Chromatographic Resolution of α-Amino Acids by (R)-(3,3'-Halogen Substituted-1,1'-binaphthyl)-20-crown-6 Stationary Phase in HPLC

Article abstract of DOI:10.1002/cjoc.201600664

Three new chiral stationary phases (CSPs) for high-performance liquid chromatography were prepared from R-(3,3'-halogen substituted-1,1'-binaphthyl)-20-crown-6 (halogen = Cl, Br and I). The experimental results showed that R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 (CSP-1) possesses more prominent enantioselectivity than the two other halogen-substituted crown ether derivatives. All twenty-one α-amino acids have different degrees of separation on R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6-based CSP-1 at room temperature. The enantioselectivity of CSP-1 is also better than those of some commercial R-(1,1'-binaphthyl)-20-crown-6 derivatives. Both the separation factors (α) and the resolution (R_s) are better than those of commercial crown ether-based CSPs [CROWNPAK CR(+) from Daicel] under the same conditions for asparagine, threonine, proline, arginine, serine, histidine and valine, which cannot be separated by commercial CR(+). This study proves the commercial usefulness of the R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 chiral stationary phase.

Full text of DOI:10.1002/cjoc.201600664

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DOI: 10.1002/cjoc.201600664
Chromatographic Resolution of α-Amino Acids by
(R)-(3,3'-Halogen Substituted-1,1'-binaphthyl)-20-crown-6
Stationary Phase in HPLC
Peng Wu,a,# Yuping Wu,a,# Junhui Zhang,a Zhenyu Lu,a Mei Zhang,b Xuexian Chen,a and
Liming Yuan*,a
a Department of Chemistry, Yunnan Normal University, Kunming, Yunnan 650500, China
b Department of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming,
Yunnan 650500, China
Three new chiral stationary phases (CSPs) for high-performance liquid chromatography were prepared from
R-(3,3'-halogen substituted-1,1'-binaphthyl)-20-crown-6 (halogen＝Cl, Br and I ). The experimental results showed
that R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 (CSP-1) possesses more prominent enantioselectivity than the
two other halogen-substituted crown ether derivatives. All twenty-one α-amino acids have different degrees of sep-
aration on R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6-based CSP-1 at room temperature. The enantioselectivity
of CSP-1 is also better than those of some commercial R-(1,1'-binaphthyl)-20-crown-6 derivatives. Both the separa-
tion factors (α) and the resolution (Rs) are better than those of commercial crown ether-based CSPs [CROWNPAK
CR(＋) from Daicel] under the same conditions for asparagine, threonine, proline, arginine, serine, histidine and va-
line, which cannot be separated by commercial CR(＋). This study proves the commercial usefulness of the
R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 chiral stationary phase.
Keywords α-amino acid, chiral crown ethers, stationary phase, high-performance liquid chromatography
Introduction
ture (5－10 ℃) is needed. To develop new and effec-
tive crown ether-based CSPs is really a challenge task.
In the present study, we report a series of stationary
phases of R-(3,3'-halogen substituted-1,1'-binaphthyl)-
20-crown-6 coated on silica gel. The preparation of
three novel chiral crown ether stationary phases,
R-(3,3'-dibromo-1,1'-binaphthyl)-20-crown-6 (CSP-1),
R-(3,3'-dichloro-1,1'-binaphthyl)-20-crown-6 (CSP-2),
and R-(3,3'-diiodo-1,1'-binaphthyl)-20-crown-6 (CSP-3)
(Figure 1) was initiated from binaphthol. The chiral
recognition ability of CSP-1 was greater than that of the
commercial CROWNPAK CR(＋) column for the reso-
lution of α-amino acids.
