Preparation and evaluation of a triazole-bridged bis(β-cyclodextrin)–bonded chiral stationary phase for HPLC

Article abstract of DOI:10.1002/chir.23147

A triazole-bridged bis(β-cyclodextrin) was synthesized via a high-yield Click Chemistry reaction between 6-azido-β-cyclodextrin and 6-propynylamino-β-cyclodextrin, and then it was bonded onto ordered silica gel SBA-15 to obtain a novel triazole-bridged bis (β-cyclodextrin)–bonded chiral stationary phase (TBCDP). The structures of the bridged cyclodextrin and TBCDP were characterized by the infrared spectroscopy, mass spectrometry, elemental analysis, and thermogravimetric analysis. The chiral performance of TBCDP was evaluated by using chiral pesticides and drugs as probes including triazoles, flavanones, dansyl amino acids and β-blockers. Some effects of the composition in mobile phase and pH value on the enantioseparations were investigated in different modes. The nine triazoles, eight flavanones, and eight dansyl amino acids were successfully resolved on TBCDP under the reversed phase with the resolutions of hexaconazole, 2′-hydroxyflavanone, and dansyl-DL-tyrosine, which were 2.49, 5.40, and 3.25 within 30 minutes, respectively. The ten β-blockers were also separated under the polar organic mode with the resolution of arotinolol reached 1.71. Some related separation mechanisms were discussed preliminary. Compared with the native cyclodextrin stationary phase (CDSP), TBCDP has higher enantioselectivity to separate more analytes, which benefited from the synergistic inclusion ability of the two adjacent cavities and bridging linker of TBCDP, thereby enabling it a promising prospect in chiral drugs and food analysis.

Full text of DOI:10.1002/chir.23147

Received: 7 September 2019
DOI: 10.1002/chir.23147
Revised: 9 October 2019
Accepted: 16 October 2019
R E G U L A R A R T I C L E
Preparation and evaluation of a triazole‐bridged bis(β‐
cyclodextrin)–bonded chiral stationary phase for HPLC
Yazhou Shuang2
| Yuqin Liao2 | Hui Wang2 | Yuanxing Wang1 | Laisheng Li1,2
1 State Key Laboratory of Food Science
and Technology, Nanchang University,
Nanchang, China
2 College of Chemistry, Nanchang
University, Nanchang, China
Abstract
A triazole‐bridged bis(β‐cyclodextrin) was synthesized via a high‐yield Click
Chemistry reaction between 6‐azido‐β‐cyclodextrin and 6‐propynylamino‐β‐
cyclodextrin, and then it was bonded onto ordered silica gel SBA‐15 to obtain
a novel triazole‐bridged bis(β‐cyclodextrin)–bonded chiral stationary phase
(TBCDP). The structures of the bridged cyclodextrin and TBCDP were charac-
terized by the infrared spectroscopy, mass spectrometry, elemental analysis,
and thermogravimetric analysis. The chiral performance of TBCDP was evalu-
ated by using chiral pesticides and drugs as probes including triazoles, flava-
nones, dansyl amino acids and β‐blockers. Some effects of the composition in
mobile phase and pH value on the enantioseparations were investigated in dif-
ferent modes. The nine triazoles, eight flavanones, and eight dansyl amino
acids were successfully resolved on TBCDP under the reversed phase with
the resolutions of hexaconazole, 2′‐hydroxyflavanone, and dansyl‐DL‐tyrosine,
which were 2.49, 5.40, and 3.25 within 30 minutes, respectively. The ten β‐
blockers were also separated under the polar organic mode with the resolution
of arotinolol reached 1.71. Some related separation mechanisms were discussed
preliminary. Compared with the native cyclodextrin stationary phase (CDSP),
TBCDP has higher enantioselectivity to separate more analytes, which
benefited from the synergistic inclusion ability of the two adjacent cavities
and bridging linker of TBCDP, thereby enabling it a promising prospect in chi-
ral drugs and food analysis.
Correspondence
Laisheng Li, State Key Laboratory of Food
Science and Technology, Nanchang
University, Nanchang 330047, China.
Email: lilaishengcn@163.com
Funding information
Open Project of State Key Laboratory of
Food Science and Technology, Grant/
Award Number: SKLF‐KF‐201605;
National Natural Science Foundation of
China, Grant/Award Numbers: 21165012
and 31860469
KEYWORDS
bridged bis(β‐cyclodextrin)–bonded chiral stationary phase, chiral drugs and pesticides, enantiomeric
separations, high‐performance liquid chromatography, preparation and evaluation
1 | INTRODUCTION
the others are not only ineffective but also harmful to
the human bodies. For example, R‐propranolol is 100
Chiral compounds have been widely used in medical
treatment and agricultural production due to their high
biological activities.1-4 Many studies have confirmed that
enantiomers of chiral drugs and pesticides have different
properties in biological activities and pharmacokinetics,
with usually only one of them has desired effects,1 and
times weaker than S‐propranolol in antihypertensive
effect and even may cause the male infertility.2 At pres-
ent, most of chiral compounds still have been using in
the racemic form due to difficulty in synthesis and purifi-
cation, which made the enantiomeric separation thriv-
ing.3,4 With effective resolution and universal
Chirality. 2019;2–16.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

