Direct enantioseparation of nitrogen-heterocyclic pesticides on cellulose-based chiral column by high-performance liquid chromatography

Article abstract of DOI:10.1002/chir.22385

The enantiomeric separation of eight pesticides including bitertanol (1), diclobutrazol (2), fenbuconazole (3), triticonazole (4), imazalil (5), triapenthenol (6), ancymidol (7), and carfentrazone-ethyl (8) was achieved, using normal-phase high-performanc

Full text of DOI:10.1002/chir.22385

CHIRALITY 27:32–38 (2015)
Direct Enantioseparation of Nitrogen-Heterocyclic Pesticides on
Cellulose-Based Chiral Column by High-Performance Liquid
Chromatography
1
TINGTING CHAI,1,2† WENWEN YANG,1,2† JING QIU,2 AND SHICONG HOU
*
1College of Science, China Agricultural University, Beijing, China
2Institute of Quality Standards & Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of
Agricultural Sciences, Beijing, China
ABSTRACT
The enantiomeric separation of eight pesticides including bitertanol (1),
diclobutrazol (2), fenbuconazole (3), triticonazole (4), imazalil (5), triapenthenol (6), ancymidol
(7), and carfentrazone-ethyl (8) was achieved, using normal-phase high-performance liquid
chromatography on two cellulosed-based chiral columns. The effects of isopropanol composition
from 2% to 30% in the mobile phase and column temperature from 5 to 40 °C were investigated.
Satisfactory resolutions were obtained for bitertanol (1), triticonazole (4), imazalil (5) with the
(+)-enantiomer eluted first and fenbuconazole (3) with the (—)-enantiomer eluted first on Lux
Cellulose-2 and Lux Cellulose-3. (+)-Enantiomers of diclobutrazol (2) and triapenthenol (6) were
first eluted on Lux Cellulose-2. (—)-Carfentrazone-ethyl (8) were eluted first on Lux Cellulose-2
and Lux Cellulose-3 with incomplete separation. Reversed elution orders were obtained for
ancymidol (7). (+)-Ancymidol was first eluted on Lux Cellulose-2 while on Lux Cellulose-3
(—)-ancymidol was first eluted. The results of the elution order at different column temperatures
suggested that column temperature did not affect the optical signals of the enantiomers. These
results will be helpful to prepare and analyze individual enantiomers of chiral pesticides.
Chirality 27:32–38, 2015. © 2014 Wiley Periodicals, Inc.
KEY WORDS: enantioseparation; chiral pesticide; chiral stationary phase; cellulose; HPLC
INTRODUCTION
EXPERIMENTAL
Chemicals and Reagents
Chirality has been a common property in the field of
commercial agrochemical compounds.1 A large number of
pesticides contain stereogenic centers and are introduced
into markets in the form of racemates. However, their enan-
tiomers often have distinct behaviors in biological and envi-
ronmental systems.2 Thus, the optically pure forms of chiral
pesticides are required to improve application efficiency,
avoid possible side effects caused by one enantiomer, and
study enantioselective toxicity, bioactivity, and environmen-
tal behaviors. Now many analytical technologies have been
applied to detect chiral pesticides such as high-performance
liquid chromatography (HPLC),3 HPLC-tandem mass spec-
trometry (LC-MS/MS),4 gas chromatography-mass spectrome-
try (GC-MS),5 and supercritical fluid chromatography (SFC).6
HPLC with chiral stationary phase (CSP) is one of the most use-
ful methods for enantioseparation at both the analytical and pre-
parative scales. Cellulose-tris(3,5-dimethylphenylcarbamate)
(CDMPC)7,8 and amylose-tris(3,5-dimethylphenyl-carbamate)
(ADMPC)9,10 belong to polysaccharide-based CSPs, which
were mostly used over the past three decades because they
exhibit excellent separation toward a large variety of chiral
compounds.11,12
In this report, two cellulose-based chiral columns including
Lux Cellulose-2 and Lux Cellulose-3 were used to separate eight
pesticides, bitertanol (1), diclobutrazol (2), fenbuconazole (3),
triticonazole (4), imazalil (5), triapenthenol (6), ancymidol (7),
and carfentrazone-ethyl (8) (Figure 1). The influence of column
temperature and mobile phase composition on resolution were
investigated. In addition, the enantiomeric elution orders on
two chiral columns were detected by optical rotation detector
(OR) in different separation conditions.
