Chiral separation of neonicotinoid insecticides by polysaccharide-type stationary phases using high-performance liquid chromatography and supercritical fluid chromatography

Article abstract of DOI:10.1002/chir.20898

The enantiomeric separations of three neonicotinoid insecticides (identified as compounds 1, 2, and 3) were performed on three polysaccharide-type chiral columns, that is, Chiralcel OD-H, Chiralpak AD-H, and Chiralpak IB, by high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Effects of the modifier percentage and column temperature on chiral recognitions of chiral stationary phases were also studied. Both 1 and 2 could be resolved on all three columns selected, with the highest R_s values obtained on Chiralpak AD-H and Chiralcel OD-H, respectively. However, satisfactory separation of the four stereoisomers of 3 was only achieved on Chiralcel OD-H. Considering the effects of ethanol on the values of k, α, and R_s, we concluded that hydrogen bonding, π-π, and/or dipole-dipole interactions might be all responsible for the chiral separation. In comparison to HPLC, a shorter run time was achieved for 1 and 2 by SFC. However, 3 could not be stereoselectively resolved using SFC. On the basis of the calculated thermodynamic parameters, we found that the separation processes of enantiomers of 1 and 2 were entropy controlled and enthalpy controlled, respectively. Copyright

Full text of DOI:10.1002/chir.20898

CHIRALITY 23:215–221 (2011)
Chiral Separation of Neonicotinoid Insecticides by Polysaccharide-
Type Stationary Phases Using High-Performance Liquid
Chromatography and Supercritical Fluid Chromatography
1
1
CHENG ZHANG,1 LIXIA JIN,1 SHANSHAN ZHOU,1 YIFAN ZHANG, SHUOLI FENG, AND QINYAN ZHOU2
*
1Research Center of Environmental Science, College of Biological and Environmental Engineering, Zhejiang University of Technology,
Hangzhou, China
2Ministry of Education Key Laboratory of Environmental Remediation and Ecosystem Health, College of Natural Resource and Environment Science,
Zhejiang University, Hangzhou, China
ABSTRACT
The enantiomeric separations of three neonicotinoid insecticides (identified as
compounds 1, 2, and 3) were performed on three polysaccharide-type chiral columns, that is,
Chiralcel OD-H, Chiralpak AD-H, and Chiralpak IB, by high-performance liquid chromatogra-
phy (HPLC) and supercritical fluid chromatography (SFC). Effects of the modifier percentage
and column temperature on chiral recognitions of chiral stationary phases were also studied.
Both 1 and 2 could be resolved on all three columns selected, with the highest Rs values
obtained on Chiralpak AD-H and Chiralcel OD-H, respectively. However, satisfactory separation
of the four stereoisomers of 3 was only achieved on Chiralcel OD-H. Considering the effects of
ethanol on the values of k, a, and Rs, we concluded that hydrogen bonding, p–p, and/or dipole–
dipole interactions might be all responsible for the chiral separation. In comparison to HPLC, a
shorter run time was achieved for 1 and 2 by SFC. However, 3 could not be stereoselectively
resolved using SFC. On the basis of the calculated thermodynamic parameters, we found that
the separation processes of enantiomers of 1 and 2 were entropy controlled and enthalpy con-
C
VV
trolled, respectively. Chirality 23:215–221, 2011.
2010 Wiley-Liss, Inc.
KEY WORDS: enantiomeric separation; neonicotinoid insecticides; polar modifier; column
temperature
INTRODUCTION
environmental friendliness. A wide range of CSPs have been
used in SFC.14–17
Chirality is the rule rather than the exception in the uni-
verse, as living organisms are anisotropic on either micro-
scales or macroscales. This molecular chirality in life science
has been well recognized and applied in the pharmaceutical
industry, where 50 of the top 100 most widely sold drugs are
marketed as single enantiomers to avoid adverse side
effects.1,2 In the last two decades, great attention has also
been paid to the stereoselective properties of currently used
pesticides, of which up to 25% were chiral in the 1990s,3 and
this ratio has now increased to about 40%, as compounds
with more complex structures are being registered for use.4
Numerous examples have demonstrated that many biological
activities such as insecticidal activity, aquatic toxicity, devel-
opment toxicity, endocrine disruption, immunotoxicity, and
microbial degradation of chiral pesticides are enantioselec-
tive.5–10 As a result, it is very important to assess the pestici-
dal activity and environmental safety of chiral pesticides in
their enantiopure forms.11
Neonicotinoids are a class of insecticides that act on insect
nicotinic acetylcholine receptors. They have outstanding
effectiveness toward the target organisms but exhibit rela-
tively low toxicity to vertebrates. They are now among the
most widely used insecticides, accounting for ꢀ17% of the
total insecticide market.18,19 Recently, Li’s group designed
and synthesized a series of neonicotinoid analogues with
high insecticidal activity.20 It should be noted that some of
the extremely efficient chemicals have chiral structures. For
example, compound 1 has an oxa-bridged structure, while
compounds 2 and 3 have one and two asymmetric carbon
atoms, respectively (Fig. 1). However, there is very little in-
formation with respect to the enantiomeric separation and
toxicity of these chiral neonicotinoid insecticides.