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SHUANG ET AL.
applicability, the high‐performance liquid chromatogra-
phy (HPLC) coupling various chiral stationary phases
(CSPs) has developed into one of the most important
methods in the chiral separation, in which the efficient
and versatile CSPs has played a crucial role in this
method.5
They attributed this to the space steric hindrance caused
by the excessive substituents, leading to the congestion
of the CD's rims and incurring difficulty for the CD's cav-
ities to include the analytes.19 Therefore, only relying on
the functionalization is limited in improving the chiral
performance of the CD‐CSPs. A promising alternative is
developing the novel bridged CD‐CSPs.
The most common CSPs derivatized the cellulose‐
based stationary phase, of which the separation capability
mainly depends upon the effects of inclusion, hydrogen
bonding, and ion‐association provided by the ordered
grooves of the polysaccharides.6 For the purpose of
protecting the ordered structure, this type of CSPs is usu-
ally used in a coating manner, with being employed
under the normal phase (NP) mode that uses the non-
aqueous hexane‐isopropanol as the mobile phase.7,8
Therefore, it is relatively insufficient for these cellulose‐
based CSPs in the compatibility of being combined with
electrospray mass spectrometry (ESI‐MS). As we know,
the exploration of the multimode CSPs (MMCSP) has an
important significance.9,10 The cyclodextrin‐based CSPs
(CD‐CSPs),11 characterized by the multimode compatibil-
ity, which is applicable to the reversed phase (RP),12
NP,13 and polar organic (PO) mode,14 have been fre-
quently used in HPLC.
The bridged cyclodextrins is a new kind of supramo-
lecular compounds coupled by two or more native cyclo-
dextrins.21 By bridging two or more cavities with a
functional linker, the bridged cyclodextrins could signifi-
cantly enhance the original binding ability and molecular
selectivity of the native cyclodextrin, thus being exten-
sively used in molecular recognition, asymmetric cataly-
sis, drug‐directed carriers, and artificial mimic
enzymes.22-24 Many studies have confirmed that the bind-
ing constant of the bridged cyclodextrins with guests was
as high as the level of biological enzymes.23 Moreover,
Liu et al24 found that in addition to the synergistic inclu-
sion, the bridging functional linker could also act as a
pseudo‐cavity21 to generate extraordinary effects.
Although the bridged cyclodextrins has attracted great
attention for a long time, few reports can be found in chi-
ral separation. Chang et al25 prepared three diamino‐
bridged bis(β‐CD)‐CSPs and separated different sets of
chiral compounds including aryl alcohols and β‐blockers,
and acquired the encouraging resolutions. Ai et al26 pre-
pared two ureido‐bridged β‐CD CSPs and achieved the
good resolutions for aromatic positional isomers and
dansyl amino acid enantiomers, which supported the syn-
ergistic inclusion of bridged cyclodextrins in separations.
Our research group27 also prepared an ethylenediamino‐
bridged bis(β‐CD)‐CSP to resolved fourteen β‐blocker
drugs, of which exhibited higher resolutions and wider
enantioselectivities than the native CD‐CSP. Obviously,
the bridged CD‐CSPs has a potential applicable value
for chiral separations.
In 1985, Armstrong15 reported the first stable β‐CD‐
CSP and applied a patent. After that, most studies have
focused on the functionalization of CD‐CSPs such as
alkylation, benzoylation, and phenylcarbamated,16-18
which introduced various interaction sites including π‐π
stacking, dipole‐dipole, ion paring, electrostatic attrac-
tion, and steric repulsive for the chiral separation. How-
ever, the performance of CD‐CSPs could be influenced
by the number of substituents on the CD's rims.19 By
far, according to the degree of substitution, the current
derivatization of CD‐CSPs has been divided into
monoderivatization, partial derivatization, and full deriv-
atization. The monoderivatized CD‐CSPs has well‐defined
structures and chromatographic stability, but the single
substituent may be inefficient to improve the
enantioselectivity.20 The partial‐derivatized CD‐CSPs has
suitable substituents, but with incapability to ensure the
number and the position of substituents that caused the
poor chromatographic reproducibility. The full‐
derivatized CD‐CSPs could readily be obtained by adding
excess modifiers. Armstrong research group12,14,17,18 has
prepared a series of fully aromatic‐derivatized CD‐CSPs
and systematically investigated the chiral separation
mechanism of them. They separated a large spectrum of
enantiomers including the derivatized chiral amines,
alcohols, and amino acids. However, the dansyl amino
acids, crown ethers, and some biologically active com-
pounds were not resolved on their phenethyl‐ and
naphthylethyl‐carbamate fully derivatized CD‐CSPs.17
The bridged bis(β‐cyclodextrin) is usually synthesized
by the bifunctional reagents such as diisocyanates,
dicarboxylic acids, and diacid chlorides.22,25,28 But the
steric hindrance and excessive reaction sites (–OH) of
the bulky cyclodextrins would lead to the above‐
mentioned synthetic approaches inclining a lower yield
(5‐15%), thus having to purify by time‐consuming and
complicated process.22 A satisfactory alternative is the
Click Chemistry reaction proposed by Sharpless for the
first time.29 As a powerful, highly reliable and selective
reaction to rapidly synthesize various new compounds,
the [3
+
2] dipolar cycloaddition between
azides/alkynes catalyzed by copper (I) is a primary Click
Chemistry reaction.30
We herein described a new approach to prepare the
triazole‐bridged bis(β‐CD)–bonded CSP (TBCDP) via

SHUANG ET AL.
3
Click Chemistry reaction between 6‐azido‐β‐cyclodextrin
(N3‐CD) and 6‐propynylamino‐β‐CD (PA‐β‐CD) and then
bonding the resulted bridged cyclodexrins onto the sur-
face of SBA‐15. After chemical structure characterization,
the chiral performance of TBCDP was systematically
evaluated by using different racemic drugs and pesticides
as analytes in the RP and PO modes. Some chromato-
graphic conditions such as mobile phase composition,
pH values, and column temperature on the
enantioselectivity were optimized, and some related chi-
ral separation mechanisms of the TBCDP were prelimi-
narily discussed.
acid
(HOAc),
triethylamine
(TEA),
N,N‐
dimethylformamide (DMF), acetone, and other chemicals
were all analytical reagents purchased from the
Sinopharm Chemical Reagent Co, Ltd (Shanghai, China).
DMF was treated with CaH2 to remove water before use.
All HPLC chiral separations were performed at a
ZQ4000/2695 LC/MS equipped with 2966 diode array
detector (Waters, USA). Infrared characterization was
performed on a Nicolet 5700 Fourier‐transform infrared
spectrometer (Thermo, USA). Elemental analysis was
performed on a Vorio EL III (Elementar, Germany).
Thermogravimetric analysis was performed on a Dia-
mond TG/DTA (Perkin, Elmer, USA). The HPLC column
was packed by an AW‐60 chromatographic packing
device (Haskel, USA). Ultra‐pure water (resistivity >18.2
MΩ.cm) was prepared by a Milli‐Q water purification sys-
tem (Millipore, USA).
2 | MATERIALS AND METHODS
2.1 | Materials and instruments
Triblock polymers
orthosilicate (TEOS), 1,3,5‐trimethyl‐benzene (TMB), β‐
cyclodextrin (β‐CD), and 3‐
P 123 (MW ~5800), tetraethyl
2.2 | Preparation of triazole‐bridged bis(β‐
CD)‐CSP
isocyanatopropyltriethoxysilane were purchased from
Sigma‐Aldrich. p‐Toluenesulfonyl chloride (p‐TsCl),
propargylamine, triphenylphosphine, sodium azide, and
cuprous iodide (CuI) were analytical grade and purchased
from Aladdin‐Reagent (Shanghai, China). Racemic stan-
dards (flavanones, amino acids, and β‐blockers) were pur-
chased from Sigma‐Aldrich. Triazole racemic standards
(purity ≥ 98%) were purchased from Shanghai Pesticide
Research Institute (Shanghai, China). HPLC‐grade meth-
anol (MeOH) and acetonitrile (ACN) were purchased
from Tedia Company (Inc, USA). Formic acid (FA), acetic
The synthetic route of TBCDP was shown in Figure 1,
including the preparation of triazole‐bridged bis(β‐CD)
and its immobilization.
2.2.1 | Preparation of SBA‐15
According to the improved method of Zhao et al,31
a
spherical and ordered mesoporous materials of SBA‐15
was prepared by our group16 and used as the silica gel
matrix for TBCDP, which has the particle diameter of
FIGURE 1 Synthetic route of triazole‐bridged bis(β‐cyclodextrin)–bonded chiral stationary phase (TBCDP)

