Preparation and enantioseparation of a mixed selector chiral stationary phase derived from benzoylated tartaric acid and 1,2-diphenylethylenediamine

Article abstract of DOI:10.1002/chir.20799

L-Dibenzoyl tartaric acid was mono-esterified with benzyl alcohol, and then chlorinated with SOCl₂ to give (2S,3S)-1-(benzyloxy)-4-chloro-1,4- dioxobutane-2,3-diyl dibenzoate (Selector 1). (1R,2R)-1,2- Diphenylethylenediamine was mono-functionalized with phenyl isocyanate and phenylene diisocyanate in sequence to give (1R,2R)-1,2-diphenyl-2-(3- phenylureido) ethyl 4- isocyanatophenylurea (Selector 2). Two brush-type chiral stationary phases (CSPs) of single selector were prepared by separately immobilizing selectors 1 and 2 on aminated silica gel. Selectors 1 and 2 were simultaneously immobilized on aminated silica gel to give a mixed selector CSP. The enantioseparation ability of these CSPs was studied. The CSP of selector 1 has strongest separation ability, while the enantioseparation ability of the mixed selector CSP is relatively lower.

Full text of DOI:10.1002/chir.20799

CHIRALITY 22:604–611 (2010)
Preparation and Enantioseparation of a Mixed Selector Chiral
Stationary Phase Derived from Benzoylated Tartaric Acid
and 1,2-Diphenylethylenediamine
WEN-JUAN WEI,1 HUI-WEN DENG,1 WEI CHEN,1 ZHENG-WU BAI,1 AND SHI-RONG LI2
1Key Laboratory of Green Chemical Process of Ministry of Education, Hubei Key Laboratory of Novel Chemical Reactor
and Green Chemical Technology, Wuhan Institute of Technology, Wuhan, China
2Hubei Key Laboratory of Biologic Resources Protection and Utilization, Hubei Institute for Nationalities, Enshi, Hubei, China
ABSTRACT
L-Dibenzoyl tartaric acid was mono-esterified with benzyl alcohol, and
then chlorinated with SOCl2 to give (2S,3S)-1-(benzyloxy)-4-chloro-1,4-dioxobutane-2,3-
diyl dibenzoate (Selector 1). (1R,2R)-1,2-Diphenylethylenediamine was mono-functional-
ized with phenyl isocyanate and phenylene diisocyanate in sequence to give (1R,2R)-1,2-
diphenyl-2-(3-phenylureido)ethyl 4- isocyanatophenylurea (Selector 2). Two brush-type
chiral stationary phases (CSPs) of single selector were prepared by separately immobi-
lizing selectors 1 and 2 on aminated silica gel. Selectors 1 and 2 were simultaneously
immobilized on aminated silica gel to give a mixed selector CSP. The enantioseparation
ability of these CSPs was studied. The CSP of selector 1 has strongest separation abil-
ity, while the enantioseparation ability of the mixed selector CSP is relatively lower.
C
VV
Chirality 22:604–611, 2010.
2009 Wiley-Liss, Inc.
KEY WORDS: brush-type; mixed selector; chiral stationary phase; enantioseparation;
high-performance liquid chromatography
INTRODUCTION
commercially available Kromasil CHI-DMB [O,O0-bis
(dimethyl)benzoyl tartaric diamide] CSP.20 They found
that the resolution ability of the twin selector CSP is lower
than that of Kromasil CHI-DMB. In the above-mentioned
biselector CSPs, each selector is prepared from two chiral
compounds. Kraak et al. established a method to prepare
mixed selector CSPs, where the selectors were the deriva-
tives of phenylglycine which were ionically bonded on ami-
nopropyl silica gel.21 Obviously, the CSP cannot tolerate a
dramatic change in the acidity of mobile phases because
the selectors were immobilized by acid-base reactions.
It has not been reported that a CSP can be prepared by
covalently immobilizing two separate selectors on a sup-
port. This work aims at establishing an approach to prepare
a mixed selector CSP and investigating its enantiosepara-
tion ability in comparison with the single selector CSPs.