This study is the first report on the enantiomeric separa-
tion of these three neonicotinoid insecticides using HPLC
and SFC. Three commercial polysaccharide-type chiral col-
umns, Chiralpak AD-H, a 3, 5-dimethylphenyl-carbamate
One of the biggest challenges in the study of the enantio-
selective profiles of chiral pesticides is the separation and
analysis of pure enantiomers. With various inherent advan-
tages such as rapid analysis, reproducibility, and flexibility,
high-performance liquid chromatography (HPLC) with differ-
ent chiral stationary phases (CSPs) has become one of the
most widely used methods for today’s enantiomeric separa-
tions.12,13 Nevertheless, in the last few years, supercritical
fluid chromatography (SFC) has emerged as a powerful alter-
native. The physicochemical properties of the supercritical
fluids allow to obtain separations with short analysis time,
high separation efficiency, low solvent consumption, and
Contract grant sponsor: National Natural Science Foundation of China;
Contract grant numbers: 40973077, 20837002, 20707021
Contract grant sponsor: National Basic Research Program of China;
Contract grant number: 2009CB421603
Contract grant sponsor: Program for Changjiang Scholars and Innovative
Research Teams in Chinese Universities; Contract grant number: IRT 0653
*Correspondence to: Shanshan Zhou, Zhejiang University of Technology,
Hangzhou 310014, China. E-mail: sszhou@zjut.edu.cn
Received for publication 10 December 2009; Accepted 3 May 2010
DOI: 10.1002/chir.20898
Published online 16 September 2010 in Wiley Online Library
(wileyonlinelibrary.com).
C
VV
2010 Wiley-Liss, Inc.

216
ZHANG ET AL.
The SFC tests were carried out by a Thar SD-ASFC-2 system (Thar
Technologies, Pittsburgh, PA) equipped with a UV/vis-151 detector and
a Rheodyne 7410 injector. The flow rate, injection volume, and UV detec-
tion wavelength were fixed at 2.0 ml min21, 5 ll, and 254 nm, respec-
tively. The column temperature was 358C and the outlet pressure was
set at 150 bar.
Chromatographic Characterization
The chromatographic parameters, including retention factor (k), sepa-
ration factor (a), and resolution (Rs) for the resolved enantiomers of the
three neonicotinoid insecticides were calculated and used to evaluate
the enantioselectivities of the CSPs. The retention factor (k) was calcu-
lated by the following formula:
Fig. 1. Structures of neonicotinoid pesticides; *Denotes chiral center.
derivative of amylose, coated onto 5-lm silica gel, Chiralcel
OD-H, a 3,5-dimethylphenyl-carbamate derivative of cellu-
lose, coated onto 5-lm silica gel, and Chiralpak IB, a 3,5-
dimethylphenyl-carbamate derivative of cellulose, immobi-
lized onto 5-lm silica gel, which have been demonstrated to
be highly effective not only for HPLC but also for SFC, were
used.21–25 Results of this study may be useful not only to pre-
pare small quantities of the enantiomers but also to correctly
determine their target activities and nontarget toxicities.
tR ꢁ t0
k ¼
ð1Þ
t0
where t0 is the column void time determined by recording the first base-
line perturbation. tR is the average retention time of duplicate injections
of the analytes taken at the peak maxima. The separation factor a was
calculated as:
MATERIALS AND METHODS
Chemicals and Reagents
k2
a ¼
ð2Þ
k1
Three neonicotinoid insecticides (1, 2, and 3) with purity >98.0%
were kindly donated by the East China University of Science and Tech-
nology (Shanghai, China). Stock solutions of all analytes were prepared
by dissolution in ethanol at a concentration of ꢀ500 mg L21. The mobile
phases used for chromatography were of HPLC grade and purchased
from Tedia Company (Fairfield, OH). Carbon dioxide with a purity of
99.995% was purchased from Jingong Specialty Gas (Hangzhou, China).
