HPLC with cellulose Tris (3,5-DimethylPhenylcarbamate) chiral stationary phase: Influence of coating times and coating amount on chiral discrimination

Article abstract of DOI:10.1002/chir.23044

Coating cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) on silica gels with large pores have been demonstrated as an efficient way for the preparation of chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC). During the process, a number of parameters, including the type of coating solvent, amount of coating, and the method for subsequent solvent removing, have been proved to affect the performance of the resultant CSPs. Coating times and the concentration of coating solution, however, also makes a difference to CSPs' performance by changing the arrangement of cellulose derivatives while remaining the coating amount constant, have much less been studied before, and thereby, were systematically investigated in this work. Results showed that CSPs with more coating times exhibited higher chiral recognition and column efficiency, suggesting that resolution was determined by column efficiency herein. Afterwards, we also investigated the effect of coating amount on the performance of CSPs, and it was shown that the ability of enantio-recognition did not increase all the time as the coating amount; and four of seven racemates achieved best resolution when the coating amount reached to 18.37%. At the end, the reproducibility of CDMPC-coated CSPs were further confirmed by two methods, ie, reprepared the CSP-0.15-3 and reevaluated the effect of coating times.

Full text of DOI:10.1002/chir.23044

Received: 5 September 2018
DOI: 10.1002/chir.23044
Revised: 4 December 2018
Accepted: 12 December 2018
R E G U L A R A R T I C L E
HPLC with cellulose Tris (3,5‐DimethylPhenylcarbamate)
chiral stationary phase: Influence of coating times and
coating amount on chiral discrimination
Qiuhong Wei1,2
| Hongjiu Su1 | Diannan Gao1 | Shudong Wang1
1 Dalian National Laboratory for Clean
Energy, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences,
Dalian, P. R. China
2 University of Chinese Academy of
Sciences, Beijing, P. R. China
Abstract
Coating cellulose tris (3,5‐dimethylphenylcarbamate) (CDMPC) on silica gels
with large pores have been demonstrated as an efficient way for the prepara-
tion of chiral stationary phase (CSP) for high‐performance liquid chromatogra-
phy (HPLC). During the process, a number of parameters, including the type of
coating solvent, amount of coating, and the method for subsequent solvent
removing, have been proved to affect the performance of the resultant CSPs.
Coating times and the concentration of coating solution, however, also makes
a difference to CSPs' performance by changing the arrangement of cellulose
derivatives while remaining the coating amount constant, have much less been
studied before, and thereby, were systematically investigated in this work.
Results showed that CSPs with more coating times exhibited higher chiral
recognition and column efficiency, suggesting that resolution was determined
by column efficiency herein. Afterwards, we also investigated the effect of
coating amount on the performance of CSPs, and it was shown that the ability
of enantio‐recognition did not increase all the time as the coating amount; and
four of seven racemates achieved best resolution when the coating amount
reached to 18.37%. At the end, the reproducibility of CDMPC‐coated CSPs
were further confirmed by two methods, ie, reprepared the CSP‐0.15‐3 and
reevaluated the effect of coating times.
Correspondence
Hongjiu Su and Shudong Wang, Dalian
National Laboratory for Clean Energy,
Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, 457
Zhongshan Road, Dalian 116023,
P. R. China.
Emails: suhj@dicp.ac.cn;
wangsd@dicp.ac.cn
KEYWORDS
cellulose tris (3,5‐dimethylphenylcarbamate), chiral recognition, chiral stationary phase,
enantioseparation, high‐performance liquid chromatography, resolution
1 | INTRODUCTION
of their high selectivity, sensitivity, and reproducibility.
Among the derivatives of polysaccharide, cellulose tris
Optical resolution of racemic compounds by high‐
performance liquid chromatography (HPLC) using chiral
stationary phases (CSPs) is one of the most popular
methods for determining optical purity and obtaining
optically pure enantiomers.1-3 Polysaccharide‐based CSP
developed by Okamoto and co‐workers4,5 had achieved
a great reputation in the area of chiral resolution because
(3,5‐dimethylphenylcarbamate) (CDMPC), with two
electron‐donating groups at 3‐ and 5‐position, shows very
high chiral recognition abilities to many racemates.6-8
The CDMPC‐CSP was typically prepared by coating
CDMPC onto large pore silica gel with the coating
amount of 20 wt%.4 The influences of silica gel, CDMPC,
and coating process on chiral discrimination have been
Chirality. 2019;1–10.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

