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High-performance liquid chromatographic separation of imidazolinone herbicide enantiomers and their methyl derivatives on polysaccharide-coated chiral stationary phases
Journal of Chromatography A, 1117 (2006) 184–193
High-performance liquid chromatographic separation of imidazolinone herbicide enantiomers and their methyl derivatives on polysaccharide-coated chiral stationary phases Wenjian Lao, Jay Gan ∗ Department of Environmental Sciences, University of California, Riverside, CA 92521, USA Received 5 December 2005; received in revised form 23 March 2006; accepted 24 March 2006 Available online 18 April 2006
Abstract Many chiral pesticides exhibit enantioselectivity in biotransformation and ecotoxicity in the environment. A significant class of chiral pesticides is imidazolinone herbicides, of which enantioselectivity has not been well studied. Development of efficient chiral separation methods is the first step for allowing characterization of enantioselectivity in environmental processes. In this study, we attempted to resolve enantiomers of imidazolinone herbicides using reversed-phase and normal-phase high-performance liquid chromatography with polysaccharide-type chiral columns. Enantiomers of imazethapyr, imazaquin, and imazamox were separated on a Chiralcel OD-R column using 50 mM phosphate buffer-acetonitrile as mobile phase. Enantiomers of imazapyr, imazapic, imazethapyr, imazamox and imazaquin were resolved on a Chiralcel OJ column using n-hexane (0.1% trifluoroacetic acid)-alcohol as mobile phase. The enantiomers of five methyl derivatives of imidazolinone herbicides were also resolved on the Chiralcel OJ column. The k values revealed a structure-enantioselectivity relationship for the separation behaviors of the enantiomers on the OJ column. The described method was successfully applied for chiral analysis of two imidazolinone herbicides (imazapyr and imazaquin) in spiked soil samples. © 2006 Elsevier B.V. All rights reserved. Keywords: Imidazolinone herbicides; Enantiomer separation; Chiral stationary phases; Chiral contaminants
1. Introduction High-performance liquid chromatography (HPLC) has become one of the most powerful techniques in the field of enantioselective separation. Although a large number of chiral compounds, including pharmaceuticals, food additives, natural products, synthetic intermediates, pollutants and pesticides have been separated by HPLC, separation of enantiomers is still one of the most challenging tasks. Up to 25% of pesticides are chiral compounds, and most of them are currently used as mixtures of stereoisomers or enantiomers. Enantioselectivity in degradation, toxicity and biological activity has been reported for various chiral pesticides, such as synthetic pyrethroids, organophosphate insecticides, and some chiral herbicides [1–3]. The need for characterizing enantioselective environmental behaviors of chiral pesticides has spurred the development of analytical methods for enantiomer separation. Gas chromatography (GC)
∗
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and capillary electrophoresis (CE) techniques have shown high efficiency for separating enantiomers of some chiral compounds [4–6]. However, capillary GC and CE have drawbacks for preparative separation due to the small injection quantity and concentrations [7–9]. In contrast, HPLC is currently the best option for analytical and small-scale preparative separation through which enantiomerically pure chemicals may be obtained and used for evaluation of environmental processes and toxicities. Chiral stationary phases (CSPs) play a key role in enantiomer separation by HPLC. The chiral selectors used in CSPs include polysaccharide derivatives, cyclodextrin derivatives, macrocyclic antibiotics, proteins, ligand exchange complex, crown ether, imprinted polymers and some low-molecular-mass selectors such as Pirkle-type compounds [10]. Polysaccharidetype CSPs developed by Okamoto’s group have been shown to be widely useful for chiral separation [11]. More than 80% of chiral drugs currently available on the market can be separated by three kinds of polysaccharide CSPs that have complementary characteristics. The commercial columns containing these CSPs include Chiralcel OD [cellu-
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Fig. 1. Chemical structures of imidazolinone herbicides and their methyl derivatives. (1) Imazapyr (R1 = H, R2 = R3 = H, R4 = N); (2) imazapic (R1 = –CH3 , R2 = R3 = H, R4 = N); (3) imazethapyr (R1 = –CH2 CH3 , R2 = R3 = H, R4 = N); (4) imazamox (R1 = –CH2 OCH3 , R2 = R3 = H, R4 = N); (5) imazamethabenz-methyl (R1 = –CH3 , R2 = CH3 , R3 = H, R4 = C); (6) imazapyr derivative (R1 = H, R2 = R3 = CH3 , R4 = N); (7) imazapic derivative (R1 = –CH3 , R2 = R3 = CH3 , R4 = N); (8) imazethapyr derivative (R1 = –CH2 CH3 , R2 = R3 = CH3 , R4 = N); (9) imazamox derivative (R1 = –CH2 OCH3 , R2 = R3 = CH3 , R4 = N); (10) imazamethabenz-methyl derivative (R1 = –CH3 , R2 = R3 = CH3 , R4 = C); (11) imazaquin (R5 = R6 = H); (12) imazaquin derivative (R5 = R6 = CH3 ).