4
SHUANG ET AL.
about 3.2 μm and the pore size of about 24 nm with the
specific surface area of about 400 m2/g. The procedure
was briefly described as follows: 5.6‐g P123 (template)
and 12‐g TEOS (silicon source) dissolved into 250 mL of
2.0 mol/L of HCl, after adding of 6.0‐g TMB (porogen)
and appropriate amount KCl, stirred at 38°C for 24 hours.
Then the mixture was transferred to a hydrothermal
autoclave and reacted statically at 110°C for 48 hours,
and white solid was collected and calcined at 550°C for
10 hours to obtain the SBA‐15.
2.2.2 | Synthesis of triazole‐bridged bis(β‐
CD)
Triazole‐bridged bis(β‐CD) was synthesized by the Click
Chemistry reaction. Mono‐6‐deoxy‐6‐azido‐β‐cyclodextrin
(N3‐CD) and mono‐6‐deoxy‐6‐propynylamino‐β‐cyclodex-
trin (PA‐CD) were synthesized according to the previous
reports,20,30 respectively. Starting from the readily
obtained mono‐6‐(p‐tosylsulfonyl)‐β‐CD (TsO‐CD).
To a solution of TsO‐CD (5.0 g, 3.9 mmol) in 50 mL
of water was added sodium azide (1.26 g, 19.4 mmol).
The mixture was stirred at 70°C for 24 hours under
the atmosphere of nitrogen. After filtration, the filtrate
was poured into 200‐mL acetone. Then the obtained
white precipitate was collected and washed with ace-
tone for several times and dried at 50°C to obtain the
product N3‐CD with a yield of 92.8%. ESI‐MS: m/z, [M
+ Na]+ calculated for 1183.01 and found at 1182.94
shown in Figure 2A.
TsO‐CD (7.0 g, 5.4 mmol) was added to a 50‐mL round
bottom flask, and then 15‐mL propargylamine was added
dropwise. The mixture was stirred at 75°C for 20 hours
before poured into 200‐mL acetone. After filtration, the
yellow precipitate was recrystallized twice from methanol
and dried at 40°C to give the product of PA‐β‐CD with a
yield of about 88.6%. ESI‐MS: m/z, [M + H]+ was calcu-
lated for 1172.08 and found at 1171.60 shown in
Figure 2B.
FIGURE 2 The electrospray mass spectrometry (ESI‐MS) spectra
of azido‐β‐cyclodextrin A propargylated β‐cyclodextrin B and
bridged bis(β‐cyclodextrin) C
The triazole‐bridged bis(β‐CD) was synthesis by the
Click Chemistry reaction of the above resulted N3‐CD
and PA‐CD. To a solution of PA‐CD (2.3 g, 19.6 mmol)
in 50 mL of DMF was added CuI (PPh3)20 (0.025 g, 0.06
mmol) and N3‐CD (2.3 g, 19.8 mmol). After stirring at
80°C for 24 hours, the solvent was removed under a
reduced pressure and then precipitated by adding ace-
tone. The obtained precipitates were filtered and washed
with acetone to give the triazole‐bridged bis(β‐CD) ligand.
After dried at 50°C, the yield was calculated for about
62.0%. ESI‐MS [M + H]+ was calculated for 2332.07 and
found at 2331.77 shown in Figure 2C.
2.2.3 | Preparation of TBCDP
TBCDP was prepared by immobilizing the above synthe-
sized triazole‐bridged bis(β‐CD) onto the silica gel (SBA‐
15). To a solution of triazole bridged β‐CD (1.5 g) in 40
mL
of
DMF
was
added
0.1
mL
3‐
isocyanatepropyltriethoxysilane by dropwise. The mix-
ture was stirred at 80°C for 3 hours under the atmosphere
of nitrogen. After adding of 3.0‐g SBA‐15 and reacted
with stirring at 110°C for 24 hours. The mixture was fil-
tered and washed repeatedly by DMF, MeOH, and ace-
tone to give the final TBCDP.