where k1 and k2 are the respective retention factors for the first and sec-
ond elution peaks. The resolution was calculated as:
1:18ðt2 ꢁ t1Þ
Rs ¼
ð3Þ
ðw1=2;1 þ w1=2;2
Þ
Column Information
where t1 and t2 are the peak retention time for the first and second enan-
tiomers eluted, respectively, and w1/2,1 and w1/2,2 are the half peak
widths of the first and second peaks, respectively.
Three commercial Daicel chiral columns were used in both HPLC and
SFC tests. They were Chiralpak AD-H (4.6 3 250 mm ID coated on 5-lm
silica gel), Chiralcel OD-H (4.6 3 250 mm ID coated on 5-lm silica gel),
and Chiralpak IB (4.6 3 250 mm ID immobilized on 5-lm silica gel).
RESULTS AND DISCUSSION
Chiral Separation by HPLC
Apparatus and Chromatographic Conditions
Enantioseparation is highly specific to CSPs in HPLC anal-
ysis. As shown in Tables 1 and 2, baseline resolution of the
enantiomers was obtained for both compounds 1 and 2 on
both of the coated CSPs. When the ethanol content of the
mobile phase was reduced, the values of k increased. How-
Liquid chromatography was performed on a Jasco LC-2000 series
HPLC system (Jasco Corporation, Tokyo, Japan) equipped with a PU-
2089 quaternary gradient pump, a mobile phase vacuum degasser, an
As-2055 automated microliter syringe with a 25-ll loop, a CO-2060 col-
umn thermostat, a UV-2075 detector, a variable-wavelength CD-2095 cir-
cular dichroism (CD), and an LC-Net II/ADC signal delivery system.
Chromatographic data were acquired and processed by computer-based
ChromPass software (version 1.7.403.1, Jasco). For the separation
experiments on various columns, the flow rate of mobile phase, the injec-
tion volume, and the UV detection wavelength were fixed at 1.0 ml
min21, 5 ll, and 254 nm, respectively. The oven temperature was set at
258C unless noted otherwise to determine the temperature dependence
of enantiomeric separation. In terms of the pressure drop, ethanol
proved to be more suitable as the polar modifier than 2-propanol in this
study. The initial column screening was conducted at 258C with a mobile
ever, the corresponding
a
values were essentially
unchanged, resulting in successful separation of 1 and 2
with all n-hexane/ethanol combinations used (hexane/etha-
nol from 10/90 to 50/50). These results suggested that the
conformations and adsorption sites of the modified polysac-
charide CSPs, as well as the selectand/selector associations
between the analytes and the CSPs are probably not affected
by the polar components of 1 and 2.26 However, stereoselec-
tive recognition of 3 on the chiral CSPs was significantly
influenced by the ethanol content. As listed in Table 2, satis-
phase of hexane/ethanol (40/60 v/v) at a flow rate of 1.0 ml min21
.
TABLE 1. Chromatographic separation results for 1 and 2 on Chiralpak AD-H column
Compound
1
2
Hexane/ethanol (v/v)
k1
10/90
0.51
20/80
0.67
30/70
0.90
40/60
1.33
50/50
1.77
10/90
0.80
20/80
1.05
30/70
1.44
40/60
2.22
50/50
3.41
k2
2.08
2.66
3.64
5.28
7.36
1.76
1.93
2.65
4.11
6.24
a
Rs
4.14
5.27
1/2
4.05
5.29
1/2
4.02
5.33
1/2
3.97
5.52
1/2
4.16
6.27
1/2
2.20
1.70
1/2
1.84
1.51
1/2
1.83
1.57
1/2
1.86
1.85
1/2
1.83
1.95
1/2
CD signala
aUV wavelength 5 254 nm.