lose tri(3,5-dimethylphenylcarbamate)], Chiralcel OJ [cellulose tri(4-methylbenzoate)], and Chiralpak AD [amylose tri(3,5dimethylphenylcarbamate)] [12]. Polysaccharide CSPs can be used in polar-organic, polar aqueous-organic and normal-phase modes [13]. The polysaccharide CSPs have relatively high loading capacities and are the most used CSPs for preparative separations [14]. A great number of pesticides, including organophosphorus pesticides [15], synthetic pyrethroids [16] and organochlorine compounds [17], have been enantiomerically resolved using columns coated with polysaccharide CSPs. Imidazolinone herbicides are widely used for broad-spectrum weed control. This class of herbicides currently consists of six commercially available members: imazapyr (1), imazapic (2), imazethapyr (3), imazamox (4), imazaquin (11) and imazamethabenz-methyl (5). The structures of the six imidazolinone herbicides and their methyl derivatives are shown in Fig. 1. The imidazolinone herbicides are chiral compounds in which a stereogenic center is located at the imidazolinone ring. Enantioselectivity in herbicidal activity was recognized for these herbicides in a previous study, in which the R enantiomer of imazethapyr was found to be 10-fold more inhibitory to acetohydroxy acid synthase (AHAS) than the S enantiomer [18]. However, so far only very limited research has been reported on the separation of imidazolinone enantiomers. Cyclodextrinmodified capillary zone electrophoresis was applied to separate enantiomers of imazaquin (11) and imazamethabenz-methyl (5) [19]. The enantioselective transformation of imazaquin in soil slurries was investigated using a CE method with dimethyl -cyclodextrin as the chiral selector [20]. In this study, we evaluated the use of HPLC with polysaccharide CSPs to resolve enantiomers of imidazolinone herbicides. Two different columns were tested in the reversed and normal-phase mode, respectively, and were shown to enantiomerically resolve these imidazolinone herbicides. The retention behavior and effect of mobile phase modifiers and compositions were investigated. Conditions for chiral separation of the methylation derivatives of these herbicides were also evaluated in the normal-phase mode, as methyl derivatization is often used prior to GC analysis of imidazolinone herbicide residues in environmental samples.
2. Experimental 2.1. Equipment Two HPLC systems were used. A modular Dionex HPLC system (Dionex, Sunnyvale, CA, USA) was used for the reversed-phase analysis. The Dionex system was equipped with a GP50 gradient pump, an AS50 thermal compartment, an AS50 autosampler, a PDA-100 photodiode array detector, and Chromeleon software for data acquisition and processing. An Agilent 1100 HPLC system (Agilent, Wilmington, DE, USA) equipped with a vacuum degasser, a quaternary pump, an autosampler, a thermostatic column compartment, and a multiple wavelength detector was used for the normal-phase analysis. An Agilent 6890N GC-5973 mass selective detector system with a DB-1701 column (30 m × 0.25 mm, film thickness: 0.25 m) was used for structural confirmation of methyl derivatives of imidazolinone herbicides. Two chiral columns were evaluated in this study. A Chiralcel OJ [cellulose tri(4-methylbenzoate)] column (250 mm × 4.6 mm I.D. with 10 m particle size) was used for normal-phase analysis, and a Chiralcel OD-R [cellulose tri(3,5-dimethylphenylcarbamate)] column (250 mm × 4.6 mm I.D. with 10 m particle size) was used for reversed-phase analysis. Both columns were purchased from Chiral Technologies (West Chester, PA, USA). 2.2. Chemicals and reagents Racemic standards of imazapyr (1) (99%) (Generic name: 2-(4-Isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid; trade names include Arsenal, Chopper, Contain), imazapic (2) (99%) (Generic name: 2-[4,5-dihydro-4-methyl-4(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-methyl-3-pyridinecarboxylic acid; trade names include Plateau, Cadre, Plateau Eco-Paks), imazethapyr (3) (99%) (Generic name: (2[4.5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-lH-imidazol2-yl]-5-ethyl-3-pyridinecarboxylic acid; trade names include Contour, Hammer, Overtop, Passport, Pivot, Pursuit, Pursuit
