Immobilization and chromatographic evaluation of novel regioselectively substituted amylose-based chiral packing materials for HPLC

Article abstract of DOI:10.1002/chir.21000

The regioselectively substituted amylose derivatives bearing a 4-tert-butylbenzoate or 4-chlorobenzoate group at 2-position, and 3,5-dichlorophenylcarbamate and a small amount of 3-(triethoxysilyl) propylcarbamate groups at 3- and 6-positions were synthesized by a two-step process based on the esterification of 2-position of a glucose unit. The obtained derivatives were effectively immobilized onto macroporous silica gel by intermolecular polycondensation of triethoxysilyl groups. Their chiral recognition abilities were evaluated as chiral packing materials (CPMs) for high-performance liquid chromatography. These CPMs showed high chiral recognition as well as the conventional coated-type CPM, and can be used with the eluents-containing chloroform and tetrahydrofuran. With the extended use of these eluents, improvement of chiral recognition and reversed elution orders were realized. For some racemates, the immobilized CPM exhibited ability comparable or better to the commercial immobilized amylose- or cellulose-based columns,

Full text of DOI:10.1002/chir.21000

CHIRALITY 23:878–886 (2011)
Immobilization and Chromatographic Evaluation of Novel
Regioselectively Substituted Amylose-based Chiral Packing Materials
for HPLC
JUN SHEN, PENGFEI LI, SHUANGYAN LIU, XIANDE SHEN, AND YOSHIO OKAMOTO1,2*
1
1
1
1
1
Polymer Materials Research Center, College of Material Science and Chemical Engineering, Harbin Engineering University, Harbin 150001, China
2
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan
ABSTRACT
The regioselectively substituted amylose derivatives bearing a 4-tert-butylben-
zoate or 4-chlorobenzoate group at 2-position, and 3,5-dichlorophenylcarbamate and a small
amount of 3-(triethoxysilyl)propylcarbamate groups at 3- and 6-positions were synthesized by a
two-step process based on the esterification of 2-position of a glucose unit. The obtained deriva-
tives were effectively immobilized onto macroporous silica gel by intermolecular polycondensa-
tion of triethoxysilyl groups. Their chiral recognition abilities were evaluated as chiral packing
materials (CPMs) for high-performance liquid chromatography. These CPMs showed high chi-
ral recognition as well as the conventional coated-type CPM, and can be used with the eluents-
containing chloroform and tetrahydrofuran. With the extended use of these eluents, improve-
ment of chiral recognition and reversed elution orders were realized. For some racemates, the
immobilized CPM exhibited ability comparable or better to the commercial immobilized amy-
lose- or cellulose-based columns, Chiralpak IA, IB, and IC. Chirality 23:878–886, 2011. VV C 2011
Wiley-Liss, Inc.
KEY WORDS: regioselective substitution; resolution; enantioseparation; chiral stationary
phases; high-performance liquid chromatography; amylose; immobilization;
chiral packing materials
INTRODUCTION
(THF), chloroform, ethyl acetate, toluene, acetone, and so
on, which can damage the packed columns by dissolving or
swelling the polymer inside. These solvents may lead to a
better solubility of racemates and more efficient enantiosepa-
ration in both analytical and large-scale preparative HPLC.
Therefore, the immobilization of the regioselectively substi-
tuted polysaccharide derivatives is highly desired to
enhance their solvent versatility and stability as well as reso-
lution efficiency.
The goal of this study is to develop the efficient CPMs
based on the novel regioselective polysaccharide derivatives
with a high chiral recognition ability and universal solvent
durability. In continuation of our previous study with coated
CPMs prepared from the amylose derivatized by regioselec-
tively substituted groups, amylose derivatives having 4-tert-
butylbenzoate and 4-chlorobenzoate at 2-position and
3,5-dichlorophenylcarbamate and a controlled small amount
Polysaccharide-based derivatives, especially phenylcarba-
mate and benzoate derivatives of cellulose and amylose, are
among the most efficient chiral stationary phase (CSP) for
high-performance liquid chromatography (HPLC) and a
broad range of chiral compounds have been sufficiently sepa-
rated by the chiral packing materials (CPMs) based on these
1
–11
CSPs.
To derivatize the polysaccharide, the same sub-
stituents are usually introduced onto the three positions of
the glucose unit. And the selective substitution have been
1
2
only restricted between the 6-position and 2-, 3-positions.
