Talanta 220 (2020) 121419

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Study on improvement of chiral separation of capillary electrophoresis

based on cyclodextrin by deep eutectic solvents
Suya Deng, Jingmiao Pan, Min Wang, Yike Huang, Zhining Xia *
School of Pharmaceutical Sciences, Chongqing University, Chongqing, 401331, China

A R T I C L E I N F O A B S T R A C T

Keywords: For the first time, the mechanism of deep eutectic solvents (DESs) improving chiral separation by capillary
Capillary electrophoresis electrophoresis has been studied. The capillary electrophoresis chiral separation has been improved by using
Chiral separation DESs as the auxiliary additive. Taking tropicamide, homatropine hydrochloride, ofloxacin, atenolol and pro­
Cyclodextrin
pranolol hydrochloride as model chiral separation targets and cyclodextrin (CD) as the chiral selector, and the
Deep eutectic solvents
Coordination effect
effects of DESs on the chiral separation resolution were investigated on the basis of optimized conditions. The
results of fluorescence spectrophotometry and non-aqueous capillary electrophoresis showed that DESs can
improve the resolution of the enantiomers, and the coordination mechanism of DESs was also explored. After
DESs were added, the resolution of the above enantiomers increased from 1.26, 1.70, 5.10, 1.90, 2.02 to 3.02,
4.10, 6.86, 2.84 and 5.51 respectively, and the binding constant of CD with propranolol hydrochloride increased
from 23 M 1 to 142 M 1. The results showed that DESs is an effective auxiliary additive to improve the sepa­
ration efficiency of capillary electrophoresis with CD as a basic chiral additive.

1. Introduction
Chiral drugs often have similar physical and chemical properties, but
they also have different activities in pharmacology and toxicology. For
example, one enantiomer is valid, and the other is ineffective or even has
toxic side effects [1]. Therefore, it is of considerable significance to
establish some efficient methods for the separation analysis of chiral
enantiomers. At present, the most commonly used methods for separa­
tion and analysis of enantiomers are high performance liquid chroma­
tography (HPLC), gas chromatography (GC), capillary electrophoresis
(CE), supercritical fluid chromatography (SFC), etc [2]. CE as a chiral
separation analysis method, has many advantages such as short analysis
time, high separation efficiency, and low cost, and it plays an increas­
ingly important role in chiral separation [3]. CD is one of the most
commonly used and effective chiral selectors for capillary electropho­
resis. The relatively hydrophobic structure inside the cavity and the
relatively hydrophilic outside the cavity make CD and chiral molecules
form inclusion complexes with different binding constant to achieve
chiral separation [4,5]. However, in some cases, the use of CD alone as a
chiral selector does not always provide satisfactory separation effects
[6]. The technology of adding another additive (such as an ionic liquid)
on the basis of CD has received widespread attention.
Ionic liquids (ILs) are a class of salts composed of organic cations and
inorganic/organic anions that are liquid at or near room temperature.
They have the characteristics of low vapor pressure, excellent solubility,
high electrical conductivity, and good thermal stability [7,8]. Some ILs
structures usually have a chiral center, also known as chiral ionic liquids
(CILs), which can be combined with CD through some force such as
hydrogen bonding, electrostatic force, dipole-dipole interaction, and π-π
interaction to produce a synergistic effect [9,10]. The adsorption of
cations of CILs on the capillary will inhibit electroosmotic flow (EOF),
which will prolong the migration time of the enantiomers, thus
increasing the enantioselectivity and improving the resolution [10–12].
However, due to the strong thermal and chemical stability of CILs, it is
difficult to degrade in the environment, and can only be recovered by
chemical degradation. Some studies have proved that CILs are toxic to