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Plus, and Resolve), imazamox (4) (99%) (Generic name: (2[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol2-yl]-5-(methoxymethyl)-3-pyridinecarboxylic acid; trade name: Raptor), imazamethabenz-methyl (5) (99%) (reaction mixture of (RS)-6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin2-yl)-m-toluic acid and (RS)-2-(4-isopropyl-4-methyl-5-oxo2-imidazolin-2-yl)-p-toluic acid; trade name: Assert), and imazaquin (11) (99%) (Generic name: (2-[4,5-dihydro-4methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-y1]-3-quinolinecarboxylic; trade names include Ala-Scept, Scepter, Squadron, Tri-Scept, and Partner), were purchased from Chem Service (West Chester, PA, USA). Mobile phases used in this study were prepared from HPLC grade solvents. Acetonitrile, n-hexane, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butanol, acetone, H3 PO4 , Na2 HPO4 , and NaH2 PO4 were purchased from Fisher (Springfield, NJ, USA). Trifluoroacetic acid (TFA, an acidic mobile phase additive), 1,3,5-tri-tert-butylbenzene (a void time marker), iodomethane, and tetrabutylammonium hydroxide (1.0 M solution in methanol) were purchased from Sigma–Aldrich (Milwaukee, WI, USA). The water used in mobile phases was prepared with a Milli-Q water purification system. 2.3. Chromatographic conditions The herbicides were dissolved in acetonitrile or 2-propanol at 0.2 mg/ml, and 20 l was injected for analysis. The signals of HPLC UV detector were recorded at 254 nm. GC–MS analysis was performed with injection of 1 l in the splitless mode (260 ◦ C, 60 s splitless). The oven temperature program was as follows: 70 ◦ C held 1 min, ramped to 150 ◦ C at 10 ◦ C/min, held at 150 ◦ C for 5 min, ramped to 280 ◦ C at 10 ◦ C/min, and held at 280 ◦ C for 2 min. The transfer line temperature was 280 ◦ C, and the ion source temperature was 230 ◦ C. 2.4. Preparation and characterization of methylated derivatives of herbicides The derivatives of imidazolinone herbicides were prepared using a published method with some modifications [21]. The general procedure was as follows: racemic herbicide (5 mg) was added to 2 ml acetone in a 4.6 ml vial with a small magnetic stir bar. The vial was sealed by a screw cap and kept in a 40 ◦ C water bath. Tetrabutylammonium hydroxide (200 l, 1.0 M solution in methanol) and 400 l of iodomethane were added into the solution. The solution was stirred at 40 ◦ C for 1.5 h, and then blown to dryness under a gentle nitrogen stream. After water was added to the reaction mixture, the solution was extracted with ethyl ether-n-hexane (1:2, v/v, 30 ml) for three times. The extract was dried with sodium sulfate and concentrated on a rotary evaporator to dryness. The residue was recovered in 2propanol for HPLC normal-phase analysis or in n-hexane for GC–MS analysis. The derivatives of imidazolinone herbicides were characterized by GC–MS analysis with the following identifiers [21]: imazapyr derivative (6): retention time 24.53 min, and MS (EI) M+ m/z 289 (relative intensity 5.28%); imazapic deriva-
tive (7): retention time 25.64 min, and MS (EI) M+ m/z 303 (6.78%); imazethapyr derivative (8): retention time 25.26 min, and MS (EI) M+ m/z 317 (7.82%); imazamox derivative (9): retention time 27.31 min, and MS (EI) M+ m/z 333 (11.15%); imazamethabenz-methyl derivative (10): retention times 25.19 and 25.28 min, respectively, and MS (EI) M+ m/z 302 (1.68% and 1.72%, respectively); and imazaquin derivative (12): retention time 29.46 min, and MS (EI) M+ m/z 339 (9.02%). 2.5. Calculation The hold-up time (t0 ) was measured for every mobile phase with 1,3,5-tri-tert-butylbenzene as the tracer. The retention factor (k ) was calculated using the equation k = (tr − t0 )/t0 where tr is the retention time. The separation factor (α) was calculated as the ratio α = k2 /k1 , where k1 is the retention factor of the first eluted enantiomer and k2 is the retention factor of the second eluted enantiomer. Two different equations for resolution calculation were adopted due to the different softwares available on the two HPLC systems. The resolution factor (Rs ) for the reversed-phase mode was calculated using the equation Rs = 2 × (tr2 − tr1 )/(w1 + w2 ), where tr1 and tr2 are the retention times of the first and second eluted enantiomer, respectively, and w1 and w2 are the corresponding base peak widths. The resolution factor (Rs ) for the normal-phase mode was calculated using the equation Rs = 1.18 × (tr2 − tr1 )/((w1/2 )1 + (w1/2 )2 ), where (w1/2 )1 and (w1/2 )2 are the widths at half peak height. The structures of six imidazolinone herbicides and their methyl derivatives were calculated using Chem 3D Ultra version 7.0.0 (CambridgeSoft, Cambridge, MA, USA). The minimized energy of molecule was calculated by molecular mechanics using MM2 force field (minimum root-mean-square gradient was 0.01), following the semi-empirical AM1 (minimum rootmean-square gradient was 0.1). 