The regioselective substitution at all three positions has not
been realized until Dicke reported the method of selective
1
3
esterification at 2-position of amylase. Several series of
polysaccharide derivatives bearing different substituents on
2
-, 3-, and 6-positions have been successfully developed by
1
4–16
our group recently.
These regioselective substituents at
different position seem to change the structure and local po-
9
,11
larity of the polysaccharide derivatives,
whose chiral rec-
ognition abilities will then be significantly influenced by the
nature of the substituents on the aromatic moieties.
Contract grant sponsor: National Natural Science Foundation of China;
Contract grant numbers: 51073046.
Contract grant sponsor: Natural Science Foundation of Heilongjiang Prov-
ince; Contract grant numbers: E201012.
Contract grant sponsor: Special Fund for Scientific and Technological Innova-
tive Talents in Harbin City; Contract grant numbers: 2011RFLXG009.
Contract grant sponsor: Fundamental Research Funds for the Central Univer-
sities; Contract grant numbers: HEUCFT1009.
9
,17–26
Among the coated-type CPM that we previously prepared,
those bearing 4-tert-butylbenzoate and 4-chlorobenzoate
groups at 2-position and 3,5-dichlorophenylcarbamate at 3-
and 6-positions (1 and 3) (Fig. 1) exhibited a higher chiral
recognition than the others with different substituents at
Contract grant sponsor: Daicel Chemical Industries (Tokyo, Japan).
1
5
*
Correspondence to: Yoshio Okamoto, Polymer Materials Research Center,
three positions. Some racemates could be more efficiently
resolved on them than on the commercially available col-
umns.
College of Material Science and Chemical Engineering, Harbin Engineering
University, Harbin 150001, China; Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8603, Japan. E-mail: okamoto@apchem.nagoya-u.ac.jp
Received for publication 5 May 2011; Accepted 21 June 2011
DOI: 10.1002/chir.21000
On the other hand, the coated-type CPMs prepared by
the conventional coating procedure have much limitation in
the usage of organic solvents, such as tetrahydrofuran
Published online 20 September 2011 in Wiley Online Library
(wileyonlinelibrary.com).
VV C 2011 Wiley-Liss, Inc.

CHROMATOGRAPHIC ABILITY OF NOVEL AMYLOSE DERIVATIVES
879
Fig. 1. Structures of amylose derivatives.
of 3-(triethoxysilyl)propyl residue as a crosslinkable group at
- and 6-positions were prepared (Fig. 1). Then the immobili-
(Bruker). The thermogravimetric analyses (TGA) was performed using
TGA Q 50 (TA).
3
zation of the amylose derivatives onto the surface of silica
gel were efficiently performed via the intermolecular poly-
condensation of the triethoxysilyl groups as reported by our
Synthesis of Regioselective Amylose Derivatives Bearing 2-
(
4-Tert-butylbenzoate) and 2-(4-Chlorobenzoate) at 2-
Position and 3,5-Dichlorophenylcarbamate/3-
1
6,27
group.
The chiral recognition abilities of the coated-
CPMs and immobilized-CPMs were evaluated by HPLC and
compared with those of commercially available columns. The
influence of the amount of 3-(triethoxysilyl)propyl residues
introduced onto 3- and 6-position of the amylose derivatives
on their chiral recognition abilities was also investigated.
(
Triethoxysilyl)propyl Residues at 3- and 6-Positions
The amylose derivatives 2a–c bearing 4-tert-butylbenzoate at 2-posi-
tion and 3,5-dichlorophenylcarbamate/3-(triethoxysilyl)propylcarbamate
residues at 3- and 6-positions were synthesized by the regioselective
esterification of 2-position of amylose using the method reported by
Dicke, followed by one-pot procedure reported by our group
). The regioselective esterification of the 2-hydroxy group was realized
by the following steps. First, the amylose (3.0 g) was dissolved in DMSO
60 ml) at 808C. Second, vinyl 4-tert-butylbenzoate (2.3 equiv to 2-posi-
tion) and Na HPO (2 wt% of amylose, as the catalyst) were added to the
solution at 408C, and the reaction was continued with stirring for 21 days
until the esterification of the 2-position was complete. The reaction mix-
ture was added in a large excess of 2-propanol, and the product was
recovered as an insoluble fraction in a 98% yield. The obtained mono-
ester was then dissolved in a mixture of N,N-dimethylacetamide, lithium
chloride, and pyridine. After completely dissolved, it was allowed to react
with 3,5-dichlorophenyl isocyanate at 808C, which not only converted
most of the hydroxyl groups at 3- and 6-positions into carbamates but
also eliminated the water in the reaction system at the same time. After
reacted for 6 h, the calculated amount of 3-(triethoxysilyl)propyl isocya-
nate was then added, and the reaction was continued for 16 h at 808C.