Abbreviations: DESs, deep eutectic solvents; CD, cyclodextrin; HPLC, high performance liquid chromatography; CE, GC; gas chromatography, capillary electro­
phoresis; SFC, supercritical fluid chromatography; ILs, ionic liquids; CILs, chiral ionic liquids; FT-IR, Fourier Transform Infrared Spectroscopy; 1H NMR, 1H Nuclear
Magnetic Resonance; EOF, electroosmotic flow; ChCl, choline chloride; LA, lactic acid; EG, ethylene glycol; PG, 1; 2-propylene glycol, G; glycerol, H3PO4; phosphoric
acid, NaH2PO4; sodium dihydrogen phosphate, Na2HPO4; disodium hydrogen phosphate, HP-β-CD; hydroxypropyl beta cyclodextrin, CM-β-CD carboxymethyl beta
cyclodextrin; β-CD, beta cyclodextrin; dimethyl sulfoxide, DMSO; B–H, benesi-hilebrand; PPL, propranolol.
* Corresponding author. School of Pharmaceutical Sciences, Chongqing University, Chongqing, 401331, PR China.
E-mail address: tcm_anal_cqu@163.com (Z. Xia).

https://doi.org/10.1016/j.talanta.2020.121419
Received 25 May 2020; Received in revised form 10 July 2020; Accepted 14 July 2020
Available online 20 July 2020
0039-9140/© 2020 Elsevier B.V. All rights reserved.
S. Deng et al.
cells [13–15]. The above disadvantages and high cost limit the appli­
cation of CILs in chiral separation. Therefore, more and more efficient
synergistic additives need to be explored for application on CD chiral
capillary electrophoresis.
DESs are eutectic mixtures composed of hydrogen bonding acceptors
and hydrogen bonding donors with a certain stoichiometric ratio.
Because their physical and chemical properties are very similar to ILs,
some people classified them as new ionic liquids [16–18]. Compared
with ILs, the source of DESs is more environmentally friendly and low
cost [19]. At present, there is a little literature that uses DESs as addi­
tives in chiral separation of capillary electrophoresis and proves that
DESs can improve the enantiomeric resolution [20].
In this work, DESs were applied as auxiliary additives for the chiral
separation of five enantiomers, and the separation conditions of capil­
lary electrophoresis were optimized. To further verify the effect and
mechanism of DESs increasing the resolution, a fluorescence spectro­
photometer was used to determine the binding constants of enantiomers
and CDs (with or without DESs). To the best of our knowledge, there has
been little research on the principle of DESs as auxiliary additives to
improve the efficiency of chiral separation of capillary electrophoresis.
In addition, we also used DESs for chiral separation by nonaqueous
capillary electrophoresis. In order to explore the effect of changes in
ionic strength on the increase in resolution, we compared the separation
effects of different additives with the same ionic strength. By studying
the mechanism of DESs improving the chiral separation effect of capil­
lary electrophoresis, the basis for expanding the application of DESs in
capillary electrophoresis was provided.
2. Experimental
2.1. Instrumentation
Rotary evaporator from RV10 (IKA, Germany). The Fourier Trans­
form Infrared Spectroscopy (FT-IR) characterizations of DESs were
carried out on an IR-Affinity-1 Fourier transform infrared spectrometer
(Shimadzu, Japan). The 1H Nuclear Magnetic Resonance (1H NMR)
analysis of DESs was detected by Agilent DD2 400-MR (Agilent Tech­
nologies, USA). All the CE experiments were performed on an Agilent
7100 3D CE system (Agilent Technologies, Waldbronn, Germany)
equipped with a diode array detector and an Agilent ChemStation
software. The temperature of the sample tray was always maintained at
25 � C. The inner diameter of the uncoated fused silica capillary was 50
μm, the outer diameter was 365 μm, and the total length was 50 cm,
while the effective length was 41.5 cm (Yongnian Optical Fiber Factory,
Hebei, China). The new capillary was first rinsed with 0.1 M NaOH for
30 min for activation. Between experiments, the capillary was sequen­
tially washed with 0.1 M HCl, water, 0.1 M NaOH, and running buffer
for 3 min. Binding constant measured by RF-5301PC (Shimadzu, Japan).
