Metolachlor stereoisomers: Enantioseparation, identification and chiral stability

Accepted Manuscript Title: Metolachlor Stereoisomers: Enantioseparation, Identification and Chiral Stability Author: Jingqian Xie Lijuan Zhang Lu Zhao Qiaozhi Tang Kai Liu Weiping Liu PII: DOI: Reference:

S0021-9673(16)30967-0 http://dx.doi.org/doi:10.1016/j.chroma.2016.07.045 CHROMA 357758

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

26-5-2016 9-7-2016 16-7-2016

Please cite this article as: Jingqian Xie, Lijuan Zhang, Lu Zhao, Qiaozhi Tang, Kai Liu, Weiping Liu, Metolachlor Stereoisomers: Enantioseparation, Identification and Chiral Stability, Journal of Chromatography A http://dx.doi.org/10.1016/j.chroma.2016.07.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Metolachlor Stereoisomers: Enantioseparation, Identification and Chiral Stability

Jingqian Xie, Lijuan Zhang, Lu Zhao, Qiaozhi Tang, Kai Liu and Weiping Liu*

MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China

*

To whom correspondence should be addressed.

Tel.: +86-571-8898-2740; Fax: +86-571-8898-2740. E-mail address: [email protected].

Highlights 1



Metolachlor was successfully entioseparated



Absolute configuration of the four stereoisomers was identified



The elution order was αSS, αRS, αSR, αRR

2

1

Abstract: Metolachlor is a chiral herbicide consisting of four stereoisomers, which is typically

2

used as a racemic mixture or is enriched with the herbicidally active 1’S-isomers. Because studies

3

on the enantioselective behavior of phyto-biochemical processes and the environmental fate of

4

metolachlor have become significant, a practical method for analyzing and separating

5

metolachlor stereoisomers must be developed. In the present study, the enantiomeric separation

6

of metolachlor was achieved using OD-H, AS-H, OJ-H and AY-H chiral columns. The effects of

7

different organic modifiers in an n-hexane-based mobile phase were investigated, and various

8

temperatures and flow rates, which may influence metolachlor separation, were also explored.

9

The optimal resolution was obtained using an AY-H column with n-hexane/EtOH (96/4) as the

10

mobile phase at a rate and temperature of 0.6 ml min-1 and 25 C, respectively. The absolute

11

configuration of the four stereoisomers was identified as αSS, αRS, αSR, αRR using computed and

12

experimentally measured ECD and VCD spectra. Thermal interconversion and solvent stability

13

experiments were also performed. Pure metolachlor stereoisomers in different organic solvents

14

and water at 4 C or 30 C were stable. These results were used to establish a sound method for

15

analyzing, preparing, characterizing, and preserving individual metolachlor stereoisomers in most

16

natural environments.

17

Key words: metolachlor stereoisomers; enantioseparation; electronic circular dichroism;

18

vibrational circular dichroism; chiral stability

19

3

20 21

1. Introduction Metolachlor (2-chloro-N-[2-ethyl-6-methylphentl]-N-[2-methoxy-1-methylethyl] acetamide)

22

is a pre-emergent selective herbicide for the control of a variety of annual grass and broad leaf

23

weeds in corn and other crops [1]. An asymmetric carbon atom in the alkyl moiety of metolachlor,

24

as well as hindered rotation about the Ar-N bond, yields two sets of enantiomers: αSS/ αRR and

25

αRS/αSR. Metolachlor was initially placed on the market as a racemic product. However, Moser

26

et al. [2] found that C*S-metolachlor (with respect to the C chiral center, αRS and αSS) showed

27

the highest herbicidal activity, while C*R-metolachlor (αRR and αSR) possessed superior

28

antifungal properties. Although racemic metolachlor was replaced by C*S-metolachlor-enriched

29

products in many places to reduce herbicide usage and eschew the side effects of unnecessary

30

enantiomers, C*R-metolachlor has been identified in water [3, 4] and other media.