Chiral stationary phases (CSPs) have been estab-
lished as the most accurate and convenient means for
the exact determination of the enantiomeric composition
of chiral compounds in high-performance liquid chro-
matography (HPLC). Various CSPs have been devel-
oped.[1,2] The crown ether CSPs have attracted the most
attention since they were developed by Sogah and Cram
in the 1970s.[3] (3,3'-Diphenyl-1,1-binaphthyl)-20-
crown-6,[4-8] (＋)-(18-crown-6)-2,3,11,12-tetracarbox-
ylic acid,[9,10] and phenolic pseudo chiral crown
ethers[11,12] are the most effective CSPs of the crown
ether type.[13] A series of CSPs based on immobilized
chiral crown ethers [(＋)-18-crown-6 tetracarboxylic
acid] and (3,3-diphenyl-1,1-binaphthyl)-20-crown-6 on
polystyrene or silica gel showed good resolution ability
for α-amino acids and their derivatives.[14-21] The related
commercialized CSP known as CROWNPAK CR(＋)
(Daicel Chemical Industries) has proven to be useful for
the chromatographic resolution of chiral compounds
that contain a primary amino group.[22-24] However, the
enantioseparation of some α-amino acids on commer-
cialized CSPs is inconvenient because a lower tempera-
Experimental
Apparatus
The HPLC system consisted of a Shimadzu LC-15C
HPLC pump and an SPD-15C UV/V detector (Japan)
operating at 210 nm. Data acquisition and processing
were performed with an N2000 chromatography data
system. An Auto science AT-330 column heater
(±0.1 ℃) was used to control the column temperature
1
during the HPLC separations. H NMR and 13C NMR
*
E-mail: yuan_limingpd@126.com; Tel: 0086-0871-65941088, Fax: 0086-0871-65941088; #These Authors equally contributed to the work.
Received October 17, 2016; accepted January 14, 2017; published online XXXX, 2017.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201600664 or from the author.
Chin. J. Chem. 2017, XX, 1—6
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1

Wu et al.
COMMUNICATION
spectra were obtained with a Varian Mercury Plus 300
and a Unity-Inova 500 spectrometer. Scanning electron
microscopy (SEM) images were recorded on an FEI
Quanta FEG 650 scanning electron microscope (USA).
tracted by dichloromethane. The organic phase was
washed, dried and evaporated at 50 ℃ to afford (R)-B
(10.51 g, 95.4% yield). (NMR in Supplementary Infor-
mation S1)
Scheme 1 Synthetic route of R-(3,3'-dibromo-1,1'-binaph-
thyl)-20-crown-6
Cl
Br
O
O
O
O
O
O
O
O
O
O
CH3I, K2CO3
OH
OH
OCH3
OCH3
O
O
Cl
CsCO3, acetonitrile
Br
CSP-2
CSP-1
A
B
Br
I
Br
O
O
O
O
O
O
O
C4H9Li, Br2
Ether
BBr3
CH2Cl2
OCH3
OCH3
O
O
O
OH
OH
O
O
Br
Br
I
C
D
CSP-3
CSP-4
Br
Figure 1 The structure of the crown ether chiral stationary
phases (CSPs).
O
O
O
O
TsO
O
O
O
O
OTs
KOH, THF
O
O
A stainless steel empty column (250 mm long×4.6
mm i.d.) and a 1/3 HP liquid pump were purchased from
Alltech (USA). A CROWNPAK CR (＋) chiral crown
ether column (150 mm long×4.0 mm i.d.) was pur-
chased from Daicel (Japan).
Br
R-1
n-Butyllithium (23.5 mL, 37.6 mmol) was added
dropwise to a stirred solution of tetramethylethylene-
diamine (3.8 mL, 26.6 mmol) in dry diethyl ether (150
mL). After the mixture was stirred for 15 min at 25 ℃,
(R)-B (5.0 g, 16 mmol) was added, and the suspension
was stirred for 3 h. The mixture was cooled to 75 ℃,
and 3 mL of bromine (9.6 g, 60 mmol) in 10 mL of
pentane was added over a 10 min period. The mixture
was warmed to 25 ℃ and stirred for 3 h. Then, 300 mL
of a saturated solution of Na2SO3 was added, and the
mixture was stirred for an additional 2 h. The mixture
was then extracted with dichloromethane, and the or-
ganic layer was dried and evaporated. The residue was
purified by column chromatography (silica gel, petro-
leum ether/ethyl acetate, 20∶1) to give (R)-C (3.8 g,
50.3% yield). (NMR in Supplementary Information S2)
BBr3 (2 mL, 32.3 mmol) was added to a solution of
(R)-C (3.0 g, 6.36 mmol) in dry dichloromethane (150
mL) at 0 ℃, and the mixture was stirred for 24 h at
25 ℃. The reaction was quenched by the slow addition
of water. The organic layer was washed with water,
dried, evaporated, and chromatographed on silica gel
(silica gel, petroleum ether/acetone, 30∶1, V/V) to ob-
tain (R)-D (2.31 g, 81.2% yield). (NMR in Supplemen-
tary Information S3)
Materials
All chemicals were at least analytical grade, includ-
ing (R)-(＋)-1,1'-bi-2-naphthol (C20H14O2, 99%, Daicel,
China), methyl iodide (CH3I, 99%, Xiya Reagent, Chi-
na), n-butyllithium (n-C4H9Li, 1.6 mol/L in hexanes,
Adamas-beta, China), hexachloroethane (C2Cl6, 98%,
Adamas-beta, China), iodine (I2, 96%, Adamas-beta,
China), phenylboronic acid (C6H7BO2, 98%, Ada-
mas-beta, China), pentaethylene glycol ditosylate
(C24H34O10S2, 95%, Alfa Aesar, USA), boron tribromide
(BBr3, 95%, Adamas-beta, China), and C18 silica gel
(average particle size: 5 μm, average pore diameter: 12
nm) from the Sepax of America. All reactions were
performed in purified and dried solvents and glass dried
under nitrogen gas.