Acidity of background buffer was adjusted by Delta 320 pH meter
(Mettler-Toledo Instruments, Shanghai, China).
2.2. Materials
The choline chloride (ChCl, 99%), urea, lactic acid (LA, �85.0%),
ethylene glycol (EG, �99.5%), 1, 2-propylene glycol (PG, �99%), and
glycerol (G, �99%) were purchased from Solarbio Science & Technology
Co., Ltd. (Beijing, China). DESs were prepared by our laboratory, and the
molar ratios of various DESs hydrogen bond acceptors and hydrogen
bond donors are ChCl/urea ¼ 1/2, ChCl/LA ¼ 1/2, ChCl/EG ¼ 1/2,
ChCl/PG ¼ 1/2, ChCl/G ¼ 1/2. Phosphoric acid (H3PO4, �99%), sodium
dihydrogen phosphate (NaH2PO4, �99%) and disodium hydrogen
phosphate (Na2HPO4, �98%) were all provided by Aladdin (Shanghai,
China). Hydroxypropyl beta cyclodextrin (HP-β-CD, �97%), carbox­
ymethyl beta cyclodextrin (CM-β-CD, �98%), beta cyclodextrin (β-CD,
�98%) were obtained from Shandong Binzhou Zhiyuan Biological
Technology Co., Ltd. (Shandong, China). Tropicamide (�99%),
2
Talanta 220 (2020) 121419
homatropine hydrochloride (�98%), ofloxacin (�98%), atenolol
(�98%) and propranolol hydrochloride (�98%) were supplied by
Chengdu Kelong Chemical Co., Ltd. (Chengdu, China). Deionized water
was obtained from A.S. Watson Group (Hong Kong) Ltd. (Hong Kong,
China). Dimethyl sulfoxide (DMSO) was used as a neutral marker to
determine the EOF, which was purchased from Sigma-Aldrich
(Shanghai) Trading Co., Ltd. (Shanghai, China).
2.3. Preparation and structural characterization of DESs
Accurately weighed the ChCl and different types of hydrogen bond
donors with the molar ratio of 1:2, mixed in a 50 mL round bottom flask,
and heated in a rotary evaporator at 80 � C water bath for 2 h until a clear
and transparent liquid was obtained. In order to verify whether the DESs
prepared in our laboratory were formed, this study carried out Fourier
Transform Infrared Spectroscopy (FT-IR) analysis and 1H Nuclear
Magnetic Resonance (1H NMR) analysis on five DESs and individual
components, and determined the density and pH of various DESs.
2.4. Enantiomeric separation of multiple classes of chiral enantiomers
We performed separation conditions for topiramates such as tropi­
camide and homatropine hydrochloride [21,22], fluoroquinolones such
as ofloxacin [23,24], beta receptor inhibitors such as atenolol and pro­
pranolol hydrochloride [25–27]. The separation conditions (CD species,
CD concentration, buffer pH, buffer ionic strength, DESs type, DESs
concentration) of these enantiomers were optimized and the comparison
was made with or without DESs as an additive.
2.5. Determination of the binding constant of propranolol hydrochloride
and CD in the presence or absence of DES by fluorescence
spectrophotometry
In order to further prove that DESs can improve the efficiency of
chiral separation in capillary electrophoresis, the fluorescence in­
tensities of propranolol hydrochloride and CD with or without DESs as
additive were measured by fluorescence spectrophotometer, and the
binding constants of propranolol hydrochloride and CD were calculated
and compared by the double reciprocal method of Benesi-Hilebrand
(B–H) [28,29].
2.6. Non-aqueous capillary electrophoresis for the separation of
tropicamide enantiomers in the presence of DESs
To demonstrate the applicability of DESs as an additive for chiral
separations, we separated the tropicamide enantiomers in 20 mM
ammonium acetate-methanol buffer containing 20 mM HP-β-CD [30].
2.7. Preliminary exploration of the reasons for the increased resolution of
DESs
DESs were replaced by choline chloride (ChCl) with the same ionic
strength to be an additive for the enantiomeric separation of topiramate,
homatropine hydrochloride and ofloxacin, and respective resolutions of
them were compared. It was investigated whether the improved reso­
lution of DESs originated from the increase of ionic strength.
3. Results and discussion
3.1. DESs characterization results and discussion
3.1.1. Fourier transform infrared spectroscopy characterization of DESs
Fig. S1 was the FT-IR spectrum of ChCl, urea and the formed DES