Racemic bitertanol (1), diclobutrazol (2), fenbuconazole (3), triticonazole
(4), imazalil (5), triapenthenol (6), ancymidol (7), carfentrazone-ethyl (8)
were purchased from Dr. Ehrenstorfer (Germany) with purity of >95%. Stock
solutions of all eight compounds were prepared at 1000 mg/L in 2-propanol
(IPA). 1,3,5-Tri-tert-butylbenzene (100 mg/L) was used to determine the void
time (t0). n-hexane and IPA (HPLC grade) were obtained from Fisher
Scientific (UK).
Instrumentation
Chromatographic separation was carried out on an Agilent 1200 series
HPLC equipped with a G1322A degasser, G1311A QuatPump, G1316B
column compartment, G1315C diode array detector, G1329A
autosampler, and a 20-μl sample loop (Wilmington, DE). UV signals were
acquired and manipulated using an Agilent Chemstation. The enantio-
meric optical rotations of chiral analytes were determined at 426 nm using
a
CHIRALYSER-MP optical rotation detector produced by
IBZMesstechnik (Germany) and provided by the Beijing Separation Sci-
ence & Technology Development Co. (Beijing, China). Optical signals
were acquired and processed using an Agilent Chemstation with signal
transformation using an Agilent 35900E A/D converter.
Chromatographic Conditions
Enantioseparation was obtained on chiral columns including Lux
Cellulose-2 (cellulose tris-(3-chloro-4-methylphenylcarbamate) (CCMPC)
and Lux Cellulose-3 (cellulose tris-(4-methylbenzoate) (CTMB),
*Correspondence to: Shicong Hou; Professor; College of Science, China Agri-
cultural University, Beijing 100193, China. E-mail: houshc@cau.edu.cn
†The first two authors contributed equally to this work.
Received for publication 28 March 2014; Accepted 10 August 2014
DOI: 10.1002/chir.22385
Published online 20 October 2014 in Wiley Online Library
(wileyonlinelibrary.com).
© 2014 Wiley Periodicals, Inc.

ENANTIOSEPARATION OF NITROGEN-HETEROCYCLIC PESTICIDES
33
Fig. 1. Molecular structures of eight chiral pesticides.
manufactured by Phenomenex (Torrance, CA), and obtained from
Guangzhou FLM Scientific Instrument Co. The structures of chiral col-
umns are shown in Figure 2. The columns were 250 mm × 4.6 mm (i.d., in-
ner diameter) and packed with 5-μm particles. The mobile phases were
composed of different proportions of n-hexane and IPA. The flow rate
was 0.8 mL/min. The column temperature was in the range of 5–40 °C.
The UV detection wavelength for bitertanol (1) and diclobutrazol (2)
was set at 210 nm, and for other target analytes at 220 nm. The injection
volume was 10 μL.
compositions of mobile phase (IPA:hexane). With the de-
crease of IPA from 30% to 2%, the separation factor (α) in-
creased slightly and better resolution factors (Rs) were
achieved on both chiral columns for bitertanol (1),
diclobutrazol (2), fenbuconazole (3), triticonazole (4),
imazalil (5), triapenthenol (6) and carfentrazone-ethyl (8)
on Lux Cellulose-2 and Lux Cellulose-3. For example, the Rs
value of triticonazole (4) on Lux Cellulose-2 was 25.20 when
the percentage of IPA was 10%, while the Rs value of
triticonazole (4) decreased to 15.25 when the composition
of IPA increased to 30%. For triapenthenol (6), incomplete
resolution was obtained with 10% of IPA in mobile phase,
while complete separation was obtained with 2% of IPA. But
ancymidol (7) was an exception, as with the decrease of
IPA from 20% to 5%,the Rs value decreased from 1.01 to 0.68.
