compared with CSP 3, CSP 4 was substituted by tert‐
butyl carbamoyl group on oxygen. The separation factors
and plate numbers on CSP 4 are much lower than those
on CSP 3, which indicates that π‐π interaction provided
by the substitution group is favorable for the chiral
discrimination of the analytes, as shown in Figure 3B. In
summary, the CSP 3 showed the best ability of separation.
In addition, compared with the results obtained under nor-
mal phase mode (8H/2I/50‐mmol HAc/50‐mmol TEA),
separation factors and plate numbers of the enantiomers
are much higher under polar organic mode (ACN/50‐mmol
HAc/50‐mmol TEA), as shown in Figure 4B. Therefore, the
next study was based on the polar organic mode.
3.3 | The influence of counterion
concentration
3.2 | General comparison of CSP 1 to CSP 4
In this paper, four stationary phases were evaluated,
mainly to study the effect of steric hindrance, π‐π
The retention of acidic analyte is primarily controlled by
ion exchange, which is a characteristic of the investigated

HE ET AL.
5
TABLE 1 Enantioseparation of AR 1 to AR 10 on chiral stationary phase (CSP) 1 to CSP 4
CSP
Analyte
1
2
3
4
AR1
k1
α
13.24
5.71
1
15,900
—
14.13
6.62
1.15
21,260
1.77
B
15.52
3.82
1
2,993
—
17.65
1.02
3,833
—
7.83
1.04
3,873
—
1
6,493
—
1
7,013
—
1
6,673
—
N1 (m−1
Rs
)
)
)
)
)
)
MP
A
B
A
A
B
A
B
AR2
AR3
AR4
AR5
AR6
AR7
AR8
AR9
AR10
k1
α
7.62
1
7,713
—
2.81
1
20,640
—
8.45
1
13,647
—
3.03
1.1
22,640
1.08
B
9.89
1
11,693
—
1.69
1
3,060
—
11.92
1.01
4,993
—
3.52
1.05
3,333
—
N1 (m−1
Rs
MP
A
B
A
A
B
A
B
k1
α
13.62
1
6,647
—
5.75
1
15,893
—
15.21
1
6,673
—
6.66
1.15
23,853
1.8
16.81
1
6,507
—
3.91
1
2,727
—
18.21
1.02
3,660
—
8.05
1.05
3,800
—
N1 (m−1
Rs
MP
A
B
A
B
A
B
A
B
k1
α
9.83
1.05
27,680
0.83
A
15.02
1
8,847
—
7.21
1.06
13,247
1.02
A
17.46
1
6,767
—
9.12
1.11
26,813
1.61
A
12.75
1.14
4,687
0.86
C
10.02
1.02
9,833
—
11.41
1.1
10,680
0.92
C
N1 (m−1
Rs
MP
C
C
A
k1
α
2.01
1
14,027
—
6.28
1
10,720
—
1.52
1
18,873
—
4.74
1
16,307
—
1.79
1.14
34,087
1.57
A
3.95
1.15
5,667
0.83
C
3.64
1.04.
8,233
—
7.8
1.12
10,080
0.95
C
N1 (m−1
Rs
MP
A
C
A
C
A
k1
α
2.76
1
11,613
—
9.35
1
10,120
—
2.21
1
21,037
—
6.17
1
10,640
—
2.65
1.17
39,433
2.23
A
5.27
1.18
6,080
0.92
C
3.73
1.03
8,587
—
9.5
1.15
5,967
1.01
C
N1 (m−1
Rs
MP
A
C
A
C
A
k1
α
3.52
1
8,427
—
12.01
1
8,993
—
2.09
1
19,507
—
7.57
1
9,713
—
2.75
1.27
31,993
3.27
A
5.74
1.16
4,247
0.86
C
4
1.06
8,507
—
10.9
1.17
4,667
1.03
C
N1(m−1
Rs
)
MP
A
C
A
C
A
k1
α
2.54
1
13,447
—
11.41
1
9,813
—
2.34
1
18,640
—
6.74
1
10,427
—
2.05
1.19
33,920
2.17
A
5.68
1.17
4,607
0.95
C
3.85
1.01
8,300
—
11.05
1.15
6,213
1.05
C
N1(m−1
Rs
)
MP
A
C
A
C
A
k1
α
2.65
1
14,240
—
13.1
1
8,320
—
2.54
1
17,927
—
8.01
1
10,127
—
2.47
1.21
29,200
2.52
A
5.18
1.19
14,040
1.01
C
4.07
1.03
8,427
—
10.82
1.14
7,027
1.04
C
N1 (m−1
Rs
)
MP
A
C
A
C
A
k1
α
2.68
1
14,767
—
12.52
1
8,747
—
2.46
1
18,327
—
7.68
1
10,893
—
2.41
1.15
31,720
1.65
A
5.45
1.15
4,773
0.85
C
4.14
1.05
7,853
—
10.62
1.14
7,047
1.04
C
N1 (m−1
Rs
)
MP
A
C
A
C
A
MP: mobile phase; A: acetonitrile/50‐mmol HAc/50‐mmol triethylamine (TEA); B: 6H/4I/0.2 trifluoroacetic acid (vol/vol/vol); C: 8H/2I/50‐mmol HAc/50‐mmol
TEA.