(ChCl-urea). It could be seen from the figure that no new chemical bond
was formed. The urea’s (νNH2) characteristic peak was red-shifted from
1668 cm 1 to 1654 cm 1, and the characteristic peak (δNH2) was blue-
 S. Deng et al.
shifted from 1624 cm 1 to 1639 cm 1. In the formed DESs, the char­
acteristic peak of ChCl (δCH3) blue-shifted from 1479 cm 1 to 1483
cm 1, and the absorption peak of ChCl (δOH) blue-shifted from 950
cm 1 to 956 cm 1. The hydroxy stretching vibration absorption peak
range of the solvent (3400-3440 cm 1) was obviously widened. These
results indicated that there was a strong hydrogen bonding effect in
ChCl-urea. Fig. S2 was the FT-IR spectrum of ChCl, LA and DES (ChCl-
LA). From the figure, it could be discoverd that 1735 cm 1 was the C–
–O
1
absorption peak of LA, and blue-shifted to 1737 cm in the formed
ChCl-LA. ChCl’s characteristic peak (δCH3) was red-shifted from 1479
cm 1 to 1477 cm 1, the absorption peak of ChCl (δOH) was blue-shifted
from 950 cm 1 to 954 cm 1; the hydroxy stretching vibration range of
the solvent was changed from 3400 to 3440 cm 1 to 3348-3537 cm 1,
the above results explained that ChCl-LA was formed by hydrogen
bonding. The FT-IR diagram of ChCl, EG and the formed DES (ChCl-EG)
was shown in Fig. S3, where 1041 cm 1 and 1083 cm 1 were the EG’s
CH2 in-plane rocking vibration (ρCH2) and C–OH stretching vibration
(νC-OH), the characteristic peak did not change in the formed DES.
1641 cm 1 was the CH2 bending vibration (δCH2) of EG, in DES, the
characteristic peak red-shifted to 1639 cm 12953 cm 1 and 2885 cm 1
were EG’s CH2 stretching vibration (νCH2); ChCl’s characteristic peak
(δCH3) was red-shifted from 1479 cm 1 to 1477 cm 1, and the ab­
sorption peak of ChCl (δOH) was blue-shifted from 950 cm 1 to 966
cm 1. These datas proved that the formed ChCl-EG did not generate new
chemical bonds but was bonded by hydrogen bonding. Fig. S4 was the
FT-IR spectrum of ChCl-PG and its individual components. Among them,
993 cm 1 and 1045 cm 1 were the C–O symmetric stretching vibration
of the secondary alcohol (νsC-O-A) in PG, while in DES were blue-shifted
to 995 cm 1 and 1082 cm 1, respectively. 1134 cm 1 was the asym­
metric stretching vibration of the secondary alcohol C–O (νasC-O-A) of
PG, and red-shifted to 1132 cm 1 in DES, and 3471 cm 1 was the OH
stretching vibration of PG; the characteristic peak of ChCl (δCH3) blue-
shifted from 1479 cm 1 to 1487 cm 1, and the absorption peak (δOH)
blue-shifted from 950 cm 1 to 995 cm 1. The hydroxy stretching vi­
bration range of the solvent was from 3400 to 3440 cm 1 to 3404-3506
cm 1, the above phenomenons indicated that ChCl-PG was associated
through hydrogen bonding. Fig. S5 was the FT-IR diagram of ChCl-G and
its individual components. It would be observed from the figure that
989 cm 1 and 1043 cm 1 were the symmetric stretching vibration of the
primary alcohol C–O of G (νsC-O-1-A, νsC-O-2-A), and moved to 962
cm 1 and 1045 cm 1 in DES respectively, 1107 cm 1 was the asym­
metric stretching vibration of secondary alcohol C–O of G (νasC-O-A),
red-shifted to 1101 cm 1 in DES. 1408 cm 1 was G’s CH2 bending vi­
bration (δCH2), in DES, blue-shifted to 1477 cm 12887 cm 1 and 2943
cm 1 were PG’s CH2 symmetric stretching vibrations (νsCH2) and CH2
asymmetrical stretching vibration (νasCH2), there were no changes in
DES. 3400 cm 1 was G’s OH stretching vibration (νasOH). ChCl’s char­
acteristic peak (δCH3) red-shifted from 1479 cm 1 to 1477 cm 1, and
the absorption peak of ChCl (δOH) blue-shifted from 950 cm 1 to 962
cm 1. The hydroxy stretching vibration range of the solvent changed
from 3400 to 3440 cm 1 to 3340-3466 cm 1, these results indicated that
ChCl-G was formed by hydrogen bond between ChCl and G.
3.1.2. 1H NMR characterization of DESs
The 1H NMR charts of the five DESs were shown in Figs. S6–S10. It
could be seen from the figures that the chemical shift values of the five
DESs have not changed compared with the individual components.
Taking ChCl-G as an example, Fig. S10 was the 1H NMR chart of ChCl, G,
and the formed DES (ChCl-G). From the figure, it could be observed that