SHUANG ET AL.
5
The cyclodextrin stationary phase (CDSP) was pre-
The retention factor (k′) calculated by the fomular of k′
= (tR − t0)/t0, respectively, where t0 is the retention time
at which the first baseline disturbance by the solvent
peak appeared and tR is the retention time of the each
enantiomer. The separation factor (α) calculated accord-
ing to the formula of α = k′2/k′1, where k′1 and k′2 are
the retention factor of the first and second enantiomer,
respectively. The chiral resolution (Rs) evaluated by the
equation of Rs = 1.18(tR2 − tR1)/(wh1 + wh2), where tR1
and tR2 are the retention time of the first and second
enantiomers and wh1 and wh2 are the half‐peak width of
each enantiomer, respectively.
pared by the similar method of TBCDP. To a solution of
β‐CD (0.8 g) in 40 mL of DMF was added 0.3 mL 3‐
isocyanatepropyltriethoxysilane by dropwise. Then the
3.0 g of SBA as silica gel support was added. The rest of
preparation steps was similar to of the TBCDP. The
native cyclodextrin‐bonded phase (CDSP) was obtained
for the following comparative studies.
2.2.4 | Packing of the columns
The above‐resulted TBCDP (3.5 g) and CDSP were packed
into HPLC column in the conventional slurry approach,
where methanol (30 mL) served as the packing solvent.
The CSPs of TBCDP and CDSP were respectively
suspended in acetone by ultrasonicating for 20 minutes
and then packed into stainless steel columns (4.6 mm ×
150 mm) under the constant pressure (about 34.5 MPa)
maintained for 40 minutes by the packing system
(Haskel, USA). Finally, the packing pressure was released
to ambient gradually.
3 | RESULTS AND DISCUSSION
3.1 | Characterization of TBCDP
The prepared TBCDP was characterized by infrared spec-
troscopy, elemental analysis, and thermogravimetric
analysis.
The infrared spectra of the bridged β‐CD ligand and
TBCDP were shown in Figure 3, where the representative
peaks of cyclodextrin at νO–H (3365 cm−1), νC–H (2926 and
2869 cm−1), and νC–O (1160, 1082, and 1030 cm−1) could
be observed. The peak cluster of 1660, 1595, and 1465
cm−1 were attributed to νC═N, νN═N, and νC–N stretching
vibration, which indicated that the triazole bridging
linker was attached on the β‐CD that implied the success-
ful synthesis of the bridged β‐CD ligand.
2.3 | HPLC procedures
2.3.1 | HPLC conditions
The mobile phases were prepared by mixing appropriate
amounts of acetonitrile (ACN) or methanol (MeOH) into
water. For the separations of some specific analytes, the
mobile phases were revised by employing 1% (v/v)
triethylammonium acetate buffer (1% TEAA, pH was
adjusted by acetic acid) or 0.1% (v/v) FA (0.1% FA) in
place of water. All analytes were dissolved in methanol
to prepare stock solutions with the concentration of 100
to 200 μg/mL and stored at 4°C. Both of the mobile
phases and stock solutions were filtered with 0.22‐μm
membrane and ultrasonicated for 30 minutes before use.
The above stock solutions were diluted to appropriate
concentration accordingly occasionally. The flow rate of
mobile phase was 0.8 mL/min, the column temperature
was selected at 20°C, which was easy to be controlled.
According to the spectral properties of the analytes, the
detection wavelength range was set 200 to 380 nm (flava-
nones at 280 nm, dansyl amino acids at 254 nm, and tri-
azole pesticides at 220 nm). The injection volume was
adjusted as 3 to 10 μL.
The infrared spectra of TBCDP were similar to ligand,
with the characteristic peaks of triazole group at 1629.34
and 1543.51 cm−1, indicating that the bridged β‐CD
ligand was bonded onto the silica gel. Successful immobi-
lization of triazole‐bridged bis(β‐cyclodextrin) on SBA‐15
was evidenced by the following adsorption peaks:
3341.63 cm−1 (νO–H of β‐CD and silica gel) and 2933.48
cm−1 (νC–H of β‐CD), which indicated that the organic
layer was contained on silica gel. The characteristic
absorption peaks of νN═N═N at 2124 cm−1 and νC–H and
νC–C at 3249 and 2112 cm−1 almost disappeared, hinting
that both of N═N═N and C≡C groups were exhausted
by reacting with each other. The new peaks at 1629.34
and 1543.51 cm−1 respectively belong to the vibration of
the C=N and triazole ring, indicating that the azido
groups reacted with the terminal‐alkynes via the “Click
Chemistry” reaction. The peak cluster at 1237.00,
1155.26, 1102.14, and 1032.66 cm−1 were the typical
absorptionsνC–O of the β‐CD and νSi–O–Si of the SBA‐15.
The elemental analysis results were shown in Table S1.
According to the carbon content (6.02%), the average
loading of TBCDP was about 0.14 μmol/m2 calculated
by the formula: C%/(12 × Nc × S) × 106, where Nc is
2.3.2 | HPLC evaluation parameters
Chromatographic performance of TBCDP was evaluated
by following parameters (according to USP standards):

6
SHUANG ET AL.
FIGURE 3 The FTIR spectra of bridged
bis(β‐cyclodextrin) and triazole‐bridged bis
(β‐cyclodextrin)–bonded chiral stationary
phase (TBCDP)
the number of carbon atoms in per ligand molecule and S
is the surface area of SBA‐15 (400 m2/g).
Likewise, according to the carbon content (6.18%), the
average loading of CDSP was about 0.28 μmol/m2.
The weight loss of TBCDP was determined by the ther-
mogravimetric analysis in the range of recording temper-
ature from 50°C to 900°C (10°C/min as the heating rate),
and the weight loss result was 10.5% (w/w). The average
loading of TBCDP was estimated to be 0.11 μmol/m2
according to the weight loss result, which was corre-
sponding to the loading result calculated on the basis of
elemental analysis. Moreover, the excellent chiral chro-
matographic properties of TBCDP also evidenced the fea-
sibility of the above proposed preparation method. In
addition to chiral separations, TBCDP has also excellent
selectivity to positional isomers (Figure S1).
3.2 | Chiral performance of TBCDP
FIGURE 4 The chemical structures of nine triazole pesticides
3.2.1 | Chiral separation of triazole
pesticides
conditions were shown in Table 1, as well as some chro-
matograms in Figure 5.
The triazole chiral pesticide is one of the most widely
used high‐efficiency fungicides and plant growth regula-
tors in the world. At present, more than 30 varieties of
triazoles have been used in agricultural production and
the preservation of fruits and vegetables. Since enantio-
mers of each chiral triazole pesticide has big difference
in biological activity, toxicity, and residue behavior in
animals and plants, the triazole pesticides in the United
States have been classified as a potential chirality carcin-
ogens requiring high monitor. Therefore, we choose com-
mon nine triazole pesticides (Figure 4) as chiral probes to
evaluate the chromatographic performance of TBCDP.
The corresponding results and chromatographic
Both the TBCDP and CDSP were not derivatized; the
inclusion effects of them should be responsible for their
separation abilities in the RP chromatography.14,17,18 In
this experiment, we used simple methanol‐ and
acetonitrile‐water as mobile phase to separate the
selected triazole pesticides under the RP mode. As shown
in Table 1, nine triazoles were successfully resolved on
the TBCDP with the resolutions (Rs) from 0.50 to 2.49
within 30 minutes (Figure 5). The highest
enantioresolution (Rs = 2.49) for hexaconazole was due
to the minimal steric hindrance near its chiral carbon,
making the anayte convenient to enter or approach to
the cavity of the cyclodextrin. However, CDSP only