6
HE ET AL.
FIGURE 3 High‐performance liquid chromatography of AR 1 and AR 7: A, mobile phase: 6 hexane/4 isopropanol/0.2 trifluoroacetic acid,
analyte: AR 1; B, mobile phase: acetonitrile/50‐mM HAc/50‐mM triethylamine, analyte: AR 7. Flow rate: 1.0 mL/min, T: 25°C, UV detection
at 254 nm
FIGURE 4 High‐performance liquid chromatography of AR 7: A, mobile phase: 8H/2I/50‐mmol HAc/50‐mmol triethylamine (TEA);
B, stationary phase: CSP 3, mobile phase: acetonitrile/50‐mmol HAc/50‐mmol TEA and 8H/2I/50‐mmol HAc/50‐mmol TEA. Flow rate:
1.0 mL/min, temperature: 25°C, UV detection at 254 nm
CSPs. A near neutral polar organic mobile phase system
was applied to investigate the ion‐exchange property of
the CSPs. Equivalent molar ratio of acid and basic additive
were added to the mobile phase to evaluate the stationary
phase. Under such conditions, the acidic analytes are pro-
tonated while the CSPs are cationic. Thus, the ion exchange
between the cationic stationary phase and the anionic ana-
lyte is the predominant interaction leading to the retention
and separation of the analytes. Chiral stationary phase 3
was taken as an example to explore the existence of ion
exchange. Counterion concentrations were varied in the
range of 20 to 50 mmol. The results obtained were depicted
as plots of the logarithm of the retention factors (log k1)
versus the logarithm of counterion concentration of the first
eluted enantiomer (log c) (Figure 5A).
As the concentration of counterions increased, the
retention time decreased. The linear relationship between
log k and log c can be observed. This phenomenon clearly
indicates that the retention of these analytes on the CSP
was mainly due to the effect of ion exchange interaction
according to a stoichiometric displacement model.33 The
ratio of solute to counterion effective charge determines
the slope of the curve. Under the given mobile phase con-
ditions, it is expected that both the analyte and the coun-
terion are protonated and have equivalent isoelectric
charge. Chromatographic data demonstrated that the plot
showed very similar slopes of log k1 versus log c plots of
0.95 to 1.05. The first and second eluted enantiomers
exhibited almost the same slope (data not shown), indicat-
ing that the two isomers had the same type of ion
exchange process in the column and respond equally to
changes in counterion concentration. Thus, the selectivity
does not change significantly with the change of the con-
centration of the counterion (Figure 5B). The indepen-
dence of selective on counterion concentration is a
unique feature of these novel stationary phases that
depend on the ion exchange process and can adjust the
retention time without affecting the selectivity.
3.4 | Variation of the acid‐to‐base ratio
The proton activity of the mobile phase can also affect the
ion exchange process by adjusting the degree of ionization
of the analyte. To explore the appropriate operating

HE ET AL.
7
FIGURE 5 Influence of the counterion concentration on (A) retention of the first eluting enantiomer and (B) selectivity. The data of five
representative chiral analytes are shown
conditions, the dependence of CSP 3 on the mobile phase
3.5 | Variation of the type of acidic
with different acid‐to‐base ratio was studied. For this pur-
pose, six different acid‐to‐base molar ratios of the mobile
phase additive components were selected by using HAc
and TEA in an ACN solvent system. Besides equimolar con-
ditions, the acidic conditions are expressed by the ratio of
acid to base 1.2/1, 1.4/1, 1.6/1, 1.8/1, 2/1 (60, 70, 80, 90,
and 100‐mmol HAc and 50‐mmol TEA). The retention
and the selectivity of representative acidic species for differ-
ent acid‐base ratio mobile phases were shown in Figure 6.
As the alkaline mobile phase affects the life of the sta-
tionary phase, it is not used. It can be seen that the reten-
tion of analytes had a slight decrease as the proportion of
acid additives in the mobile phase increased but constant
concentration of the basic counterion. This is because the
extra acidic additives inhibited the ionization of the acidic
sample, resulting in the ion‐exchange interaction between
the sample and the stationary phase reducing (Figure 6A).
The capacity of the strongly basic ion exchange sites on
CSP 3 did not decrease. And there was also a slight reduc-
tion in selectivity (Figure 6B). Thus, the equivalent phase
of the acid‐base ratio of the mobile phase was selected to
use for the further study. It can be concluded from
Figure 6 that the stationary phase of a strong anion
exchange mode such as CSP3 has a broad prospect in
resolving acidic compounds.
counterion
The counterion in the mobile phase is a competitor to the
analyte and stationary phase in the ion exchange equilib-
rium, and therefore, it plays an important role in ion
exchange reaction. It is for this reason that, as described
in the previous section, the retention time of the analyte
FIGURE 7 Effect of the nature of the counterion being formic
acid, acetic acid, trifluoroacetic acid, n‐hexanoic acid, and
trimethylacetic acid. Data of a representative chiral analyte AR 7 are
shown. CSP 3; T: 25°C, flow 1.0 mL/min, UV detection at 254 nm,
mobile phase: formic acid, HAc, n‐hexanoic acid, trifluoroacetic acid
(50 mM), and diethylamine (50 mM) in acetonitrile
FIGURE 6 Effect of the acid‐to‐base molar ratio on (A) retention and (B) selectivity. Data of five representative chiral analytes are shown.
CSP 3: T: 20°C, flow 1.0 mL/min, UV detection at 254 nm, mobile phase: triethylamine (50 mM), and HAc (60, 70, 80, 90, and 100 mM) in
acetonitrile

