Functionalities tuned enantioselectivity of phenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC

Article abstract of DOI:10.1002/chir.22732

The mixed chloro- and methyl- functionalities can greatly modulate the enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs). A comparison study is herein reported for per(4-chloro-3-methyl)phenylcarbamate and per(2-chloro-5-methyl)phenylcarbamate β-CD clicked CSPs (i.e., CCC4M3-CSP and CCC2M5-CSP). The enantioselectivity dependence on column temperature was studied in both normal-phase and reversed-phase mode high performance liquid chromatography (HPLC). The thermodynamic study revealed that the stronger intermolecular interactions can be formed between CCC4M3-CSP and chiral solutes to drive the chiral separation. The higher enantioselectivities of CCC4M3-CSP were further demonstrated with the enantioseparation of 17 model racemates in HPLC.

Full text of DOI:10.1002/chir.22732

Received: 4 April 2017
DOI: 10.1002/chir.22732
Revised: 11 May 2017
Accepted: 24 May 2017
R E G U L A R A R T I C L E
Functionalities tuned enantioselectivity of phenylcarbamate
cyclodextrin clicked chiral stationary phases in HPLC
Jian Tang
| Yuzhou Lin | Bo Yang | Jie Zhou | Weihua Tang
School of Chemical Engineering, Nanjing
University of Science and Technology,
Nanjing, China
Abstract
The mixed chloro‐ and methyl‐ functionalities can greatly modulate the
enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary
phases (CSPs). A comparison study is herein reported for per(4‐chloro‐3‐methyl)
phenylcarbamate and per(2‐chloro‐5‐methyl)phenylcarbamate β‐CD clicked CSPs
(i.e., CCC4M3‐CSP and CCC2M5‐CSP). The enantioselectivity dependence on
column temperature was studied in both normal‐phase and reversed‐phase mode high
performance liquid chromatography (HPLC). The thermodynamic study revealed
that the stronger intermolecular interactions can be formed between CCC4M3‐CSP
and chiral solutes to drive the chiral separation. The higher enantioselectivities of
CCC4M3‐CSP were further demonstrated with the enantioseparation of 17 model
racemates in HPLC.
Correspondence
Weihua Tang, School of Chemical
Engineering, Nanjing University of Science
and Technology, 200 Xiaolingwei Street,
Nanjing 210094, China.
Email: whtang@njust.edu.cn
Funding information
National Natural Science Foundation of
China, Grant/Award Number: 21305066;
Doctoral Fund of Ministry of Education
of China, Grant/Award Number:
20103219120008
KEYWORDS
chiral separation, enantioselectivity, high‐performance liquid chromatography, phenylcarbamate
cyclodextrin
1 | INTRODUCTION
prepared CSPs exhibited much improved enantioselectivities
in both normal‐phase (NP) and reversed‐phase (RP) HPLC.
The safety concern of racemic pharmaceuticals has been
awakened since the thalidomide tragedy. Chiral analysis and
purification have become a prerequisite process in drug dis-
covery.1 High‐performance liquid chromatography (HPLC)
has been employed as an important option for both chiral
analysis and manufacturing by the use of chiral stationary
phases (CSPs).2-13 To date, chemically bonded cyclodextrins
(CDs) CSPs have aroused prominent interest due to their
practicality in different modes of HPLC.14,15 To form
noncovalent “host–guest” inclusion complexes, CDs and
their derivatives have found wide applications in drug
delivery and enantioseparation. The “click chemistry”16 has
recently evolved as a facile and efficient synthesis approach
in the preparation of CSPs,17-22 with thiol‐ene clicked
perphenylcarbamate β‐CD CSP demonstrating good
enantioselectivities in HPLC.20
The enhanced enantioseparations in RP‐HPLC were attrib-
uted to the formation of extra hydrogen bonding between
‐NH‐ and C=O in phenylcarbamate moieties, which facili-
tated the formation of the inclusion complex with enantio-
mers adopting a favorable conformation. In addition, π–π
stacking and dipole–dipole interactions also contributed to
the enantioseparation.4,6,23,24
The functionalities modulated enantioselectives of CD
clicked CSPs have been a research theme in our group.19,22,24
A
series of mixed chloro‐ and methyl‐substituted
phenylcarbamate β‐CD clicked CSPs has been devel-
oped.18,19 The introduction of an electron‐donating methyl
group or an electron‐withdrawing halogen at the 3‐ and/or
4‐position of the phenyl ring on phenylcarbamate polysac-
charide (cellulose and amylose) CSPs was found to improve
their chiral recognition ability towards many racemates.25
In order to reveal the functionalities‐tuned
enantioselectivities in multiple modes HPLC, per(4‐chloro‐
We recently explored the perphenylcarbamate β‐CD
derived CSPs via azide‐yne click reaction.19,22 The as‐
Chirality. 2017;1–8.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

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TANG ET AL.