the chemical shift values of ChCl were 3.87 ppm, 3.35 ppm, 3.01 ppm,
and the chemical shift values of G were 3.60 ppm, 3.47 ppm, and 3.01
ppm. After comparison, the chemical shift values of ChCl-G did not
change, which indicating that no proton rearrangement occurred in the
formed DES, and each component remained independent.
3
­
Talanta 220 (2020) 121419
3.1.3. Density and pH of DESs
This paper tested the density and pH of five DESs, and the results
were shown in Table 1. And after adding DESs to the running buffer, the
pH of the buffer did not change.
3.2. Chiral separation results of various enantiomers
The enantiomeric separation conditions were optimized which
included CD species, CD concentration, buffer pH, buffer ionic strength,
and types and concentrations of DESs by using the topiramate enantio­
mers tropicamide, homatropine hydrochloride, the fluoroquinolone
enantiomer ofloxacin, the beta receptor inhibitors atenolol, and pro­
pranolol hydrochloride as the separation targets. The separation of
various enantiomers in the presence or absence of DESs was also
compared.
According to the resolution and separation time, the best separation
conditions for tropicamide before DESs adding (Fig. S11): uncoated
fused silica capillary (50/41.5 cm, 50 μm), 40 mM NaH2PO4–H3PO4
buffer (pH ¼ 2.5) containing 10 mM HP-β-CD, UV detection wavelength
was 210 nm, the separation voltage was 20 kV, and the concentration of
tropicamide was 1 mM. The optimal separation conditions for homa­
tropine hydrochloride without DESs are as follows (Fig. S12): uncoated
fused silica capillary (50/41.5 cm, 50 μm), 40 mM NaH2PO4–H3PO4
buffer (pH ¼ 3) containing 10 mM HP-β-CD, UV detection wavelength
was 210 nm, the separation voltage was 20 kV, and the concentration of
homatropine hydrochloride was 1 mM. For ofloxacin, the most satis­
fying separation conditions without DESs (Fig. S13): uncoated fused
silica capillary (50/41.5 cm, 50 μm), 30 mM NaH2PO4–H3PO4 buffer
(pH ¼ 4) containing 6.5 mM CM-β-CD, UV detection wavelength was
294. Nm, separation voltage was 20 kV, and ofloxacin concentration was
1 mM. The best separation conditions for atenolol without DESs
(Fig. S14): uncoated fused silica capillary (50/41.5 cm, 50 μm), 30 mM
NaH2PO4–H3PO4 buffer (pH ¼ 4) containing 6.5 mM CM-β-CD, UV
detection wavelength was 235 nm, separation voltage was 20 kV,
atenolol concentration is 2 mM. For propranolol hydrochloride, the most
gratifying conditions without DESs (Fig. S15) are 30 mM NaH2
PO4–H3PO4 buffer (pH ¼ 4) solution containing 6.5 mM CM-β-CD, UV
detection wavelength was 230 nm. The separation voltage was 20 kV,
and the concentration of propranolol hydrochloride was 0.5 mM.
Based on the obtained optimal conditions, we examined the types
and concentrations of DESs, and the results are shown in Fig. 1.
Considering the resolution and separation time, the optimal DESs’
concentrations and types of tropicamide, homatropine hydrochloride,
ofloxacin, atenolol, propranolol hydrochloride were 1.5% (v/v) ChCL-
EG, 1.5% (v/v) ChCL-EG, 1% (v/v) ChCL-G, 1% (v/v) ChCL-urea,
1.5% (v/v) ChCL-LA respectively. The resolutions of various enantio­
mers were shown in Table 2. It could be seen from Table 2 That after
adding DESs, the resolution of five enantiomers has been improved,
among them tropicamide achieved baseline separation, which proved
that DESs can apply on varied enantiomers separation. It was caused by
increasing of CD packetization by DESs [31]. Physical and chemical
properties of DESs are similar to ILs including the adsorption of ILs
cations on the capillary wall to shield part of EOF [32,33]. This would
make the migration time of the enantiomer obviously increased after
adding DESs, which is beneficial for the separation of the enantiomers. It
can be seen that as the concentration of DESs increased, the resolution of
Table 1
The density and pH of five DESs.
Analytes Density (g/mL) pH
ChCl-urea 1.162 10.16
ChCl-LA 1.071 4.36
ChCl-EG 1.104 6.94
ChCl-PG 1.064 6.22
ChCl-G 1.135 6.37
S. Deng et al. Talanta 220 (2020) 121419