As shown in Figure 1, all eight pesticides contain a
nitrogen-heterocyclic ring. Theoretically, bitertanol (1) and
diclobutrazol (2) have four enantiomers for the existing two
chiral carbons. However, in a previous study,13 two peaks of
bitertanol (1) on Lux Cellulose-1 could be separated with Rs
1.5 when acetonitrile was used as an organic modifier com-
bined with an aqueous solution. This separation also can be
obtained by capillary electrophoresis (CE) when an organic
solvent such as methanol or acetonitrile is added.14
Fenbuconazole (3) could be separated with excellent resolu-
tion on Lux Cellulose-2 in both normal-phase and reverse-
phase HPLC systems, which suggested that the change of
mobile phases from IPA to ethanol or acetonitrile did not
Chromatographic parameters were calculated using the following
equations. The retention factor was calculated as:
k′ ¼ ðtR ꢀ t0Þ=t0
(1)
where tR is retention time and t0 is void time. The separation factor was
calculated as:
α ¼ k′2=k′1
(2)
where k′1 and k′2 are retention factors of the first and second eluting enan-
tiomers, respectively. The resolution factor was calculated as:
Rs ¼ 2ðt2 ꢀ t1Þ=ðW2 þ W1Þ
(3)
where W1 and W2 are the base peak widths of the first and second eluting
enantiomers, respectively.
RESULTS AND DISCUSSION
Enantiomeric Separation on Lux Cellulose-2 and Lux
Cellulose-3
The data of enantioseparation both on Lux Cellulose-2 and
Lux Cellulose-3 are summarized in Table 1 with different
Fig. 2. Structures of CCMPC and CTMB.
Chirality DOI 10.1002/chir

34
CHAI ET AL.
TABLE 1. Stereoselective separations of chiral pesticides on Lux Cellulose-2 and Lux Cellulose-3 with different contents of IPA
Lux Cellulose-2 Lux Cellulose-3
Wavelength
(nm)
Content
of IPA(%)
Elution
order
Content
of IPA(%)
Elution
order
Pesticides
k1
k2
α
Rs
k1
k2
α
Rs
bitertanol (1)
210
5
10
20
5
10
20
20
30
10
20
30
9.41 14.42
1.53
1.30
1.13
1.19
1.15
1.15
1.26
1.12
3.62
3.66
3.56
7.75 (+)/(-)
4.04
1.66
2.52 (+)/(-)
2.13
1.63
3.43 (-)/(+)
1.95
25.20 (+)/(-)
24.11
5
10
20
5
10
20
20
30
5
10
20
5
10
20
5
10
20
5
10
20
5
6.79 13.85 2.04 12.13 (+)/(-)
3.95
1.69
3.34
1.46
0.66
5.14
1.91
3.96
1.68
0.76
3.05
1.09
—
5.79 1.90
1.97 1.81
7.09
3.92
—
diclobutrazol (2)
220
—
—
—
—
—
—
—
—
—
—
fenbuconazole (3)
triticonazole (4)
220
220
10.11 12.69
10.70 12.00
9.86 35.71
3.94 14.41
7.18 15.01 2.09
6.86 (-)/(+)
6.71
8.49 (+)/(-)
5.19
4.07
2.86
1.22
0.53
3.89
1.80
0.80
—
8.51 2.09
6.04 2.11
2.41 1.98
0.98 1.86
8.58 2.20 12.68 (+)/(-)
3.91 2.18 10.79
2.41
8.57
15.25
3.49
imazalil (5)
220
220
220
220
10
20
2
5
10
10.18 13.36
1.31
1.32
1.16
1.12
1.10
4.75 (+)/(-)
2.93
2.62 (+)/(-)
1.62
4.28
8.17
2.60
1.16
5.65
9.48
2.93
1.28
1.71 2.13
7.31
—
triapenthenol (6)
ancymidol (7)
—
—
—
—
—
—
—
—
—
—
1.10
9.10
3.65
1.52
3.25
2.13
1.34
9.25 1.02
3.77 1.03
1.60 1.05
3.79 1.17
2.45 1.15
1.54 1.14
0.68 (-)/(+)
0.94
1.01
3.08 (-)/(+)
2.72
2.20
10
20
2
10
20
5.29
—
5.50
—
1.04
—
0.86 (+)/(-)
—
carfentrazone-ethyl (8)
3.09
1.48
0.98
3.30
1.59
1.02
1.07
1.07
1.04
1.15 (-)/(+)
1.15
0.80
10
20
affect the enantioselective separation of fenbuconazole (3) on
Lux Cellulose-2.1 However, it can be partially separated
on Lux Cellulose-115 by reverse-phase HPLC. This indicates
that Lux Cellulose-2 and Lux Cellulose-3 showed better