Synthesis of R-(3,3'-dibromo-1,1'-binaphthyl)-20-
crown-6 (R-1)
R-(3,3'-Dibromo-1,1'-binaphthyl)-20-crown-6 (R-1)
was synthesized using (R)-(＋)-1,1'-bi-2-naphthol as the
starting material. The synthetic route is shown in
Scheme 1.
K2CO3 (20.0 g, 145 mmol) and CsCO3 (0.50 g, 1.5
mmol) were added to a solution of (R)-(＋)-1,1'-bi-2-
naphthol (10.0 g, 35 mmol) in 250 mL of acetonitrile,
and the solution was stirred under nitrogen at 70 ℃.
After 10 min, methyl iodide (6.5 mL, 0.1 mol) was
added dropwise over a period of 15 min. Then, the mix-
ture was refluxed for 4 h. The reaction mixture was ex-
(R)-D (2.0 g, 4.46 mmol) and pentaethylene glycol
ditosylate (2.5 g, 4.5 mmol) in 250 mL dry tetrahydro-
furan were stirred under nitrogen, and KOH (0.24 g, 4.3
mmol) was added. The mixture was refluxed for 72 h at
60 ℃. The mixture was extracted with 300 mL di-
chloromethane/water (1∶1, V/V), and the organic layer
2
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Chromatographic Resolution of α-Amino Acids
was dried and evaporated. The residue was purified by
column chromatography (silica gel, petroleum ether/
ethyl acetate, 10∶1, V/V) to obtain a viscous yellow oil
of the desired compound R-1 (2.38 g, 83.1% yield)
(NMR in Supplementary Information S4 and Figures S1
and S2)
Synthesis of R-(3,3'-dichloro-1,1'-binaphthyl)-20-
crown-6 (R-2) and R-(3,3'-diiodo-1,1'-binaphthyl)-
20-crown-6 (R-3)
The synthesis of the other two halogen substituted
crown ethers (R-2, R-3) was similar to that of R-1. The
details are described in the Supplementary Information
(Scheme S1 and Scheme S2).
Figure 2 (a) SEM image of silica spheres; (b) SEM image of
the C18 silica before coating its surface; (c) SEM image of the
coated surface; (d) a close view of the detailed features of the
CSP-1 electrode, along with the (Si, C, O, Br) corresponding
EDS element mapping of the silica composite.
Preparation of CSPs and column packing
The preparation of R-1-coated CSP-1 followed the
traditional coating method: compound R-1 (0.40 g, 0.71
mmol) was dissolved in 10 mL of dichloromethane.
Then, the solution was added slowly to the C18 silica gel
(3.60 g) through a dropper. When the dichloromethane
was removed, the residue of R-1 was coated on the C18
silica gel. The stationary phase was through a sieve and
the CSP-1 was prepared. CSP-2 and CSP-3 were pre-
pared using the same method.
A 4.0 g mass of the prepared CSPs was dispersed in
a mixture of methanol/water (1∶10, V/V). Then, the
suspension was packed into a stainless steel column
using the conventional slurry packing method with an
Alltech slurry packer at 40－50 MPa. The water was
used as the pressuring solvent to avoid the dissolution of
crown ethers (Supplementary Information Figure S10).
The column was conditioned for 20－30 min using the
perchloric acid solution (10 mmol/L, pH＝2) before use.
The t0 was estimated using sodium nitrate salt in HPLC.
