Preparation of a novel hydroxypropyl-γ-cyclodextrin functionalized monolith for separation of chiral drugs in capillary electrochromatography

Article abstract of DOI:10.1002/chir.23300

In this study, a novel hydroxypropyl-γ-cyclodextrin (HP-γ-CD) functionalized monolithic capillary column was prepared by one-pot sequential strategy and used for chiral separation in capillary electrochromatography for the first time. In one pot, GMA-HP-γ-CD as functional monomer was allowed to be formed via the ring opening reaction between HP-γ-CD and glycidyl methacrylate (GMA) catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and then copolymerized directly with ethylene dimethacrylate (EDMA) and 2-acrylamido-2-methyl propane sulfonic acid (AMPS) in the presence of porogenic solvents via thermally initiated free radical polymerization. The preparation conditions of monoliths were optimized. Enantiomer separations of six chiral drugs including pindolol, clorprenaline, tulobuterol, clenbuterol, propranolol, and tropicamide were achieved on the monolith. Among them, pindolol, clorprenaline, and tropicamide were baseline separated with resolution values of 1.62, 1.73, and 1.55, respectively. The mechanism of enantiomer separation was discussed by comparison of the HP-γ-CD and HP-β-CD functionalized monoliths.

Full text of DOI:10.1002/chir.23300

Received: 1 July 2020
Revised: 2 December 2020
Accepted: 19 January 2021
DOI: 10.1002/chir.23300
S H O R T C O M M U N I C A T I O N
Preparation of a novel hydroxypropyl-γ-cyclodextrin
functionalized monolith for separation of chiral drugs in
capillary electrochromatography
Miaoduo Deng | Mengyao Xue | Yanru Liu | Min Zhao
College of Pharmacy, Shenyang
Abstract
Pharmaceutical University, Shenyang,
China
In this study, a novel hydroxypropyl-γ-cyclodextrin (HP-γ-CD) functionalized
monolithic capillary column was prepared by one-pot sequential strategy and
used for chiral separation in capillary electrochromatography for the first time.
In one pot, GMA-HP-γ-CD as functional monomer was allowed to be formed
via the ring opening reaction between HP-γ-CD and glycidyl methacrylate
(GMA) catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and then cop-
olymerized directly with ethylene dimethacrylate (EDMA) and 2-acrylamido-
2-methyl propane sulfonic acid (AMPS) in the presence of porogenic solvents
via thermally initiated free radical polymerization. The preparation conditions
of monoliths were optimized. Enantiomer separations of six chiral drugs
including pindolol, clorprenaline, tulobuterol, clenbuterol, propranolol, and
tropicamide were achieved on the monolith. Among them, pindolol,
clorprenaline, and tropicamide were baseline separated with resolution values
of 1.62, 1.73, and 1.55, respectively. The mechanism of enantiomer separation
was discussed by comparison of the HP-γ-CD and HP-β-CD functionalized
monoliths.
Correspondence
Min Zhao, College of Pharmacy,
Shenyang Pharmaceutical University,
No. 103, Wenhua Road, Shenyang, China.
Email: zm19871224@sina.com
K E Y W O R D S
chiral drugs, enantiomer separation, hydroxypropyl-γ-cyclodextrin, monolith, one-pot strategy
1 | INTRODUCTION
the best features of capillary electrophoresis and high-
performance liquid chromatography (HPLC). The core of
The separation of enantiomers is an important topic dur-
ing the past three decades. It is well known that one
enantiomer of a racemic drug may exhibit pharmacologi-
cal activities and/or side effects different from the other
one1; therefore, it is important to develop an analytical
method for enantiomer separation of chiral drugs with
different chemical structures. So far, chromatographic
approaches as efficient tools for enantiomer separation
have been widely utilized.
CEC is the development of column technology which
comprises packed, monolithic, and open-tubular col-
umns. Among the three types of columns, monolithic col-
umns are slowly attracting more and more attention. In
the case of chiral separation, much efforts have been
devoted to design and fabricate novel monolithic station-
ary phase.2–4
Over the past few decades, monolithic separation
media have emerged as an advantageous alternative to
the conventional column packing material in chromatog-
raphy and electrochromatography. The widespread
Capillary electrochromatography (CEC) is an electro-
migration separation technique which strives to combine
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Chirality. 2021;33:188–195.