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WEI ET AL.
investigated by many researchers. Much work so far has
focused on the effect of the physical and chemical prop-
erty of silica, including particle size,9 pore size,5,10-12
and functional groups on the silica gel surface.13
Okamoto14 and Zhang15 have reported the influence of
the properties of CDMPC, for instance, the degree of
polymerization or the molecular weight of the cellulose
used for preparing CDMPC and the substitution degree
of the prepared CDMPC. Considerable research efforts
have also been devoted to the influence of the parameters
of coating process, such as the type of coating solvent,5,16
coating amount,5,11 and the method of removing sol-
vent.17 However, few studies have been reported about
the influence of coating times or concentration of coating
solution on the performance of the CDMPC‐CSP.
According to the mechanism of CDMPC‐CSP for chiral
separation proposed by Okamoto et al,18 whether the
arrangement of cellulose derivatives was ordered may
affect the separation performance, and the coating times
and the concentration of the coating solution might affect
the arrangement of cellulose derivatives. On the other
hand, the CDMPC‐CSP had been commercially available
from Daicel Chemical Industries, Ltd. and Chiral Tech-
nologies. Since the expiration of the patents covering
these CSPs, generic materials have become available from
a number of suppliers under different tradenames.
However, the overall performance of these generics often
differ significantly from the original products in terms of
chiral recognition capability, retention behavior, and
column efficiency, because distinct supports and coating
protocols are used for their fabrication (just as all C18
RPC columns are not equivalent).19
size of 10 nm was prepared by agglutination of silica sols
in presence of a polymer, followed by calcination of the
beads. Then, the silica gel with a mean pore size of
80 nm was gained by a certain concentration of NaCl
solution impregnation of small pore silica gel and calcina-
tion of the beads, followed by pickling with hydrochloric
acid. Cellulose was purchased from Sigma‐Aldrich
(Shanghai, China). 3,5‐dimethylphenyl isocyanate was
purchased from InnoChem (Beijing, China). Pyridine,
methanol, tetrahydrofuran (THF), and xylene were ana-
lytical grade, and were purchased from Sinopharm
Chemical Reagent (Shanghai, China). N‐hexane and
isopropanol were HPLC grade, and were purchased from
Spectrum (Shanghai, China) and Sinopharm Chemical
Reagent (Shanghai, China), respectively.
IR spectra were recorded on a Nicolet‐6700 instru-
ment (Thermo Fisher Scientific, USA). The morphology
analysis of the samples was obtained by using a JEOL
JSM‐7800F scanning electron microscope (SEM). The
particle size distribution was determined using the
Malvern Mastersizer 2000 (Malvern Instruments Ltd.,
UK). The pore size distribution was conducted on an
AutoPore IV 9500 (Micromeritics Instrument Corpora-
tion, USA). The specific surface areas were measured
using
a BET surface area analyzer (NOVA‐2000,
Quantachrome, USA). Elemental analysis measurement
was conducted on a CHNS‐analyzer (Elementar Vario
III, Elementar Analysensysteme GmbH, Hanau, Ger-
1
many). The H NMR spectra (700 MHz) were recorded
in tetrahydrofuran‐d8 using a Bruker‐700 spectrometer
(Bruker, USA). Molecular weight was determined by a
Viscotek GPC
liquid chromatograph. THF was used
MAX
In the present study, the influence of the coating
times and coating amount on the chiral discrimination
of the CDMPC‐CSP was investigated. We prepared
CDMPC‐CSP with different coating times and different
concentration of coating solution, and compared the
chromatographic performance of these chiral stationary
phases. The CSP with excellent performance was pre-
pared through optimization, and the plate numbers for
( ) trans‐stilbene oxide exceed 40 000/m. This indicates
coating times or concentration of coating solution should
be taken into account in the preparation process of
CDMPC‐CSP.
as the eluent. Polystyrene standards (Aldrich) were
used for calibration. Chromatographic experiments were
performed using a SHIMADZU HPLC system consisting
of a UV detector, a quaternary pump, a column oven,
and an auto sampler.
2.2 | Preparation of CDMPC‐coated CSP
Tris (3,5‐dimethylphenylcarbamate) cellulose was pre-
pared according to the reported method.4
The different amount of CDMPC (i = 0.05, 0.1, 0.15,
0.2, 0.25 g) was dissolved in 3.5 mL THF. The solution
was added to the silica gel (2 g), and the wetted silica
gel was dried under vacuum after it was dried at ambient
temperature. This coating process was repeated for j
( j = 1, 2, 3, 4, 5) times to get the achieved amount.
According to the above coating method, a series of
CSP‐i‐j samples were prepared when the amount of
CDMPC and coating times were different. The theoretical
coating amount was shown in Table 1
2 | MATERIALS AND METHODS
2.1 | Chemicals and apparatuses
Spherical silica gel, with a particle diameter of 7 μm and
a pore size of 80 nm, was prepared by our group in the
laboratory. Specifically, the silica gel with a mean pore