Finally, an excess of 3,5-dichlorophenyl isocyanate was added into reac-
tion mixture and reacted for 7 h at 808C to complete the carbamoylation
of the underivatized hydroxyl groups. Three amylose derivatives 2a–c
bearing different amounts of the 3-(triethoxysilyl)propylcarbamate resi-
dues at 3- and 6-positions were isolated as a methanol-insoluble fraction.
The amylose derivatives 4a–d bearing 4-chlorobenzoate at 2-position
and 3,5-dichlorophenylcarbamate/3-(triethoxysilyl)propylcarbamate resi-
dues at 3- and 6-positions were synthesized by similar procedure. The
degree of substitution at three different positions and yields of 2a–c and
4a–d are summarized in Table 1, respectively.
13
27,28
(Fig.
2
EXPERIMENTAL
Materials
(
Amylose (DP-300) and 3,5-dichlorophenyl isocyanate were gifts from
Daicel Chemical Industries (Tokyo, Japan). Vinyl acetate and dimethyl
sulfoxide (DMSO) were purchased from Guangfu (Tianjin, China).
Other solvents, such as acetonitrile, THF, and pyridine, were purchased
from Kermel (Tianjin, China), which were dehydrated by distillation
before use. Vinyl 4-tert-butylbenzoate and vinyl 4-chlorobenzoate were
obtained from Wako (Tokyo, Japan). 3-(Triethoxysilyl)propyl isocyanate
was obtained from Azmax (Chiba, Japan). Wide-pore silica gel (Daiso gel
SP-1000) with a mean particle size of 7 lm and a mean pore diameter of
2
4
100 nm was kindly supplied by Daiso Chemical (Osaka, Japan). The sol-
vents used in chromatographic experiments were of HPLC grade. The
racemates were commercially available or prepared by the usual meth-
ods.
Apparatus and Chromatography
Chromatographic experiments were performed using a JASCO PU-
2089 chromatograph equipped with UV/vis (JASCO UV-2070) and circu-
lar dichroism (JASCO CD-2095) detectors at room temperature. A solu-
tion of a racemate (3 mg/ml) was injected into the chromatographic sys-
1
tem through an intelligent sampler (JASCO AS-2055). The H NMR
spectra (500 MHz) were recorded using a Bruker-500 spectrometer
Chirality DOI 10.1002/chir

8
80
SHEN ET AL.
Fig. 2. Scheme of the synthesis of amylose derivatives 2 and 4.
Immobilization of Amylose Derivatives by Intermolecular
Polycondensation of Triethoxysilyl Groups
in vacuo. The immobilization efficiency was estimated by thermogravi-
metric analysis with a TGA instrument.
Seven amylose derivatives 2a–c and 4a–d (0.35 g) were first coated
Preparation and Evaluation of Packed Columns
on the plain silica gel (1.40 g) after completely dissolving in THF (8 ml)
9
according to the previous way. Then the 2-coated and 4-coated silica
The 2-immobilized and 4-immobilized silica gels were packed in a
stainless-steel tube (25 3 0.20 cm i.d.) by a slurry technique. The plate
numbers of the packed columns were 1800–2700 for benzene using hex-
ane/2-propanol (90/10, v/v) mixture as the eluent at the flow rate of 0.1
gels (0.65 g) were added into a mixture of ethanol, water, and trimethyl-
silyl chloride (6/1.5/0.1, v/v/v). After stirring for 10 min at 1108C, the
immobilized CPMs were sufficiently washed with THF and dried at 608C
TABLE 1. Synthesis of regioselective amylose derivatives 2a-c and 4a-d bearing a benzoate group at 2-position and 3,5-dichloro-
phenylcarbamate/3-(triethoxysilyl)propylcarbamate groups at 3- and 6-positions
Amount of 3-(triethoxysilyl)
propyl isocyanate(mol%)
Degree of
substitution(R )
Degree of
substitution(R /R )
a
1 b
2
3 b
Amylose derivatives
Yield(%)
2
2
2
4
4
4
4
a
b
c
a
b
c
d
0.8
2.3
3.5
1.0
2.1
3.4
4.2
1.0
1.0
1.0
1.0
1.0
1.0
1.0
99.4/0.6
98.0/2.0
97.2/2.8
99.2/0.8
98.4/1.6
97.3/2.7
96.5/3.5
95
98
91
88
88
84
87
a
On the basis of hydroxyl groups of amylose.