DENG ET AL.
189
acceptance of monolithic separation media has been
facilitated by the ease of preparation, favorable mass
transfer properties, low back pressure, and high separa-
tion efficiency. Moreover, the availability of diverse kinds
of functional monomers or reactive precursors from a
rich chemistry bank and a wide range of porosity control
during the preparation process provide solutions for a
variety of separation problems.5–7 In the early stage, the
polymer monoliths are based on soft hydrophilic poly-
acrylamide gels.8–10 Svec's group pioneered the fabrica-
tion of the polymethacrylate-based monoliths as HPLC
separation media.11–13 In contrast to polyacrylamide gels,
the polymethacrylate-based monoliths as rigid polymers
are not compressible and do not change their size sub-
stantially on swelling. Due to the significant advantage,
the rigid polymer monoliths have gained continuously in
popularity.14
Chiral functionalities on the surface of monolith are
the prerequisite for enantiomer separations.15 Cyclodex-
trins (CDs) are a family of cyclic oligosaccharides, which
possess the truncated cone-shaped structure with rela-
tively hydrophilic exteriors and hydrophobic internal cav-
ity. The unique structure enables their ability to
selectively interact with chiral compounds, and then,
they can be widely applied in enantiomer separation.16–18
In the early stages of chiral separation technologies, a
number of CDs as chiral mobile phase additives were
successfully utilized in CEC. However, CDs in the mobile
phase result in relatively low sensitivity with a high
consumption of chemicals. Since the discovery of the
advantageous property of monoliths, attempts have
been made to explore β-CD-based monolithic stationary
phase for enantiomer separation.19–26 With the promising
development, new CD derivatives functionalized mono-
liths need to be prepared in order to achieve altered
enantioselectivities and further broaden the application
range. Compared with β-CD and its derivatives,
hydroxypropyl-γ-CD (HP-γ-CD) provides different
enantioselectivities and may serve as a good candidate
for enantiomer separation of multiring compounds with
rigid structure due to its larger hydrophobic cavity
size.27–29 However, to the best of our knowledge, there is
no report on preparing HP-γ-CD functionalized station-
ary phase.
monolith. The newly developed monolithic column
showed enantioselectivity toward six chiral compounds
including pindolol, clorprenaline, tulobuterol, clenbuterol,
propranolol, and tropicamide. The mechanism of
enantiomer separation was discussed by comparison of
the HP-γ-CD and HP-β-CD functionalized monolith.
2 | EXPERIMENTAL PART
Ammonium acetate (NH4OAc) was obtained from Tian-
Jin Bodi Chemical Holding (Tianjin, China). HP-γ-CD
and n-hexanol were purchased from Energy Chemical
(Shanghai, China). Ethylene dimethacrylate (EDMA),
GMA, γ-methacryloxy propyltrimethoxysilane (γ-MAPS),
2,20-azobis(2-methylpropionitrile)
(AIBN),
and
2-acrylamido-2-methyl propane sulfonic acid (AMPS)
were purchased from Tokyo Chemical Industry (Tokyo,
Japan). Cyclohexanol was obtained from Acros (Geel,
Belgium). 1-Dodecanol was obtained from China
National Pharmaceutical Group Co., Ltd. (Beijing,
China).
n-Butanol
(NBA),
sodium
hydroxide,
hydrochloric acid, acetone, toluene, and diethyl ether of
analytical grade were purchased from Shandong Yuwang
Industrial Co., Ltd (Shandong, China). Acetonitrile
(ACN), glacial acetic acid, and triethylamine (TEA) of
HPLC grade were purchased from Concord Technology
Co., Ltd (Tianjin, China). Anhydrous dimethyl sulfoxide
(DMSO) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
were obtained from Aladdin Bio-Chem Technology Co.,
Ltd (Shanghai, China). All chiral analytes were pur-
chased from National Institutes for Food and Drug Con-
trol. The chemical structures of all analytes are shown in
Figure 3. Doubly distilled water was used throughout all
experiments. All solutions were filtered with a 0.22-μm
syringe filter.