2.6. Analysis of soil samples Soil samples (Arlington sandy loam with organic matter content of 0.92%) were collected from a field located at the Agricultural Experiment Station on the campus of University of California in Riverside, CA, USA. To prepare herbicide-spiked soil samples, 1.0 ml of stock solution of imazapyr (1) or imazaquin (11) in acetone-water was added to 30 g of soil in 200-ml flasks, which resulted in an initial herbicide concentration of 10.0 mg/kg. The flasks were allowed to sit for 2 h (to allow acetone to evaporate) and then were mixed thoroughly by rotation and shaking. The soil sample was then transferred into a 200ml propylene centrifuge bottle, and sodium hydroxide solution (0.5 M, 100 ml) was added. The mixture was shaken for 20 min on a mechanical shaker followed by centrifugation at 3000 rpm for 20 min. The supernatant was decanted into a 1000 ml separation funnel. The soil sample was extracted with fresh sodium hydroxide solution for two additional times, and the extracts were combined. The alkaline extract was adjusted to pH 2.8 with 6 M hydrochloric acid and centrifuged at 9000 rpm for 20 min. The supernatant was decanted into a 1000-ml separation funnel, and extracted with 60 ml of dichloromethane for three times. The
W. Lao, J. Gan / J. Chromatogr. A 1117 (2006) 184–193
combined dichloromethane extract was transferred into a 200ml centrifuge bottle, and centrifuged at 9000 rpm for 20 min (to eliminate emulsion). The dichloromethane layer was dried with anhydrous sodium sulfate, and then concentrated on a rotary evaporator to near dryness. The residue was dissolved in 0.5 ml of 2-propanol and an aliquot (50 l) was injected into the HPLC for analysis. 3. Results and discussion Imidazolinone molecules with a free carboxylic acid group and an imine group exhibit ampholytic nature. As shown by the equilibrium of imazaquin (11) (Fig. 2), imidazolinone pesticides can be in the neutral form (II), in the deprotonated form (III) on the carboxyl, or in the protonated form (I) on the imidazolinone nitrogen. The pKa1 and pKa2 values of imazaquin (11) are 1.8 and 3.7. The corresponding values are 1.9 and 3.6 for imazapyr (1), 2.1 and 3.9 for imazethapyr (3), and 3.1 and 3.9 for imazapic (2). The pKa1 value of imazamethabenz-methyl (5) is 2.9 and that of imazamox (4) 3.3. Imidazolinones were reacted with methyl iodide in the presence of tetrabutylammonium hydroxide to obtain methylated derivatives. The structures of methyl derivatives of the six imidazolinone herbicides were confirmed by GC–MS analysis [21]. A large number of compounds were reported to be enantiomerically separated on tribenzoate and triphenylisocyanate derivatized polysaccharide CSPs. The interactions such as hydrogen bonding, – interaction and dipole–dipole stacking are believed to be responsible for the chiral discrimination. With
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these interactions, the degree of steric fit of the enantiomers in the “chiral cavity” plays a fundamental role in chiral separation. For normal-phase mode, alkane-alcohol solvents are mainly used as the mobile phase for polysaccharide CSPs. Acetic acid, TFA, ethanesulfonic acid [22,23], diethyl amine and triethylamine [24,25] are commonly used as mobile phase additives to minimize peak broadening arising from unwanted interactions between polar solutes and the stationary phase. Neutral compounds have been shown to be unaffected by the acidic or basic additives, and can be chromatographed with or without the additives. For the reversed-phase mode, aqueous-alcohol or aqueousacetonitrile are commonly used as eluents. Several studies have shown that the effect of pH of the aqueous mobile phase on the separation is significant for ionizable compounds [24,26]. Polysaccharide CSPs do not have any ionic interaction sites. Analytes should be kept neutral to achieve better separation. 3.1. Reversed-phase mode The phosphate buffer solutions (PBS) were selected as mobile phase components due to their demonstrated usefulness for separating ionizable compounds in the reversed-phase mode on Chiralcel OD-R column [26]. Methanol as organic modifier in PBS (50 mM) was first tested. No resolution was observed for any of the six imidazolinone herbicides at pH 2.0 or 3.0. The PBS (50 mM)-acetonitrile mobile phase systems were then tested at pH from 2.0 to 7.0. Enantiomers of imazethapyr (3), imazaquin (11) and imazamox (4) were resolved at pH 2.0 and 1.0 ml/min flow rate with 70/30 (v/v) PBS-acetonitrile as the