SHUANG ET AL.
7
TABLE 1 The enantioseparation results of triazole pesticides and better chromatographic conditions by HPLC
Compounds
k′1
k′2
α
Rs
Mobile Phases
Columns
Hexaconazole
2.93
4.31
3.56
3.54
5.26
5.01
6.24
6.68
7.08
5.97
6.81
7.52
4.40
7.45
7.89
6.54
4.56
5.32
7.04
5.33
3.22
2.89
4.01
5.27
3.55
5.02
4.17
3.94
5.85
5.41
6.81
7.34
7.58
6.35
7.27
8.20
4.68
7.90
8.21
7.70
5.21
6.01
7.50
5.53
3.29
/
1.21
1.16
1.17
1.11
1.11
1.08
1.09
1.10
1.07
1.06
1.07
1.09
1.06
1.06
1.04
1.18
1.14
1.13
1.07
1.04
1.02
/
2.49
1.95
1.18
1.37
1.61
1.27
1.12
1.42
1.25
0.65
1.19
1.00
0.89
1.23
0.98
1.15
1.28
0.93
1.14
0.53
<0.5
0
MeOH/H2O (40/60,v/v)
TBCDP
TBCDP
CDSP
ACN/H2O (20/80, v/v)
MeOH/H2O (40/60,v/v)
Flutriafol
MeOH/H2O (30/70, v/v)
TBCDP
TBCDP
CDSP
MeOH/ACN/H2O (15/5/80, v/v/v)
MeOH/H2O (30/70, v/v)
Diniconazole
Tebuconazole
Uniconazole
Paclobutrazol
MeOH/H2O (35/65, v/v)
TBCDP
TBCDP
CDSP
MeOH/ACN/H2O (25/5/70, v/v/v)
MeOH/ACN/H2O (25/5/70, v/v/v)
MeOH/H2O (40/60, v/v)
TBCDP
TBCDP
CDSP
ACN/H2O (20/80, v/v)
ACN/H2O (20/80, v/v)
MeOH/H2O (40/60, v/v)
TBCDP
TBCDP
CDSP
MeOH/ACN/H2O (25/5/70, v/v/v)
MeOH/ACN/H2O (25/5/70, v/v/v)
MeOH/H2O (40/60, v/v)
TBCDP
TBCDP
CDSP
ACN/H2O (30/70, v/v)
ACN/H2O (30/70, v/v)
Triticonazole
Myclobutanil
Triadimenol
MeOH/H2O (40/60, v/v)
TBCDP
CDSP
MeOH/H2O (40/60, v/v)
MeOH/ACN/H2O (25/5/70, v/v/v)
MeOH/ACN/H2O (25/5/70, v/v/v)
MeOH/H2O (35/65, v/v)
TBCDP
CDSP
4.20
/
1.05
/
0.75
0
TBCDP
CDSP
MeOH/H2O (35/65, v/v)
Abbreviations: CDSP, cyclodextrin stationary phase; HPLC, high‐performance liquid chromatography; TBCDP, triazole‐bridged bis(β‐cyclodextrin)–bonded chi-
ral stationary phase.
separated seven types and with lower resolutions (Rs,
0.53‐1.28). This indicated that the two stationary phases
were different in their chiral recognition under the RP
mode. CDSP relies on a single cavity with small size
(0.65 nm) that only benzene ring of the analytes was
encapsulated by it so that most of solute molecules were
exposed to the achiral mobile phase, thereby the chiral
recognition of CDSP was insufficient. The two cavities
of TBCDP integrated with bridging group, and the whole
molecule of analytes colud be embedded in the bridged
cyclodextrins, which could more accurately recognize
the R‐ and S‐configurations of analytes, resulting the sep-
aration capacity was broadened and enantioselectivity
was improved, which was consistent with the synergistic
inclusion confirmed by other means by Liu et al.21,24,28
In addition to inclusion, the hydrogen bonding is also
important in chiral recognition. For example, seven
triazoles containing –OH on their chiral carbons
(Figure 4) had relatively high enantioresolutions. It was
indicated that the hydrogen bonding between the –OH
of the chiral carbon and the portal –OH of the cyclodex-
trins could contribute to chiral recognition.13 Taking
hexaconazole (Rs = 2.49) and myclobutan (Rs < 0.5) as
an example, when the –OH was replaced by a cyano
group (–CN), the resolution greatly decreased.
TBCDP has an advantage in separating the selected tri-
azole pesticides only with using simple mobile phase
composited by methanol or acetonitrile‐water. From Fig-
ure S2, it could be seen the selected triazole analytes had
stronger retention on TBCDP (about 30 min eluted by
30%‐40% (v/v) of methanol in mobile phases). We found
that by using high content of methanol mobile, the
triazoles were eluted with faster speed companied with
the decreasing of resolutions. In contrast, by using low
content of methanol, the retention times of analytes were
extremely prolonged leading to the broadening and asym-
metrical peaks that was also unfavorable to separation.
Considering that methanol was a protic solvent, which

8
SHUANG ET AL.
FIGURE 5 The chromatograms of some triazole pesticide enantiomers on triazole‐bridged bis(β‐cyclodextrin)–bonded chiral stationary
phase (TBCDP)
weakened the hydrogen bonding, but acetonitrile was
aprotic solvent, so that the mixed organic modifiers meth-
anol and acetonitrile could further the separation, but the
ratio of methanol to acetonitrile required to be adjusted
properly (Table 1).
Based on the chromatographic behavior of the above
triazole pesticides and compared with CDSP, it can be
found that TBCDP has a better chiral chromatographic
performance for the selected triazole pesticides due to
its synergistic inclusion ability.

SHUANG ET AL.
9
in separation performance of TBCDP and CDSP should
be related to different inclusion modes.27 TBCDP could
form an organic whole with clip‐like shape through its
two cavities and a flexible bridging group.17 When the
guest interacted with the bridged cyclodextrins, the entire
the guest was enclosed by the two cavities and a bridging
group of TBCDP, which facilitated the finer identification
of the guest molecules as a whole, including the recogni-
tion of the spatial structure differences between the R‐
and S‐enantiomers.28 However, the cavity of the native
β‐CD was limited in size (0.65 nm) at most capable of
encasing a naphthalene ring, while the bridged cyclodex-
trins was free from this confine and could form a syner-
gistic inclusion complex with a larger volume of the
guest, thus possessing stronger chiral recognition than
the single cyclodextrins.
As can be seen from Table 2 and Figure 7, TBCDP
could resolve all of the selected flavanones, including
the parent flavanone (Rs = 1.37), but the resolutions were
significantly improved when the parent ring was replaced
by the more hydroxyl groups. By comparing 2′‐
hydroxyflavanone (Rs = 5.40), 4′‐hydroxyflavanone (Rs
= 2.37), catechin (Rs = 2.60), and naringin (Rs = 3.02),
it could be obviously found that the hydroxyl groups on
the aromatic ring participated in chiral recognition.
When this kind of analytes was encapsulated by the
TBCDP, the hydrogen bonding would form between the
hydroxyl groups of analytes and of the cyclodextrins'
ports, which could help to make bigger difference in sta-
bility between R‐ or S‐flavanone complexes and cyclodex-
trins to achieve better enantioseparation. However, CDSP
was quite different in this case because it relied
completely on a single and small cavity (0.65 nm), which
3.2.2 | Chiral separation of flavanones
Chiral flavanones have extensively existed in medicinal
herbs and possessed various therapeutic effects on
inflammation, tumor, HIV virus, cardiovascular diseases,
etc. It is of great significance to resolve natural chiral
active ingredients. We here applied TBCDP to separate
eight flavanones (Figure 6). Because this kind of analytes
contain acidic phenolic hydroxyl groups, we found that
the mobile phase containing 0.1% FA was the better elu-
ent for their chiral resolutions. By changing the content
of methanol or acetonitrile to optimize the resolutions
of flavanones, it can be observed that chiral separation
can be achieved in both organic modifiers, but acetoni-
trile was better than methanol, which is related to the
structures of different flavanones. Table 2 listed the sepa-
ration results and optimized chromatographic conditions,
as well as some representative chromatograms were
shown in Figure 7.
As shown in Table 2, the TBCDP had high
enantioresolution (Rs, 1.35‐5.40) for all of the selected
eight flavanones by using simple mobile phase in RP
mode. Among them, the resolutions of 2′‐
hydroxyflavanone, 4′‐hydroxyflavanone, catechin, and
naringin were up to 5.40, 2.37, 2.60, and 3.02, respec-
tively. However, CDSP only separated four flavanones
with lower resolutions (1.12‐1.69) than TBCDP. Despite
higher bonding amount of CDSP (0.28 μmol/m2) than
TBCDP (0.14 μmol/m2), its chiral separation ability was
poor. In addition, because both of the two CD‐CSPs were
not derivatized, the chiral separation driving force should
be the inclusion effects of the cavity and the hydrogen
bonding of the ports of cyclodextrins.17,18 Such difference
FIGURE 6 The chemical structures of
eight flavanones