chiral discriminability for fenbuconazole (3) than Lux
Cellulose-1. Imazalil (5) was separated on amylose
tris-(S)-1-phenylethylcarbamate (AS)16 with Rs in the range
of 1.08-1.12, using normal-phase HPLC. The enantioselective
separation of imazalil (5) was investigated on various types
of CSPs including cellulose tribenzoate (CTB), CTMB with
the maximal resolution of 4.83, cellulose triphenylcarbamate
(CTPC), and cellulose tris(3,5-dimethylphenyl carbamate)
(CDMPC).17 In our study, imazalil (5) was completely sep-
arated on Lux Cellulose-3, identical to CTMB, with a maxi-
mal resolution of 12.68 and on Lux Cellulose-2, identical to
CCMPC, with a maximal resolution of 4.75. Comparatively,
imazalil (5) got better separation on CTMB, compared to
other types of CSPs. Carfentrazone-ethyl (8) was partially
separated on CDMPC and on Lux Cellulose-2 with IPA as
mobile phase,18 but satisfactory separation on Lux
Cellulose-3.
In our study, different separation capacities for the eight
analytes on Lux Cellulose-2 and Lux Cellulose-3 were exhibited
differently. Under a suitable percentage of IPA, all chiral
analytes could obtain baseline separation (Rs> 1.5) except
ancymidol (7) and carfentrazone-ethyl (8) on Lux Cellulose-
2, and for diclobutrazol (2), triapenthenol (6), and
andancymidol (7) on Lux Cellulose-3. Generally, Lux
Cellulose-3 with a substituted group of 4-methylbenzoate on
cellulose-based chiral stationary showed better chiral
discriminability for bitertanol (1), fenbuconazole (3), imazalil
(5), and carfentrazone-ethyl (8) than Lux Cellulose-2 with 3-
chloro-4-methylphenylcarbamate. But for diclobutrazol (2),
Chirality DOI 10.1002/chir
triticonazole (4), triapenthenol (6), Lux Cellulose-2 showed
better chiral discriminability than Lux Cellulose-3. The differ-
ences of the substituted groups on cellulose-based chiral sta-
tionary might result in the change of enantiomer–CSP
interactions and recognition mechanisms. In the enantiomer–
CSP interactions and recognition mechanisms, enantiomers
with different stereochemistries were inserted into chiral
grooves of CSPs in the process of solute-CSP complexes forma-
tion, and then enantioseparation on CSPs occurs,19 OH groups
on chiral centers are available to participate in hydrogen bond-
ing with the C = O group of the CSP. Additionally, charge
transfer π–π interactions between the CSP and the aromatic
rings of the racemates can also provide stabilization of the
solute–CSP complexes20.
Effects of Column Temperature on Enantioseparation
The effects of column temperatures from 5 to 40 °C on Lux
Cellulose-2 and Lux Cellulose-3 were investigated and the
results are summarized in Table 2. The column temperature
was found to influence the enantiomer–CSPs interactions.
At rising temperature, k1 and k2 gradually decreased on Lux
Cellulose-2 and Lux Cellulose-3 for all eight analytes.
However, α and Rs values did not follow consistent trends.
On Lux Cellulose-2, α and Rs consistently declined for
bitertanol (1), diclobutrazol (2), triticonazole (4), imazalil
(5), and carfentrazone-ethyl (8) with an increase of column
temperature. But for fenbuconazole (3), triapenthenol (6),
and ancymidol (7), α and Rs increased with temperature.