The enantiomers of a chiral drug are usually different in
their physiological activities.1,2 One enantiomer can be
effective to cure a disease, while another one may be less
effective, or ineffective, or even toxic. Accordingly, chiral
drugs need to be separated into single enantiomers during
their development and production process. Thus, enantio-
separation is very important for the pharmaceutical indus-
try, and it attracts many researchers’ attention to develop
effective enantioseparation techniques.
High-performance liquid chromatography (HPLC)
based on chiral stationary phase (CSP) is a very useful
technique to separate enantiomers.3–6In combination with
the simulating moving bed technique, chiral HPLC is ap-
plicable to separate enantiomers for industrial purposes.7–9
However, one CSP can separate a limited number of chiral
compounds. Since 1980s, considerable effort has been
made to develop new CSPs with the goal of separating as
many chiral compounds as possible. These CSPs were pre-
pared from natural polymers,10–12 oligomers,13 synthetic
polymers, etc.14–18
MATERIALS AND METHODS
Materials and Chemicals
L-Dibenzoyl tartaric acid (DBTA) and (1R,2R)-1,2-diphe-
To broaden the chiral recognition spectrum, several
biselector CSPs were developed. Salvadori and coworkers
used (S)-leucine and [(S)-1-(1-naphthyl)ethyl]amine as chi-
ral building blocks to construct biselector CSPs through s-
trichlorotriazine.19 The enantioseparation demonstrated
that the CSP bears the function of an efficient biselector
system. Oxelbark and coworkers prepared a twin selector
CSP based on O,O0-bis(dimethyl)benzoyl tartaric diamide
and a comparison was made between this CSP and the
C
nylethylenediamine (DPEDA) were purchased from
Contract grant sponsor: National Natural Science Foundation of China
(NSFC); Contract grant number: 20675061
*Correspondence to: Zheng-Wu Bai, Key Laboratory of Green Chemical
Process of Ministry of Education, Wuhan Institute of Technology, Wuhan
430073, China. E-mail: zhengwu_bai@yahoo.com
Received for publication 26 February 2009; Accepted 13 August 2009
DOI: 10.1002/chir.20799
Published online 6 November 2009 in Wiley InterScience
(www.interscience.wiley.com).
VV
2009 Wiley-Liss, Inc.

MIXED SELECTOR CHIRAL STATIONARY PHASE
605
Fig. 1. The synthetic scheme of Selectors 1 and 2.
Preparation of Chiral Stationary Phases
Chengdu Likai Chiral Tech. Co., Ltd. (China). 1,4-Phenyl-
ene diisocyanate (PDI) was from Xinyi Pesticide Factory
of Jiangsu (China) and recrystallized before use. 3-Amino-
propyltriethoxysilane (APTES) was obtained from Novel
Organic Silicon Materials Co., Ltd. of Wuhan University
(China) and redistilled before use. Silica gel (Lichrosorb
Si 100) was obtained from Merck (Germany) with a parti-
Preparation of CSP 1. L-DBTA (3.58 g, 10.0 mmol),
benzyl alcohol (1.10 g, 10.1 mmol), and catalytic amount
of p-toluenesulfonic acid were added in benzene (30 ml).
The resulting mixture was heated to reflux, during which
the water produced was removed continuously. After the
completion of the reaction, the mixture was cooled down,
washed with water three times, and the organic phase was
dried over anhydrous MgSO4. The volatiles were removed
on a rotary evaporator in vacuo to give a pale yellow solid,
which was subjected to silica gel column separation elut-
ing with cyclohexane/ethyl acetate (2:1, v/v). Compound
1 (1.95 g) was a white solid after the removal of the elu-
ent. Yield: 42%; m.p.: 153–155 8C; [a]2D0: 2408 (C 1.0, meth-
anol); IR(KBr, cm–1) t: 3187 (COOH, st), 2975 (CꢀꢀH, w),
1778, 1721 (ꢀꢀCO2ꢀꢀ, st), 1252, 1183 (ꢀꢀCO2ꢀꢀ, m); EA
˚
cle size of 5 lm, a pore size of 100 A and a surface area of
300 m2/g. Triethylamine (TEA, used for CSP preparation)
was dried over phosphorous pentoxide and redistilled. Tol-
uene was dried over sodium and redistilled. All other
chemicals used for the CSP synthesis were of analytical
grade and used as received.