WEI ET AL.
3
TABLE 1 The coating times and amount per coating of chiral
stationary phases (CSP)‐i‐j and their total coating amount
(Rs), and column efficiency (N) were calculated in the
same way as previous literature.20
Amount
per
Coating times j
coating
i/g
3 | RESULTS AND DISCUSSION
3.1 | Characterizations of prepared CSP
1
2
3
4
5
a
0.05
0.1
‐
4.76
9.09
6.98
9.09
11.11
20
4.76
6.98
9.09
13.04
18.37
23.08
16.67
23.08
As described in Section 2, the wide‐pore silica micro-
spheres were prepared by calcinating silica after NaCl
impregnation. Figure 2A‐C showed the pore size distribu-
tion, particle size distribution, and SEM images of silica
gel, respectively. It was shown that the silica gel had a
pore size of 80 nm and a particle diameter size of 7 μm,
and they were consistent with the results shown in the
SEM image. In addition, it can be seen that the pore size
distribution and particle size distribution of silica gel used
in this study were relatively narrow.
0.15
0.2
13.04
16.67
20
b
b
c
‐
‐
b
c
c
0.25
11.11
‐
‐
‐
aCSP with too low coating amount has almost no chiral recognition ability.
bWhen the coating amount of CDMPC exceeds 25%, the stationary phases
aggregated and could not be packed well.
cCSPs with too high coating amount were also not prepared.
2.3 | Column packing and
The cellulose derivative plays important roles in
realizing the chiral discrimination of enantiomers.
The successful synthesis of CDMPC in this study was
confirmed by Fourier‐transform infrared (FT‐IR), ele-
chromatography
CSP‐i‐j was suspended in a solution consisting of
n‐hexane and isopropanol (n‐hexane/isopropanol, 90/10,
v/v). The suspension was sonicated to form slurries, and
then was packed into empty stainless‐steel column
(150 × 4.6 mm ID) at 40 MPa pressure, with n‐hexane
as the displacing solvent. All the columns were packed
with the same method. Enantioseparation evaluations
were performed in the mobile phases of n‐hexane/
isopropanol (90/10), and the dead times (t0) were deter-
mined with 1, 3, 5‐tris‐ (tert‐butyl) benzene in the same
mobile phase. All of the mobile phases were filtered and
degassed before use. The chiral sample (as shown in
Figure 1) solutions were prepared by dissolving the
analytes in mobile phase(C, D, E, and G) or isopropanol
(A, B, and F) (1 mg mL−1). All the samples were filtered
through 0.45 μm filters. The injection volume was set as
10 μL. The column temperature was set at 30°C, and
the flow rate was 0.5 mL min−1 for the detection of
all analytes. All analytes were measured at 254 nm.
Retention factor (k′), separation factor (α), resolution
1
ment analysis and H NMR. As shown in Figure 3, the
peaks of 3500‐3200 cm−1 in the line of cellulose was
attributed to the stretching vibration of the hydroxyl
group of cellulose, and the peaks of 3000‐2800 cm−1 in
the same line represented the antisymmetric stretching
peak of methylene. The peaks of 3500‐3200 cm−1 in the
line of CDMPC was narrowed, and the strength was
weakened because the hydroxyl group of cellulose was
reacted and a secondary amide appeared. The presence
of new peaks at 1750 cm−1 and 1600‐1450 cm−1 in the
line of CDMPC can be attributed to the carbonyl group
and frame vibration of benzene, respectively, which con-
firmed the successful addition of 3,5‐dimethylphenyl
isocyanate.
Elemental analysis (C33H37N3O8)n found: C 64.04, H
6.28, N 6.75 and calculated: C 65.66, H 6.18, N 6.98.
According to this result, the degree of substitution (DS)
of the hydroxyl group in the cellulose skeleton was 2.68.
FIGURE 1 Structures of racemates