31

Enantiomers possess identical physico-chemical properties, making them difficult to

32

separate and analyze. Nevertheless, a variety of biological metabolic pathways have shown

33

stereoselectivity. Although reports on the successful separation of metolachlor enantiomers have

34

been limited, GC-CSP (gas chromatography-chiral phase separation) [5] and γ-CD-MEKC

35

(cyclodextrin-modified micellar electrokinetic chromatography) [6] have provided partial

36

resolution of the four isomers of metolachlor. Muller et al. [7] reported on the application of the

37

achiral Hypercarb HPLC column and the chiral Chiralcel OD-H HPLC column for the separation

38

and isolation of two metolachlor isomers in high enantiomeric purity. The absolute

39

configurations of the four stereoisomers were assigned through polarimetric measurements in

40

reference to previous data, and the kinetics of thermal interconversion was also studied. Polcaro

41

et al. developed a method using a mixture of diethyl ether (DEE) and n-hexane as the mobile

42

phase on an OD-H column, allowing the full separation of all four stereoisomers, which have not

43

been previously resolved on any other chiral stationary phases [8]. However, DEE must be 4

44

freshly prepared prior to HPLC analysis. And conformation transformations may occur under

45

certain circumstances, such as exposure to heat and polar solvents, promoting isomer inversion

46

and racemization. Jayasundera et al. identified the labile sites of metolachlor, as well as

47

conformational and configurational changes in different chemical environments [9].

48

Herein, we focused on developing a simple method for the separation of all four stereoisomers of

49

metolachlor. The resolution of Chiracel OD-H, Chiracel AS-H, Chiracel OJ-H and Chiracel AY-

50

H was compared, and the effects of the mobile phase composition, column temperature, and flow

51

rates on the resolution were also investigated. The absolute configurations were assigned based

52

on their optical rotation in ECD and VCD spectra, using both experimental and calculated data.

53

The chiral stability of the four stereoisomers at 4 C and 30 C in different organic solvents and

54

water was also determined. The current study provided an optimal approach for the acquisition of

55

pure stereoisomers and the characterization of the absolute configurations of metolachlor. The

56

results also offered further clarification on the thermal interconversion and solvent stability of the

57

stereoisomers.

58

2. Materials and Methods

59

2.1 Chemicals and Reagents

60

Racemic metolachlor (>97%) and C*S-metolachlor were provided by Shandong Qiaochang

61

Chemical (QCC). All HPLC grade solvents were obtained from Sigma. Stock solutions were

62

prepared at a concentration of 1 mg ml-1 in n-hexane and were stored at 4 C.

63

2.2 Chiral Separation of Metolachlor by HPLC

64

Metolachlor was separated on an enantioselective HPLC system consisting of a pump,

65

autosampler, column oven, photo diode array detector, and circular dichroism detector (Jasco

66

models PU-4180, AS-4050, Co-4061, MD-4010 and CD-4095, respectively). Individual

5

67

stereoisomers were acquired on commercial chiral columns, including a Chiralpak AY-H (5 µm,

68

4.6 mm i.d.×250 mm), Chiralcel AS-H (5 µm, 4.6 mm i.d.×250 mm), Chiralcel OD-H (5 µm,

69

4.6 mm i.d.×250 mm) and Chiralcel OJ-H column (5 µm, 4.6 mm i.d.×250 mm). The structures

70

of the four chiral stationary phases on columns were in Figure 1. UV and CD detection were

71

performed at 230 nm. Racemic metolachlor was dissolved in n-hexane to a concentration of 1 mg

72

mL-1. An injection volume of 10 µl was used in the enantioseparation of racemic metolachlor and

73

the subsequent analytical runs to determine the purity of the stereoisomers. Stereoisomers

74

obtained via HPLC were used to characterize the chiral configuration and evaluate the stability.

