Facile one-pot preparation of chiral monoliths with a well-defined framework based on the thiol-ene click reaction for capillary liquid chromatography

Article abstract of DOI:10.1039/c6ra01370h

A novel chiral cyclodextrin (CD) monolith was easily prepared via a one-pot process based on the thiol-ene click reaction of allyl-β-CD with pentaerythritol tetra-(3-mercaptopropionate) in a fused-silica capillary. The effects of both the composition of prepolymerization solution and reaction temperature on the morphology, permeability, and selectivity of the β-CD chiral monolith were investigated in detail. The conditions were optimized to fabricate a homogeneous and permeable chiral monolith. In this study, the β-CD monolith was used as the stationary phase of capillary liquid chromatography for the chiral separation of several pharmaceutical enantiomers including flavanone, flurbiprofen, naproxen, synephrine, isoprenaline sulfate, ketoprofen, and atropine sulfate monohydrate. Compared to the previously reported two-step method, this one-pot method for the preparation of a β-CD chiral monolith is simple and time-saving. Moreover, good resolutions were obtained for chiral isomers in a shorter analysis time compared to that reported in the literatures. These results indicate that the thiol-ene click chemistry provides a simple and robust method for the preparation of a chiral β-CD monolith.

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ARTICLE TYPE
Facile oneꢀpot preparation of chiral monoliths with a wellꢀdefined
framework based on thiol–ene click reaction for capillary liquid
chromatography
Peng Zhang, Jiannan Wang, Haiguan Yang, Linjing Su,Yuhao Xiong and Fanggui Ye*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A novel chiral cyclodextrin (CD) monolith was easily prepared via a oneꢀpot process based on the thiol–
ene click reaction of allylꢀβꢀCD with pentaerythritol tetraꢀ(3ꢀmercaptopropionate) in a fusedꢀsilica
capillary. The effects of both the composition of prepolymerization solution and reaction temperature on
10 the morphology, permeability, and selectivity of the βꢀCD chiral monolith were investigated in detail. The
conditions were optimized to fabricate a homogeneous and permeable chiral monolith. In this study, the
βꢀCD monolith was used as the stationary phase of capillary liquid chromatography for the chiral
separation of several pharmaceutical enantiomers including flavanone, flubiprofen, naproxen, synephrine,
isoprenaline sulfate, ketoprofen, and atropine sulfate monohydrate. Compared to the previously reported
15 twoꢀstep method, this oneꢀpot method for the preparation of a βꢀCD chiral monolith is simple and timeꢀ
saving. Moreover, good resolutions were obtained for chiral isomers in a shorter analysis time compared
to that reported in the literatures. These results indicate that the thiol–ene click chemistry provides a
simple and robust method for the preparation of a chiral βꢀCD monolith.
functionalized polymethacrylateꢀbased monolithic column for
enantioseparation of ibuprofen and naproxen by capillary
electrochromatography.