Comparison of CSP-1 with structurally similar CSPs
for the separation of α-amino acids
To investigate the effect of different CSP structures
on the recognition of racemic amino acids, we prepared
a series of new CSPs, CSP-1, CSP-2 and CSP-3. Their
structures are shown in Figure 1, and the synthetic
methods are provided in the supporting information
(Scheme S1 and Scheme S2). The CSPs were evaluated
for the separation of various α-amino acid enantiomers,
and the resolution results are summarized in Table 1.
The experimental results indicate that all twenty-one
α-amino acids have different degrees of separation on
CSP-1 at room temperature. The chromatograms are
shown in Figure 3 and Figure S7 (Supplementary In-
formation). Consequently, CSP-1 possesses prominent
enantioselectivity. Most α-amino acids could be sepa-
rated on CSP-2, except tryptophan, glutamine, aspartic
acid, alanine, proline, threonine and histidine (Table 1).
However, CSP-3 only separated three amino acids, leu-
cine, cysteine, and alanine. As shown in Table 1, the
enantioselectivity of the three crown ether-based CSPs
is ranked as CSP-1＞CSP-2＞CSP-3 under the same
conditions.
Results and Discussion
Characterization of CSPs
The prepared CSP-1, CSP-2, and CSP-3 were char-
acterized by elemental analysis (Supplementary Infor-
mation Table S1). As shown in Table S1, the carbon,
nitrogen and hydrogen contents of the C18 silica gel
were 16.84%, ＜0.5%, and 19%, respectively. The car-
bon and hydrogen contents of the coated CSPs increased,
indicating the crown ethers were successfully coated on
the C18 silica gel. To further investigate the coating
properties of the surface wall of the C18 silica, SEM was
performed. The surface of the C18 silica spheres was
smooth, and the hybrid particles had a uniform spherical
morphology (Figures 2a, b and c). In addition, the coat-
ing composition of CSP-1 was studied by ener-
gy-dispersive spectrometry (EDS) analysis. The spatial
distribution of the Si, C, O and Br of the CSP-1 was
examined by elemental mapping analysis (Figure 2d).
Br was distributed homogeneously throughout the C18
silica. Therefore, R-1 was successfully coated on the C18
silica gel.
Comparison of a CSP-1 column with a commercial
CROWNPAK CR(＋) column for the separation of
α-amino acids
The resolution of 21 α-amino acids on CSP-1 and
commercial CSP CR(＋) using perchloric acid solution
(pH＝2) as a mobile phase with a flow rate of 0.5
mL•min1 at 25 ℃ is summarized in Table 1. The re-
sults showed that the chiral recognition ability of CSP-1
was excellent; all the α-amino acids could have different
degrees of separation on the CSP-1 packed column,
whereas only 15 analytes were separated on commercial
CSP CR(＋). Proline, histidine, valine, asparagine,
threonine, arginine, and serine were not separated on the
CR(＋) column, which is basically consistent with the
Chin. J. Chem. 2017, XX, 1—6
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Wu et al.
COMMUNICATION
Table 1 Comparison of the resolutions of α-amino acids on CSP-1, CSP-2 and CSP-3 based columns
CR(＋)
α
CSP-1
CSP-2
CSP-3
Racemates
k1
Rs
k1
α
Rs
k1
α
Rs
k1
α
Rs
Phenylglycine
4-Hydroxyphenylglycine
Methionine
Tyrosine
1.62
1.08
1.25
5.04
3.84
0.37
21.8
2.62
1.58
1.23
0.32
0.25
0.27
0.39
0.22
0.20
0.23
0.21
0.23
0.26
0.61
4.01
6.14
1.98
1.58
1.36
1.00
1.25
1.30
1.68
1.25
3.03
2.19
1.53
2.34
2.21
1.00
1.00
1.00
1.00
1.00
1.00
7.52
11.5
2.56
4.32
0.90
－
5.20 1.48
4.73 1.57
2.85 1.34
8.83 1.26
12.8 1.26
0.51 1.38
4.86 2.39
0.54 1.26
3.81 1.39
3.86 1.24
0.65 1.53
0.34 1.55
0.45 1.10
0.46 1.26
0.33 1.43
0.19 1.81
0.38 1.42
0.22 2.11
0.43 1.30
1.71 1.26
2.55 1.15
6.82
8.28
2.64
2.25
1.35
1.75
1.83
1.48
1.83
1.03
2.52
2.61
0.73
2.12
1.92
0.80
0.92
1.67
0.86
2.95
1.30
7.15 1.70
6.46 1.45
4.19 1.23
8.85 1.25
3.31 2.12
0.94 1.15
7.58 1.00
1.06 1.12
4.84 1.28
4.28 1.25
0.96 1.30
0.47 1.00
0.54 1.00
1.34 1.09
0.47 1.00
0.49 1.00
2.53 2.55
0.53 1.00