Chirality DOI 10.1002/chir

CHIRAL NEONICOTINOID INSECTICIDES
217
factory separation of all four stereoisomers of 3 was only
achieved on the Chiralcel OD-H column when hexane/etha-
nol 5 90/10 (v/v) was used as the mobile phase. When the
percentage of ethanol in the mobile phase was increased
from 20 to 50%, peaks of the first and second eluted isomers
were consistently overlapped. On the other hand, when the
concentration of ethanol was decreased to 5%, no peak was
observed within 100 min (data not shown). Typical separa-
tion chromatograms of 1, 2, and 3 are shown in Figure 2. In
addition, as identified by the CD signals, it is interesting to
note that the elution order of the enantiomers of 2 was
reversed on Chiralcel OD-H in comparison to Chiralpak AD-
H, demonstrating that the chiral recognition interactions of
the two CSPs for compound 2 were different.
The chiral selector in Chiralpak IB is of the same nature
as that in Chiralcel OD-H, i.e., tris (3,5-dimethylphenylcarba-
mate) derivatized cellulose. Chiralcel OD-H is made by phys-
ical coating of the polymer on a silica gel, whereas the chiral
selector in Chiralpak IB is immobilized on the support. It is
well known that the immobilization of polysaccharide deriva-
tives on a matrix is an evolutionary approach that uses the
universal solvent compatibility of these highly selective CSPs
for enantioseparation. Besides the standard mobile phases
such as alkane/alcohols, pure alcohols, acetonitrile (ACN) or
their mixtures, the so-called nonstandard solvents including
dichloromethane (DCM), acetone, tetrahydrofuran, dimethyl-
formamide, or methyl tert-butyl ether (MTBE) can be safely
and effectively used as sample diluents. As a result, there is
no limitation on the sample injection solvents and racemiza-
tion of the enantiomers can be avoided if a suitable mobile
phase is chosen.27 More importantly, as the solubilities of 1,
2, and 3 in hexane are all extremely limited, the expansion
of the mobile phase selection opens up the possibility of
improving the performance for both the analytical and prepa-
rative resolutions.27 Table 3 summarizes the chromato-
graphic data achieved on the Chiralpak IB column using six
eluents: hexane, ethanol, methanol, ACN, MTBE, and DCM.
The results showed that the coated-type Chiralcel OD-H
showed a better chiral recognition for both 1 and 2 (Table
2) than the corresponding immobilized-type Chiralpak IB
(Table 3) under the same standard conditions. Moreover,
the stereoisomers of compound 3 were only partially sepa-
rated on the Chiralcel IB column with a mixture of n-hex-
ane/ethanol 5 90/10 (v/v) as the mobile phase (table not
shown). However, when the Chiralcel OD-H column was
used, nearly baseline separation of compound 3 was
obtained under the same conditions (Table 2). This is mainly
because the immobilized CSPs are chemically bonded to
silica gel through the hydroxyl groups of the polysaccha-
rides, resulting in an alteration in the high-order structure
and configuration of the polymers.28 For the various combi-
nations of solvents listed in Table 3, only a mixture of ethanol
and methanol as mobile phase offered successful separation
for both the enantiomers of 1 and 2. Moreover, compound 2
was also enantiomerically separated with a mobile phase of
100% ACN. However, with normal phases and nonstandard
solvents such as DCM and MTBE, no enantioseparation was
observed, even with a retention time over 60 min (results not
shown).
Chiral Separation by SFC
SFC is generally considered to be a normal phase separa-
tion technology. A polar organic solvent such as ethanol is
Chirality DOI 10.1002/chir

218
ZHANG ET AL.
Fig. 2. Optimal chromatograms of compounds 1, 2, and 3. Chromatographic conditions: Chiralcel OD-H column with n-hexane/ethanol: A: compound 1, (70/
30 v/v); B: compound 2, (70/30 v/v); C: compound 3, (90/10 v/v). Chiralpak AD-H column with n-hexane/ethanol; D: compound 1, (40/60 v/v); E: compound
2, (30/70 v/v); F: compound 3, (80/20 v/v). Flow rate: 1.0 ml min21; column temperature: 258C.
used as the CO2 modifier to increase the polarity of the mo-
bile phase. Table 4 gives the chromatographic separation
results for SFC. It was found that on the Chiralpak AD-H col-
umn, the Rs value of compound 1 was 6.42 for the SFC
mode, while the highest Rs value obtained by HPLC was 6.27
(Table 1). Regarding the analysis time, a much shorter elu-
tion time was required with SFC than HPLC [less than 15
min compared with 100 min (Fig. 3)]. For the enantiomers of
compound 2, the optimized resolution (Rs) in SFC was 5.23,
which was much higher than that (Rs 5 1.95) in HPLC.