Fig. 2. The dissociation equilibrium of imazaquin (11). Table 1 Effect of mobile phase compositions and pH on chiral separation of imidazolinone herbicides on Chiralcel OD-R column pH 2.0
Imazethapyr (3)a Imazaquin (11)a Imazamox (4)a Imazapyr (1)b Imazethapyr (3)c Imazaquin (11)c Imazamox (4)c a b c
pH 3.0
pH 4.0
k1
α
Rs
k1
α
Rs
k1
α
Rs
8.50 26.37 4.20 37.85 16.25 55.68 7.92
1.10 1.25 1.13 1.09 1.12 1.25 1.16
0.58 2.1 0.49 0.27 1.01 2.44 0.89
4.77 11.38 2.25 9.38 14.75 36.34 3.65
1.0 1.23 1.0 1.0 1.12 1.22 1.12
0 1.49 0 0 0.34 1.87 0.31
2.25 11.17 1.12 5.85 14.68 36.37 1.9
1.0 1.24 1.0 1.26 1.12 1.20 1.0
0 1.49 0 0.49 0.35 1.92 0
Mobile phase A: 70/30 (v/v) (50 mM phosphate buffer)-acetonitrile, flow rate 1.0 ml/min. Mobile phase B: 80/20 (v/v) (50 mM phosphate buffer)-acetonitrile, flow rate 0.5 ml/min. Mobile phase C: 80/20 (v/v) (50 mM phosphate buffer)-acetonitrile, flow rate 1.0 ml/min.
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imazamox (4). Imazapyr (1) could be resolved at a flow rate of 0.5 ml/min using 85/15 (v/v) PBS-acetonitrile as the mobile phase. Imazamethabenz-methyl (5) is a mixture of 5-methyl (meta) isomer and 4-methyl (para) isomer, and the complete resolution of which would result in four peaks. However, only initial isomeric separation was found for imazamethabenz-methyl (5) on OD-R column at pH 2.0 by decreasing the ratio of acetonitrile from 30 to 20% (α = 1.03, Rs = 0.06). Imazethapyr (3), imazaquin (11) and imazamox (4) were separated under the same mobile phase conditions without overlapping peaks in the reversed-phase mode. This suggests that the determination of the enantiomers of these three herbicides may be achieved in one sample matrix. 3.2. Normal-phase mode
Fig. 3. Chromatograms of chiral separation on Chiralcel OD-R column. Mobile phase, 80/20 (v/v) (50 mM phosphate buffer)-acetonitrile; flow rate, 1.0 ml/min.
mobile phase (Table 1). Under these conditions, baseline separation was achieved only for imazaquin. However, when the ratio of PBS and acetonitrile was changed to 80/20 (v/v), the resolution of imazethapyr (3) and imazamox (4) were improved but not to baseline separation (Fig. 3). Baseline separation of imazaquin (11) was preserved at pH 3.0 and 4.0 with slight changes in the separation factor as compared to pH 2.0. At pH 5.0, only initial separation (Rs = 0.13) was observed for imazaquin (11). At pH 6.0 and 7.0, imazaquin (11) lost its separation. Enantiomers of imazethapyr (3) and imazamox (4) were not resolved at pH 3.0 or 4.0 using 70/30 (v/v) PBS-acetonitrile as the mobile phase. Modifying chromatographic conditions to 80/20 (v/v) PBS-acetonitrile (pH 4.0) resulted in resolution of enantiomers of imazethapyr (3) and imazaquin (11), but not
3.2.1. Effect of molecular structures A n-hexane-2-propanol mixture was first used as the mobile phase on Chiralcel OJ column for the separation of imidazolinone herbicides and their methyl derivatives. Trifluoroacetic acid (0.1%, v/v) was added to n-hexane to allow elution of imidazolinone herbicides with free carboxyl group [13,27]. Enantiomers of all herbicides except imazamethabenz-methyl (5) were resolved on the OJ column using 75/25 (v/v) n-hexane (0.1%, TFA)-2-propanol (Table 2). Imazapyr (1) gave the highest separation factor, while imazethapyr (3), imazamox (4) and imazapic (2) were also resolved at the baseline (Fig. 4). In contrast, imazaquin (11) exhibited the lowest resolution with the smallest separation factor. No chiral separation of imazamethabenz-methyl (5) was achieved under these conditions. However, when the content of 2-propanol was reduced from 25 to 10% by volume, separation of imazamethabenzmethyl isomers was observed. Imazethapyr (3), imazamox (4) and imazapic (2) have different substituents at the 5 position of the pyridine moiety from imazapyr (1). Although this substituent is far away from the chiral center and may not change the spatial conformation, the substituent made obvious differences in retention and separation factors. The k2 and k1 of imazapic (2) and imazethapyr (3) were both smaller than imazapyr (1). Imazapic (2) was added