All CEC experiments were performed on an HPCE
apparatus (CL1030, Beijing Huayanglimin Instrument
Co., Ltd, Beijing, China) equipped with a UV detector, a
high-voltage power supply, and a HW-2000 chromatogra-
phy workstation. The polyimide-coated fused silica capil-
laries (375 μm o.d. × 75 μm i.d.) were obtained from
Ruifeng Chromatography Ltd. (Yongnian, Hebei, China).
A syringe pump (SPLab04, Shenchen Precision Pump
Co., Ltd, Baoding, China) was used to introduce mono-
mer solutions into the previnylized capillaries for poly-
merization. An HPLC pump (PU-1580, Jasco
Corporation, Japan) was used to flush and condition the
monolithic capillary. Optical microscopic images of
monolithic columns were taken with an Olympus micro-
scope (BX51, Olympus, Germany). Scanning electron
microscopy (SEM) images of monoliths were obtained by
using a Hitachi S4800 scanning electron microscope
In this work, a novel HP-γ-CD functionalized mono-
lith was developed and tested in CEC chiral separation
for the first time. A simple and efficient one-pot sequen-
tial strategy previously reported by our group24,25
was used for preparation of the monolith. In one pot,
HP-γ-CD functional monomer was allowed to be formed
via the ring opening reaction of HP-γ-CD with glycidyl
methacrylate (GMA) and then directly copolymerized
with comonomer and crosslinker to form the final

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DENG ET AL.
(Hitachi, Ltd., Japan). Fourier transform infrared (FT-IR)
spectra of the monolithic columns were recorded using a
Bruker EQUINOX55 FT-IR spectrophotometer (Brucker
Corporation, Germany). Nuclear magnetic resonance
exhaustively with ACN by an HPLC pump to remove
unreacted chemicals and porogens.
(NMR) spectroscopy was obtained with
a
Bruker
3 | RESULTS AND DISCUSSION
Ultrashield Plus 600-MHz spectrometer (Bruker Corpora-
tion, Germany). The specific surface area was determined
on a Micromeritics ASAP 2460 apparatus (Micromeritics
Instrument Ltd, USA).
As illustrated in Figure 1, the scheme for the one-pot
preparation of poly-(GMA-HP-γ-CD-co-EDMA) mono-
lithic column involves two sequential reactions: (1)
formation of functional monomer GMA-HP-γ-CD via the
DBU-catalyzed ring opening reaction between GMA and
HP-γ-CD and (2) free radical polymerization of functional
monomer and crosslinker for the formation of poly-
(GMA-HP-γ-CD-co-EDMA) monolith (Figure 2). In order
to obtain the proper monolithic column for chiral separa-
tion, the chemical structure of the product of ring opening
reaction was confirmed by NMR spectroscopy. After that,
the type and composition of porogenic solvents, ratio of
functional monomer to crosslinker, and polymerization
temperature were systematically investigated.
The mobile phases were a mixture of ACN and water
containing 5 mmol L−1 NH4OAc and 0.4% TEA. The
desired pH adjustment was carried out with glacial
acetic acid. Electrokinetic injection was used for all
samples. The applied voltage was 5 kV, and the injection
time was 3 s. The UV detection wavelength was set at
214 nm for clorprenaline, homatropine methylbromide,
and tulobuterol; 243 nm for clenbuterol; 254 nm for
tropicamide and chlorpheniramine; 259 nm for pro-
caterol; 276 nm for salbutamol and terbutaline; 280 nm
for pindolol, propranolol, and tetrahydropalmatine; and
294 nm for ofloxacin.