1. Introduction
20 Cyclodextrin (CD) is naturally occurring cyclic oligosaccharide
consisting of several (6, 7, 8) glucose units. Notably, the
secondary 2ꢀ and 3ꢀhydroxyl groups of βꢀCD lie along the mouth
of the cavity, and the primary 6ꢀhydroxyl groups are loated at the
rear of the molecule. The βꢀCD is one of the most commonly
25 used chiral selectors for enantioseparation. Because of their
ability to form host–guest inclusion complexes with all types of
chiral compounds, and the the advantages of monolithic columns
including a continuous porous structure, high porosity, and large
pores with smallꢀsize skeletons, native or derivatized βꢀCDꢀbased
30 monolithic columns have been successfully prepared and used for
enantioseparation. In most instances, silicaꢀbased monolithic
matrices are mainly prepared by the sol–gel technology, and then
the βꢀCD is attached by covalent bonding,1ꢀ4 physical adsorption,5
or encapsulation.6,7 To date, some studies have also been reported
35 on the preparation of chiral βꢀCD polymer monoliths. For
instance, Ahmed et al.8 prepared a chiral βꢀCDꢀfunctionalized
polymer monolith via the copolymerization of βꢀCD methacrylate
and ethylene glycol dimethacrylate in a fused silica capillary for
the enantioseparation of racemic pharmaceuticals in reversedꢀ
40 phase nano liquid chromatography. Zhang et al.9 prepared
perphenylcarbamoylated βꢀCDꢀsilica hybrid monolithic columns
for enantioseparations of 13 racemates by capillary liquid
chromatography. Zhao et al.10 developed a vancomycinꢀcapped βꢀ
CDꢀbonded chiral stationary phase for the separation of aromatic
45 positional isomers and the enantiomers of chiral compounds by
liquid chromatography. Tian et al.11 fabricated a novel βꢀCDꢀ
50 Chiral separations are crucial for drug discovery research and
pharmaceutical industry because the enantiomers of a drug
compound can differ significantly in potency, toxicity, and
biological activity.12,13 Therefore, the development of novel chiral
stationary phases (CSPs) for enantioseparation is still a hot
55 research topic.14ꢀ16In recent years, “click chemistry,” especially,
thiol–ene click reaction, has attracted much attention because of
some advantages. For example, this reaction tolerates water,
oxygen, and many functional groups, can be carried out under
mild reaction conditions, and without using a metal catalyst.17
60 The formation of a thiol ether linkage provides a firm and stable
covalent bond, which can tolerate tough conditions. Thiol–ene
click reaction has been widely used in dendrimer synthesis, new
polymer synthesis, biomolecules functionalization and surface
modification.18ꢀ20 Because of the abovementioned advantages,
65 CDꢀbased CSPs have been prepared by thiol–ene click reaction.
Yao et al.21 synthesized a novel cationic CD chiral stationary
phase by immobilizing vinyl imidazoliumꢀβꢀCD onto thiol silica
based on thiol–ene click reaction for the separation of dansyl
amino acids, carboxylic aryl compounds and flavonoids. Huang
70 et al.22 synthesized a novel perphenylcarbamated βꢀCDꢀbased
CSP
by
immobilizing
βꢀCD
mono/di(10ꢀ
the 3ꢀ
undecenoyl)perphenylaminocarbonyl
on
mercaptopropylꢀfunctionalized silica gel via thiol–ene click
reaction. Notably, it is tedious and timeꢀconsuming to prepare βꢀ
75 CDꢀbased CSPs by postmodification. To the best of our
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knowledge, chiral monolithic columns have not been prepared by
a oneꢀpot thiol–ene click reaction.
MilliꢀQ water system (Millipore, Bedford, MA, USA). Fused
30 silica capillary of 75ꢀꢁm i.d. was obtained from Hebei Yongnian
Optical Fiber Factory (Hebei, China).
In this study, a new chiral stationary phase composed of allylꢀβꢀ
CD and pentaerythritol tetra(3ꢀmercaptopropionate) (PETMP)
was prepared by thiol–ene click reaction, and used for the rapid
resolution of several pharmaceutical enantiomers by capillary
liquid chromatography (cLC). The effects of both composition of
prepolymerization mixture and reaction temperature on the
morphology, permeability, and selectivity of the chiral monolith
2.2. Instrumentation
5
A TriSepꢀ2100 pressurized CEC system was used to perform the
chromatographic experiments. A PE Spectrum One FTꢀIR
35 spectrometer (PE, USA) was used to perform Fourierꢀtransform
infrared spectroscopy (FTꢀIR) in the range (4000–400 cmꢀ1) using
KBr pellets. The microstructure of monolith was examined using
a FEI Quanta 200 FEG SEM (Philips, Netherlands). A Pyris
Diamond TG (PE, USA) was used to perform thermogravimetric
40 analysis (TGA).
10 were studied in detail. The prepared monolith exhibited good
mechanical strength and thermal stability.
2. Experimental
2.3. Preparation of chiral monolith via thiol–ene click
reaction
2.1 Reagents and materials
AllylꢀβꢀCD was purchased from Shandong Binzhou Zhiyuan
15 Biotechnology Corporation. Diethylene glycol diethyl ether
(DEGDE, 98%) and polyethylene glycol 200 (PEGꢀ200) were
obtained from Sigma (St. Louis, MO, USA). γꢀ
Methacryloxypropyltrimethoxysilane (γꢀMAPS) was purchased
from Alfa Aesar (Ward Hill, MA, USA). Azobisisobutyronitrile
20 (AIBN) was purchased from Tianjin Chemistry Reagent Factory
(Tianjin, China) and recrystallized from ethanol before use.