Table 2 The resolution (Rs ) of enantiomers of imidazolinone herbicides and their methyl derivatives on the Chiralcel OJ column in the normal-phase mode Alcohol modifier
Imazapyr (1)a Imazapic (2)a Imazethapyr (3)a Imazamox (4)a Imazaquin (11)a Imazapyr derivative (6)b Imazapic derivative (7)b Imazethapyr derivative (9)b Imazamox derivative (10)b Imazaquin derivative (12)b a b
Ethanol
1-Propanol
2-Propanol
1-Butanol
2-Butanol
t-Butanol
4.13 3.87 3.85 3.41 1.41 0.40 0.81 0.57 2.61 1.10
4.18 3.04 2.98 2.87 0.45 0.98 0.00 0.00 0.27 0.00
2.67 2.02 1.51 2.00 0.95 2.02 0.84 0.74 0.96 0.51
3.18 2.05 1.36 1.99 0.00 0.97 0 0 0 0
2.40 1.06 0.91 1.39 0.00 0.80 0.54 0 0 0
1.37 0.70 0.55 0.99 0.25 2.74 1.42 0 1.72 0.54
Mobile phase A: 75/25 (v/v) n-hexane (0.1% TFA)/alcohol modifier Mobile phase B: 90/10 (v/v) n-hexane (0.1% TFA)/alcohol modifier.
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Fig. 4. Chromatograms of chiral separation on Chiralcel OJ column. Mobile phase for imidazolinone herbicides: 75/25 (v/v) n-hexane (0.1%TFA)/alcohol modifier; and mobile phase for imidazolinone herbicides methyl derivatives: 90/10 (v/v) n-hexane (0.1%TFA)/alcohol modifier.
a methylene group to become imazethapyr (3) which exhibited a decrement in the retention factor and enantioselectivity. However, in imazamox (4) with an alkoxyl group replacing the methyl group of imazethapyr (3), the retention and separation factor were both enhanced. These results suggest that the alkoxyl group likely increased the dipole–dipole interaction with CSP. Wainer et al. [28] demonstrated that steric factor of an enantiomer played an important role in its fitting into chiral cavities of cellulose CSPs. The aromatic portion of imidazolinone herbicides may fit into the chiral cavity to form complex with CSP [29]. The bulkiness of imazaquin (11) may have resulted in a
lower degree of steric fit in the chiral cavities and consequently the poor resolution. To better understand this assumption, the optimization structures of imidazolinone herbicides and their derivatives were calculated by using the AM1 semi-empirical quantum mechanics method [30]. The values of 1-2-2 -1 dihedral angles of aromatic ring and imidazol ring reflect differences in structural characteristics (Table 3). The values of dihedral angles were negative for all compounds, indicating that the twisting directions of aromatic rings were on the same side of the imidazolinone ring. From the values of the 1-2-2 -1 dihedral angle, imidazolinone compounds could be divided into
Table 3 Calculated dihedral angles of 1-2-2 -1 of imidazolinone herbicides and their methyl derivatives Dihedral angel of 1-2-2 -1 (degree) Imazapyr (1)
Imazapic (2)
Imazethapyr (3)
Imazamox (4)
Imazaquin (11)
Imazamethabenz-methyl (5) meta isomer
Derivative
para isomer
−44.9
−43.8
−43.4
−45.1
−26.0
−63.6
−68.8
−58.9
−58.3
−58.6
−59.1
−5.8
−65.7
−84.2
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three groups. Imazapyr (1), imazapic (2), imazethapyr (3) and imazamox (4) are in the first group with almost identical 1-2-2 1 dihedral angles. Imazaquin (11) and imazamethabenz-methyl (5) are in the second and third group, respectively. The absolute values of the dihedral angle for the first group are greater than the absolute value for imazaquin (11). A higher absolute value of the dihedral angle implies that the twisting of the aromatic ring out of the plane of the imidazol ring is greater. It is therefore possible that one of the reasons for the lower separation factor of imazaquin (11) stems from its particular conformation. All of the methyl derivatives of imidazolinone compounds except imazamethabenz-methyl (10) were enantiomerically resolved using 90/10 (v/v) n-hexane-2-propanol as the mobile phase (Table 2). These derivatives evidently do not offer any hydrogen bonding interaction with the OJ column. Okamoto et al. confirmed that hydrophobic interactions could be a major force in the chiral separation on OJ column [13]. The hydrophobic interactions between the enantiomers of these derivatives and CSP seem to be of importance for the formation of the solute-