10
SHUANG ET AL.
TABLE 2 The enantioseparation results of flavanones and better chromatographic conditions by HPLC
Compounds
k′1
k′2
α
Rs
Mobile Phases
Columns
Flavanone
6.46
2.83
6.97
2.98
1.08
1.05
1.37
/
0.1% HCOOH/ACN (85/15, v/v)
0.1% HCOOH/ACN (80/20, v/v)
TBCDP
CDSP
2′‐Hydroxy flavanone
4′‐Hydroxy flavanone
6‐Hydroxy flavanone
6‐Methoxy flavanone
Catechin
3.88
2.54
5.40
2.98
1.39
1.17
5.40
1.69
0.1% HCOOH/MeOH (65/35, v/v)
0.1% HCOOH/MeOH (67/33, v/v)
TBCDP
CDSP
5.44
3.87
6.30
4.54
1.16
1.17
2.37
1.39
0.1% HCOOH/ACN (85/15, v/v)
0.1% HCOOH/ACN (80/20, v/v)
TBCDP
CDSP
6.15
2.89
6.47
3.03
1.05
1.05
1.43
/
0.1% HCOOH/MeOH (70/30, v/v)
0.1% HCOOH/MeOH (65/35, v/v)
TBCDP
CDSP
8.75
2.57
9.38
2.70
1.07
1.05
1.38
/
0.1% HCOOH/MeOH (70/30, v/v)
0.1% HCOOH/MeOH (65/35, v/v)
TBCDP
CDSP
5.27
4.46
6.07
5.32
1.15
1.19
2.60
1.36
0.1% HCOOH/ACN (87/13, v/v)
0.1% HCOOH/ACN (85/15, v/v)
TBCDP
CDSP
Hesperidin
6.01
4.93
6.54
5.08
1.09
1.03
1.35
/
0.1% HCOOH/ACN (93/7, v/v)
0.1% HCOOH/MeOH (80/20, v/v)
TBCDP
CDSP
Naringin
3.54
2.54
4.32
3.12
1.22
1.23
3.02
1.12
0.1% HCOOH/ACN (93/7, v/v)
0.1% HCOOH/MeOH (80/20, v/v)
TBCDP
CDSP
Abbreviations: CDSP, cyclodextrin stationary phase; HPLC, high‐performance liquid chromatography; TBCDP, triazole‐bridged bis(β‐cyclodextrin)–bonded chi-
ral stationary phase.
made it difficult in the encapsulating of larger‐volume
analytes. For example, CDSP could not resolve 6‐
hydroxyflavanone, 6‐methoxyflavanone, and hesperidin.
Conversely, the resolutions (Rs, 1.43, 1.38, and 1.35) for
6‐hydroxyflavanone, 6‐methoxyflavanone, and hesperidin
on TBCDP were achieved, respectively (Table 2). There-
fore, the bridged β‐CDs has advantages in improving chi-
ral recognition by synergistic inclusion.
the proper ionization states for separations. The
triethylammonium acetate (TEAA) is the most common
buffers in the separations of dansyl amino acids. Wang
et al30 successfully resolved some of dansyl amino acids
on the derivatized CD‐CSPs by using TEAA‐containing
mobile phase with appropriate pH values. They believed
that the native CD‐CSPs could provide only insufficient
separations for dansyl amino acids, and an effective reso-
lution mainly depended upon the synergistic interactions
of the derivatization groups and cavities.
According to the above reports, 1% TEAA‐MeOH was
selected as mobile phase to separate dansyl amino acids
on TBCDP. The chromatographic conditions were opti-
mized through the further investigation of some factors
such as the concentration of organic solvents and pH
values in mobile phases shown in Figure S3. Taking the
acidic Dns‐Asp as an example, it could be found that both
the methanol content and pH values in mobile phase had
optimal ranges (25% (v/v) MeOH, pH = 5.0) in terms of
the Dns‐Asp. Other amino acids were optimized by simi-
lar methods. The results are shown in Table 3.
As shown in Table 3, TBCDP could separate all of the
selected eight dansyl amino acids (Rs, 1.07‐3.25) within
20 minutes (Figure 9), whereas the CDSP only separated
six dansyl amino acids and with the lower resolutions
(Rs, 0.65‐0.88). Obviously, TBCDP was more efficient in
separating these analytes than CDSP. For examples,
when used the TBCDP, the resolutions of Dns‐Leu, Dns‐
Phe and Dns‐Tyr respectively arrived at the 2.66, 2.48,
and 3.25. These three Dns‐amino acids were typical
hydrophobic amino acids with fatty chains or benzene
3.2.3 | Chiral separation of dansyl amino
acids
The amino acids, containing an acidic carboxyl groups
and basic amino groups, are a kind of typical amphoteric
chiral compounds not only serving as the basic blocks to
constitute proteins but also serving as the intermediates
of many important synthetic chiral drugs. Therefore,
developing the new methods for the resolution and deter-
mination of amino acid enantiomers are of great signifi-
cance. For the purpose of being easily resolved and
detected, the amino acid is usually dansylated.18 In this
study, eight common DL‐dansylated amino acids were
used as probes to further investigate the chromatographic
properties of TBCDP for amphoteric chiral compounds.
The chemical structures of the selected dansyl amino
acids were shown in Figure 8. The separation results
and some chromatograms were shown in Table 3 and
Figure 9.
It was usually necessary to use the buffer solutions in
the mobile phase, which allowed the dansyl amino acids