Fig. 1. The effect of the types and concentrations of DESs

on the enantiomer separation of tropicamide, homatropine
hydrochloride, ofloxacin, atenolol, and propranolol hydro­
chloride. (A) and (B) are electropherograms of tropicamide
when the type and concentration of DESs are changed. (C)
and (D) are the electrophoresis images of homatropine hy­
drochloride when the type and concentration of DESs are
changed. (E) and (F) are the electrophoresis pictures of
ofloxacin when the type and concentration of DESs are
changed. (G) and (H) are electropherograms of atenolol
when the type and concentration of DESs are changed. (I)
and (J) are electropherograms of propranolol hydrochloride
when the type and concentration of DESs are changed,
respectively.

4
S. Deng et al. Talanta 220 (2020) 121419

Table 2
Resolution results f tropicamide, homatropine hydrochloride, ofloxacin, ateno­
PPL þ CD ↔ PPL CD
lol, propranolol hydrochloride with or without DESs. Then the binding constant was:
Analytes T 1 (min) T 2 (min) R
½PPL CD�
Tropicamide (1 mM) No DESs 10.472 11.347 1.26 K¼
½PPL�½CD�
1.5% ChCl- 26.269 32.982 3.02
EG
Homatropine hydrochloride (1 No DESs 8.982 9.415 1.70
where [PPL], [CD], and [PPL-CD] represented the equilibrium concen­
mM) 1.5% ChCl- 17.899 19.703 4.10 trations of the guest propranolol (PPL), the host CD, and the inclusion
EG complex PPL-CD, respectively. According to the double reciprocal
Ofloxacin (1 mM) No DESs 11.784 15.184 5.10 method of Benesi-Hilebrand (BH), get the following equation:
1% ChCl-G 17.587 25.825 6.86
Atenolol (2 mM) No DESs 15.877 17.737 1.90 1 1 1
1% ChCl- 21.503 25.050 2.84 ¼ þ
F F0 ðF∞ F0 Þ⋅KCCD F∞ F0
urea
Propranolol hydrochloride (0.5 No DESs 13.511 14.701 2.02
mM) 1.5% ChCl- 26.859 30.842 5.51
where F was the maximum fluorescence intensity of the guest at a
LA certain wavelength at different CD concentrations, F0 and F∞ were the
fluorescence intensity when no CD was added and all the guests were
enclosed by the CD cavity. By plotting 1/(F–F0) on 1/CCD, a linear
each enantiomer grew, but at the same time, the augment in the con­ relationship straight line can be obtained [39,40]. From the ratio of the
centration of DESs also increased the viscosity of the buffer, which intercept and the slope of the straight line, the binding constant of the
would increase the current and eventually cause a lot of Joule heat CM-β-CD-propranolol inclusion compound can be obtained.
during the electrophoresis process, so the concentration of DESs added By substituting the obtained fluorescence data into the equation, K0
should be controlled not be too high. ¼ 23 M 1 without DESs, and KDES ¼ 142 M 1 with 5% ChCl-LA. The
binding constant increased significantly, which indicated that the in­
3.3. Binding constants of propranolol hydrochloride and CM-β-CD clusion of CD was improved after the addition of DESs. More and more
PPL was squeezed into the cavity of the CD, so that CD and PPL could
It had been reported that the separation mechanism of CD as the form bigger inclusion complexes, and the free PPL decreased, which