PETMP, flavanone, flubiprofen, naproxen, synephrine,
isoprenaline sulfate, ketoprofen, atropine sulfate monohydrate
were obtained from J&K Chemical Ltd (Beijing, China). HPLCꢀ
25 grade methanol (MeOH) and acetonitrile (ACN) were obtained
from Shanghai Chemical Reagents Corporation (Shanghai,
China). Other reagents were of analytical grade. Ultrapure water
was used for the preparation of solutions and produced using a
The vinylized capillary was prepared as follows: A 50% γꢀ
MAPS MeOH (v/v) solution was used to introduce vinyl groups
45 onto the inner surface of the capillary following a previously
reported procedure.23 The chiral monolith was prepared via an
oneꢀpot thiol–ene click reaction (Fig. 1). AllylꢀβꢀCD, PETMP,
DEGDE, PEGꢀ200, and AIBN with different quantities (as listed
in Table 1) were mixed to form a uniform solution under
50 ultrasound irradiation. After sonication for 20 min, the mixture
was artificially injected into the vinylized capillary with an
effective length of 30 cm using a syringe, and then the ends of the
capillary were sealed with rubbers. The capillary was immersed
in a water bath (50ꢀ60 °C) for 22 h. Finally, the acquired
55 monolithic capillary columns were flushed with MeOH to remove
the unreacted chemicals.
O
O
OH
O
O
O
OH
O
HO
HO
OH
HO
HO
OH
O
O
OH
HO
O
O
OH
O
OH
O
O
OH
OH
OH
OH
O
O
O
click reaction
O
HO
AllylꢀβꢀCD
O
O
O
HS
HS
SH
O
O
O
O
SH
O
PETMP
Fig. 1 Preparation of a chiral monolith via thiol–ene click reaction of allylꢀβꢀCD with PETMP.
the polyimide coating at the end of the separation monolithic
column. The mobile phase used was mixture of
2.4 Standard solutions and sample preparation
a
60 Standard solutions of several pharmaceutical enantiomers in
MeOH with concentrations of 1 mg/mL were prepared. Before
the injection, the standard solutions were diluted by 100× and
filtered through a 0.22ꢀꢁm membrane. Fig. 2 shows the structures
of the racemates.
triethylammonium acetate (TEAA) buffer/ACN with different
70 volume ratios. Based on the previous report,22 the triethylamine
acetate buffer was prepared by dissolving the required amount of
pure triethylamine in water to obtain 0.1% concentration, and
then glacial acetic acid was added to obtain the required pH.
mobile phase were filtered through 0.22ꢀꢁm membrane and
75 sonicated for 20 min before use. The analyses were performed at
their optimal wavelength.
65 2.5 cLC conditions
A detection window of 2ꢀmm length was prepared by removing
2
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Table. 1 Detail preparation conditions for chiral monolithic column and their permeability
Column
DEGDE
(mg)
PEGꢀ200
(mg)
T
(°C)
Backpressure
(MPa)
Permeability
allylꢀβꢀCD/PETMP
(mol/mol)
(×10ꢀ14 m2 )
ꢀꢀꢀ
A
B
1 : 1.0
120
120
80
80
55
55
>25(too hard to pump)
15.6
1 : 2.0
1 : 3.0
1 : 4.0
1 : 3.0
1 : 3.0
1 : 3.0
1 : 3.0
1 : 3.0
2.96
C
D
E
F
120
120
120
120
60
80
80
55
55
50
60
55
55
55
12.8
3.61
32.99
8.55
2.6
1.4
80
5.4
80
17.6
G
H
I
140
120
60
0.8
9.8
57.75
4.7
80
140
>25(too hard to pump)
ꢀꢀꢀ
a Other preparation conditions: allylꢀβꢀCD 25 mg; AIBN 6.0 mg.
OH
O
F
O
OH
OH
O
OH
O
CH3
CH3
O
NH
flavanone
ketoprofen
synephrine
flubiprofen
OH
H
CH3
OH
H3C
N
N
H3C
OH
OH
CH3
CH2OH
O
H3CO
O
H2SO4
H2O
H2SO4
2H2O
O
2
2
naproxen
atropinesulfatemonohydrate
isoprenalinesulfate
5
Fig. 2 Structures of racemates.
PETMP by a oneꢀpot thiol–ene click reaction. Because the
composition of reactants significantly affects the morphology,
permeability and selectivity of the chiral monolith, several
20 parameters, the ratio of monomer/crosslinker, ratio of
porogens, and reaction temperature were optimized as shown
in Table 1. Different molar ratios of monomer/crosslinker
were investigated for the prepolymerization solutions
(columns A–D, as shown in Table 1). The backpressure of
25 the chiral monolith gradually decreased with the decrease in
the ratio of monomer/crosslinker; however, the permeability
of the corresponding monoliths increased from 3.61×10ꢀ14 to
2.6 Calculations
Permeability (K) was calculated using the following formula: 24
F ×
ꢀP ×
η
π
× L
γ 2
K =
×
where F is the volume flow rate of the eluent, η is the viscosity of
10 the mobile phase, L is the length of the monolithic column, and
ꢂP is the pressure drop across the monolith. In this study, MeOH
was used as the mobile phase, and the corresponding value of
dynamic viscosity was 0.580 × 10ꢀ3kg (ms) at 25 °C.25
32.99×10ꢀ14 m2 As shown in Fig. 3, when the molar ratio of
.