0.54 1.35
0.62 1.00
1.61 1.20
5.52
4.33
1.38
2.86
1.80
0.67
－
2.91
1.33
2.47
2.92
3.11
0.52
3.78
0.33
1.38
3.34
0.65
0.51
0.49
0.49
0.40
0.73
0.46
0.52
0.54
0.48
1.34
1.00
1.00
－
－
－
－
－
－
－
－
1.00
1.00
1.00
1.00
1.00
1.00
Phenylalanine
Arginine
Tryptophan
Lysine
1.23
1.61
0.88
0.50
1.69
1.28
0.66
2.77
0.62
－
0.64
1.51
1.17
1.61
－
Leucine
2.25 0.92
Isoleucine
Glutamic acid
Glutamine
Aspartic acid
Cysteine
1.00
1.00
1.00
1.00
－
－
－
－
－
0.54
－
1.17 0.31
1.32 0.41
Alanine
Proline
－
1.00
1.00
1.00
1.00
1.00
1.00
－
－
－
－
－
－
Serine
－
1.89
－
Threonine
Asparagine
Histidine
－
－
0.75
－
－
Valine
－
0.92
Chromatographic conditions: CSP-1 (250 mm long×4.6 mm i.d.), CSP-2 (250 mm long×4.6 mm i.d.), CSP-3 (250 mm long×4.6 mm
i.d.). Mobile phase: perchloric acid (10 mmol/L, pH＝2). Flow rate: 0.5 mL•min1. Detection: 210 nm UV. Temperature: 25 ℃. k1: reten-
tion factor of the first eluted enantiomer. α: separation factor. RS: resolution. －: could not be separated.
Figure 3 Representative chromatograms of α-amino acids on the CSP-1 (250 mm long×4.6 mm i.d.): (a) phenylglycine, (b)
4-hydroxyphenylglycine, (c) tyrosine, (d) methionine, (e) glutamic acid, and (f) histidine using a perchloric acid solution (10 mmol/L, pH
＝2) as the mobile phase at a flow rate of 0.5 mL•min−1 at 25 ℃. The signals were monitored with a UV detector at 210 nm.
4
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Chromatographic Resolution of α-Amino Acids
released application notes of commercial CROWNPAK
CR(＋) column. The representative chromatograms for
comparison are shown in Supplementary Information
Figure S8. In addition, the Crownpak CR-I(＋) has been
reported[25] which has the similar stationary phase with
CROWNPAK CR(＋) or CR(). However, the synthesis
of Crownpak CR-I is very complicated than that of the
CROWNPAK CR(＋) or CR(),[26] especially more
complicated than that of CSP-1. On the other hand, the
proline is really resolved on CSP-1, which is not re-
solved on Crownpak CR-I column.
Table 2 are consistent with Cram and co-workers’ ob-
servations.
The ring of crown ethers can form complexes with
＋
R-NH3 guests, and the direction and extent of config-
urational bias in the crown ether are important for enan-
tioseparation. Under acidic conditions at pH＝2, amino
acids are protonated and converted into RNH3 cations.
＋
Figure 4 Chromatograms on the CSP-1 packed column for the
separation of (A) histidine and (B) serine at 8－24 ℃. Perchloric
acid solution (10 mmol/L, pH＝2) as the mobile phase, flow rate
of 0.5 mL•min−1 and with UV detection at 210 nm.
When the 3,3'-position of R-(1,1'-binaphthyl)-20-crown-
6 was disubstituted with bromine, chlorine, iodine and
phenyl, different directions and extents of configura-
tional bias for the crown ether were observed. The sub-
stituent identification ability followed the order Br＞Ph
＞Cl＞I; these selectivity trends can be explained by
both position and species of the substituent. Considering
that binaphthyl crown ether skeleton prefers to adopt a
twisted conformation, the conformational complemen-
tarity between binaphthyl crown ethers and substituent
is related to the Br and phenyl selectivity. The
3,3'-dibromo substituents exhibit stronger basicity than
other substituents and have the proper size for the bind-
ing cleft. The reason for this selectivity towards dibro-
mo is a result of steric hindrance. Because the 1,1'-bi-
naphthyl group of the crown ether and the α-amino acid
possess chirality, chiral recognition is obtained on the
crown ether column. However, the influence of the chi-
ral microenvironment on the chiral properties of chro-
matographic systems is complicated; it is difficult to
completely understand the chiral recognition mecha-
nism of enantioseparation. To determine the mechanism
of chiral recognition, including the exact role of the two
bromine groups of CSP-1, further studies are needed
and are currently underway in our laboratory.