CSP complex and may contribute to proper steric fit to the cavity of CSP. The enantiomers of imazamethabenz-methyl derivatives (10) were not separated using n-hexane-2-propanol 90/10 (v/v) as the mobile phase, although isomeric separation was obtained. From the structures of imazapic (6) and imazamethabenz-methyl derivatives (10), it is likely that the nitrogen of the pyridine ring is beneficial for the chiral separation through dipole–dipole interactions. 3.2.2. Effect of alcohol modifier The effect of polar alcohol modifiers on enantiomer separation was investigated, where ethanol, 1-propanol, 2propanol, 1-butanol, 2-butanol or t-butanol was tested as the polar modifier. For all the alcohol modifiers, k1 consistently decreased in the order imazaquin (11) > imazamox (4) > imazapyr (1) > imazapic (2) > imazethapyr (3), while the order for k2 followed imazamox (4) > imazapyr (1) > imazaquin (11) > imazapic (2) > imazethapyr (3). Therefore, only the position of imazaquin (11) changed in the two orders. The reten-
Fig. 5. Influence of alcohol modifier on retention factor of imidazolinone herbicides and their methyl derivatives. (a) The first eluted enantiomer of imidazolinone herbicide; (b) the second eluted enantiomer of imidazolinone herbicide; (c) the first eluted enantiomer of imidazolinone herbicide methyl derivative; (d) the second eluted enantiomer of weimidazolinone herbicide methyl derivative. Chromatographic conditions were the same as given in Fig. 4.
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tion factors increased generally with increasing length of carbon chain of linear or branched alcohols (Fig. 5a and b). The change in the separation factor for imazapyr (1) was small with ethanol or 2-butanol as modifier instead of 2-propanol. However, with 1-propanol or 1-butanol as modifier, the separation factor of imazapyr (1) increased, but the magnitude of enhancement arisen from 1-butanol was smaller than with 1-propanol. The use of t-butanol as modifier deteriorated enantioselectivity of imazapyr (1). The separation factors of imazapic (2), imazethapyr (3) and imazamox (4) consistently decreased with the order of alcohol modifiers ethanol > 1-propanol > 2propanol > 1-butanol > 2-butanol > t-butanol. The enantioselectivity of imazaquin (11) on the OJ column was different from the other imidazolinone herbicides. First, the effect on separation factors of imazapyr (1) and imazaquin (11) was reversed when 1-propanol replaced 2-propanol as modifier. Second, using 1butanol or 2-butanol as modifier, the enantiomers of imazaquin (11) lost chiral separation. Third, when t-butanol was used as the modifier, enantioselectivity was recovered to a certain degree.
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It is likely that the imazaquin (11) enantiomers carrying a large quinoline group were in unfavorable situation to compete with the linear alcohol molecules, but with t-butanol as modifier, the enantiomers may access chiral interaction sites more easily. The combination of higher separation factor and lower retention factor indicated that ethanol was a more favorable mobile phase modifier for the separation of imidazolinone compounds on cellulose ester-type CSP [29]. However, with consideration of both enantioselective effect and cost, 2-propanol should be a good selection as the modifier. The alcohol modifiers showed different effects on retention factors and separation factors of imidazolinone herbicides and their methyl derivatives (Fig. 5c and d). The t-butanol as modifier gave the largest retention factors to enantiomers of imidazolinone herbicides and their methyl derivatives. However, retention factors with 2-propanol as modifier were larger than from ethanol, 1-propanol, 1-butanol and 2-butanol. Only imazapyr (6) methyl derivative showed enantiomeric separation with all of the alcohol modifiers, whereas the results on the other
Fig. 6. Influence of alcohol modifier on separation factor and k of imidazolinone herbicides and their methyl derivatives. (a) Separation factor of imidazolinone herbicide; (b) k of imidazolinone herbicide; (c) separation factor of imidazolinone herbicide methyl derivatives; (d) k of imidazolinone herbicide methyl derivatives. The x-axis for (a) and (b) are: imazapyr 1, imazapic 2, imazethapyr 3, imazamox 4 and imazaquin 5. The x-axis for (c) and (d) are: methyl derivative of imazapyr 1, imazapic 2, imazethapyr 3, imazamox 4 and imazaquin 5. Chromatographic conditions were the same as in Fig. 5.