These results suggested that the enantiomers of compound
2 could be baseline separated by either SFC or HPLC with
similar retention time (Fig. 3). However, compound 3 could
not be chirally resolved on Chiralpak AD-H by either SFC or
HPLC. According to the a values, the potential of Chiralcel
OD-H for chiral discrimination of compound 1 was poorer
when using SFC than when using HPLC (Tables 2 and 4).
For instance, the separation factor (a) was 1.08 for SFC
when 30% ethanol was used as the polar modifier, while that
for HPLC increased to 1.34 in a mobile phase with similar po-
TABLE 3. Chromatographic separation results for 1 and 2 on Chiralcel IB column
Compound
1
2
Hexane
Ethanol
Methanol
ACNa
DCMb
MTBEc
k1
k2
a
Rs
/
/
/
/
/
/
70
30
/
/
/
/
50
50
/
/
/
/
30
70 100
/
/
/
/
/
/
10
/
/
/
90
/
/
/
/
30 60
40
60
/
/
/
/
60
40
/
/
/
/
/
/
/
/
/
/
70
30
/
/
/
/
50
50
/
/
/
/
30
70 100
/
/
/
/
/
/
10
/
/
/
90
/
/
/
/
30 60
40
60
/
/
/
/
1.12 3.08
1.42 3.70
1.27 1.20
0.93 1.01
60
40
/
/
/
/
50 100
/
/
/
/
/
/
/
50 100
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
100 50
/
/
0.83
0.90
1.08
0.86
/
/
/
100 50
/
/
/
/
/
/
/
/
/
/
/
70 40
/
/
/
/
/
/
70 40
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
2.01 1.03 0.87 0.74 0.66
2.20 1.15 0.99 0.84 0.74
1.09 1.12 1.13 1.13 1.12
0.58 0.94 1.31 1.23 1.13
0.61 3.47 0.17
1.78 3.76 0.29
2.89 1.08 1.70
0.68 0.61 1.60
1.36 0.83 0.58 0.50 0.44
1.69 1.03 0.72 0.62 0.52
1.24 1.25 1.24 1.23 1.20
0.99 1.59 1.52 1.44 1.30
aAcetonitrile.
bDichloromethane.
cMethyl tert-butyl ether.
Chirality DOI 10.1002/chir

CHIRAL NEONICOTINOID INSECTICIDES
TABLE 4. Chromatographic separation results for 1, 2, and 3
219
by SFC
1
2
3
Compound
Column
AD
30
OD
30
AD
30
OD
30
OD
20
Ethanol (%)
20
20
30
10
k1
k2
2.50 3.42 9.29 3.99 4.77 16.78 1.82 5.05 30.38
5.80 3.69 9.98 9.68 6.15 22.64 2.00 5.37 32.04
k3
k4
/
/
/
/
/
/
/
/
/
/
/
/
3.02 7.79 46.16
3.49 9.41 55.1
a1
2.32 1.08 1.07 2.42 1.29 1.35 1.10 1.06 1.05
a2
a3
/
/
/
/
/
/
/
/
/
/
/
/
1.51 1.45 1.44
1.16 1.21 1.19
Rs12
Rs23
Rs34
6.42 0.92 1.05 5.23 1.70 2.99 0.76 0.76 0.80
Fig. 3. Optimal chromatograms of compounds 1 and 2. Chromatographic
conditions: A: compound 1, Chiralpak AD-H using CO2/ethanol (70/30, v/v)
as mobile phase; B: compound 2 using CO2/ethanol (80/20, v/v) as mobile
phase. Flow rate: 2.0 ml min21; column temperature: 358C.