chiral selector was that the CD cavity can combine the chiral molecule to made the suppression of fluorescence intensity more sufficient. The
form inclusion compounds with different binding constants [34]. So as decrease of the fluorescence intensity and the increase of the binding
to investigate the mechanism of increasing the enantiomeric separation constant proved that there was a squeezing effect between the CD and
of DESs, fluorescence spectrophotometry was used to determine the the enantiomer after the DESs were added, so that more enantiomers
binding constants of propranolol hydrochloride and CM-β-CD in the entered the CD cavity, thereby increasing the resolution.
presence or absence of DESs. The reason why we chose propranolol as
the research object was that the tropicamide, homatropine hydrochlo­
ride and ofloxacin were not fluorescent in the chiral separation buffer. 3.4. Non-aqueous capillary electrophoresis for separation of the
The fluorescence conditions were EX ¼ 298 nm, EM ¼ 340 nm; slits were enantiomers of tropicamide
3 nm; scanning speed was fast; buffer solution was 30 mM pH ¼ 3.07
NaH2PO4–H3PO4 buffer; DES was ChCl-LA. Fig. 2A shows the fluores­ For further demonstrating the mechanism of DESs for enhancing the
cence spectra of propranolol hydrochloride enantiomer and CM-β-CD in chiral separation, the non-aqueous capillary electrophoresis was used to
the absence of DESs, and Fig. 2B is the fluorescence spectrum of pro­ separate the enantiomers of tropicamide. As shown in Fig. 3, under non-
pranolol hydrochloride enantiomer and CM-β-CD when 5% ChCl-LA was aqueous conditions, the addition of 0.1% ChCl-EG increased the reso­
added. It can be seen from Fig. 2 that CM-β-CD could inhibit the fluo­ lution of tropicamide from 0.9 to 1.8, achieving baseline separation,
rescence intensity of propranolol hydrochloride, and the degree of in­ which proved that the enantiomeric resolution of DESs increases. This
hibition of the fluorescence intensity was even greater when DESs were phenomenon claimed that DESs could still increase the resolution of
added. In the inclusion complex formed, the stoichiometric ratio of host enantiomers under non-aqueous conditions.
and guest is 1: 1 [35–38], and the process of forming clathrates was: It could also be seen from Fig. 3 that the amount of DESs added to the
non-aqueous buffer solution was much smaller than that of the aqueous

Fig. 2. Fluorescence spectrum of propranolol hydrochloride and CM-β-CD inclusion complex. (A) The fluorescence spectra of propranolol hydrochloride and CM-
β-CD inclusion complex without DESs (1 → 6 indicated that the concentration of CM-β-CD was 0, 1.625, 3.25, 6.5, 13, and 26 mM). (B) The fluorescence spectra of
propranolol hydrochloride and CM-β-CD inclusion complex when 5% ChCl-LA was added (1 → 6 indicated that the concentration of CM-β-CD was 0, 1.625, 3.25, 6.5,
13, and 26 mM).

5
S. Deng et al. Talanta 220 (2020) 121419

of the second peak.