Instruments and Measurements
Elemental analysis (EA) was conducted on an Elemental (%): Calcd C 66.96, H 4.50; Found C 67.32, H 4.51; 1H
VarioEL III CHNOS apparatus (Germany). IR spectra were NMR (CDCl3, 25 8C) d: 5.08–5.10 (d, 1H, ꢀꢀCHꢀꢀ), 5.21–
recorded on a Nicolet FT-IR instrument (USA) with KBr 5.23 (d, 1H, ꢀꢀCHꢀꢀ), 5.99 (s, 2H, ꢀꢀCH2ꢀꢀ), 7.17–8.13
pellets. 1H NMR spectra were performed on a Varian (m, 15H, Ar-H).
INOVA 500 spectrometer (USA). The stainless steel HPLC
Compound 1 (2.86 g, 6.3 mmol) was dissolved in tolu-
empty columns (250 mm 3 4.6 mm) were purchased from ene (8 ml), to which SOCl2 (2 ml) was added. The reaction
Hypsil (UK). The CSPs were packed into the empty col- solution was stirred at room temperature for 2 h, and then
umns with an Alltech Model 1666 slurry packer (USA). at 708C for 6 h. The toluene and SOCl2 were removed by
The enantioseparation was implemented on an Agilent distillation in vacuo to afford yellow viscous oil (Selector
1100 chromatograph, which consisted of an Agilent 1). A small amount of Selector 1 (ꢁ0.2 g) was retained for
G1365B DAD, an Agilent G1311A Quat Pump, an Agilent characterization, and the rest was used for the immobiliza-
G1379A degasser, and an Agilent G1313A ALS autosam- tion reaction without further purification. [a]1D5: 233.58 (C
pler.
0.4, toluene); IR (KBr, cm–1) t: 2921 (CꢀꢀH, w), 1822
Chirality DOI 10.1002/chir

606
WEI ET AL.
Fig. 2. The synthetic scheme of CSPs 1–3.
(ꢀꢀCOꢀꢀCl, st), 1731, 1632 (ꢀꢀCO2ꢀꢀ, st), 1280, 1213 the immobilization reaction, the solid was filtered and
(ꢀꢀCO2ꢀꢀ, m). EA (%): Calcd C 64.32, H 4.10; Found C washed thoroughly with THF to afford CSP 1 (4.60 g). IR
1
63.83, H 3.64; H NMR (CDCl3, 258C) d: 5.12–5.14 (d, 1H, (KBr, cm–1) t: 3444 (NꢀꢀH), 1707 (ꢀꢀCO2ꢀꢀ), 1663
ꢀꢀCHꢀꢀ), 5.28–5.30 (d, 1H, ꢀꢀCHꢀꢀ), 6.13 (s, 2H, (ꢀꢀNHꢀꢀCOꢀꢀ), 1094 (SiꢀꢀO); EA (%): C 13.20, H 2.01,
ꢀꢀCH2ꢀꢀ), 7.04–8.03 (m, 15H, Ar-H).
3-Aminopropyl silica gel was prepared with dried silica
gel and APTES.22
N 1.70.
Preparation of CSP 2. A solution of phenyl isocya-
Dry 3-aminopropyl silica gel (4.00 g) was suspended in nate (1.30 g, 10.9 mmol) in pyridine (6 ml) was added
pyridine (20 ml), to which the above freshly prepared dropwise in the solution of (1R,2R)-1,2-DPEDA (2.32 g,
Selector 1 and TEA (3 ml) were added. The suspension 10.9 mmol) dissolved in pyridine (15 ml). The solution
was gently stirred at 808C for 12 h. After the completion of was then stirred at room temperature for 0.5 h, and then
Chirality DOI 10.1002/chir

MIXED SELECTOR CHIRAL STATIONARY PHASE
607
mixture was concentrated and cooled. A white solid was
obtained by dropping the concentrated solution into a solu-
tion of toluene/cyclohexane (1:1, v/v). The solid was col-
lected by centrifugation and was further purified by repre-
cipitation twice, with pyridine as the solvent to dissolve
the solid and a mixture of toluene/cyclohexane as the pre-
cipitant. Selector 2 (3.52 g) was obtained after the removal
of the volatiles. Yield: 71%; m.p.: 218–2208C; [a]2D6: 135.88
(C 0.6, DMF); IR (KBr, cm–1) t: 3311 (NꢀꢀH, st), 2922
(CꢀꢀH, w), 2268 (ꢀꢀNCO, st), 1643, 1598 (ꢀꢀNHꢀꢀCOꢀꢀ,
st); EA (%): Calcd C 70.86, H 5.13, N 14.25; Found C 71.14,
1
H 5.48, N 14.79; H NMR (DMSO, 258C) d: 5.02 (d, 2H,
ꢀꢀCHꢀꢀ), 6.88–7.38 (m, 21H, Ar-H, ꢀꢀNHꢀꢀ), 8.64 (s, 2H,
ꢀꢀNHꢀꢀ).