See Figure 1.
b
Chirality DOI 10.1002/chir

CHROMATOGRAPHIC ABILITY OF NOVEL AMYLOSE DERIVATIVES
881
1
Fig. 3. H NMR spectrum of the amylose derivative 4a in DMSO-d
6
at 808C.
ml/min. The dead time (t
zene as a nonretained compound.
0
) was determined using 1,3,5-tri-tert-butylben-
amylose solution to selectively esterify only the hydroxyl
group at 2-position of amylose. The monoester A-2 was
obtained with a high yield shown in Table 1. The 3,5-dichlor-
ophenyl isocyanate was first added to partly convert 3- and 6-
hydroxy groups of amylose into 3,5-dichlorophenylcarbamate
residues and to eliminate water from the reaction system. A
desired amount of 3-(triethoxysilyl)propyl isocyanate was
then added, and finally, the underivatized hydroxy groups
were treated with an excess of 3,5-dichlorophenyl isocyanate.
The content ratio of the 3,5-dichlorophenylcarbamate to
29
RESULTS AND DISCUSSION
Synthesis of Regioselective Amylose Derivatives Bearing A
Controlled Amount of 3-(Triethoxysilyl)propyl Residues at
3
- and 6-Positions
The regioselectively substituted amylose derivatives 2a2c
were synthesized by a sequential procedure as shown in Fig-
ure 2. The vinyl 4-tert-butylbenzoate was first added into the
Fig. 4. Immobilization scheme of the amylose derivatives. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Chirality DOI 10.1002/chir

8
82
SHEN ET AL.
a
b
TABLE 2. Immobilization of 2a-c and separation factors (a) on 2-immobilized CPMs , 1-coated CPM and Daicel
c
Chiralpak columns
CPMs
d
2
aI
2bI
2cI
2
3
e
2
3
e
2
3
e
b
c
Racemates
(R /R 599.4/0.6) 32%
(R /R 598.0/2.0) 89%
(R /R 597.2/2.8) 97%
1-Coated CPM
Daicel chiralpak columns
5
6
7
8
9
1
1
1
1
1
1.56 (1)
1.39 (2)
1.40 (2)
1.31 (1)
1.19 (2)
1.14 (1)
1.20 (2)
ꢀ 1 (1)
2.00 (1)
1.11 (2)
1.20 (1)
2.25 (2)
1.45 (2)
1.21 (1)
2.08 (2)
ꢀ 1 (1)
1.28 (2)
1.22 (1)
1.84 (1)
1.13 (2)
1.21 (1)
1.29 (2)
1.41 (2)
1.30 (1)
1.34 (2)
1.20 (1)
ꢀ 1 (2)
1.41 (1)
2.51 (1)
1.13 (2)
1.19 (1)
2.50 (2)
1.51 (2)
1.20 (1)
2.19 (2)
ꢀ 1
1.52 (1) IC
2.71 (1) IA
1.33 (1) IB
2.07 (1) IA
1.28 (2) IC
2.42 (2) IB
1.96 (1) IC
1.26 (2) IB
2.06 (1) IA
0
1
2
3
4
1.20 (2)
1.21 (1)
1.92 (1)
1.16 (2)
a
Column: 25 3 0.20 cm(i.d.); flow rate: 0.1 mL/min; eluent: hexane/2-propanol (90/10,v/v).
Data taken from Ref. 15.
Data of IA and IB taken from Ref. 27, data of IC was obtained under the same conditions as IA and IB. Column:25 3 0.46 cm(i.d.); flow rate: 0.5ml/min; eluent:
b
c
hexane/2-propanol(90/10,v/v). The signs in parentheses indicate the optical rotation of the first-eluted enantiomer.
d
Data taken from Ref. 16. The signs in parentheses indicate the CD detection at 254 nm of the first-eluted enantiomer.
e
Immobilization efficiency.