8
HE ET AL.
FIGURE 8 The effect of the acetonitrile‐to‐MeOH ratio on (A) retention and (B) selectivity. Data of AR 7 are shown. CSP 3: T: 20°C, flow
1.0 mL/min, UV detection at 254 nm, mobile phase: triethylamine (50 mM) and HAc (50 mM) in acetonitrile/methanol
can be varied by changing the ion concentration. How-
ever, not only the concentration, the type of counterion
will also affect the chromatographic behavior. In the
study of anion exchange stationary phase such as quinine,
the type of acid additive also affects the retention of acidic
samples.34 In this experiment, formic acid (pKa = 3.74),
HAc (pKa = 4.74), trifluoroacetic acid (pKa = 0.23), and
n‐hexanoic acid (pKa = 4.86) as counterion were selected
to investigate the effect of the type of ions on the retention
and selectivity of acidic samples.
It can be deduced from Figure 7 that the type of coun-
terion did change the retention of the analyte, and as the
retention time increased, the peaks were wider and the
tailings more severe. Furthermore, as a consequence of
the ion exchange process, the retention of the analyte
can be regulated either with changes of the counterion
type or its concentration. Except the retention time, the
selectivity and resolution were affected by the variation
of the type of counterion, although just a small extent.
Selectivity and resolution of the best is acetic acid as a
counter ion, the selectivity of 1.27, the resolution of 3.27.
increase in methanol content, and the selectivity of the
analyte exhibits a linear decrease with the increase of
the methanol content (Figure 8B). This indicates that
methanol significantly affects the hydrogen bond
between the analyte and the stationary phase, so meth-
anol should be avoided when selecting the mobile
phase. In addition, the retention time decreases as the
methanol content increases, because the polarity of the
methanol is greater than that of ACN, increasing the
strength of the elution, and therefore, the retention is
reduced (Figure 8A).
4 | CONCLUSION
In this study, a type of novel CIL stationary phase based
on chiral imidazole was designed. The CSPs showed effi-
cient enantioseparation effect on a number of chiral acids.
Evaluation of chromatographic data revealed that even
fine changes in the CS structure and mobile phase proper-
ties can lead to large differences in retention and
enantioselectivity of the applied systems. The
imidazolium cation site in IL CS played a key role in the
process of chiral separation, and its ion exchange charac-
teristic was evaluated. The chromatographic evaluation
also proved that other interactions such as steric hin-
drance, π‐π interaction, and hydrogen bonding are essen-
tial in the process of chiral separation besides ion
exchange interaction. In addition, based on the observa-
tions of the preliminary HPLC runs, the best suitable
mobile phase for this type of CSPs was polar organic
mode, which can provided much better peak resolutions
for the enantiomers.
3.6 | Influence of protic and aprotic
solvent components
It is well known that methanol and ACN are the preferred
organic solvents for nonpolar organic models. Methanol is
a proton solvent that affects the hydrogen bond between
the analyte and the stationary phase, while ACN is an
aprotic solvent which has a π‐π shielding effect. There-
fore, it is very meaningful to examine the effect of organic
solvents on the behavior of such stationary phase
chromatography.
For this purpose, the proportions of ACN and MeOH
in the mobile phase were varied (10/0, 9/1, 8/2, 7/3, 6/4,
and 0/10, ACN/MeOH) under otherwise identical condi-
tions. From Figure 8, we can conclude that the selectiv-
ity of the analyte is greatly reduced along with the
ACKNOWLEDGMENT
This work was supported by the NSF of China (grant no.
21375038).

HE ET AL.
9
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How to cite this article: He S, He Y, Cheng L,
Wu Y, Ke Y. Novel chiral ionic liquids stationary
phases for the enantiomer separation of chiral acid
by high‐performance liquid chromatography.
Chirality. 2018;1–10. https://doi.org/10.1002/
chir.22839
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