Selector 2 (3.60 g, 7.3 mmol) and 3-aminopropyl silica
gel (3.62 g) were mixed in pyridine (17 ml). The suspen-
sion was stirred at room temperature for 2 h, and then at
808C for 12 h. The solid was collected by filtration and
washed thoroughly with DMF and THF, respectively, to
yield pale yellow CSP 2 (3.99 g), after removing the sol-
vents. IR (KBr, cm–1) t: 3414 (NꢀꢀH), 1661, 1605
(ꢀꢀNHꢀꢀCOꢀꢀ), 1557, 1513 (ꢀꢀNHꢀꢀCOꢀꢀ), 1117
(SiꢀꢀO); EA (%) C 17.43, H 2.25, N 4.55.
Preparation of CSP 3. Compound 1 (2.25 g, 5.0
mmol) was converted into corresponding acyl chloride by
the method described for the preparation of CSP 1. This
chloride, Selector 2 (3.60 g, 7.3 mmol), TEA (3 ml) and 3-
aminopropyl silica gel (3.72 g) were added in pyridine (40
ml). The resulting mixture was gradually heated to 808C,
and stirred for 12 h. The solid was collected by filtration
and was washed, respectively, with DMF and THF to
afford CSP 3 (4.68 g). IR (KBr, cm–1) t: 3381 (NꢀꢀH),
1601 (ꢀꢀNHꢀꢀCOꢀꢀ), 1669 (ꢀꢀNHꢀꢀCOꢀꢀ), 1111 (SiꢀꢀO);
EA (%): C 19.39, H 2.44, N 3.96.
Fig. 3. The FT-IR spectra of 3-aminopropyl silica gel (a), CSP 1 (b),
CSP 2 (c), and CSP 3 (d).
at 808C for an additional 5 h to ensure the completion of
the reaction. The pyridine was removed by distillation in
vacuo to give a pale yellow solid, followed by recrystalliza-
tion from ethanol/water (8:1, v/v) to give Compound 2
(1.68 g) as a white powder. Yield: 47%; m.p.: 234–2368C; IR
(KBr, cm–1) t: 3356 (NꢀꢀH, st), 2982 (CꢀꢀH, w), 1650
(NꢀꢀH, w); [a]2D0: 257.58 (C 1.0, THF); EA (%): Calcd C
Column Packing and Chromatographic Parameters
Each prepared CSP (3.30 g) was mixed with chloroform
(30 ml), and then was sonicated for 7 min to form a slurry.
The slurry was poured into the chamber of a column
packer and was packed into empty HPLC columns with
methanol as packing solvent, under the pressure no more
than 7000 psi. The packed columns were flushed with iso-
propanol and isopropanol/n-hexane in turn. The column
efficiency was determined using biphenyl, with isopropa-
nol/n-hexane (90/10, v/v) as the mobile phase.
1
76.11, H 6.39, N 12.68; Found C 75.86, H 5.97, N 12.82; H
NMR (DMSO, 258C) d: 5.01–5.04 (m, 2H, ꢀꢀCHꢀꢀ), 6.80
(m, 1H, ꢀꢀNHꢀꢀ), 7.07–7.36 (m, 15H, Ar-H), 8.63 (s, 2H,
ꢀꢀNHꢀꢀ).