3
-(triethoxysilyl)propylcarbamate was controlled by the
3- and 6- positions. After the immobilization, the 2-immobi-
lized and 4-immobilized silica gels were thoroughly washed
and then dried. The immobilization efficiency is defined as
the ratio of the (immobilized amylose derivative)/(coated
amylose derivative), which was estimated from the organic
content in the CPMs by thermogravimetric analysis.
amount of 3-(triethoxysilyl)propyl isocyanate. The regioselec-
tive amylose derivatives 4a-d were synthesized by similar
procedure as shown in Figure 2. Figure 3 shows the
1
H
NMR spectrum of the amylose derivative 4a. The ratio of
3,5-dichlorophenylcarbamate)/(3-(triethoxysilyl)propylcar-
bamate) was estimated from (aromatic proton)/(SiCH ) in-
(
Table 2 shows the immobilization efficiencies of deriva-
tives 2a–c on silica gel. Compared with the amylose deriva-
tives 2b–c, which have a relatively higher amount of the 3-
2
2
7
tensity ratio.
Immobilization of Regioselective Amylose Derivatives
2
3
(
triethoxysilyl)propyl group (R /R 5 98.0/2.0 and 97.2/2.8,
The immobilization process is illustrated in Figure 4. The
derivatives 2 and 4 were first coated on the plain silica gel
and then was dispersed into a mixture of ethanol, water, and
trimethylsilyl chloride to be immobilized onto the surface of
silica gel by polycondensation of the triethoxysilyl groups at
respectively) at 3- and 6-positions, less amount of 2a was im-
mobilized on the silica gel during the immobilization pro-
cess. As shown in Table 3, similar phenomena can be
observed when compared the immobilization efficiency of
derivatives 4a with those of 4b2d. In 2a and 4a, some amy-
a
TABLE 3. Immobilization of 4a-d and separation factors (a) on 4-immobilized CPMs , 3-coated CPM, and Daicel
b
Chiralpak columns
CPMs
4
aI
4bI
4cI
4dI
2
3
2
3
2
3
2
3
(
R /R = 99.2/0.8)
(R /R = 98.4/1.6)
(R /R = 97.3/2.7)
(R /R = 96.5/3.5)
3-Coated
CPM
Daicel chiralpak
c
c
c
c
b
Racemates
53%
64%
70%
73%
columns
5
6
7
8
9
1.54 (1)
1.90 (2)
1.84 (1)
1.31 (1)
ꢀ 1 (2)
1.14 (1)
1.18 (1)
1.92 (1)
1.71 (1)
1.04 (2)
1.37 (1)
1.73 (2)
1.42 (1)
1.32 (1)
ꢀ 1 (2)
1.23 (1)
ꢀ 1 (1)
1.76 (1)
2.05 (1)
1.04 (2)
1.29 (1)
1.30 (2)
1.07 (1)
1.32 (1)
ꢀ 1 (2)
1.34 (1)
ꢀ 1 (1)
1.82 (1)
2.03 (1)
1.10 (2)
1.23 (1)
1.27 (2)
1.07 (1)
1.25 (1)
ꢀ 1 (2)
1.28 (1)
ꢀ 1 (1)
1.61 (1)
1.97 (1)
1.06 (2)
1.91 (1)
2.15 (2)
2.97 (1)
1.60 (1)
ꢀ 1 (2)
1.0 (1)
1.68 (1)
1.32 (1)
2.21 (1)
2.17 (2)
1.52 (1) IC
2.71 (1) IA
1.33 (1) IB
2.07 (1) IA
1.28 (2) IC
2.42 (2) IB
1.96 (1) IC
1.26 (2) IB
2.06 (1) IA
10
11
12
13
14
a
Column: 25 3 0.20 cm (i.d.); flow rate: 0.1 ml/min; eluent: hexane/2-propanol (90/10, v/v).
b
Data of IA and IB taken from Ref. 27, data of IC was obtained under the same conditions as IA and IB. Column: 253i.d.); flow rate: 0.5 ml/min; eluent: hexane/2-
propanol(90/10, v/v). The signsin parentheses indicate the optical rotation of the first-eluted enantiomer. Column: 253i.d.); flow rate: 0.5ml/min; eluent: hexane/
-propanol(90/10, v/v). The signs in parentheses indicate the CD detection at 254 nm of the first-eluted enantiomer.
2
c
Immobilization efficiency.
Chirality DOI 10.1002/chir

CHROMATOGRAPHIC ABILITY OF NOVEL AMYLOSE DERIVATIVES
883
Fig. 5. Structures of racemates 5–14.
lose chains may have no or only a few 3-(triethoxysilyl)propyl
residues, which must make efficient immobilization difficult.