4
WEI ET AL.
FIGURE 2 (A) pore size distribution, (B) particle size distribution, and (C) SEM image of the wide pore silica
FIGURE
cellulose and CDMPC
3
Fourier‐transform infrared (FT‐IR) spectra of
FIGURE 4 1H NMR spectrum of CDMPC in tetrahydrofuran‐d8
(700 MHz)
The 1H NMR spectrum of the obtained cellulose
derivative was shown in Figure 4. The peaks in the range
of δ = 8.0‐8.8, 6.1‐7.2, and 1.8‐2.4 were attributed to the
amino protons, aromatic protons, and methyl protons of
3,5‐dimethylphenylcarbamate, respectively. The peaks in
the range of δ = 2.8‐5.5 were attributed to the protons
of cellulose backbone. Those peaks proved that the
carbanilation of cellulose was achieved successfully.
The peak shape was similar with that cellulose derivative
with DS of 3.0 in previous study.21
The molecular weight of the obtained cellulose deriv-
ative was measured by the gel permeation chromatogra-
phy (GPC), and the result was shown in Table 2.
In order to characterize CSPs, the specific surface area
(SBET), particle size distribution, pore size distribution,
and SEM of CSPs were investigated for selected CSPs.
It was shown in Table 3 that the uncoated silica gel had a
specific surface area of 29.86 m2 g−1, and the CSPs had a
TABLE 2 The molecular weight of the cellulose derivative mea-
sured by gel permeation chromatography (GPC)
Mn‐
Mw‐
Mz‐
Mp‐
Mw/Mn
(Daltons) (Daltons) (Daltons) (Daltons)
172 067 254 679 534 127 161 878
1.480
TABLE 3 The specific surface area and particle size distribution
of chiral stationary phases (CSPs) and uncoated silica
SBET(m2 g−1
)
Particle size (μm)
Uncoated silica
CSP‐0.20‐3
29.86
25.37
24.69
7.13
7.89
7.80
CSP‐0.15‐4
specific surface area of 25.37 m2 g−1 and 24.69 m2 g−1,
respectively. The particle diameter of the uncoated silica
gel was 7.13 μm, and the particle diameters of the two CSPs