75

2.3 Electronic Circular Dichroism (ECD) and Vibrational Circular Dichroism (VCD)

76

Experiments and Computations

77

The combination of ECD and VCD was used to assign the configuration of the separated

78

stereoisomers. The ECD spectra of the four stereoisomers dissolved in n-hexane were obtained

79

on a Jasco J-1500 CD spectrometer, and the VCD spectra of the stereoisomers dissolved in CCl4

80

was evaluated on a Jasco FVS-6000 VCD spectrometer. ECD was performed at 210-450 nm and

81

25 C under a constant flow of nitrogen. The bandwidth and speed were set to 1.00 nm and 200

82

nm min-1, respectively. A quartz cuvette with a path length of 10 mm was utilized. The VCD

83

spectra were acquired at 2000 - 850 cm-1, and the path length of the quartz cuvette was 0.05 mm.

84

Each VCD measurement was performed for approximately 1 h. The concentrations of the

85

stereoisomers were adjusted to obtain the highest quality ECD and VCD spectra [10]. For the

86

ECD and VCD experiments, sample concentrations of 0.05 mg mL-1 and 100 mg mL-1 were

87

employed, and the spectra were baseline-corrected against n-hexane and CCl4, respectively.

88

The energies, oscillations and rotational strengths of the stereoisomers were calculated using

89

TDDFT (time-dependent density functional theory) for ECD and DFT (density functional theory)

6

90

for VCD by Gaussian 09 [11]. A conformational search was carried out with ComputeVOA for

91

each stereoisomer configuration at the molecular mechanics level, the ten lowest energy

92

conformers were further geometry optimized and frequency calculated with Gaussian09 (pbepbe

93

functional/6-31+g (d,p) basis set). During this step, only some conformers remained due to the

94

absence of vibrations with imaginary frequencies. And then the VCD spectra and ECD spectra of

95

the remained conformers were predicted using Gaussian09 (pbepbe functional/6-31+g(d,p) basis

96

set and bvp86 functional /6-31+g(d,p) basis set respectively) [12]. Lastly, the Boltzmann-

97

population-weighted calculated spectra for each configuration were obtained by the remained

98

conformers. All calculation was conducted without considering solvent effects.

99

2.4 Equilibration Experiments

100

The potential for conversion at different temperatures and in diverse solvents were further

101

researched. The initial stereoisomer solvent was removed using a nitrogen purge, and isometric

102

ethanol, isopropanol, acetone, ethyl acetate, and n-hexane were subsequently added. Specifically,

103

0.8 mL of the stereoisomer stock solution was dried, and the resulting residue was dissolved in an

104

equal volume of acetone and diluted with ultrapure water to a total volume of 40 mL. After 2, 5,

105

10, and 15 days, solutions of the above-mentioned solvents at 4 and 30 C were analyzed via

106

normal-phase HPLC. Each time, 8 mL of an aqueous sample were extracted three times with 8

107

mL of ethyl acetate. The extract was dried and recovered in 0.8 mL of n-hexane for the stability

108

study.

109

3. Results and Discussion

110

3.1 Separation and Isolation of Metolachlor Stereoisomers

111

Separation is considered optimal when the best compromise among all variables related to

112

separation is achieved, including retention time, chromatographic runtime, and cost of solvents

7

113

[10]. Metolachlor possesses two chiral centers; thus, four stereoisomers are possible (Figure 2).

114

On achiral columns, only one single peak was observed. Therefore, the enantiomeric separation

115

of metolachlor was evaluated on OD-H, OJ-H, AS-H and AY-H chiral columns. Regarding the

116

chiral recognition of the columns, AY-H provided the best separation and resolution. The mobile

117

phase in nonpolar organic mode is often composed of ethanol (EtOH) and isopropanol (IPA).

118

Regardless of the solvent (EtOH, IPA or freshly prepared DEE), full enantiomer resolution was

119

not achieved on OD-H, and the stereoisomers were only partially separated (Figure 3). According

120

to the literature [13], only three peaks appeared when n-hexane and IPA were used as the eluent

121

on OD-H. In the present experiments, although four peaks were observed, baseline separation

122

was not achieved on an OD-H column (Figure 3). Moreover, the separation results on AS-H and

123

OJ-H were also not satisfactory. As shown in Figure 3, AS-H provided the best separation effect

124

among the three chiral columns, expect for AY-H. OJ-H provided the poorest separation of

125

metolachlor, showing a lack of baseline separation. In contrast, AY-H has been successfully used