On the other hand, the high immobilization efficiencies of
tiomers on them were the same. The a values on 2aI–2cI
were slightly larger for racemates 5, 8, 10, 11, 12, and 13
and slightly smaller for 6, 7, 9, and 14 than on 1-coated
CPM, implying that the procedure used in the study is effi-
cient for the immobilization of amylose derivatives enabling
to maintain their chiral recognition abilities and elution order
of enantiomers. Among three immobilized CPMs, 2bI CPM
showed chiral recognition ability comparable to the 1-coated
CPM. The higher order structure of 2b after the immobiliza-
tion seems to be similar to that of 1-coated on the silica gel.
The slightly lower recognition of 2aI may be ascribed to the
low 2a content on silica gel.
2
b2c and 4b2d indicate that only a few percent of 3-(trie-
thoxysilyl)propyl residues is sufficient for efficient immobili-
zation.
The obtained immobilized CPMs were packed into HPLC
columns, and their chiral recognition abilities were evaluated
with 10 racemates (5-14, Fig. 5). Figure 6 shows the chro-
matogram of the resolution of racemic trans-stilbene oxide
(
6) on the immobilized CPM 4aI. The enantiomers were
eluted at the retention time t and t , respectively. The dead
1
2
time (t ) was determined for 1,3,5-tri-tert-butylbenzene to be
The highest a values for 10 racemates on three commer-
cial immobilized-type chiral columns, Chiralpak IA, IB, and
IC, consisting of amylose tris(3,5-dimethylphenylcarbamate),
cellulose tris(3,5-dimethylphenylcarbamate), and cellulose
tris(3,5-dichlorophenylcarbamate) as the CSPs, respectively,
are selected and also included in Table 2 for comparison. In
general, the derivatives 2aI-2cI bearing a 4-tert-butylbenzoyl
group at 2-position showed an equivalent or higher chiral
recognition abilities for some racemates compared to Chiral-
pak IA, IB, and IC using the hexane-2-propanol (90/10, v/v)
mixture as the standard eluent. Especially, 2aI–2cI obtained
much more efficient resolution for racemates 7 and 9, which
cannot be resolved efficiently on the three commercial immo-
bilized columns. And the a value for racemate 9 on 2b-im-
mobilized CPM has appeared to be the highest one com-
0
0
8
.65 min. After the retention factors, k ((t – t )/t ) and
1
1
0
0
0
k ((t – t )/t ), were estimated to be 0.82 and 1.76, respec-
2
2
0
0
0
0
tively, the separation factor a (k /k ) was then readily
2
1
defined to be 1.90, and a baseline separation of racemate 6
was attained.
Evaluation of Chiral Recognition Ability of Immobilized
CPMs
The results of chromatographic resolutions of 10 race-
mates (5–14) on the immobilized CPMs 2aI–2cI are sum-
marized in Table 2, which also included the resolution
results on 1-coated CPM. The 2-immobilized CPMs exhib-
ited similar or slightly different chiral recognition ability com-
pared to 1-coated one, and the elution orders of the enan-
16
pared to several commercially available columns. Race-
mates 12 and 13 were both better resolved on 2cI than the
three commercial columns.
3
The content of the R residues introduced onto the glu-
cose ring plays a critical role in the immobilization process.
To efficiently prepare an immobilized CPM without losing its
3
high chiral recognition, the content of the R residues should
3
be as low as possible, because the R residues may disturb
the regular structure of the polysaccharide derivative more
or less. Nevertheless, the immobilization efficiency
3
decreased when the R content became lower, as shown in
Table 2. Considering the two sides of its effect, the CPM
3
obtained from 2b bearing 2.0% R seems to be preferable
than those from the other two derivatives although the three
2
-immobilized CPMs have the same 4-tert-butylbenzoate
group at 2-positioin and small difference in amounts of 3-(trie-
thoxysilyl)propyl group at 3- and 6-positions, indicating that
Fig. 6. Chromatogram for the resolution of 6 on 4aI CPM. Column: 25 3
.20 cm (i.d.); flow rate: 0.1 ml/min; eluent: hexane/2-propanol (90/10, v/v).
0
Chirality DOI 10.1002/chir

8
84
SHEN ET AL.