On Lux Cellulose-3, α generally decreased with increasing
temperature, while Rs increased for all the analytes except
for ancymidol (7).
The van’t Hoff equations21 as follows were used
to calculate the thermodynamics parameters shown in

ENANTIOSEPARATION OF NITROGEN-HETEROCYCLIC PESTICIDES
35
TABLE 2. Effects of column temperatures on separation of chiral pesticides on Lux Cellulose-2 and Lux Cellulose-3
Lux Cellulose-2
Lux Cellulose-3
Pesticides
Temperature
(°C)
Hexane/IPA
95/5
k1
k2
α
Rs
Hexane/IPA
95/5
k1
k2
α
Rs
bitertanol (1)
5
10
20
30
40
5
10
20
30
40
5
10
20
30
40
5
10
20
30
40
5
10
20
30
40
5
10
20
30
40
5
10.76
10.17
9.41
8.73
7.99
17.09
15.97
14.42
13.16
11.96
3.90
1.59
1.57
1.53
1.51
1.50
1.19
1.26
1.17
1.17
1.17
1.09
1.11
1.26
1.14
1.16
3.86
3.76
3.66
3.36
3.11
1.34
1.32
1.31
1.29
1.18
1.11
1.12
1.12
1.14
1.16
1.02
1.02
1.07
9.23
8.11
7.75
7.48
7.34
2.52
3.15
2.49
2.56
2.52
1.76
2.86
3.43
3.24
3.38
20.95
21.36
24.11
20.72
20.22
5.20
4.66
4.75
3.85
2.38
1.39
1.52
1.62
1.83
2.00
0.58
0.58
1.52
8.07
7.52
6.84
6.22
5.73
—
16.50
15.48
13.93
12.53
11.24
—
2.04
2.06
2.04
2.01
1.96
—
7.21
8.57
12.13
13.76
15.70
—
diclobutrazol (2)
fenbuconazole (3)
triticonazole (4)
imazalil (5)
95/5
3.29
3.28
3.12
4.11
3.67
—
—
—
—
—
—
—
—
3.01
2.92
3.53
3.42
—
—
—
—
—
—
—
—
80/20
80/20
90/10
95/5
26.02
23.15
10.11
14.43
11.32
4.56
4.37
3.94
3.44
3.07
28.46
25.65
12.69
16.52
13.12
17.60
16.42
14.41
11.55
9.52
70/30
95/5
95/5
10.53
8.38
4.10
3.49
2.98
3.51
3.13
2.88
2.67
2.59
5.95
4.96
3.92
3.71
2.33
—
26.71
19.07
8.56
6.46
4.90
7.45
6.78
6.08
5.34
4.94
13.99
11.59
8.63
7.48
4.97
—
2.54
2.28
2.09
1.85
1.65
2.12
2.16
2.11
2.00
1.91
2.35
2.34
2.20
2.02
2.13
—
3.80
4.70
6.71
8.19
8.81
5.64
6.45
8.49
10.06
11.21
12.36
11.80
12.68
13.71
19.72
—
12.74
12.17
10.18
9.01
17.11
16.12
13.36
11.61
9.82
8.34
triapenthenol (6)
ancymidol (7)
2.79
2.70
3.10
3.02
—
—
—
—
—
—
—
—
2.60
2.47
2.93
2.82
—
—
—
—
—
—
—
—
2.27
2.65
95/5
16.46
15.43
12.66
16.81
15.76
13.50
95/5
12.03
10.41
9.15
12.67
10.80
9.31
1.05
1.04
1.02
1.29
1.08
0.68
10
20
Table 3 to better understand the thermodynamic effects on
enantioseparation.
bitertanol (1), diclobutrazol (2), triticonazole (4), imazalil
(5), and carfentrazone-ethyl (8), which meant a weaker
combination of first-eluted enantiomer with CSPs. The posi-
tive ΔΔH° values of fenbuconazole (3), triapenthenol (6),
and ancymidol (7) on Lux Cellulose-2 suggested that a
higher temperature was helpful to get better resolution. On
the other hand, negative ΔΔH° values of other analytes
meant a lower temperature was helpful to get better resolu-
tion. The ΔS° values of all target compounds were positive
on Lux Cellulose-2 and Lux Cellulose-3. The ΔΔS° values
of eight compounds were positive, which indicated that the
enantioselectivity was not an enthalpy-driven course.