The detailed preparation procedures of the HP-γ-CD
functionalized monolith are described. At first, polymeri-
zation mixture was prepared. HP-γ-CD (60 mg), GMA
(16 mg), DBU (6 mg), and anhydrous DMSO (0.15 g)
were weighed into a 3-ml vial sealed with a rubber
septum-lined screw cap. Then, the mixture was stirred at
100ꢀC for 30 min. After cooling to room temperature,
n-hexanol (0.25 g), EDMA (24 mg), AMPS (7.5 mg), and
AIBN (2.5 mg) were added into mixture. In order to
remove dissolved air, the resultant solution was sonicated
at around 15ꢀC for 15 min and purged with nitrogen for
5 min; then, the polymerization mixture was introduced
into the vinylized capillary with an effective length of
30 cm according the reported procedures.30 The capillary
was sealed at both ends with silicon stoppers and sub-
merged into a preadjusted water bath at 60ꢀC for 20 h.
Finally, the obtained monolithic column was washed
The novel functional monomer GMA-HP-γ-CD was
formed by the ring opening reaction between HP-γ-CD
and GMA catalyzed by DBU at a temperature of 100ꢀC
for 30 min; then, the products of ring opening reaction
were isolated from the reaction mixture for 1H NMR
characterization (detailed procedures are described in the
1
supplementary material). Figure S2 shows the H NMR
spectra of HP-γ-CD and GMA-HP-γ-CD. As expected,
new signals for GMA-HP-γ-CD are observed. The peaks
at 5.79 and 6.19 ppm can be assigned to the methylene
protons, which demonstrated that hydroxyl groups of
HP-γ-CD were successfully substituted with glycidyl
methacrylate groups.31 The average degree of substitution
(ADS) of GMA-HP-γ-CD was calculated to be 1.16 by
comparison of the corresponding signal areas of HP-γ-CD
signals and substituent signals.
For CD-based monoliths, DMSO was found to be a
suitable solvent which can offer good solubility for native
FIGURE 1 One-pot sequential strategy for the preparation of the HP-γ-CD functionalized monolith

DENG ET AL.
191
FIGURE 2 Two major reactions in one pot: (1) the ring opening reaction between HP-γ-CD and GMA catalyzed by DBU and (2) the
copolymerization reaction of GMA-HP-γ-CD, EDMA, and AMPS
CDs and their derivatives. However, when DMSO was
used as a single porogenic solvent, the obtained mono-
lithic matrix was in gel state with low permeability.
Therefore, a solvent mixture of DMSO and an alkanol as
binary porogens was attempted. After a lot of experi-
ments, DMSO/cyclohexanol, DMSO/1-dodecanol, and
DMSO/NBA were found not suitable to produce a mono-
lithic column because they cause the formation of semi-
transparent gels, whereas an opaque white monolith was
formed in the porogenic system of DMSO/n-hexanol.
Hence, DMSO and n-hexanol were finally screened out
as optimized binary porogenic solvents to prepare mono-
lith. The multicomponent porogens allow for fine tuning
of the monolith morphology by simply changing the
ratios of its constituents. Table S1 shows the effect of the
component ratio changes of porogenic solvents on mor-
phology and permeability of monoliths. In the case of
Column A1, the monolith bed was inhomogeneous with
gaps. Most likely 67.5% n-hexanol led to so loose mono-
lithic structure and then the monolith collapsed. On the
other hand, Column A3 with 57.5% n-hexanol was too
hard to pump through even though the homogeneous
morphology was observed in the microscopic image. Only
62.5% n-hexanol in the porogenic system provided the
monolith (Column A2) with good balance of permeability
and homogeneous morphology.
that when the weight percentage of EDMA was 30% or
36% (Columns B2 and B3), the monolithic columns were
impermeable to ACN. This is because the higher
crosslinker concentration favors higher crosslinking
level, resulting in a lower porosity. An EDMA proportion
of 24% (Column A2) provided a homogeneous monolithic
matrix with acceptable permeability. However, a further
decrease in the EDMA content caused many light and
shade stripes in the microscopic image of Column B1. It
may be explained by the low crosslinking and mechanical
strength resulted from lower EDMA content.
Temperatures for copolymerization during the prepa-
ration of the monolithic columns were also examined.