TABLE 4. Separation factors (a) on 2bI CPM and 4bI CPM
bI CPM
a
2
4bI CPM
b
b
Racemates
H/I (90/10)
H/C/I(90/10/1)
H/C (70/30)
H/T (70/30)
H/I (90/10)
5
6
7
8
9
1
1
1
1
1
1.20 (1)
2.25 (2)
1.45 (2)
1.21 (1)
2.08 (2)
ꢀ 1 (1)
1.28 (2)
1.22 (1)
1.84 (1)
1.13 (2)
1.51 (1)
1.28 (2)
1.24 (2)
1.21 (1)
1.29 (2)
ꢀ 1
1.22 (1)
ꢀ 1
1.17 (1)
1.36 (2)
1.17 (2)
1.57 (1)
1.38 (1)
1.00
ꢀ 1 (1)
ꢀ 1 (1)
1.09 (1)
1.06 (2)
1.37 (1)
1.73 (2)
1.42 (1)
1.32 (1)
ꢀ 1 (2)
1.23 (1)
ꢀ 1 (1)
1.76 (1)
2.05 (1)
1.04 (2)
1.56 (1)
1.22 (2)
ꢀ 1 (2)
1.13 (1)
ꢀ 1 (2)
1.11 (2)
ꢀ 1
0
1
2
3
4
1.12 (2)
1.15 (1)
ꢀ 1 (1)
1.12 (2)
1.13 (2)
a
Column:25 30.20 cm (i.d.); flow rate: 0.1 ml/min; eluent: H, hexane; I,2-propanol; c,chloroform; T, tetrahydrofuran
Data taken from Ref. 16 except for data of racemate 14.The signs in paretheses indicate CD detection at 254 nm of the first-eluted enantiomer.
b
the immobilization efficiency has a great effect on the chiral
recognition abilities of these immobilized CPMs. As already
mentioned, 2a showed low immobilization efficiency (32 %),
and therefore, its immobilized CPM 2aI should have a rather
high bare silica gel surface without immobilized 2a, which
must reduce its chiral recognition.
The chromatographic resolutions of 10 racemates (5–14)
on the immobilized CPMs 4aI–4dI are summarized in Ta-
ble 3, which also includes the data on 3-coated CPM and
the commercial Chiralpak IA, IB, and IC. Using the hexane/
Influence of Eluents on Chiral Recognition Ability of the
Immobilized CPMs
The chiral recognition ability of 2b-immobilized CPM
under nonstandard eluents containing chloroform and THF
was investigated, and the results are shown in Table 4. By
using chloroform and THF, which are the prohibited eluents
for 1-coated CPM, the resolution for racemates 5, 7, 8, and
0 was much improved on 2bI in comparison with that on
-coated CPM in Table 2. Especially, the extended use of
chloroform with different ratio can lead to different contribu-
tion to the improvement of the chiral recognition ability. For
example, 10% chloroform containing eluent (H/C/I 5 90/
1
1
2
-propanol (90/10, v/v) as the eluent, four immobilized
CPMs 4aI–4dI showed similar or slight variation in chiral
recognition abilities compared to those of 3-coated one,
such as the larger a values for racemates 10 and 12 and
slightly smaller values for the others except for 9. In con-
trast to Chiralpak IA, IB, and IC under the standard eluent,
racemates 5 and 7 were equally or better resolved on 4aI
and 4bI. And racemate 12 was more efficiently resolved on
1
0/1, v/v/v) can lead to a more efficient enantioseparation
for racemate 5, and better resolution for 7, 8, and 10 could
be obtained when increased chloroform ratio to 30% (H/C 5
7
0/30, v/v). In the separation of 7, 8, and 12, the sign of the
CD detection was reversed by changing eluents from hex-
ane/2-propanol (90/10, v/v) to hexane/chloroform (70/30,
v/v) as shown in Table 4. This result may be ascribed to the
reversed elution order of enantiomers, although the possibil-
ity of the reversal in the CD sign of some compounds caused
by the change of solvents cannot completely excluded. The
reverse elution order of 7 was actually confirmed using both
pure enantiomers of 7 on 2bI CPM. This indicates that the
stereoselectivity can be very sensitive and varied depending
on changes of the composition of the mobile phase, which is
4
aI–4dI than the three commercial immobilized columns.
The immobilization efficiency gradually increased when the
3
R content increased from left (4aI, 53%) to right (4dI, 73%).
3
The CPM obtained from 4a bearing 0.8% R content seems
to be desirable than those from the other three derivatives.