SHUANG ET AL.
11
FIGURE 7 The chromatograms of some flavanone enantiomers on triazole‐bridged bis(β‐cyclodextrin)–bonded chiral stationary phase
(TBCDP)
rings that tend to be encapsulated by hydrophobic cavi-
ties of the cyclodextrin, thus facilitating chiral separa-
tions. This was consistent with the reported results of
Wang research group,32,33 which indicated that the sepa-
ration of dansyl amino acids should mainly depend on
the inclusion effects. In addition, encouraging results
was that the TBCDP exhibited higher enantioresolutions
for hydrophilic amino acids such as Dns‐Ser, Dns‐Thr,
and Dns‐Asp, containing polar hydroxymethyl,
hydroxyethyl, and carboxymethyl groups, respectively.
In general, these hydrophilic analytes are not easy to be
encapsulated by hydrophobic cavities of the cyclodextrin;
it is common to see that the derivatized CD‐CSPs has
incapable to separate these compounds.30 However, the
above Dns‐amino acids were separated at the level of
baseline resolutions by TBCDP (Figure 9), which further
confirmed the advantage of the bridged cyclodextrins in
chiral separation.

12
SHUANG ET AL.
FIGURE 8 The chemical structures of
eight dansyl amino acids
used TBCDP to separate ten chiral β‐blockers (Figure 10).
Initially, the chiral separation performed in RP mode, but
it was found that no chiral separation occurred in this
case, which mainly caused by the water of mobile phase
that would destroy the hydrogen bonds. After switching
to the RP mode, the selected β‐blockers were successfully
resolved on TBCDP. The results and the optimized sepa-
ration conditions were listed in Table 4, as well as some
chiral chromatograms in Figure 11.
TABLE 3 The enantioseparation results of dansyl amino acids
and better chromatographic conditions by HPLC
Compounds k′1 k′2
α
Rs Mobile Phases
CSPs
Dns‐DL‐Ala 1.64 1.75 1.07 1.07 1% TEAA/MeOH, TBCDP
2.19 2.21 1.01 0.65 65/35, pH 5.0 CDSP
Dns‐DL‐Leu 2.00 2.59 1.30 2.66 1% TEAA/MeOH, TBCDP
1.16 1.21 1.04 0.97 60/40, pH 5.0 CDSP
Dns‐DL‐Met 2.03 2.22 1.09 1.28 1% TEAA/MeOH, TBCDP
Armstrong et al14 firstly discovered the PO mode
with using ACN/MeOH/HOAc/TEA as the mobile
phase to separate anticholinergic, non‐steroidal anti‐
inflammatory drugs and other chiral drugs that were
difficult to be resolved under the aqueous RP mode.
In this experiment, the effects of the ratio of
ACN/MeOH and HOAc/TEA on the enantiomeric sep-
arations of these β‐blockers were investigated (Figure
S4), the results indicated that the methanol could
accelerate the elution rate, but excess methanol could
reduce the resolution significantly. In addition, adding
a small amount of TEA could reduce the solute reten-
tion and make the shapes of enantioseparation peaks
of these selected alkaline β‐blockers more symmetrical;
as well as an appropriate amount of HOAc could
improve resolution. Therefore, the content of
HOAc/TEA should be adjusted in an appropriate ratio.
After optimizing chromatographic conditions, the good
enantioseparations of the ten selected blockers on
TBCDP were successfully achieved in the PO mode
(Rs, 0.50‐1.71) (Table 4), while the CDSP only sepa-
rated six β‐blockers with lower resolutions (Rs, 0.55‐
0.96).
65/35, pH 5.0
1.49 1.58 1.06 0.70
Dns‐DL‐Ser 1.41 1.57 1.11 1.52 1% TEAA/MeOH, TBCDP
1.47 65/35, pH 5.0 CDSP
Dns‐DL‐Thr 2.74 3.17 1.16 2.11 1% TEAA/MeOH, TBCDP
1.37 75/25, pH 5.0 CDSP
Dns‐DL‐Asp 3.55 3.88 1.09 1.45 1% TEAA/MeOH, TBCDP
1.24 1.34 1.08 0.82 75/25, pH 5.0 CDSP
Dns‐DL‐Phe 2.74 3.40 1.24 2.48 1% TEAA/MeOH, TBCDP
1.75 1.82 1.04 0.87 60/40, pH 5.0 CDSP
Dns‐DL‐Tyr 3.34 4.61 1.38 3.25 1% TEAA/MeOH, TBCDP
1.45 1.62 1.11 0.88 70/30, pH 5.0 CDSP
CDSP
/
/
/
/
/
/
Abbreviations: CDSP, cyclodextrin stationary phase; CSPs, chiral stationary
phases; HPLC, high‐performance liquid chromatography; TBCDP, triazole‐
bridged bis(β‐cyclodextrin)–bonded chiral stationary phase.
3.2.4 | Chiral separation of β‐blockers
The β‐blocker is a class of chiral drugs that has been
widely used in the treatment of cardiovascular diseases.
Due to the rapid development of the species, there is no
systematic enantiomeric monitoring method. Therefore,
it is necessary to develop new materials to separate and
determine the enantiomers of β‐blockers. We herewith
The chiral carbon of β‐blockers possesses the
aminopropanol structure (–N–CH2–C*HOH–CH2–). The

SHUANG ET AL.
13
FIGURE 9 The chromatograms of some dansyl amino acid enantiomers on triazole‐bridged bis(β‐cyclodextrin)–bonded chiral stationary
phase (TBCDP)
–OH on their chiral carbons could form hydrogen bond-
ing with –OH on the cyclodextrins, which was the key
effects in the chiral separation. Since water was a power-
ful hydrogen bond donor that would greatly weaken the
hydrogen bonding, the RP chromatographic mode had
incapability to separate β‐blockers.18,20 The PO mode
with free from water was favorable for hydrogen bonding
for chiral separation of β‐blockers. For example, both
atenolol (Rs = 1.46) and arotinolol (Rs = 1.71) contained
amide groups that had stronger hydrogen bonding with
TBCDP, thus prolonging the retention times (elution of
methanol required about 10%), as well as increasing the
resolutions.
From the resolutions of atenolol (Rs = 1.46), arotinolol
(Rs = 1.71), metoprolol (Rs = 1.56), and esmolol (Rs =
1.47) (Figure 10), we concluded that these linear struc-
tures would easily to enter the cavities of TBCDP.
Although the similarity in structure of bisoprolol and
metoprolol, there was a significant gap in resolutions of
bisoprolol (Rs < 0.5) and metoprolol (Rs = 1.56). The rea-
son may be due to the substituent chain on the benzene
ring of bisoprolol was too long to be encapsulated by
the cyclodextrins, because its enantiomer‐resolution sig-
nificantly decreased (<0.5). Likewise, except for aromatic
ring, propranolol was very similar to carteolol in the
structure (Figure 10), but the resolution (Rs = 1.33) of
propranolol was significantly higher than that of carteolol
(Rs = 0.57). This may be attributed to the fact that
In addition to hydrogen bonding, inclusion also con-
tributed to the above resolutions significantly.26,32,33