3‐methyl)phenylcarbamate CD clicked CSP (CCC4M3‐CSP)
and per(3‐chloro‐4‐methyl)phenylcarbamate one (CCC3M4‐
CSP) as well native CD clicked CSP were developed for
the enantioseparation of a large library of racemates.18 We
herein further report the comparison study of CCC4M3‐CSP
and per(2‐chloro‐5‐methyl)phenylcarbamate clicked CSP
(CCC2M5‐CSP) for enantioseparation in both NP and RP
modes of HPLC. The effect of flow rate and column temper-
ature on the enantioseparation was studied. The thermody-
namic study revealed that the functionalities on CD rims can
greatly affect the intermolecular interactions between CSPs
and chiral solutes for chiral separation. The better
enantioselectivities of CCC4M3‐CSP were further demon-
strated with 17 racemates in both NP and RP HPLC. The
enantioselectivities can be tuned by optimizing the composi-
tion of mobile phases.
lab and packed into columns (4.6 × 250 mm) accord-
ingly.15,18 Typically, 6A–azido‐β‐cyclodextrin was function-
alized by reacting with 4‐chloro‐3‐methylphenylisocyanate
or 5‐chloro‐2‐methyl phenylisocyanate in dry pyridine. The
CD derivatives were then clicked onto an alkynyl surface‐
modified silica support to afford target CSPs. The structures
of racemates and two CSPs are depicted in Figure 1.
Kromasil spherical silica gel (5 μm, 100 Å) from Eka
Chemicals (Bohus, Sweden) was used as the support. The
CCM3C4‐CSP possessed a content of 12.28% for C atom,
0.822% for N atom, and 2.064% for H atom; while
CCC2M5‐CSP had 10.78% C, 0.35% N, and 1.835% H. the
contents of CNH atoms were greatly increased when
compared with the corresponding 6.90% C, 0.271% N, and
1.526% H for alkynyl silica. The surface loading of
CCC4M3‐CSP and CCC2M5‐CSP was calculated to be
0.21 and 0.18 μmol/ m2.
All HPLC chiral separations were performed at Agilent
1260 system (Agilent Technologies, Palo Alto, CA). The
system is equipped with a G1315D diode array detection
(DAD) system, G1329B quaternary pump, G1331C auto-
matic injector, G1316A temperature controller, and Agilent
Chem Station data manager software (v. No.C.01. 04).
For NP HPLC, all analytes were prepared into 200 μg/mL
solutions in IPA. For RP HPLC, all analytes were prepared
into 200 μg/mL solutions in MeOH and stored at 4 °C. Every
2 | MATERIALS AND METHODS
HPLC‐grade solvents including n‐hexane (Hex), 2‐propanol
(IPA) methanol (MeOH), ethanol (EtOH), and acetonitrile
(ACN) were purchased from Tedia (Fairfield, OH). HPLC‐
grade trifluoroacetic acid (TFA) and triethylamine (TEA)
were purchased from J&K (Shanghai, China). All racemates
were procured from J&K. Two CSPs were prepared in our
FIGURE 1 Chemical structure of 17 analytes and CSPs studied

TANG ET AL.