From Table S1, it can be observed that Column C1 was
inhomogeneous and poorly permeable. The reason may
be that the formed monolithic structures at relatively
lower temperature (50ꢀC) are unstable, prone to collapse
with pore clogging during the polymerization and/or sub-
sequent workup. When the temperature increased to
60ꢀC, Column A2 showed homogeneous morphology and
good permeability. Further increasing the copolymeriza-
tion temperature to 70ꢀC (Column C2), it was difficult to
pump through the column, which may be because the
increased temperature leads to a reduction in the average
pore diameter.
On the basis of these observations, a polymerization
mixture consisting of HP-γ-CD (60 mg), GMA (16 mg),
DBU (6 mg), anhydrous DMSO (0.15 g), n-hexanol
(0.25 g), EDMA (24 mg), AMPS (7.5 mg), and AIBN
(2.5 mg) was chosen and polymerized at 60ꢀC to
The content of crosslinker (EDMA) in polymerization
mixture was then investigated because it could affect the
polymer crosslinking and therefore the permeability,
homogeneity, and rigidity. From Table S1, it can be found

192
DENG ET AL.
obtain the homogeneous and permeable HP-γ-CD
functionalized monolithic column.
The spectrum of HP-γ-CD shows bands at 943 and
1083 cm−1. These bands also exist in the spectrum of
HP-γ-CD functionalized monolith but are absent in the
spectrum of blank monolith. The FT-IR spectra provide
the evidence of the presence of HP-γ-CD on the mono-
lithic material.
In CEC experiment, we attempted to separate 13 com-
pounds by use of the HP-γ-CD functionalized monolith.
Their chemical structures are shown in Figure 3. Of the
13 compounds, pindolol, propranolol, clorprenaline,
tulobuterol, clenbuterol, and tropicamide could be sepa-
rated. After the optimization of separation conditions,
baseline separation was achieved for pindolol,
clorprenaline, and tropicamide with Rs values of 1.62,
1.73, and 1.55, and partial separation of propranolol,
clenbuterol, and tulobuterol was obtained with Rs values
of 0.53, 0.84, and 0.53. Figure 4 shows the electro-
chromatograms of the six analytes and the corresponding
Rs values, selectivity factors (α), and column efficiencies
(n) at the optimum conditions.
The cross-section morphology of the prepared
HP-γ-CD functionalized monolithic column (Column
A2) was observed by SEM (SEM sample preparations are
described in the Supporting Information), and the
obtained SEM images are shown in Figure S1 with two
different magnifications. As shown in Figure S1a, a uni-
form HP-γ-CD functional monolith with large through
pores is observed and tightly anchored to the capillary
wall. In Figure S1b, it can be seen that many small pores
are contained in the monolithic bed which most signifi-
cantly contributes to the surface area. Furthermore, the
specific surface area of the resultant material was
43.3 m2 g−1 determined by nitrogen adsorption–desorption
experiments (sample preparations are described in the
Supporting Information).
The chemical groups of the monolith were confirmed
by FT-IR spectroscopy (FT-IR sample preparations are
described in the Supporting Information). Figure S3
shows the FT-IR spectra of HP-γ-CD, HP-γ-CD
functionalized monolith, and blank monolith. The FT-IR
spectrum of the HP-γ-CD functionalized monolith
(Figure S3b) combines features of the HP-γ-CD
(Figure S3c) and the blank monolith (Figure S3a) spectra.
Our group has previously reported the fabrication of
the HP-β-CD functionalized monolith by the same strat-
egy.25 Table S2 summarizes enantioseparation results of
chiral drugs on the HP-γ-CD and HP-β-CD functionalized
monoliths. Compared with HP-β-CD, HP-γ-CD composed
FIGURE 3 Chemical structures of (A) β-adrenergic receptor blockers, (B) anticholinergic agents, (C) β-adrenergic receptor agonist, and
(D) others

DENG ET AL.
193
FIGURE 4 Electrochromatograms of six analytes under the optimum conditions on the poly-(GMA-HP-γ-CD-co-EDMA) monolith.