monomer/crosslinker was 1:1 (column A) or 1:2 (column B),
30 the generated monolith was extremely dense, basically
imperforate, and had no separation effect. However, the
reactants reacted well when the molar ratio of
monomer/crosslinker was 1:4 (column D); however, the
generated monolith was significantly macroporous, which
3. Results and discussion
15 3.1 Preparation of chiral monolith
The chiral monolith was prepared from allylꢀβꢀCD and
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Fig. 3 SEM images of chiral monoliths of column A (a, b), column B (c, d), column C (e, f), and column D (g, h).
was bad for the retention of analytes and reduced the separation
of the enantiomers (Fig. 4). To our delight, when the molar ratio
of monomer/crosslinker was 1:3 (column C), the generated
Therefore, 1ꢀpropanol was replaced with PEGꢀ200. An opaque
white monolith was obtained in the porogenic system of
DEGDE/PEGꢀ200. Next, the effect of the ratio (w/w) of
5
monolith had
a wellꢀdefined framework, and satisfactory 30 DEGDE/PEGꢀ200 on the permeability and morphology of the
separations were obtained. Finally, a monomer/crosslinker ratio
of 1:3 was selected to perform the following experiments. The
10 type and ratio of porogenic solvents in the prepolymerization
solution play an important role in the formation of monolith.
resulting monolith was investigated. An increase in the content of
PEGꢀ200 from 60 mg to 140 mg increased the permeability. This
phenomenon is related to the relative speed of phase separation.
Although PEGꢀ200 shows excellent compatibility with DEGDE
Therefore, several types of porogenic systems were selected to 35 and thiolꢀcontaining monomer, it served as a macroporogenic
prepare a chiral monolith, including nꢀpropanol/1,4ꢀbutanediol
and cyclohexanol/decanol. Unfortunately, satisfactory monoliths
15 could not be obtained. According to the results recently reported
by group of Zou,26 we also used DEGDE/1ꢀpropanol as the binary
solvent for both the hydrophobic eneꢀcontaining monomer and
the sulfoether oligomer generated via thiol–ene reaction.27 The
above results are consistent with the fact that a poor solvent leads
to an earlier phase separation and forms larger pores in an organic
porogen. However, a satisfactory monolith could also not be 40 monolith.28 As shown in Table 1, the ratio of DEGDE/PEGꢀ200
obtained, indicating that DEGDE/1ꢀpropanol was not suitable to
form the monolith with allylꢀβꢀCD and PETMP as monomers.
was evaluated from 60:140 to 140:60 (w/w). When the ratio of
DEGDE/PEGꢀ200 was >140:60 (w/w), a high backpressure (>
25.0 MPa) was obtained; when the ratio of DEGDE/PEGꢀ200
was < 80:120 (w/w), macropores were obtained, not suitable for
45 separation. However, an appropriate permeability was observed
when the ratio of DEGDE/PEGꢀ200 was 120:80 (w/w), good for
separating enantiomers.
Thiol–ene click reaction is similar to other radical polymerization
reactions, where the reaction temperature significantly affects the
50 morphology of the prepared monoliths. Table 1 shows the effects
of reaction temperature on permeability and backpressure. The
permeability of the chiral monolith decreased from 8.55×10ꢀ14 to
2.6×10ꢀ14 m2, when the reaction temperature was increased from
50 °C to 60 °C. This is mainly because a higher reaction
55 temperature would change the solubility of the porogenic system,
thus delaying the phase separation process.29 When the click
reaction temperature was set at 50 °C and 60 °C, a low column
efficiency and resolution were obtained (Fig. 5). However, when
the reaction temperature was 55 °C, column C with a reticular
1.6
1.4
(c) Column B
1.2
1.0
0.8
0.6
0.4
(b) Column C
(a) Column D
0.2
0.0
ꢀ0.2
0
2
4
6
8
10
Time (min)
20
Fig. 4 Effect of the molar ratio of monomer/crosslinker of (a) 1:4 (column
D) (b) 1:3 (column C) and (c) 1:2 (column B) on the separation of
flavanone using the chiral monolith. Conditions: mobile phase, ACN
/0.1%TEAA (70:30, v/v) , pH=4.0; flow rate, 0.05mL/min; detection
25 wavelength, 214 nm; chiral monolith: 55 cm × 75 ꢁm i.d. (30 cm effective
length). supplement pressure, 500 psi.