A solution of PDI (1.78 g, 11.1 mmol) in pyridine (7 ml)
was added dropwise to a solution of Compound 2 (3.34 g,
10.1 mmol) in pyridine (20 ml). The solution was stirred at
room temperature for 0.5 h, and then at 808C for 6 h. The
TABLE 1. The elemental analysis and selector loading of CSPs 1–3
CSP 1 CSP 2
CSP 3
Element, selector,
Content
Content
Content
and selector loading
Content (%)
increment (%)
Content (%)
increment (%)
Content (%)
increment (%)
C
H
N
13.20
2.01
1.70
7.67
0.03
20.18
17.43
2.25
4.55
12.49
0.33
2.69
19.39
2.44
3.96
13.09
0.60
1.58
Selector
Selector loading
(mmol/g)
Selector 1
0.26
Selector 2
0.36
Selector 1
0.17
Selector 2
0.23
Chirality DOI 10.1002/chir

608
WEI ET AL.
Fig. 4. The structures of chiral analytes (1–31) separated by CSPs 1–3.
RESULTS AND DISCUSSION
The sample solutions (1 mg/ml) were prepared by dis-
Preparation and Characterization of CSPs 1–3
solving the chiral analytes in acetonitrile and were filtered
before injection. The injection volume was 15 ll. Triethy-
lammonium acetate buffer was prepared by adjusting the
pH value of 0.1 mol/l aqueous glacial acetic acid to the
desired value by addition of triethylamine. All mobile
phases were filtered and degassed before use. Except
when indicated, the flow rates were set at 1 ml/min; col-
umn temperature was set at 258C.
The selectors and CSPs 1–3 were synthesized accord-
ing to the scheme described in Figures 1 and 2. The IR
spectra of CSPs 1–3 are presented in Figure 3, where the
absorbance of amide bonds substantiates the successful
immobilization of Selectors 1 and 2. Meanwhile, the con-
tents of carbon, nitrogen (except in CSP 1), and hydrogen
Chirality DOI 10.1002/chir

MIXED SELECTOR CHIRAL STATIONARY PHASE
609
Fig. 5. The structures of chiral analytes (32–56) separated by CSPs 1–3.
Enantioseparation Ability of CSPs 1–3
of the CSPs are higher than those of 3-aminopropyl
silica gel. The content increments of these elements on
the surface of the silica gel further confirm the selectors
have been immobilized on the aminated silica gel (Table
1). According to the formula 1, the selector loadings are
calculated, in which DC is the content increment of carbon
The column efficiency of CSPs 1–3 was measured as
37,100, 27,000, and 47,100 plates per meter, respectively.
The enantioseparation by CSPs 1–3 was evaluated with
structurally different chiral analytes under both normal
and reversed phase modes. Figures 4 and 5 show 56 chiral
analytes, which were separated by CSPs 1–3. CSPs 1, 2,
and 3, respectively, separated 46, 30, and 26 chiral analy-
tes. The chromatographic data are presented in Table 2.
In general, although the resolutions are low, CSPs 1–3
can chirally recognize a wide range of enantiomers, CSP 1
in particular. CSPs 1 and 2 were prepared from single
selectors, while CSP 3 was derived from mixed selectors.
or
DC
Selector loading ¼ 10 3
mmol=g
ð1Þ
M 3 n
nitrogen; M is the relative atom mass of carbon or nitro-
gen; n is the number of the atom (carbon or nitrogen) con- Table 2 reveals that CSP 3 does not demonstrate better
tained in each selector molecule. According to the incre-
overall chiral recognition than CSPs 1 and 2; on the con-
ment of carbon content, the loadings of selectors 1 and 2
trary, its enantioseparation ability is much lower than that
on CSPs 1 and 2 were calculated, respectively. On the ba- of CSP 1, and slightly lower than that of CSP 2. This
sis of the content increments of carbon and nitrogen, the
loadings of selectors 1 and 2 on CSP 3 were, respectively,
determined (Table 1).