126

several times for enantioseparation. When AY-H was applied in the current separation, the use of

127

n-hexane and IPA as the mobile phase resulted in the appearance of four peaks. Moreover, the

128

use of ethanol as an organic modifier in n-hexane led to high selectivities on an AY-H chiral

129

column. When a suitable percentage of EtOH was used and the n-hexane/ethanol ratio ranged

130

from 85/15 to 98/2, good separation effects were observed, allowing complete baseline separation

131

(RS>1.5) of all four stereoisomers (Table 1). When EtOH was not added or the percentage of

132

EtOH was 1%, the elution time was long, but an increase in retention did not occur, which was

133

unsatisfactory. Table 1 shows the resolution of AY-H at different n-hexane/EtOH ratios. As the

134

ethanol content decreased from 20% to 4%, the retention factors RS12, RS23, and RS34 increased,

135

providing better resolution. Therefore, AY-H sufficiently resolved the four isomers of

136

metolachlor. The use of n-hexane/ethanol (96/4) on an AY-H column provided optimal 8

137

discriminability in the separation. Judging from the peak area of each stereoisomer in UV

138

absorbance, the ratio of the four stereoisomers in the racemic standard was 2:1:1:2

139

(pk1:pk2:pk3:pk4).

140

The elution of metolachlor was optimized at 20, 25, 30, 35, and 40C (Table 2) on an AY-H

141

column. The column temperature showed a strong influence on enantiomer-CSP interaction.

142

Namely, as the temperature increased from 20 to 25C, an improvement in peak resolution was

143

observed. However, when the temperature was set between 25 and 40C, Rs decreased. The vant

144

Hoff equation was used to calculate the thermodynamic parameters [14].

145

ln k  

H  S   ln  RT R

ln   

146

H  S   RT R

147

where ΔH and ΔS represent changes in the standard enthalpy and entropy as the analyte moved

148

from the mobile phase to the stationary phase. ΔΔHand ΔΔS  were calculated as ΔH2-ΔH1 and

149

ΔS 2-ΔS1. R, T, and Φ represent the gas constant, absolute temperature, and phase ratio,

150

respectively.

151

The values of ΔH, ΔS, ΔΔHand ΔΔS were all negative (Table 3), which suggested that the

152

transfer of all four enantiomers from the mobile phase to the stationary phase was enthalpy-

153

driven. The values of ΔH1, ΔH2, ΔH3 and ΔH4 decreased with an increase in their retention

154

time, indicating that compounds with earlier retention times had weaker interactions with the

155

CSPs and that the compounds became more orderly after complexation with the CSPs. The value

156

of the separation factor decreased with an increase in temperature.

157

The results shown in Table 4 suggested that when the flow rate was increased from 0.4 ml

158

min-1 to 0.6 ml min -1, the resolution also increased, providing faster elution. However, superior 9

159

resolution was observed at 0.6 ml min -1, compared to that obtained at 0.8 ml min-1 and 1 ml min -1.

160

Therefore, using an AY-H column and n-hexane/EtOH (96/4) at 0.6 ml min -1 and 25 C, optimal

161

separation was obtained (Figure 4). Fractions corresponding to individual stereoisomers were

162

automatically collected. The AY-H column was later used to analyze the purities of the isolates,

163

indicating >99% purity. However, the elution order could not be identified.

164

A variety of theoretical mechanisms for enantiomer separation have been suggested, but

165

most scholars accept the three-point rule [15]. Although the exact mechanism is not known,

166

enantiomer separation may have occurred due to the following forces: the carbonyl (C=O) group

167

in metolachlor and –NH group in Chiralcel AS-H, OD-H and AY-H form a hydrogen bond, and

168

the C=O of metolachlor forms a dipole–dipole bond with the C=O of Chiralcel AS-H, OD-H, OJ-

169

H and AY-H. Moreover, the phenyl moiety in metolachlor and the aromatic ring on AS-H, OD-H,

170

OJ-H and AY-H form π-π interactions. The Cl atom in metolachlor hydrogen bond with the –NH