60 monolithic network exhibited
a
good chromatographic
performance. Based on the above investigations, the monolith
4
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(column C in Table 1) prepared was used for further experiments.
occurred.
The thermal stability of the chiral monolith was studied by
TGA. As shown in Fig. 7a, because of the pyrolysis of organic
moieties, an endothermic mass loss started at 230 °C and
35 continued up to 400 °C, indicating that chiral monolith had good
thermostability. Moreover, the mechanical strength of the
prepared chiral monolith was investigated using MeOH as the
mobile phase to measure the decrease in column pressure. As
shown in Fig. 7b, a good linear relationship was observed
40 between the flow rate and backpressure drop even at a high
pressure, indicating good mechanical stabilities of the prepared
chiral monoliths.
1.6
1.4
1.2
(c) Column E
1.0
0.8
0.6
(b) Column C
0.4
0.2
(a) Column F
0.0
Reproducibility and stability are important factors for the
efficiency and practicality of the synthesized chiral monolith. In
45 this study, the reproducibility and stability of the chiral monolith
were evaluated in terms of retention times of ketoprofen on
column C. The relative standard deviations (RSDs) for the
retention time of ketoprofen were less than 1.8% (n = 6) and
2.1% (n = 6) for runꢀtoꢀrun and dayꢀtoꢀday, respectively. The
50 RSD of columnꢀtoꢀcolumn reproducibility with three batches of
the column C was less than 6.8% (n = 3). In addition, Figs. 8a, 8b
and 8c show the separation of ketoprofen every 15 days. The
baseline was very stable, and the enantioseparation results were
readily reproducible. The above results indicated that the
55 prepared chiral monolith have good reproducibility and stability.
ꢀ0.2
0
2
4
6
8
10
12
Time (min)
Fig. 5 Effect of thiol–ene click reaction temperature of (a) 60 °C (column
F), (b) 55 °C (column C) and (c) 50 °C (column E) on the separation of
flavanone using the chiral monolith. The conditions are the same as in Fig.
4.
5
3.2 Characterization of chiral monolith
Fig. 3 shows the SEM images of columns A–D. The prepared
monolithic matrix attached well to the inner wall of the capillary.
10 This is because the alkylene group at the inner wall of the
capillary took part in the polymerization during the formation of
chiral monolith. Fig. 3 shows that the morphologies of columns A
and B are dense. When the content of crosslinker was very high
(column D), the prepared monolith had larger pores, reducing
120
(a)
25
100
(b)
20
15
80
60
15 separation. However,
obtained for column C.
a continuous reticular skeleton was
R=0.9997
10
5
40
20
0
The monolithic material formed in the centrifuge tube was
characterized by FTꢀIR; this provided a strong evidence for the
thiol–ene click reaction of allylꢀβꢀCD and PETMP. The peaks at
20 1643 cm−1 can be attributed to C=C signals (Fig. 6a), indicating
the presence of vinyl groups of in the allylꢀβꢀCD monomer. The
peak at 2560 cm−1 was assigned to the thiol group and the peak at
1740 cm−1 was assigned to the C=O stretching in the PETMP
crosslinker (Fig. 6b). A comparison of Figs. 6a, 6b and 6c and the
25 disappearance of the peak at 2560 cm−1 and the decrease in the
intensity of the peak at 1643 cm−1 shown in Fig. 6c confirmed
that the thiol–ene click reaction of allylꢀβꢀCD with PETMP
0
0
200 400 600 800 1000
Temperature
1
2
3
4
5
6
7
(
℃
)
Flow rate (µL/min)
Fig. 7 (a) TGA of chiral monolith (column C) at a heating rate of 10 °C
/min in nitrogen. (b) Relationship between the flow rate and backpressure
drop of chiral monolith, Conditions: flow rate, 1–6 µL/min for MeOH.
Ketoprofen
0.6
0.4
(a)
80
70
60
50
40
30
20
10
0
allylꢀβꢀCD
c
30 days
15 days
0.2
0.0
1643
b
(b)
PETMP
a
1 day
2560
1740
(c)
0
2
4
6
8
10
chiral monolith
Time (min)
60
Fig. 8 Comparison of enantioseparation results of ketoprofen on the chiral
monolith obtained after (a) 1 day, (b) 15 days and (c) 30 days. Detection
wavelength, 254 nm, other conditions are the same as in Fig. 4.