result is different from earlier reports, where the two selec-
tors are covalently bonded within a new molecule, which
is immobilized on support to form a twin selector CSP.23
Chirality DOI 10.1002/chir

TABLE 2. Chromatographic resolution of racemates on CSPs 1–3
CSP 1
CSP 2
CSP 3
Chiral
analytes
Separation
conditions
Separation
conditions
Separation
conditions
k
a
Rs
k
a
Rs
k
a
Rs
1
2
3
4
5
6
7
8
0.79
0.66
0.44
0.75
1.07
1.24
0.96
0.32
0.30
0.98
0.66
1.00
2.01
0.84
0.63
1.12
1.36
1.77
1.11
1.23
1.32
1.69
1.27
1.32
1.10
1.18
1.07
1.12
1.48
1.30
0.51
0.81
0.75
0.41
0.62
1.07
1.06
0.54
0.52
0.48
0.19
0.64
0.62
0.80
0.77
A(70/30)a
B(10/45/45)b
C(30/70)c
A(70/30)a
D(20/80)d
C(60/40)b
C(10/90)c
E(80/20)a
F(60/40)e
A(70/30)a
C(70/30)c
D(10/90)e
C(30/70)e
D(40/60)d,i
B(20/40/40)b
0.76
0.76
0.54
0.29
1.12
1.10
1.18
1.18
0.51
0.48
0.68
0.28
L(70/30)a
L(70/30)a
M(50/50)f
N(90/10)a
0.38
0.19
0.31
0.29
0.19
0.42
1.06
1.39
1.23
1.39
1.31
1.06
0.31
0.71
0.58
0.72
0.47
0.49
U(90/10)a
B(50/25/25)f,l
K(10/90)b
B(50/25/25)b
D(50/50)e
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
C(80/20)f
1.29
1.07
0.70
B(70/15/15)f
1.47
1.14
0.45
0.37
1.15
1.08
1.38
1.48
0.60
0.47
0.53
0.72
O(10/90)a
C(50/50)f
D(30/70)a
D(5/95)d
0.67
0.33
0.51
0.62
0.44
0.24
0.43
1.62
1.17
0.50
0.98
0.87
1.28
1.38
1.10
1.12
1.13
1.17
1.21
1.16
1.22
1.13
1.09
1.10
0.73
0.75
0.42
0.32
0.52
0.38
0.53
1.00
0.65
0.51
0.51
0.49
B(30/35/35)d
G(70/30)f
E(70/30)a
A(80/20)b
E(70/30)a
H(60/40)e
E(70/30)f
B(50/25/25)b
D(70/30)a
E(70/30)a
A(70/30)a
A(70/30)a
0.34
2.42
1.20
1.02
0.38
0.25
B(30/35/35)e
L(90/10)f
0.41
1.11
0.27
K(10/90)b
0.19
1.38
0.51
K(20/80)a
0.26
0.35
0.56
1.32
1.27
1.17
0.62
0.73
0.96
V(50/50)f
W(50/50)f
K(10/90)d
0.16
0.96
0.41
1.41
1.12
2.26
0.25
0.68
0.65
N(90/10)a
B(10/50/40)a
K(40/60)a
1.79
1.00
0.42
1.02
1.58
1.09
0.10
0.83
0.67
U(90/10)a
D(10/90)f
C(65/35)f
0.62
0.56
0.44
0.43
1.47
1.08
1.19
1.12
0.76
0.51
0.61
0.81
I(30/70)a
K(10/90)d
P(50/50)d
Q(30/70)f
0.79
1.26
0.33
0.68
0.75
0.88
0.95
1.17
1.35
1.14
1.89
1.25
1.27
1.39
1.10
1.08
1.24
0.42
0.62
0.76
0.60
0.91
0.50
0.48
B(50/25/25)b
B(10/45/45)b
C(50/50)g
I(40/60)a
0.36
1.20
0.92
K(20/80)a
0.47
1.29
0.61
D(15/85)a
0.35
2.48
1.14
1.01
0.43
0.12
B(30/35/35)d
B(60/20/20)a
D(5/95)d
D(40/60)a,j
A(70/30)a
A(70/30)a
1.07
0.46
1.13
0.39
0.47
0.70
1.01
1.55
1.41
1.81
1.38
1.03
0.11
0.93
0.69
0.85
0.70
0.22
R(50/50)a
D(15/85)a
O(20/80)e
L(80/20)a
D(5/95)d
S(80/20)a
0.87
0.89
1.02
0.85
0.97
1.11
1.44
1.21
1.11
1.19
0.52
1.00
0.59
0.20
0.54
A(70/30)a
D(40/60)a,k
D(50/50)a
C(80/20)h
D(10/90)e
3.70
1.01
0.10
D(50/50)e
0.53
0.46
0.38
1.43
2.37
2.33
0.69
0.94
0.79
D(15/85)a
M(30/70)a
M(20/80)f
0.42
0.38
15.28
12.75
16.06
15.22
C(30/70)c
C(30/70)c
0.69
0.39
1.34
1.49
1.37
1.34
0.64
0.51
0.88
T(35/35/30)f
D(5/95)d
1.42
1.08
0.46
J(70/30)f
O(10/90)a
1.80
2.48
0.58
0.56
1.01
0.20
1.22
1.01
1.13
1.03
1.05
1.31
2.20
0.18
0.52
0.27
0.49
0.68
C(65/35)f
B(60/20/20)a
O(50/50)d,m
U(70/30)a
U(70/30)a
U(90/10)a
1.03
0.85
1.09
1.10
0.47
0.20
A(70/30)a
K(50/50)a
0.57