3
injection of 10 μL sample solution was adopted. The detec-
tion wavelength range was 200–300 nm. All mobile phases
were degassed with ultrasonication and all samples were
filtered through a 0.22 μm membrane before use.
time and higher Rs, while the enantioselectivity (α) remained
unchanged with varied flow rate. The highest Rs of 8.15 was
obtained with CCC4M3‐CSP at 0.4 ml/min, while only 4.24
with CCC2M5‐CSP at the flow rate of 0.4 mL/min. Taking
a close look at the enantioseparation chromatograms of
3′‐hydroxyflavanone with two CSPs under every flow rate,
one can find that CCC4M3‐CSP demonstrated all higher Rs
and α values for 3′‐hydroxyflavanone than CCC2M5‐CSP,
but with much longer retention. This may indicate that
CCC4M3‐CSP can form stronger interactions with chiral
solutes than CCC2M5‐CSP. When the flow rate of Hex/EtOH
(70:30, v/v) increased from 1.0 to 1.4 mL/min, the column
pressure increased from 8.5 to 10 MPa for CCC4M3‐CSP,
while 8 to 9 MPa for CCC2M5‐CSP.
The enantioresolution capability of two CSPs in RP
HPLC at increased flow rates was evaluated in MeOH/H2O
(90/10) mobile phase (Figure 2C,D). Both the retention time
and Rs of 3′‐hydroxyflavanone increased when the flow rate
of the mobile phase decreased from 1.4 to 0.4 mL/min. Sim-
ilarly, CCC4M3‐CSP demonstrated both higher Rs and α
values for 3′‐hydroxyflavanone than CCC2M5‐CSP in RP
mobile phases at every flow rate. The highest Rs of 8.73
was achieved with CCC4M3‐CSP, while only 2.82 with
CCC2M5‐CSP. Meanwhile, the retention of chiral solutes
with CCC4M3‐CSP became stronger than that with
CCC2M5‐CSP, reflected by much longer retention times.
3 | RESULTS AND DISCUSSION
3.1 | Effect of flow rate on enantioseparation
of two CD CSPs
The flow rate of a mobile phase is a crucial parameter for the
chromatographic analysis in HPLC.24 The flow rate can
greatly affect the column pressure, which in turn changes
the retention and peak profiles of solutes. The optimized flow
rate can afford the desired chiral resolutions (Rs) of CSPs
within suitable analysis times. The optimization of flow rate
for two clicked CSPs was thus investigated in both NP and
RP HPLC.
The enantioseparation on CCC4M3‐CSP and CCC2M5‐
CSP at various flow rates was investigated with
3′‐hydroxyflavanone as the model analyte by gradually
increasing the flow rate of the mobile phase from 0.4 to
1.4 mL/min. For NP HPLC, Hex/EtOH (70/30, v/v) was
employed as the mobile phase. The enantioseparation chro-
matograms at varied flow rates are shown in Figure 2A,B.
The decreased flow rate resulted in an increased retention
FIGURE 2 Enantioseparation chromatograms of 3′‐hydroxyflavanone at different flow rates with A, CCC4M3‐CSP and B, CCC2M5‐CSP in NP
HPLC, as well as with C, CCC4M3‐CSP, and D, CCC2M5‐CSP in RP HPLC

4
TANG ET AL.
When the flow rate of MeOH/H2O (90:10, v/v) increased
from 1.0 to 1.4 mL/min, the column pressure was found to
increase from 10 to 14 MPa for CCC4M3‐CSP, while 9 to
13.5 MPa for CCC2M5‐CSP.
Taking a close look at the peak profile of chiral solutes,
one would find that the peak tailing intensified with the
decrease of the flow rate in mobile phases. Taking
enantioselectivity, column pressure, and analysis speed into
consideration, the flow rate of 1.0 mL/min was chosen for
further study.
and Rs for chiral solutes than CCC2M5‐CSP, indicating its
stronger chiral recognition ability.