However, most compounds cannot get the linear relations
between ln(α) or ln(k) and 1/T both on Lux Cellulose-2
and Lux Cellulose-3. The effects of temperature on the
resolution might depend not only on the structure of the
CSPs but also on that of the analytes.
ΔH° ΔS°
lnk ¼ ꢀ
þ
þ lnΦ
RT
R
ΔΔH° ΔΔS°
lnα ¼ ꢀ
þ
RT
R
where ΔH° and ΔS° are the changes of standard enthalpy
and entropy for the process of analyte from the mobile
phase to the stationary phase. ΔΔH° and ΔΔS° are the
differences of ΔH2°-ΔH1° and ΔS2°-ΔS1°, respectively. R
is the gas constant, T is absolute temperature, Φ is the
phase ratio. If plots of lnk versus 1/T are linear, -ΔH°/R
and ΔS°/R+ lnΦ are the slope and intercept. For linear plots
of lnα versus 1/T, the slope and intercept are -ΔΔH°/R
and ΔΔS°/R, respectively.
The values of ΔH° were negative and between –0.0050
and –0.0412 kJ/mol both on Lux Cellulose-2 and Lux
Cellulose-3, which suggested that an enthalpy-driven
process happened with the transfer of the enantiomers from
mobile phase to the stationary phase. The ΔH2° values were
more negative than ΔH1° values on Lux Cellulose-3 for
bitertanol (1), fenbuconazole (3), triticonazole (4), imazalil
(5), and carfentrazone-ethyl (8) and on Lux Cellulose-2 for
Elution Order
The optical rotation for all the enantiomers of target
pesticides were identified with an optical rotation detector
at 426 nm in different mobile phase systems. The results
of the elution order are summarized in Table 1. Although
triapenthenol (6), ancymidol (7), and carfentrazone-ethyl
(8) could not be significantly separated on two chiral
Chirality DOI 10.1002/chir

36
CHAI ET AL.
Chirality DOI 10.1002/chir

ENANTIOSEPARATION OF NITROGEN-HETEROCYCLIC PESTICIDES
37
Fig. 3. Typical UV and OR chromatograms of bitertanol (1), diclobutrazol (2), fenbuconazole (3), triticonazole (4), imazalil (5), triapenthenol (6), ancymidol
(7), and carfentrazone-ethyl (8). Chromatographic conditions: hexane/IPA at 95/5 for (1), (2), (6), and 80/20 for (3) and (4) on Lux Cellulose-2, at 95/5 for
(5) and 80/20 for (7) and (8) on Lux Cellulose-3. All column temperatures were 20 °C.
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CONCLUSION
Eight chiral compounds were successfully separated with
satisfactory resolution on Lux Cellulose-2 and Lux Cellulose-
3, using NP-HPLC with hexane/IPA as the mobile phase.
Generally, with a decrease of IPA, better resolutions were
achieved on both chiral columns. Although most compounds
cannot achieve linear relations between ln (α) or ln (k) and
1/T on Lux Cellulose-2 and Lux Cellulose-3 when the column
temperature was in the range of 5 to 40 °C, α and Rs were
changed to some extent. The results from OR showed that
the temperature changes did not reverse the stereoisomeric
optical signals of all pesticides, but the signals of some pesti-
cides would change on different columns. This work will be
helpful to easily prepare single enantiomers from racemic
mixtures, and to establish effective analytical methods for fu-
ture study on the stereoselective behaviors of chiral pesti-
cides in the environment.
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ACKNOWLEDGMENT
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2001;906:105–125.
Contract grant sponsor: Ministry of Science and Technology
of the People’s Republic of China; Contract grant numbers:
2011BAD23B05-4.
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