4000 3500 3000 2500 2000 1500 1000
500
0
Wavenumber (cmꢀ1)
3.3. Chromatographic performance
Fig. 6 FTꢀIR spectra of (a) allylꢀβꢀCD, (b) PETMP and (c) chiral
30 monolith (column C).
65 The enantioselection performance of the optimized chiral
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monolithic column was evaluated using several pharmaceutical 35 the solute.
enantiomers. Table 2 summarizes the chromatographic results
obtained for several pharmaceutical enantiomers under the
optimal separation conditions. Compared to the results reported
in the literatures, the chiral isomers showed good resolutions in a
Tabe. 2 Enantioseparation on chiral monolith (column C) by cLC
The effects of ACN concentration on k and Rs were investigated
by varying the percentage composition of ACN in the mobile
phase from 50% to 90% (v/v). As shown in Fig. 9a, the Rs value
for the enantiomers only changed slightly over the studied ACN
40 concentration range. A slightly higher Rs was obtained when the
mobile phase contained 70% ACN; therefore, 70% ACN was
considered optimal for further experiments. Fig. 9b clearly shows
that a higher ACN content in the mobile phase resulted in the
lower retention of the analyte. This indicates a hydrophobic
45 retention mechanism on the monolithic stationary phase under the
tested separation conditions.
5
sample
t1
t2
α
N1
N2
Rs
(t2/t1)
(min)
(min)
(Plates/m) (Plates/m)
flavanone
naproxen
3.324 5.145 1.548 5133.8 3352.9 2.5
3.435 6.317 1.839 7767.5 7857.8 3.9
ketoprofen
flubiprofen
synephrine
3.362 4.916 1.489 7843.8 3020.9 2.3
2.701 10.073 3.729 3044.9 1898.5 4.1
3.156 7.047 2.232 1173.7 1313.8 2.6
6.130 6.811 1.111 5950.5 6749.5 1.6
1.2
atropine sulfate
monohydrate
0.6
0.9
Flavanone
Ketoprofen
0.4
isoprenaline sulfate 3.170 6.716 2.119 1022.5 1298.0 2.7
0.6
0.3
shorter analysis time. For instance, as shown in Table 2, the
resolution value of flavanone was 2.5, higher than that reported
by the Fanali’s group,4 and the separation time of flavanone was
shorter than theirs (>40 min). The resolution value of naproxen
0.2
0.0
0.0
ꢀ0.3
0
2
4
6
8
10
12
0
2
4
6
8
10
Time (min)
Time (min)
10 was 3.9, higher than that reported by the Zeng’s group,11 and the
separation time was shorter than theirs. The composition of the
mobile phase significantly affected the enantioseparation by cLC.
Therefore, the enantioseparation conditions including the nature
and concentration of organic additives and buffer concentration
15 were systematically optimized. Synephrine was used as the test
compound.
0.4
0.3
0.2
0.1
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
ꢀ0.2
Naproxen
Isoprenaline suflate
0
2
4
6
8
10
0
2
4
6
8
10 12 14
Time (min)
Time (min)
3.2
(b)
k
2.4
(a)
3.0
2.8
2.6
2.4
2.2
2.0
0.4
0.3
R
1.2
s
Flubiprofen
2.0
1.6
1.2
Synephrine
0.9
0.6
0.3
0.0
0.2
0.1
0.0
ꢀ0.1
ꢀ0.2
50
60
70
80
90
50
60
70
80
90
ACN
(
%)
ACN (%)
0
2
4
6
8
10 12 14
0
2
4
6
8
10 12 14 16
Time (min)
Time (min)
2.2
2.4
(d)
(c)
R
s
k
2.1
2.0
1.9
1.8
1.8
1.2
0.6
0.6
Atropine sulfate monohydrate
0.5
0.4
0.3
0.2
0.1
0.0
ꢀ0.1
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
TEAA
(%)
TEAA (%)
2
3
4
5
6
7
8
9
10
Fig.
9 Effects of ACN content and buffer concentration on the
Time (min)
50
20 enantioseparation of synephrine. Conditions: mobile phases, (a and b)
ACN/0.1%TEAA (at various volume ratios) (pH=5.0); (c and d)
ACN/TEAA (70:30, v/v) at various buffer concentrations; chiral monolith,
column C; sample, synephrine; flow rate, 0.05 mL/min.