In MeOH/H2O (90/10) mobile phase, similar behaviors
were observed for the enantioseparation of 3′‐
hydroxyflavanone on both CSPs as in NP HPLC. CCC4M3‐
CSP demonstrated much better enantioselectivities than
CCC2M5‐CSP at every column temperature. The highest Rs
of 10.26 and α of 5.03 was obtained on CCC4M3‐CSP at
5 °C. The enantioselectivities of two CSPs decreased when
increasing the column temperature, but the peak profile
improved with less peak tailing.
In order to elucidate the higher enantioselectivities of
CCC4M3‐CSP with 4‐chloro‐3‐methyl functionality to con-
struct a stronger interaction, the thermodynamics of the chiral
separation were investigated. The thermodynamic process
can be expressed using the Gibbs–Helmholtz equation:
ΔG0i ¼ ΔHi0−TΔSi0 .26 And the dependence of retention on
temperature can be described as the van't Hoff equation:
3.2 | Thermodynamic study of column
Optimization of the column temperature should be consid-
ered a basic tenet of chiral separations in HPLC since it can
influence the retention, selectivity, and resolution.13,24 It is
generally acknowledged that direct separation is, to a large
extent, dependent on the formation of diastereomeric associ-
ates via intermolecular interactions of enantiomers with CD.
For this purpose, the column temperature was optimized in
both NP and RP mode HPLC as for the flow rate discussed
above. The temperature tuned enantioselectivities of both
CCC4M3‐CSP and CCC2M5‐CSP were studied by gradually
increasing the column temperature from 5 °C to 45 °C at
intervals of 10 °C.
ΔH°i
ΔS°i
lnki ¼ − T1 ∙
þ
þ ln Φ,27 where ki, Hi, Si, R, T, and
R
R
Φ are the retention factors for the solute, the standard partial
molar enthalpy of transfer, the standard partial molar entropy
of transfer, the gas constant, the absolute temperature, and the
phase ratio (i.e., the volume of the stationary phase divided by
the volume of the mobile phase). The correlation of selectivity
and temperature can thus be deduced as follows27:
In Hex/EtOH (70/30, v/v) mobile phase, both α and Rs
values of 3′‐hydroxyflavanone on both CSPs decreased
dramatically with the increase in column temperature
(Table 1). The highest Rs of 15.26 and 6.12 were obtained
for 3′‐hydroxyflavanone at 5 °C on CCC4M3‐CSP and
CCC2M5‐CSP, respectively. However, the retention of the
second enantiomer was found to be 2‐fold longer on
CCC4M3‐CSP than CCC2M5‐CSP. With the decrease in col-
umn temperature, the peak tailing became worse. In the whole
temperature range, CCC4M3‐CSP exhibited much higher α
ð−ΔH2 þ ΔH1Þ ðΔS2−ΔS1Þ
lnα ¼ ln k2− ln k1 ¼
þ
RT
R
By plotting lnα against 1/T, one can determine the partial
À
Á
molar enthalpy of transfer [ΔHod ¼ ΔH2o−ΔH1o ꢀ thermody-
namic parameters from the fitting lines. The plots of lnα
against 1/T on both CSPs were explored for 3′‐
hydroxyflavanone in Hex:EtOH (70:30) and MeOH:H2O
(90:10) mobile phases. The correction coefficients, r, of all
TABLE 1 Thermodynamic results for 3′‐hydroxyflavanone with CCC4M3‐CSP and CCC2M5‐CSP
Cond. Ib
Cond. Ic
Temp.