The van’t Hoff plots for the separation of all analytes
show good linearity (Supplementary Information Figure
S9), suggesting no changes in the interaction mecha-
nism in relation to temperature in the studied tempera-
ture range. The thermodynamic parameters for the
transfer of all analytes from the mobile phase to the sta-
tionary phase of CSP-1 are summarized in Table 3. The
negative values of G indicate that the transfer of all
analytes from the mobile phase to the stationary phase
of CSP-1 was a thermodynamically spontaneous pro-
cess that was controlled by H and S. More negative
values of G were more favourable for the transfer of
the solute from the mobile phase to the stationary phase,
resulting in stronger retention for the solute on the sta-
tionary phase.
Conclusions
In summary, we have synthesized three chiral crown
ethers for HPLC. The R-(3,3'-dibromo-1,1'-binaph-
thyl)-20-crown-6-coated CSP-1 exhibits excellent enan-
tioselectivity; all 21 α-amino acids have different de-
grees of separation at room temperature. The resolution
ability of CSP-1 is better than those of two other CSPs.
Compared with commercial CROWNPAK CR( ＋ )
column, the CSP-1 based column had stronger chiral
recognition ability. The resolution of most α-amino ac-
ids on CSP-1 was higher than that of the commercial
crown ether-based CSPs (CROWNPAK CR(＋) from
Daicel). Proline, histidine, valine, asparagine, threonine,
arginine and serine were not separated by the commer-
cial CROWNPAK CR (＋) under the same conditions.
In addition, the synthetic steps and preparation proce-
dure of chiral crown ether CSP-1 are less complicated
than those of commercial CR(＋), which will make
CSP-1 more widely used in practice. This work sug-
gests that chiral crown ether CSP-1 will become a use-
ful chiral selector for α-amino acids in the near future.
Effect of temperature on the HPLC separation
Cram and co-workers[27,28] observed differences in
the stability of two diastereomeric (host-guest) com-
plexes formed between a chiral crown ether and two
enantiomers of an amino acid, which increases with de-
creasing temperature. To investigate the effect of tem-
perature on the enantioselectivity of CSP-1, histidine
and serine were chosen for a more detailed study. When
other chromatogram conditions did not change, we tried
to find out the optimum temperature phase for the reso-
lution of a broad spectrum of α-amino acids on CSP-1
by varying the temperature phase, as shown in Table 2
and Figure 4. When the temperature of the column de-
creased from 24 to 8 ℃, all three chromatographic pa-
rameters, i.e., the retention factors (k), separation factors
(α), and resolution (RS), increased. The results shown in
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5

Wu et al.
COMMUNICATION
Table 2 Effect of column temperature on retention and enantionselectivity of histidine and serine on CSP-1a
Histidine
Serine
Temperature/℃
k1'
k2'
α
Rs
k1'
k2'
α
Rs
8
1.39
1.19
0.87
0.77
0.62
2.37
1.84
1.33
1.10
0.89
1.72
1.56
1.51
1.43
1.42
4.42
2.25
2.22
2.18
2.06
1.33
1.19
0.87
0.69
0.61
1.71
1.49
1.03
0.79
0.65
1.29
1.25
1.18
1.13
1.09
1.90
1.54
1.05
0.62
0.50
12
16
20
24
a The chromatographic condition was same as that in Table 1 except temperature.
Table 3 Values of H, S, G and r2 (r refers to the linear correlation coefficient of ln k'1/T plot) for histidine and serine
H/(kJ•mol−1)
27.94±0.65
37.51±0.94
28.19±0.78
35.17±1.32
S/(J•mol−1•K−1)
85.74±10.44
115.09±11.86
87.65±10.77
109.23±11.45
G/(kJ•mol−1)
2.45±0.65
3.31±0.94
2.14±0.78
2.71±1.32
Analyte
L-Histidine
D-Histidine
L-Serine
r2
0.9973
0.9963
0.9924
0.9908
D-Serine
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Acknowledgement
This work was supported by the National Natural
Science Foundation of China (Nos. 21675141,
21165022).
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