171

on AS-H, OD-H and AY-H. An important difference among AS-H, OD-H, OJ-H and AY-H

172

columns is presence of a Cl atom on the CSP of AY-H, which may increase hydrogen bonding

173

interactions between metolachlor and the CSP. Moreover, the atom weight of Cl is 37.5, larger

174

than other atoms or groups in the CSPs structure. The Cl atom occurred in methyl para position

175

on AY-H would like to enhance the steric hindrance, contributing to higher selectivity. In

176

addition, a C atom with an S configuration was present on AS-H, which may have increased

177

interactions between the CSP and metolachlor, compared to OD-H and OJ-H.

178

3.2 Characterization of the Absolute Configurations of Metolachlor Stereoisomers

179

Experiments and computational simulations were performed to determine the configuration

180

of the four stereoisomers. Simple inspection and comparison of the computed ECD and VCD

181

spectra to the experimental ECD and VCD spectra showed that the experimental bands were

182

qualitatively similar with the calculated bands expect that peak 2 and peak 3 were systematically 10

183

shift a little. The convergent results illustrated in Figure 4 showed that both the ECD and VCD

184

signals of peak 1 and 4, peak 2 and 3 exhibited nearly equal magnitude and opposite sign. Mirror-

185

image enantiomers usually exhibit mirror-image ECD and VCD spectra [16]. Thus, one may

186

conclude with a high level of confidence that peak 1 and 4, peak 2 and 3 were the resolved

187

enantiomers [8].Moreover, peak 1, 2, 3, and 4 were assigned as exhibited in Figure 5: (1) αSS; (2)

188

αRS, (3) αSR,; (4) αRR,. The elution order observed herein was not in accordance with the results

189

reported by Chiara M. Polcaro, which were obtained on an OD-H column using freshly prepared

190

DEE [8]. However, the present results were consistent with the elution order obtained by Muller,

191

who used an achiral Hypercarb column and a chiral Chiralcel OD-H column, i.e., αS prior to αR,

192

and C*S prior to C*R [7].Although analytical standards for the isomers of metolachlor were not

193

available, the separation diagram of C*S-metolachlor had once again allowed the direct

194

identification of peak 1 and 2, which corresponded to the C*S diastereomers (Figure 6).

195

3.3 Thermal Conversion and Solvent Stability

196

Generally, the preservation, extraction and analysis of stereoisomers are performed in

197

organic solvents. Secondary peaks other than the analyte stereoisomer were not observed for any

198

of the isomers in the tested organic solvents in our experiments, indicating that the four

199

stereoisomers of metolachlor were stable in the presence of heat and various solvents in

200

conventional environments. In addition, isomer conversion did not occur during the storage of the

201

stereoisomers in n-hexane, methanol and acetone for more than 6 months at 4 C. In contrast,

202

most ecotoxicology simulations are performed in aquatic environments because nearly all organic

203

compounds enter the water through direct or indirect pathways. The stability of metolachlor in

204

water was further evaluated, and the results were similar to those obtained with organic solvents:

205

metolachlor stereoisomers were stable and did not engage in any detectable isomer conversion.

206

Jayasundera et al.[9] reported that pure aromatic and nonaromatic solvents can stabilize 11

207

individual metolachlor conformations. However, metolachlor isomers in Bz-d6: DMSO-d6 (40:60)

208

exchanged quickly, and a small amount of D2O slowed the velocity due to hydrogen bonding

209

between C=O and –OCH3 oxygen atoms. At the end of the article, Jayasundera suggested that

210

different metolachlor isomers were stable in most natural environments, which was in accordance

211

with the results obtained herein. Moreover, when the temperature increases, metolachlor may

212

undergo thermal interconversion. Muller [7] et al. found that metolachlor stereoisomers

213

interconverted rapidly at 200 C. During this process, C-chirality was in thermal equilibrium, and

214

the atropisomerism of the isomers depended on rotation about the phenyl-nitrogen bond. The

215

energy barrier for rotation around this bond is 154 kJ mol-1, which is relatively high [2].