The immobilization efficiency (53 %) of 4a is higher than
that (32 %) of 2a, which may be ascribed to the relatively
high recognition of 4aI.
a
TABLE 5. Separation factors (a) on 4aI CPM and 2aI CPM
2
3
2
3
4
aI CPM (R /R 599.2/0.8)
2aI CPM (R /R 599.4/0.6)
Racemates
H/I (90/10)
H/C/I (90/10/1)
H/T/I (90/10/1)
H/I (90/10)
5
6
7
8
9
1.54 (1)
1.90 (2)
1.84 (1)
1.31 (1)
ꢀ 1 (2)
1.14 (1)
1.18 (1)
1.92 (1)
1.71 (1)
1.04 (2)
1.50 (1)
1.26 (2)
1.22 (1)
1.20 (1)
ꢀ 1 (2)
1.09 (1)
1.27 (1)
1.73 (1)
ꢀ 1 (1)
ꢀ 1 (2)
1.23 (1)
1.45 (1)
1.41 (2)
1.22 (1)
ꢀ 1
1.26 (1)
1.39 (2)
1.40 (2)
1.31 (1)
1.49 (2)
1.14 (1)
1.20 (2)
ꢀ 1 (1)
2.00 (1)
1.11 (2)
10
11
12
13
14
1.05 (1)
1.25 (2)
1.38 (1)
ꢀ 1 (1)
ꢀ 1 (2)
a
Column: 25 3 0.20 cm (i.d.); flow rate: 0.1 ml/min; eluent: H, hexane; l, 2-propanol; c, chloroform; T, tetrahydrofuran. The signs in parentheses indicate the CD
detection at 254 nm of the first eluted enantiomer.
Chirality DOI 10.1002/chir

CHROMATOGRAPHIC ABILITY OF NOVEL AMYLOSE DERIVATIVES
885
2
7
in agreement with the previous results. And the conforma-
tion of the amylose derivatives may be altered in these elu-
ents, which can partially explain the improvement of chiral
recognition and reversed elution orders. The better resolu-
tion for racemate 8 and reversed elution order for racemate
ble content, the novel immobilized CPMs could achieve
more efficient resolution and reversed elution order for some
racemates, which are difficult to be resolved on the coated
CPMs or the commercially available columns, indicating that
this method for immobilization of CPMs based on the regio-
selectively substituted amylose derivatives is useful to
improve the chiral recognition ability and the solvent compat-
ibility.
1
7
1 were also observed using a hexane-THF mixture (H/T 5
0/30, v/v) as the eluent, whereas no obvious improvement
was found under the eluent composition as H/T/I 5 90/10/
, v/v/v.
The data for 4bI CPM bearing a similar R /R content ra-
1
2
3
LITERATURE CITED
tio to that of 2bI was also included in Table 4 for compari-
son. The only difference between the two CPMs is that 2bI
has an electron-donating substituent, 4-tert-butyl group,
whereas 4bI contains an electron-withdrawing group, 4-
chloro, on the benzoate group at the 2-position of glucose
unit. Five racemates, such as 6, 7, 9, 11, and 14, were bet-
ter resolved on the former under the same chromatographic
condition using hexane and 2-propanol mixture as eluent. In
particular, racemates 9 and 11 could not be separated on
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009;109:6077–6101.
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bI, but baseline separation was attained on 2bI. And race-
6
7
8
9
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The influence of eluents on the chiral recognition abilities
of 4aI CPM was also explored. As shown in Table 5, the re-
solution for racemates 10 and 12 was effectively improved
after adding 10% chloroform (H/C/I 5 90/10/1, v/v/v) or
THF (H/T/I 5 90/10/1, v/v/v) to the standard eluent in
comparison with the 3-coated CPM in Table 3. However,
more content of chloroform or THF, such as the eluent com-
position of H/C 5 70/30 and H/T 5 70/30, cannot contrib-
ute to the improvement of recognition ability. Moreover, the
extended use of THF (H/T/I 5 90/10/1, v/v/v) also
reversed the elution orders of the enantiomers 6, 7, and 11
in comparison with the coated CPM. The complementary
chiral recognition ability of 2aI and 4aI can also be
observed, especially for the resolution of racemates 9 and
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2. This also implies that the chiral recognition and higher
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CONCLUSIONS
lose derivatives bearing a 4-tert-butylbenzoate group at the 2-position and
3
3
2
,5-dichlorophenylcarbamate/3-(triethoxysilyl)propylcarbamate groups at
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Novel immobilized amylose derivatives bearing 4-tert-butyl-
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phenylcarbamate/3-(triethoxysilyl)propylcarbamate at 3- and
1
1
1
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dichlorophenylcarbamoylation and 3-(triethoxysilyl)propyl-
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silica gel via intermolecular polycondensation of triethoxy-
silyl groups introduced at 3- and 6-positions, their chiral rec-
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some racemates, the immobilized derivatives containing a
certain amount of the 3-(triethoxysilyl)propyl residue showed
high chiral recognition abilities that are comparable to the
conventional-coated CPMs and the commercially available
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