Separation conditions: (B) ACN-H2O (40:60), 5 mmol L−1 NH4OAc, and 0.4% TEA, pH 5.0, +20 kV. (A,C,D,E) ACN-H2O (30:70),
5 mmol L−1 NH4OAc, and 0.4% TEA, pH 5.0, +20 kV. (F) ACN-H2O (20:80), 5 mmol L−1 NH4OAc, and 0.4% TEA, pH 5.0, +20 kV
of eight glucose residues symmetrically arranged in a
ring, which means HP-γ-CD has a larger hydrophobic
cavity and can accommodate large-sized molecules.
Therefore, it is our interest to use bicyclic and polycyclic
compounds as model compounds to explore the HP-γ-CD
monolith to determine if the HP-γ-CD monolith behaves
better than the HP-β-CD monolith for chiral separation
of large-sized compounds. In details, the model com-
pounds include pindolol, propranolol, homatropine
methylbromide, tropicamide, procaterol, ofloxacin, chlor-
pheniramine, and tetrahydropalmatine. Unfortunately,
the resolution of most of these compounds on the
HP-γ-CD monolith was lower than that on the HP-β-CD
monolith. The reason why the selectivity is reduced may
be the large cavity of HP-γ-CD has relatively high degree
of flexibility and it is difficult to form enantioselective
inclusion complex. It is worth mentioning that the
separation performance of pindolol and propranolol was
better than that on the HP-β-CD monolith. A comparison
of chemical structures of these analytes reveals that
only pindolol and propranolol contain large planar
π-conjugation systems. Molecules of rigid planar struc-
ture are assumed to possess a limited freedom of rotation
within hydrophobic cavities of HP-γ-CD, permitting
differences in affinity for the CD cavity between
enantiomers.
Besides size, other factors such as van der Waals
forces, dipole–dipole interaction, hydrogen bonding, and
hydrophobic interactions also govern the formation and
the strength of an inclusion complex. Therefore, some
substituted benzene ring compounds including terbuta-
line, clenbuterol, clorprenaline, salbutamol, and
tulobuterol were still tested on the HP-γ-CD monolith.
The results showed that the HP-γ-CD monolith had
enantiomer selectivity toward clorprenaline, clenbuterol,
and tulobuterol, but only the resolution of clenbuterol on
the HP-γ-CD monolith was higher than that on the
HP-β-CD monolith. According to Figure 3, the 3,4,5 posi-
tion of the benzene ring was substituted by chloride,
amino group, and chloride, respectively. The presence of
the three substituted groups may enlarge the planar
conjugated systems of the molecule. To some extent, the
substituted benzene ring of clenbuterol seems to resem-
ble naphthalene ring or indole ring. This may be the rea-
son why clenbuterol obtain a better separation result on
the HP-γ-CD monolith. More model compounds should
be tested in order to demonstrate separation mechanisms
of CD functionalized monoliths better.

194
DENG ET AL.
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In this study, a novel poly-(GMA-HP-γ-CD-co-EDMA)
monolithic capillary column was prepared and found to
be efficient for chiral separation. Under the optimal
preparation conditions, the resulting monolithic column
possesses uniform structure and proper permeability.
Furthermore, six pairs of enantiomers including pindolol,
clorprenaline, tropicamide, propranolol, clenbuterol, and
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ACKNOWLEDGMENT
This research did not receive any specific grant from
funding agencies in the public, commercial, or not-
for-profit sectors.
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DATA AVAILABILITY STATEMENT
Data are available on request from the authors.
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ORCID
Min Zhao https://orcid.org/0000-0002-8312-0678
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
How to cite this article: Deng M, Xue M, Liu Y,
Zhao M. Preparation of a novel hydroxypropyl-
γ-cyclodextrin functionalized monolith for
separation of chiral drugs in capillary
electrochromatography. Chirality. 2021;33:188–195.
https://doi.org/10.1002/chir.23300
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evaluation of chiral monolithic column modified by beta-
cyclodextrin derivatives. Talanta. 2010;80(3):1378-1384.