216

Therefore, configurational changes can only occur by overcoming the energy barrier or

217

decreasing the activation energy, which explains the fixed stereoisomer configurations observed

218

in the present study.

219

4. Conclusions

220

Metolachlor was optimally separated into four stereoisomers on an AY-H chiral column

221

using n-hexane/EtOH (96/4) as the mobile phase at a flow rate of 0.6 ml min-1 and a column

222

temperature of 25 C. Complete baseline separation was achieved. The measured and calculated

223

ECD and VCD results showed that the isomers eluted according to the following sequence: αSS,

224

αRS, αSR, αRR. In most natural environments, the individual isomers were stable. The newly

225

developed coherent method described herein may be used to analyze other chiral chemicals.

226

Acknowledgements

227

The authors acknowledge financial support from the National Natural Science Foundation of

228

China (21427815 and 213320102007).

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24

Figure Captions Figure 1 The structures of the four CSPs on the chiral columns. Figure 2 Chemical structure of the four isomers of metolachlor. Figure 3 The optimum separation spectra of metolachlor on (A) AS-H, Hexane/IPA=85/15, 0.6 ml min-1, 25 C, 230 nm; (B) OD-H, Hexane/IPA=90/10, 0.6 ml min-1, 25 C, 230 nm; (C) OJ-H column, Hexane/IPA=90/10, 0.6 ml min -1, 25 C, 230 nm. Figure 4 Resolution of Metolachlor on Chiracel AY-H using 4% EtOH in n-hexane as the mobile phase with (A) UV, 230 nm; (B) CD, 230 nm. Flow rate, 0.6 ml min -1; Column temperature, 25 C. Figure 5 Measured and Computed ECD and VCD spectra. (A) ECD ; (B)VCD Figure 6 Separation of C*S-Metolachlor on Chiracel AY-H using n-hexane/EtOH (96/4) (A) UV, 230 nm; (B) CD, 230 nm. Flow rate, 0.6 ml min -1; Column temperature, 25 C. Table 1. Separation of the chiral compound metolachlor on Chiralpak AY-H with nhexane/ethanol as the mobile phase n-hexane/ethanol ratio

RS12

RS23

RS34

80/20

1.459

2.809

2.221

85/15

1.616

3.139

2.434

90/10

1.826

3.606

2.696

95/5

2.059

4.400

2.990

96/4

2.010

4.500

2.946

97/3

1.828

4.711

2.540

25

98/2

1.951

5.289

2.897

99/1

1.432

6.029

2.694

26

Table 2. Effects of column temperature on the separation of metolachlor isomers on AY-H column Temperature (C) 20 25 30 35 40

RS12 1.862 2.022 1.987 2.005 1.786

RS23 4.527 4.627 4.546 4.497 4.020

RS34 3.037 3.081 3.063 2.942 2.563

27

Table 3. Van’t hoff equations and thermodynamic parameters Pesticides

R12

ΔH°(KJ ΔS(J mol-1) mol-1 K-1)

0.999

-10.218

-54.418

lnk2=1271.7/T-3.4088

0.999

-10.573

lnk3=1461.8/T-3.8169

0.999

lnk4=1713.5/T-4.4968

0.998

lnk=-ΔH°/RT+ΔS°/R

metolachlor lnk1=1229/T-3.3673

lnα=-ΔΔH°/RT+ΔΔS°/R

R22

ΔΔH°(KJ mol-1)

ΔS°(J mol-1 K-1)

-54.763

lnα1=49.73/T-0.0640

0.988

-0.413

-0.532

-12.153

-58.156

lnα2=206.3/T-0.4630

0.971

-1.715

-3.849

-14.246

-63.809

lnα3=251.82/T-0.6801

0.974

-2.094

-5.654

28

1

Table 4. Effects of flow rate on the separation of metolachlor isomers on AY-H

2

column Flow rate (ml min-1) 0.4 0.5 0.6 0.8 1.0

RS12 1.881 1.842 2.022 1.596 1.486

RS23 3.996 3.864 4.627 3.356 3.117

RS34 3.276 3.132 3.081 2.662 2.445

3 4

29