1.20
0.74
D(65/35)d
1.07
1.12
0.36
B(50/25/25)f
Retention factors: [k1: (t1 2 t0)/t0; and k2: (t2 2 t0)/t0], where t1 and t2 are, respectively, the retention time of the first and the second-eluted enantiomer, and
t0 is determined by measuring the retention time of NaNO2 solution. Separation factor (a): k2/k1; Resolution (Rs): 2(t2 2 t1)/(w1 1 w2), where w1 and w2 are
the bandwidth of the first and the second-eluted enantiomer, respectively.
Eluent: A: Methanol/buffer (pH 8.03); B: n-Hexane/ethanol/methanol; C: Acetonitrile/water; D: n-Hexane/ethanol; E: Methanol/buffer (pH 6.09); F: Meth-
anol/buffer (pH 6.90); G: Acetonitrile/buffer (pH 8.67); H: Acetonitrile/buffer (pH 6.09); I: n-Hexane/n-propanol; J: Methanol/buffer (pH 8.67); K: Ethanol/
methanol; L: Methanol/water; M: n-Hexane/2-propanol; N: Methanol/water/TEA (0.2 ml TEA in 100 ml water); O: n-Hexane/n-butanol; P: n-Hexane/t-buta-
nol; Q: Acetonitrile/methanol; R: Acetonitrile/buffer (pH6.86); S: Methanol/buffer (pH 5.99); T: Acetonitrile/methanol/water; U: Acetonitrile/buffer (pH
5.92); V: Acetonitrile/buffer (pH 8.10); W: Acetonitrile/buffer (pH 7.52).
Wavelength of UV detection (nm): a265; b230; b214; d254; e245; f285; g218; h222.
Column temperature (8C): i45; j35; k40; l30.
Flow rate (ml/min): m0.9.

MIXED SELECTOR CHIRAL STATIONARY PHASE
611
8. Seidel-Morgenstern A, Keler LC, Kaspereit M. New developments in
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826–837.
Therefore, the loadings of the two selector units are equal.
The chiral analytes interact with the one of selector units
revealing separate chiral recognition. Regarding CSP 3,
however, the two selectors are separately immobilized on
the support. The loadings of these two selectors are
unequal, and they are lower than those in CSP 1 and 2.
Selector loading is an important factor to influence enantio-
separation. Thus, the lower enantioseparation ability of
CSP 3 may be attributed to lower individual selector
loadings. On the other hand, the lower enantiosepara-
tion ability is probably caused by the reverse contribu-
tion of the two selectors. In chiral recognition, Selectors
1 and 2, respectively, interact with a pair of enantiomers
to form two groups of temporary diastereoisomers. The
stability difference of the diastereoisomers of each
group leads to separation. If there is no stability differ-
ence between the each group of diastereoisomers, the
enantiomers will not be separated. Suppose the diaster-
eoisomer results from Selector 1 and R-form isomer of
an analyte is more stable than the one results from
Selector 1 and S-form isomer; while the diastereoisomer
formed between Selector 2 and the R-form isomer is
less stable than the one formed between Selector 2 and
S-form isomer, the contributions of Selectors 1 and 2 to
separation impairs each other, in this case, the enantio-
separation resolution decreases, or the two enantiomers
cannot be separated.
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Chirality DOI 10.1002/chir