(°C)a
Thermodynamics at
Cond. I
Thermodynamics at
Cond. II
CSP
α
Rs
α
Rs
CCC4M3‐CSP
5
15
25
35
45
6.08
4.91
3.97
3.35
2.87
15.26
12.96
8.71
7.42
7.66
5.03
4.21
3.46
2.93
2.48
10.26
8.84
7.82
6.99
6.02
lnα = 1670.20/T‐4.21
ΔHod= −13.89 kJ/mol
r = 0.99934
lnα = 1573.76/T‐4.05
ΔHod= − 13.08 kJ/mol
r = 0.99983
CCC2M5‐CSP
5
15
25
35
45
2.40
2.11
1.84
1.65
1.50
6.12
5.39
3.53
3.16
2.68
2.00
1.78
1.58
1.44
1.29
3.86
2.81
2.39
2.14
1.33
lnα = 1051.3/T‐
2.91
ΔHod= −8.74
r = 0.99929
lnα = 963.95/T‐2.77
ΔHod= −8.01
r = 0.99976
aColumn temperature for HPLC.
bHex/EtOH (70/30).
cMeOH/H2O (90/10).

TANG ET AL.
5
fitting curves were at least 0.99734 and 0.99929 for
CCC4M3‐CSP and CCC2M5‐CSP, respectively.
exhibited higher enantioselectivities to all analytes except
coumachlor in comparison to CCC2M5‐CSP when the same
HPLC conditions were employed.
Table 1 summarizes the thermodynamic results for 3′‐
hydroxyflavanone on both CSPs. In general, more negative
ΔHod values represent stronger interactions between the
isomer and CSP.28 With a close look at the thermodynamic
results of 3′‐hydroxyflavanone on two CSPs in the same
mobile phase, one can find that the ΔHod values were more
negative with CCC4M3‐CSP than with CCC2M5‐CSP in
both mobile phases studied. This indicates that CCC4M3‐
CSP can construct stronger intermolecular interactions to
drive the chiral recognition process. The chiral solutes hence
have stronger retention on CCC4M3‐CSP, which agrees well
with the longer retention time observed for enantiomers in
HPLC. The thermodynamic results are in good agreement
with the HPLC results. It should be noted that the thermody-
namic results of chiral solutes on the same CSP cannot
be used to explain its enantioselectivity difference in
different mobile phases, due to different chiral recognition
mechanisms.
3.4 | Enantioselectivities of CD clicked CSPs
in RP HPLC
To investigate the effect of mobile phase (MeOH/H2O) com-
position on the enantioseparations of flavonoids, the polarity
of the elution solvent was tuned by adjusting the methanol
content in the mobile phase for RP HPLC. Quite different
behaviors were observed for the chiral resolution of
flavonoids with two CSPs (Figure 3). In terms of flavonoids,
all analytes could be baseline‐resolved by CCC4M3‐CSP.
7‐Methoxyflavanone and 6‐methoxyflavanone achieved
better enantioseparation than 7‐hydroxyflavanone and 6‐
hydroxyflavanone by CCC4M3‐CSP and CCC2M5‐CSP,
indicating that the position of the substituent on the aromatic
ring played an important role in the enantioseparation
in RP HPLC. Compared with 7‐hydroxyflavanone, 3′‐
hydroxyflavanone possessed much better enantioresolution
(Rs > 6.5), indicating that the position of the hydroxyl group
would dramatically affect the inclusion complexation.
CCC2M5‐CSP exhibited decreased enantioselectivities
with increased methanol contents in mobile phases for
five flavonoids (including flavanone as well as 7‐methoxy‐,
6‐methoxy‐, 4′‐hydroxy‐ and 3′‐hydroxyflavanone).
CCC4M3‐CSP, however, showed continuous increased Rs
for 7‐methoxy‐, 6‐methoxy‐ and 3′‐hydroxyflavanone with
increased methanol content. The enantioseparation of
flavanone, 6‐hydroxy‐ and 4′‐hydroxyflavanone with
CCC4M3‐CSP was found to be relatively insensitive to
methanol content in the studied mobile phases.