WEI ET AL.
5
were 7.89 μm and 7.80 μm, respectively. The pore size dis-
tribution and particle size distribution of the CSP‐0.15‐4
and naked silica were shown in Figure 5. The particle size
of CSP increased slightly, and a new peak of less than
80 nm appeared in the pore size distribution of the pre-
pared CSP. Changes in specific surface area, particle size
distribution, and pore size distribution confirmed that the
CDMPC was coated on silica gel. However, there was less
obvious difference from the SEM images (shown in
Figure 6), and it was not found in the particle agglomera-
tion or the particles with a size of tens of microns from
the SEM images and the patterns of particle size distribu-
tion, which indicated the dispersion of CSPs was very well.
3.2 | Separation properties of prepared
columns
The 18 CSPs prepared in Table 1 were evaluated with
racemates in Figure 1, and the results obtained were
shown in Table S1.
3.3 | Influence of coating times and each
coating amount
Eight groups of chiral stationary phase with different total
coating amount were compared in this section, these CSPs
in each group had the same total coating amount but differ-
ent coating times. The influence of coating times on resolu-
tion Rs was illustrated in Figure 7. The separation results of
seven racemates on each CSP in the eight groups were
shown in Figure 7A‐H, respectively. To gain more insight,
Figure 7I counts the racemates number of achieving better
resolution on the two or three CSPs in each group. For
example, in Figure 7B, five of seven racemates get better
resolution on CSP‐0.05‐3 than in CSP‐0.15‐1. It was indi-
cated that the CSPs with more coating times showed higher
chiral recognition in most groups in Figure 7I, and the rea-
son for this tendency will be explained later.
Figure 8 showed the effect of coating times on separa-
tion factor α, and the data processing method of separa-
tion factor was similar to that of resolution. Different
from the resolution, the CSPs with less coating times
showed higher selectivity in most groups. The concentra-
tion of coating solution decreased with the increase of
coating times when the total coating amount was the
same. As we all know, CDMPC forms a lyotropic liquid
crystal phase at the high solution concentrations.
FIGURE
5
The pore size distribution (A) and particle
distribution (B) of uncoated silica and the CSP‐0.15‐4
FIGURE 6 SEM image of (A) uncoated silica and (B) CSP‐0.20‐3

6
WEI ET AL.
FIGURE 7 Resolution of seven racemates on chiral stationary phases (CSPs) in eight groups (A‐H), and the racemates number of
achieving better resolution on the two or three CSPs in each group (I). CA is the coating amount. The first line of the abscissa in each
figure represents the coating times, and the second line represents the racemates to be separated (A‐H) or the total coating amount (I)
FIGURE 8 The racemates number of achieving better separation
factor on the two or three chiral stationary phases (CSPs) in each
group
FIGURE 9 The retention factors of seven racemates

WEI ET AL.
7
However, when the dilute solution was used, it would
turn to be more and more denser during the evaporation
of the solvent. Ultimately, all of the coating solution was
at high concentration before it was coated on silica, so all
of the CDMPC solution could form an ordered structure.
The decrease of separation selectivity with coating times
may result from the different orientation of the CDMPC
coated by every time.
The influence of coating times on retention factors k1′
was shown in Figure 9. Almost no change of k1′ value
was observed in each group. The total coating amount of
every CSP in each group was the same, so the amount of
adsorption site was the same, thus they had similar
retention factor. The influence of coating times on column
efficiency was shown in Figure 10. It can be seen that the
CSPs with more coating times showed higher column effi-
ciency in most groups. It is easy to form a homogeneous
coating on the surface of silica with increasing the coating
times and decreasing the concentration of coating solution.
Then, it was in favor of the diffusion of the samples
between the stationary phase and the mobile phase, and
the efficiency of the column became higher.
The influence of coating times on resolution, separa-
tion factor, retention factor, and column efficiency were
summarized in Table 4. It was worthwhile mentioning
that the column efficiency and resolution displayed the
same tendency. It is well‐known that resolution can
be expressed as a function of the retention factor k1′
(term a), the separation factor α (term b), and column
efficiency or the plate number N (term c), as shown in
Equation 1. In the present work, from the results we have
obtained, it is concluded that the resolution was deter-
mined by column efficiency when the coating times of
these CSPs were different.
ꢀ ꢁꢂ
ꢃ
FIGURE 10 The racemates number of achieving higher column
efficiency on the two or three chiral stationary phases (CSPs) in
each group
1
4
k
Rs ¼
ðα − 1Þ N0:5
:
k þ 1
(1)
ðaÞ
ðbÞ
ðcÞ
TABLE 4 The influence of coating times on resolution, separation factor, and column efficiency
Coating Amount %
Coating Times
Resolution
Separation Factor
Column Efficiency
CSP‐0.10‐1
CSP‐0.05‐2
4.76
1
2
√
√
√
CSP‐0.15‐1
CSP‐0.05‐3
6.98
9.09
1
3
√
√
√
√
CSP‐0.20‐1
1
CSP‐0.10‐2
CSP‐0.05‐4
2
4
√
√
√
√
CSP‐0.25‐1
CSP‐0.05‐5
11.11
13.04
16.67
1
5
√
CSP‐0.15‐2
CSP‐0.10‐3
2
3
√
√
√
√
√
√
√
√
√
√
CSP‐0.20‐2
CSP‐0.10‐4
2
4
CSP‐0.25‐2
CSP‐0.10‐5
20
2
5
√
√
CSP‐0.20‐3
CSP‐0.15‐4
23.08
3
4