/
/
/
/
/
/
/
/
/
/
/
/
3.15 4.17 4.19
1.26 2.15 2.04
TABLE 5. Effect of temperature on the enantiomeric separation of 1, 2, and 3 by HPLC
on the Chiralcel OD-H column
Compound
1
2
3
Temperature (8C)
k1
k2
k3
k4
a1
a2
a3
15
5.88
8.11
/
/
1.38
/
/
2.67
/
20
5.65
7.61
/
/
1.32
/
/
2.82
/
25
5.25
7.34
/
/
1.34
/
/
2.87
/
30
4.62
6.15
/
/
1.33
/
/
3.12
/
35
4.18
5.53
/
/
1.32
/
/
3.20
/
15
3.83
6.96
/
/
1.82
/
/
2.45
/
20
3.52
6.19
/
/
1.76
/
/
2.66
/
25
30
2.99
4.95
/
/
1.66
/
/
2.97
/
35
2.77
4.43
/
/
1.60
/
/
3.08
/
15
1.37
1.65
3.07
4.42
1.20
1.86
1.44
1.18
4.19
2.62
20
1.27
1.49
2.74
3.92
1.17
1.84
1.43
0.94
4.08
2.80
25
30
1.09
1.24
2.21
3.10
1.14
1.78
1.40
0.81
4.33
3.02
35
1.01
1.13
1.94
2.77
1.12
1.72
1.43
0.86
5.11
3.07
3.26
5.57
/
/
1.71
/
/
2.84
/
1.18
1.39
2.52
3.47
1.17
1.82
1.37
1.19
4.52
2.61
Rs12
Rs23
Rs34
/
/
/
/
/
/
/
/
/
/
TABLE 6. Effect of temperature on the enantiomeric separation of 1, 2, and 3 by SFC
on the Chiralcel OD-H column
Compound
1
2
3
Temperature (8C)
k1
k2
k3
k4
a1
a2
a3
35
2.97
3.14
/
/
1.06
/
/
0.79
/
40
45
3.10
3.36
/
/
1.08
/
/
0.83
/
35
3.67
5.06
/
/
1.38
/
/
2.77
/
40
3.85
5.11
/
/
1.33
/
/
2.54
/
45
4.14
5.37
/
/
1.30
/
/
2.15
/
35
1.26
1.35
2.18
2.67
1.07
1.61
1.22
0.61
4.40
2.14
40
45
1.16
1.30
2.20
2.55
1.12
1.69
1.16
0.91
3.67
1.11
2.85
3.05
/
/
1.07
/
/
0.81
/
1.24
1.32
2.11
2.58
1.06
1.60
1.22
0.44
2.82
1.29
Rs12
Rs23
Rs34
/
/
/
/
/
/
TABLE 7. Summary of enthalpy, entropy, and Tiso
HPLC
SFC
Compound
DDH8 (J mol21
)
DDS8 (J mol21 K21
)
R2
Tiso (8C)
DDH8 (J mol21
)
DDS8 (J mol21 K21
)
R2
Tiso (8C)
1
2
/
/
/
/
2026.6
24872.34
7.04
213.17
1.00
0.99
14.86
96.93
24660.50
211.20
0.997
143.11
Chirality DOI 10.1002/chir

220
ZHANG ET AL.
larity. Compound 2 was found to be satisfactorily enantio-
merically separated by both HPLC and SFC when using
Chiralcel OD-H as the chiral selector, with the chiral recogni-
tion capability of the former method being slightly better (Rs
5 3.43 vs. 2.99; Tables 2 and 4). However, the stereoisomers
of 3 were only partially resolved on the Chiralcel OD-H by
SFC (Table 4). For the Chiralcel IB column, no effective sep-
aration of the stereoisomers of the three analytes was
observed under SFC conditions, which did not appear to be
any better than those of HPLC.
In summary, SFC had some advantages over HPLC in the
chiral separation of the neonicotinoid insecticides we stud-
ied. First, SFC separation needed shorter retention time than
HPLC, which greatly reduces the amount of organic solvent
required, and is helpful for environmental safety. Second,
separation of the enantiomers of 1 and 2 on Chiralpak AD-H
was more efficient by SFC than by HPLC, especially for the
preparation of optically pure isomers.
Temperature can affect chiral separation in at least two
ways. One is its effect on the viscosity and diffusion coeffi-
cient of the solute. The other is the thermodynamic effect
that changes the separation factor (a).30 Generally, the for-
mer is small enough to be negligible, whereas the latter is
more important and can be investigated using the following
three equations:
DH DS
ln k0 ¼ ꢁ
ln a ¼ ꢁ
Tiso
þ
þ ln /
R
ð4Þ
ð5Þ
ð6Þ
RT
and
and
DDHꢂ DDSꢂ
þ
RT
R
DDHꢂ
DDSꢂ
¼
Effect of Temperature
In the above equations, f, R, and T mean the phase ratio,
gas constant, and absolute temperature, respectively. DH8
and DS8 are the standard transfer enthalpy and entropy of
the analyte from the mobile phase to the stationary phase;
and DDH8 and DDS8 are the differences (DH2 2 DH1) and
(DS2 2 DS1), respectively. When the enthalpy and entropy
contributions become equal, the selectivity equals one and
coelution occurs. The temperature at which coelution occurs
is called the isoelution temperature (Tiso).