A direct comparison study on the effect of organic solvents
on the enantioselectivities of two CSPs was demonstrated with
flavanone, 6‐methoxyflavanone, and 3′‐hydroxyflavanone. As
shown in Figure 4, by using organic solvent/H2O (80/20, v/v)
as the mobile phase, all three racemates achieved excellent
enantioseparation (Rs: 4.8 ~ 7.8) by CCC4M3‐CSP with
MeOH as the organic modifier. However, flavanone and
6‐methoxyflavanone only achieved baseline separation by
CCC2M5‐CSP under the same chromatographic conditions,
while 3′‐hydroxyflavanone achieved far decreased Rs in compar-
ison to that with CCC4M3‐CSP. By using ACN/H2O (80/20), all
enantiomers showed faster elution on both CSPs but with
dramatically decreased enantioseparations. And 3′‐flavanone
even harvested slightly higher Rs on CCC2M5‐CSP with
ACN/H2O (80/20) as the mobile phase.
3.3 | Enantioseparation comparison of two
clicked CSPs in NP‐HPLC
As is known, the chiral separations of racemates are mainly
driven by the intermolecular interactions between CD func-
tionalities and chiral solutes in NP HPLC, due to the occupa-
tion of the CD cavity by less polar eluent molecules. These
interactions may include hydrogen‐bonding, dipole–dipole
and π–π interactions.19,20,29 In RP HPLC, however, the inclu-
sion complexation between CD CSPs and chiral solutes can
act as an extra driving force for the chiral separation.27 The
thermodynamic study as discussed above indicates that
CCC4M3‐CSP can form stronger intermolecular interactions
with chiral solutes than CCC2M5‐CSP in both modes HPLC.
This behavior reveals that the functionalities on CD rims can
fine‐tune the driving forces for chiral recognition by optimiz-
ing the modification of phenylcarbamate groups.
The functionality‐tuned enantioselectivities of two
phenylcarbamate CD clicked CSPs was first investigated with
NP HPLC. The optimized chiral resolutions for 17 racemates
were compared with CCC2M5‐CSP (Table 2). By varying
the volume ratios of Hex/EtOH or Hex/IPA mobile phases,
the enantioseparations of three flavonoids in Hex/EtOH
(80/20, 70/30, 60/40, and 50/50) mobile phases were
explored and compared with two CSPs.21 As we know, the
addition of alcohol additives can result in increased retention
of chiral eluents; on the one hand, it may worsen the
enantioseparation on the other. We herein further explored
the enantioseparation of the flavonoids in Hex/EtOH
(90/10), where most of them obtained their maximum
Rs values. Among the selected racemates, CCC4M3‐CSP
Based on the results discussed above, our two
chloromethylphenylcarbmated CD clicked CSPs exhibited
quite different enantioselectivities for the model racemates,
which may be explained by the difference of interactions
between CD and two enantiomers. Similar substituents

6
TANG ET AL.

TANG ET AL.
7
FIGURE 3 Chiral resolutions of
flavonoids tuned by methanol content in
mobile phase with two CSPs at 25 °C
FIGURE 4 Enantioselectivities comparison of two CSPs for three flavanoids under different mobile phases in RP HPLC at 25 °C
position‐tuned enantioselectivities were observed earlier for
chloromethylphenylcarbarmated cellulose CSPs,25 where
the meta‐ and para‐disubstituted derivatives were found to
show higher chiral recognition than ortho‐ and meta‐ or
para‐disubstituted ones. The reason was that with more
acidic N‐H protons of meta‐ and para‐disubstituted
phenylcarbamate, celluloses would interact more strongly
with appropriate enantiomers via hydrogen‐bonding. This
may explain the higher enantioselectivities of CCC4M3‐
CSP than CCC2M5‐CSP in our cases.
exhibited a much better enantioseparation ability than 5‐
chloro‐2‐methylphenylcarbarmate one under the same
mobile phase, flow rate, and column temperature. The chi-
ral resolutions of 17 racemates were explored and compared
with both CSPs in both modes HPLC.
ACKNOWLEDGMENTS
We gratefully acknowledged the funding from the National
Natural Science Foundation of China (Grant No. 21305066)
and Doctoral Fund of Ministry of Education of China (No.
20103219120008).
4 | CONCLUSION
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How to cite this article: Tang J, Lin Y, Yang B, Zhou
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