Fig. 10 Enantioseparation of seven racemic compounds on the chiral
monolith (column C). Conditions: mobile phase: ACN/0.1%TEAA (70:30,
v/v); flow rate, 0.05 mL/min; supplement pressure, 500 psi; flavanone,
atropine sulphate monohydrate, detection wavelength, 214 nm, pH=4.0;
First, the enantioseparations were first attempted using
25 MeOH/TEAA buffer as the mobile phase. This is because
although MeOH is commonly regarded as a weaker displacer of
analytes from the CD cavity,30 long retention times and small
55 flubiprofen, naproxen, ketoprofen, detection wavelength, 254 nm, pH=4.0;
synephrine, isoprenaline sulfate, detection wavelength, 230 nm, pH=5.0.
TEAA was proven to be a very good buffer for the chiral
separation of CDꢀfunctionalized stationary phases. In this study,
TEAA was also selected as the aqueous buffer. The effect of
60 buffer concentration on enantioseparation was also investigated
by adjusting the value from 0.1% to 1.0%. As shown in Figs. 9c
and 9d, both the k and Rs values of synephrine slightly decreased
with increasing buffer concentration from 0.1% to 1.0%. This
phenomenon can be attributed to two reasons: On one hand, when
resolutions are achieved. MeOH generally has
a weaker
displacing activity than ACN; this might explain why a large
30 retention time was obtained when methanol was used as the
organic modifier. Finally, when MeOH was replaced with ACN,
all the compounds separated well. Probably because ACN (an
aprotic solvent) had
a greater tendency to occupy the
hydrophobic cavity of CD than MeOH, it was less accessible to
6
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

Page 7 of 8
RSC Advances
View Article Online
DOI: 10.1039/C6RA01370H
60 10 J. Zhao, S. K. T. Chelvi, D. Tan, E. L. Yong, H. K. Lee and Y. Gong,
Chromatographia, 2010, 72, 1061ꢀ1066.
11 Y. Tian, C. Zhong, E. Fu and Z. Zeng, J. Chromatogr. A, 2009, 1216,
1000ꢀ1007.
the TEA (triethylamine) concentration increased, the electrostatic
interactions between chiral selector and analytes altered, causing
the variation in resolution; on the other hand, because of
electrostatic competition between chiral selector and TEA
towards the analytes, the decrease in resolution can be explained
12 I. Ilisz, R. Berkecz and A. Péter, J. Sep. Sci., 2006, 29, 1305ꢀ1321.
65 13 Y. H. Gong and H. K. Lee, Anal. Chem., 2003, 75, 1348ꢀ1354.
14 Y. Xiao, S.C. Ng, T. T. Y. Tan and Y. Wang, J. Chromatogr. A, 2012,
1269, 52ꢀ68.
5
by the increased competition effect with the chiral selector.11
A
higher Rs was obtained with 0.1% TEAA; therefore, 0.1% TEAA
was considered optimal for further experiments.
The chromatographic performance of the chiral monolith was
15 J. Ou, H. Lin, S. Tang, Z. Zhang, J. Dong, H. Zou, J. Chromatogr. A,
2012, 1269, 372ꢀ378.
10 investigated by separating several pharmaceutical enantiomers by
cLC. Fig. 10 shows the separation of enantiomers by cLC using
ACN/0.1%TEAA (70:30, v/v) as the mobile phase. All the tested
racemates could be enantioseparated well within 13 min. High
separation efficiencies were observed for these racemates.
70 16 B. Chankvetadze, J. Chromatogr. A, 2012,1269, 26ꢀ51.
17 C. E. Hoyle and C. N. Bowman, Angew. Chem. Int. Ed., 2010, 49,
1540ꢀ1573.
18 M. J. Kade, D. J. Burke and C. J. Hawker, J. Polym. Sci. Part A:
Polym. Chem., 2010, 48, 743ꢀ750.
75 19 A. Marechal, R. ElꢀDebs, V. Dugas and C. Demesmay, J. Sep. Sci.,
2013, 36, 2049ꢀ2062.
15 4. Conclusions
20 A. B. Lowe, Polymer, 2014, 55, 5517ꢀ5549.
21 X. Yao, T. T. Y. Tan and Y. Wang, J. Chromatogr. A, 2014, 1326, 80ꢀ
88.
In this study, a novel chiral monolith was easily prepared using
allylꢀβꢀCD and PETMP by a thiol–ene click reaction. The
synthetic method was very simple and convenient. The resulting
chiral monolith exhibited good permeability, thermal stability,
20 and mechanical stability. The chiral monolith also demonstrated
its excellent enantioseparation of selected racemic compounds by
cLC. This study provides possibilities for preparing other chiral
monolithic columns that involve click reaction using multienes
and multithiols as the precursors.