8
WEI ET AL.
As it was plotted in Figure 11A, the retention time of 1, 3,
5‐tris‐(tert‐butyl) benzene noted as dead time t0 on these
column decreased as the coating amount increased. This
was because the coating of CDMPC resulted in a smaller
pore size and a shorter diffusion distance. The influence
of coating amount on retention factor k1′ could be seen in
Figure 9. The amount of adsorption site increased
with coating amount, so the retention factor increased.
In terms of separation factor α (shown in Figure 11B),
except for B, they hardly changed with the change of
coating amount, which indicated that the coating amount
had little effect on the separation factor. As shown in
Figure 12, the effect of coating amount on column
efficiency N was the same as that of previous studies,22
and an excessive amount of CDMPC affected the intra‐
particle diffusion of the samples, lowering performance
of the columns.
3.4 | Influence of total coating amount
Overall, it was revealed that columns with low coating
amount (less than 12%) showed very poor chiral recogni-
tion, and the highest resolution for all of the racemates
never appeared in these columns. The reason for this
low resolution was that the amount of the chiral selector
was too low to offer sufficient energy difference for the
two enantiomers. On the other hand, when the coating
amount was low, the silanol group of the bare silica gel
might provide an achiral effect. However, the ability of
enantio‐recognition did not increase all the time as the
coating amount, and four of seven racemates achieved
best resolution when the coating amount was 18.37%.
To investigate the influence of coating amount on
each parameters (t0, k1′, α, and N), CSPs with the same
coating times need to be selected. In this study, the CSPs
with the coating times of three were taken as an example.
3.5 | Reproducibility of CDMPC‐coated
CSPs
The CSP‐0.15‐3 with the best separation performance was
reprepared and evaluated, and the evaluation parameters
were compared with the original one in the previous
section. Figure 13 showed the results of reprepared
CSP‐0.15‐3 with the resolution of the seven substances
on the left and the column efficiency on the right. It
can be seen from the figure that the reproducibility was
good whether it was resolution or column efficiency.
CSPs with a total coating amount of 15% and coating
times of 1, 2, 3, and 4 were prepared and evaluated, and
their separation performance was compared according
to the processing method mentioned before. Table S2
FIGURE 11 The influence of coating amount on dead time (A)
FIGURE 12 The influence of coating amount on column
and separation factor (B)
efficiency (N)

WEI ET AL.
9
CDMPC‐CSP was investigated. The coating times had
great influence on the chiral separation performance
under the same coating amount. Resolution and column
efficiency increased with the increase of coating times,
while separation factor decreased with the increase of
coating times. Resolution was determined by column
efficiency. The CSPs with the coating times of three were
selected to investigate the influence of coating amount. It
was found that the coating amount had a significant
effect on retention factor and column efficiency. The
CSP with excellent performance was prepared through
optimization. When the coating amount was 18.37%, the
plate numbers for ( ) trans‐stilbene oxide exceed
40 000/m, and four of seven racemates achieved the best
resolution. The reproducibility of CDMPC‐coated CSPs
were confirmed by repreparing the CSP‐0.15‐3 and
reevaluating the effect of coating times.
FIGURE 13 The results of reprepared CSP‐0.15‐3
ACKNOWLEDGMENT
We gratefully acknowledge support from the Youth Inno-
vation Promotion Association of CAS.
ORCID
Qiuhong Wei https://orcid.org/0000-0002-2356-5050
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