Under HPLC conditions, significant deviation from line-
arity was observed for both 1 and 3 (data not shown), it
may due to the conformational change of the CSP.31 Only
2 gave a linear response (R2 ꢃ 0.99). Under SFC condi-
tions, 1 and 2 showed a linear relationship. The calculated
results for DDH8 and DDS8 for HPLC and SFC are tabu-
lated in Table 7. In the case of HPLC, the Tiso of com-
pound 2 was above the range of temperatures examined,
suggesting that enantioseparation of 2 was enthalpy con-
trolled, and could be better achieved at a lower tempera-
ture. Under SFC conditions, compounds 1 and 2 had lin-
ear responses. The Tiso obtained for compound 1 was
below and relatively close to the temperatures assayed. Its
separation was entropy controlled. On the other hand, the
high Tiso of compound 2 indicated an enthalpy-controlled
separation, suggesting that better separation would occur
at lower temperatures.32
Temperature is an important parameter in understanding
the chromatographic behaviors of the analytes as well as the
characteristics of the stationary phase. Changes in the col-
umn temperature can also be used to improve the enantiose-
lectivity of the CSPs. Given its substantially better perform-
ance, Chiralcel OD-H was chosen to determine the effects of
temperature in both HPLC and SFC modes. In the HPLC
tests, the column temperature was set from 15 to 358C at
intervals of 58C. In the case of SFC, only three temperatures,
that is, 35, 40, and 458C were used. The ethanol content was
maintained at 30% for both HPLC and SFC analyses. The val-
ues of k, a, and Rs at various temperatures are shown in Ta-
ble 5 for HPLC and Table 6 for SFC.
Data in Table 5 show that the values of both k and a for
compounds 1 and 2 decreased notably with an increase in
column temperature, while the values of Rs increased. The
effect of temperature on chiral discrimination of the CSPs
was more complex for 3 than for 1 and 2. For example, the
maximum Rs values were obtained at 25, 35, and 358C for
Rs12, Rs23, and Rs34, respectively (Table 5). In addition, the
separation factors (a) did not always decrease with an
increased oven temperature. However, the first and second
eluted isomers did not separate successfully at any of the
temperatures used.
In the SFC test, the influence of temperature on the chiral
separation of 1 was extremely slight, especially in view of
the values of a and Rs. For compound 2, the retention time
increased with an increased temperature, which was the op-
posite result to that for HPLC. A similar result was obtained
for the enantiomeric separation of proline derivatives using
SFC.29 This phenomenon may be ascribed to the fact that
although the pressure is kept constant as the temperature
increases, the density decreases, resulting in reduced solvat-
ing power of the mobile phase, hence increasing the reten-
tion of analytes. The a and Rs values of 2 decreased slightly
when the column temperature was increased, with a better
selectivity at a lower temperature. In the cases where poor
separation was achieved for compound 3, the values of k and
a changed slightly with an increase in temperature. How-
ever, the effect of temperature was more significant on the
corresponding Rs values.
CONCLUSIONS
Results of this study demonstrated that three chiral neoni-
cotinoid insecticides (1, 2, and 3) could be successfully
enantiomerically separated on a Chiralcel OD column by
HPLC. For the two compounds with only one asymmetric
center, i.e., 1 and 2, Chiralpak AD and Chiralcel IB also
worked. Moreover, satisfactory chiral separations of 1 and 2
were also obtained by SFC with lower retention time than by
HPLC, so SFC seems to be suitable for individual enantiomer
preparation. Overall, the established method could be used
for preparing small amounts of pure enantiomers of the chi-
ral neonicotinoid insecticides studied.
Chirality DOI 10.1002/chir

CHIRAL NEONICOTINOID INSECTICIDES
221
supercritical fluid chromatography-mass spectrometry. J Chromatogr A
2003;1003:157–166.
ACKNOWLEDGMENTS
The authors thank Xusheng, Shao of the East China Uni-
versity for kindly providing the three neonicotinoid insecti-
cides.
17. del Nozal MJ, Toribio L, Bernal JL, Castano N. Separation of triadimefon
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18. Tomizawa M, Casida JE. Neonicotinoid insecticide toxicology: mecha-
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