80 22 G. Huang, J. Ou, X. Zhang, Y. Ji, X. Peng and H. Zou,
Electrophoresis, 2014, 35, 2752ꢀ2758.
23 J. Ou, G. T. T. Gibson and R. D. Oleschuk, J. Chromatogr. A, 2010,
1217, 3628ꢀ3634.
24 S. Yang, F. Ye, Q. Lv, C. Zhang, S. Shen and S. Zhao, J. Chromatogr.
85
A, 2014, 1360, 143–149.
25 P. S. Nikam, L. N. Shirsat and M. Hasan, J. Chem. Eng. Data., 1998,
43, 732ꢀ737.
25 Acknowledgements
26 Z. Liu, J. Ou, H. Lin, Z. Liu, H. Wang, J. Dong and H. Zou, Chem.
Commun., 2014, 50, 9288ꢀ9290.
The financial support from the National Natural Science
Foundation of China (21365005), Guangxi Natural Science
Foundation of China (2014GXNSFGA118002), Key Laboratory
for the Chemistry and Molecular Engineering of Medicinal
30 Resources (Guangxi Normal University), Ministry of Educationof
China (CMEMR2014ꢀA09) and Guangxi Pharmaceutical
Industry Talent Highland Project (1414) is gratefully
acknowledged.
90 27 L. Chen, J. Ou, Z. Liu , H. Lin, H. Wang, J. Dong and H. Zou, J.
Chromatogr. A, 2015, 1394, 103ꢀ110.
28 F. Svec and J. M. J. Fréchet, Chem. Mater., 1995, 7, 707ꢀ715.
29 H. Lin, J. Ou, Z. Liu, H. Wang, J. Dong and H. Zou, J. Chromatogr.
A, 2015, 1379, 34ꢀ42.
95 30 S. C. Ng, T. T. Ong, P. Fu and C. B. Ching, J. Chromatogr. A, 2002,
968, 31ꢀ40.
Notes and references
35 Key Laboratory for the Chemistry and Molecular Engineering of
Medicinal Resources (Ministry of Educationof China), College of
Chemistry and Pharmaceutical Science of Guangxi Normal University,
Guilin 541004, P. R. China.
100
Corresponding author: Prof. Fanggui Ye
40 Tel: +86ꢀ773ꢀ5856104; fax: +86ꢀ773ꢀ5832294
E-mail address: fangguiye@163.com
105
1 Z. Chen and T. Hobo, Anal. Chem., 2001, 73, 3348ꢀ3357.
2 J. Kang, D. Wistuba and V. Schurig, Electrophoresis, 2002, 23, 1116ꢀ
45
50
55
1120.
3 Z. Guo, Y. Jin, T. Liang, Y. Liu, Q. Xu, X. Liang and A. Lei, J.
Chromatogr. A, 2009, 1216, 257ꢀ263.
4 K. SiꢀAhmed, F. Tazerouti, A. Y. BadjahꢀHadjꢀAhmed, Z. Aturki, G.
D’Orazio, A. Rocco and S. Fanali, J. Chromatogr. A, 2010, 1217,
1175ꢀ1182.
Z. Liu, K. Otsuka, S. Terabe, M. Motokawa and N. Tanaka,
Electrophoresis, 2002, 23, 2973ꢀ2981.
6 M. Kato, K. SakaiꢀKato, N. Matsumoto and T. Toyo’oka, Anal. Chem.,
2002, 74, 1915ꢀ1921.
M. Kato, H. Saruwatari, K. SakaiꢀKato and T. Toyo’oka, J.
Chromatogr. A, 2004, 1044, 267ꢀ270.
5
7
8 M. Ahmed and A. Ghanem, J. Chromatogr. A, 2014, 1345, 115ꢀ127.
9 Z. Zhang, M. Wu, R. Wu, J. Dong, J. Ou and H. Zou, Anal. Chem.,
2011, 83, 3616ꢀ3622.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 7

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Page 8 of 8
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DOI: 10.1039/C6RA01370H
Graphical Abstract
O
O
OH
O
O
O
HO
OH
OH
O
HO
HO
O
HO
OH
O
OH
O
HO
O
OH
O
OH
O
O
OH
OH
OH
OH
O
O
O
O
HO
click reaction
AllylꢀβꢀCD
O
O
O
O
HS
HS
SH
O
O
O
O
SH
PETMP