14
SHUANG ET AL.
FIGURE 10 The chemical structures of
ten chiral β‐blocker drugs
TABLE 4 The enantioseparation results of β‐blockers and better chromatographic conditions by HPLC
Compounds
k'1
k'2
α
Rs
Mobile Phases
CSPs
Atenolol
7.88
2.67
8.96
4.99
1.14
1.07
1.46
0.55
ACN/MeOH/HAc/TEA (90/10/1/0.9)
TBCDP
CDSP
Arotinolol
Metoprolol
Esmolol
7.64
4.67
9.27
5.18
1.21
1.11
1.71
0.82
ACN/MeOH/HAc/TEA (93/7/1/1)
ACN/MeOH/HAc/TEA (95/5/1/0.4)
ACN/MeOH/HAc/TEA (95/5/0.4/0.4)
ACN/MeOH/HAc/TEA (94/6/1/1)
ACN/MeOH/HAc/TEA (99.4/0.6/1/1)
ACN/MeOH/HAc/TEA (97/3/0.5/0.6)
ACN/MeOH/HAc/TEA (95/5/0.9/0.8)
ACN/MeOH/HAc/TEA (94/6/0.9/1)
ACN/MeOH/HAc/TEA (96/4/1/1)
TBCDP
CDSP
3.00
1.54
3.42
1.67
1.14
1.08
1.56
0.68
TBCDP
CDSP
2.63
2.31
2.98
2.41
1.13
1.04
1.47
0.65
TBCDP
CDSP
Propranolol
Carvedilol
Carteolol
Sotalol
3.90
2.95
4.35
3.15
1.12
1.06
1.33
0.96
TBCDP
CDSP
4.80
3.75
5.49
/
1.14
/
1.30
/
TBCDP
CDSP
3.93
2.88
4.14
/
1.05
/
0.57
/
TBCDP
CDSP
5.06
3.44
5.23
/
1.03
/
<0.5
/
TBCDP
CDSP
Labetalol
Bisoprolol
3.75
2.89
3.89
/
1.04
/
0.54
/
TBCDP
CDSP
2.45
1.94
2.56
/
1.04
/
<0.5
/
TBCDP
CDSP
Abbreviations: CDSP, cyclodextrin stationary phase; HPLC, high‐performance liquid chromatography; TBCDP, triazole‐bridged bis(β‐cyclodextrin)–bonded chi-
ral stationary phase.
hydrophobic naphthalene ring of propranolol could be
easily entrapped by the cavity of TBCDP, while the polar
benzocaprolactam ring of carteolol could not match with
the hydrophobic cavity that caused the inclusion effect of
TBCDP weakened and decreased the resolution of
indicated that the synergistic inclusion effect and other
potential effects of the bridged CD‐CSPs could identify
such chiral β‐blocker drugs.
Based on the above chromatographic evaluation data, it
could be concluded that the bridged CDSP (TBCDP) had a
stronger chiral separation ability and wider separation
range than the single CDSP. When analytes interacted with
the bridged cyclodextrin, the entire guest could be
carteolol.
In
general,
TBCDP
had
potential
enantioseparation ability for some bulky and more com-
plex analytes such as carvedilol and labetalol, which

SHUANG ET AL.
15
FIGURE 11 The chromatograms of some β‐blocker drug enantiomers on triazole‐bridged bis(β‐cyclodextrin)–bonded chiral stationary
phase (TBCDP)
cooperatively encapsulated by the two cavities and a linker
group of the bridged cyclodextrin host. This facilitated the
finer identification of the guest molecules as a whole,
including the recognition of the spatial structure differences
between the R‐ and S‐enantiomers.
high chemical stability and a good chromatographic
reproducibility. The common chiral triazole pesticides
and chiral drugs including flavanones, β‐blockers, and
dansyl amino acids had been successfully resolved under
the RP and PO modes with encouraging results. By con-
structing a new supramolecular host with the two cavities
and a bridging linker without derivatization, the synergis-
tic inclusion, hydrogen bond, steric hindrance, and π‐π
were introduced to chiral separations, thus improving
the enantioselectivity of TBCDP. Therefore, the bridged
CDSP was a kind of multimode stationary phase with
potentially application value.
4 | CONCLUSION
In this paper, a novel TBCDP was prepared by a Click
Chemistry reaction. The preparation method was simple,
rapid, and efficient, and the as‐prepared TBCDP had a

16
SHUANG ET AL.
chromatographic separation of enantiomers. Anal Chem.
1990;62:1610‐1615.
ACKNOWLEDGMENTS
The work was supported by the National Natural Science
Foundation of China (no. 31860469 and 21165012) and
the Open Project of State Key Laboratory of Food Science
and Technology, Nanchang University, China (no. SKLF‐
KF‐201605).
13. Xiao Y, Ng SC, Tan TTY, Wang Y. Recent development of cyclo-
dextrin chiral stationary phases and their applications in
chromatography. J Chromatogr A. 2012;1296:52‐68.
14. Chang SC, Iii GLR, Chen S, Chang CD, Armstrong DW. Evalu-
ation of
a
new polar‐organic high‐performance liquid
chromatographic mobile phase for cyclodextrin‐bonded chiral
stationary phases. TRAC‐Trend Analy Chem. 1993;12:144‐153.
CONFLICT OF INTEREST
15. Armstrong DW. Bonded phase material for chromatographic
separations. U S Patent 4539399. 1985.
The authors have declared no conflict of interest.
16. Li L, Cheng BP, Zhou RD, Cao ZG, Zeng C, Li LS. Preparation
and evaluation of a novel N‐benzyl‐phenethylamino‐β‐cyclodex-
trin‐bonded chiral stationary phase for HPLC. Talanta.
2017;174:179‐191.
ORCID
Yazhou Shuang https://orcid.org/0000-0002-4425-3523
Laisheng Li https://orcid.org/0000-0003-4805-1264
17. Hilton ML, Chang SC, Gasper MP, Pawlowska M, Armstrong
DW, Stalcup AM. Comparison of the enantioselectivity of
phenethyl‐ and naphthylethyl‐carbamate substituted cyclodex-
trin bonded phases. J Chromatogr A. 1993;16:127‐147.
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