3.5. Preliminary exploration of the reasons for the increased resolution of

DESs

In addition to reducing EOF, increasing the CD’s packaging and

Fig. 3. Non-aqueous capillary electrophoresis for separation of tropicamide.
Electrophoresis conditions: uncoated fused silica capillary (50/41.5 cm, 50 μm),
25 � C, 20 mM ammonium acetate- Methanol containing 20 mM HP-β-CD, UV
detection at 210 nm, applied voltage was 20 kV, DES was ChCl-EG.
squeezing effect, were there other reasons why DESs increase the reso­
lution of enantiomers? For example, changes in ionic strength and po­
larity? In order to further explore the reason why DESs improve the
enantiomeric resolution, choline chloride (ChCl) with the same ionic
strength was used as an additive instead of DESs to be added to the
buffer, and examined the enantiomeric separation of tropicamide and
homatropine hydrochloride. The results were shown in Fig. 4 The action
sites of tropicamide and homatropine hydrochloride were both choline
receptors, in order to rule out the effect of this factor, the effect of flu­
oroquinolone ofloxacin was also examined with ChCl replacing DESs.
The results were shown in Fig. 5. The resolution data of each enantiomer
were shown in Table 3. From Fig. 5, after replacing DESs with ChCl of
the same ionic strength, the increasing tendency of the resolution of
fluoroquinolone enantiomers was similar to that of choline receptor
enantiomers, so the interaction between ChCl and enantiomers can be

buffer solution, and very few DESs could double the resolution of chiral
separation. This phenomenon showed that the addition of DESs had a
more obvious effect on the non-aqueous buffer, and the Joule heat
generated by the lower current of the non-aqueous buffer during elec­
trophoresis could be negligible, so it was more beneficial to study the
mechanism of DESs to increase the resolution of the enantiomers in
capillary electrophoresis under non-aqueous conditions.
In addition, as the concentration of ChCl-EG increased, the difference
between the two enantiomeric peak heights become larger, indicating
that the absorption spectrum has changed after the addition of DESs.
This phenomenon showed that DES has a squeeze-like effect on the
combination of CD and enantiomers. It could also be summarized from
Fig. 3 that increasing the concentration of ChCl-EG would prolong the
migration time of the two enantiomers and increase the asymmetry of
the second enantiomer peak. There may be two reasons for this phe­
nomenon: (1) As the concentration of ChCl-EG increased, the viscosity of
the running buffer would become larger, making the migration time
longer. As a new type of ILs, DESs had the relevant properties of ILs, so
Fig. 5. Electrophoresis of ofloxacin with ChCl replacing DESs. Electrophoresis
they could also suppress EOF and prolonged the migration time. (2) As
conditions: uncoated fused silica capillary (50/41.5 cm, 50 μm), 25 � C, 30 mM
the concentration of ChCl-EG increased, the squeezing effect of DESs on NaH2PO4–H3PO4 (pH ¼ 4) containing 6.5 mM CM-β-CD, UV detection at 294
CD and enantiomers was more obvious, causing the asymmetric tailing nm, applied voltage was 20 kV, the concentration of ofloxacin was 0.5 mM.

Fig. 4. Electrophoretic graphs of tropicamide and homatropine hydrochloride after ChCl replacing DESs. (A) The electrophoresis picture of tropicamide. Electro­
phoresis conditions: uncoated fused silica capillary (50/41.5 cm, 50 μm), 25 � C, 40 mM NaH2PO4–H3PO4 (pH ¼ 2.5) containing 10 mM HP-β-CD, UV detection at
210 nm, applied voltage was 20 kV, the concentration of tropicamide was 1 mM. (B) The electrophoresis image of homatropine hydrochloride. Electrophoresis
conditions: uncoated fused silica capillary (50/41.5 cm, 50 μm), 25 � C, 40 mM NaH2PO4–H3PO4 (pH ¼ 3) containing 10 mM HP-β-CD, UV detection at 210 nm,
applied voltage was 20 kV, the concentration of homatropine hydrochloride was 1 mM.

6
S. Deng et al. Talanta 220 (2020) 121419

Table 3 Appendix A. Supplementary data

Results of tropicamide, homatropine hydrochloride and Ofloxacin with ChCl
replacing DESs. Supplementary data to this article can be found online at https://doi.
Analytes T 1 (min) T 2 (min) R org/10.1016/j.talanta.2020.121419.
Tropicamide (1 mM) No DESs 10.472 11.347 1.26
EG 11.036 11.994 1.19 References
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