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methyl derivatives were inconsistent. Enantiomers of methyl derivatives of imazapyr (6) and imazamox (9) were resolved with 1-propanol as modifier. With 1-butanol as the modifier, however, only methyl derivative of imazapyr (6) showed enantiomeric separation. Enantiomers of methyl derivatives of imazamox (9), imazethapyr (6) and imazaquin (12) were not resolved with 2-butanol as modifier. With the exception of imazethapyr derivative (8), the methyl derivatives gave the largest separation factors when t-butanol was used as the modifier. The separation factors of imidazolinone herbicides and their methyl derivatives measured by using different alcohol modifiers are shown in Fig. 6a and c. The structure-enantioselectivity relationships were different among the different alcohol modifiers. This suggests that comparing apparent separation factors alone may not be enough to gain a better understanding of trends in the structure-enantioselectivity relationship. Therefore, the k values were further considered for the evaluation. Schurig et al. first suggested the importance to differentiate contributions to α by physical (achiral) and chemical (chiral) interactions between a chiral compound and stationary phases in complexation GC [31]. Guiochon et al. also observed that k consisted of contributions from non-enantioselective and enantioselective interactions [32]. The conventional separation factor α is an apparent separation factor aapp : αapp =
+ k kns2 s2 + k kns1 s1
(1)
and k are the contributions of non-enantioselective where kns1 ns2 interaction of the less and the more retained enantiomer, respec and k the contributions of enantioselective interactively; ks1 s2 tion of the less and the more retained enantiomer, respectively. Since the non-enantioselective interaction is identical for both enantiomers, the difference between the retention factors of two enantiomers, k , is caused by the enantioselective interaction:
k = k2 − k1
Fig. 7. Effect of mobile phase compositions on the separation factor. Chromatographic conditions were the same as in Fig. 5, except for the volume fraction of alcohol modifier.
imazamox (9) gave the highest “true selectivity”. The k values of the methyl derivatives illustrated that the t-butanol modifier led to the highest “true selectivity” in spite of no resolution of enantiomers of imazethapyr methyl derivatives (8). Ethanol as modifier showed average lower “true selectivity” than 2propanol. 1-propanol, 1-butanol and 2-butanol as mobile phase modifiers gave lower k values and did not favor the separation of imazaquin (12) and imazethapyr derivatives (8) (Fig. 6d). 3.2.3. Effect of mobile phase composition As the 2-propanol volume fraction in the n-hexane-2propanol mobile phase was increased from 20 to 35%, the
(2)
The higher k values imply that the “true selectivity” is larger. Linear chromatography does not provide enough information to distinguish the nonselective and enantioselective interactions of each enantiomer. However, some trends can still be observed from the k values (Fig. 6b). The measured k followed the order imazapyr (1) > imazamox (4) > imazapic (2) > imazethapyr (3) > imazaquin (11). This sequence revealed clearly a structure-enantioselectivity relationship: with increasing chain length of the substituent on the 5 position of pyridine ring (H < CH3 < CH2 CH3 ), the “true selectivity” decreased. However, if this substituent provides extra interaction other than pure hydrophobic interactions, such as the alkoxyl group in imazamox (4), the “true selectivity” increased. On the other hand, the bulkiness of a quinoline group appeared to result in deterioration of chiral separation. The different alcohol modifier did not affect the order of k . Therefore, this study showed that changes of separation factors for structurally related compounds may be manifested by comparison of k values. The k values further revealed a trend of chiral separation for the methyl derivatives. Methyl derivatives of imazapyr (6) and
Fig. 8. Typical chromatograms of imazapyr (1) and imazaquin (11) extracted from a sandy loam soil. Mobile phase for imazapyr: 75/25 (v/v) n-hexane (0.1%TFA)/2-propanol; and mobile phase for imazaquin: 80/20 (v/v) n-hexane (0.1%TFA)/2-propanol.
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k and Rs decreased and the enantioselectivity was essentially unchanged (Fig. 7). However, when the volume fraction of 2propanol was further increased from 35 to 40%, a more rapid decrease in separation factor was observed. As the fraction of 2-propanol was increased, it is likely that the strength of hydrogen bonds between imidazolinone compounds and CSP became weaker and the solubility of the solute increased. These results suggest that the hydrogen bonding interaction not only influenced the retention but also the enantiomeric selectivity [33]. 3.3. Applications Chromatograms on the OJ column of imazapyr (1) and imazaquin (11) extracted from spiked soil samples are shown in Fig. 8a and b, respectively. Enantiomers of imazapyr (1) were baseline separated (Fig. 8a), while enantiomers of imazaquin (11) were also adequately resolved (Fig. 8b). These results suggest that the developed method may be applied for chiral separation of imidazolinone herbicides in environmental matrices such as soil or sediment samples, and may be useful for enantioselectivity evaluation in environmental processes. 4. Conclusions Enantiomers of imazethapyr (3), imazaquin (11), and imazamox (4) were separated on Chiralcel OD-R column using PBS-acetonitrile as the mobile phase. Imazapyr (1) only showed faint enantioselectivity on this column in the reversed-phase mode, while no chiral separation was observed for imazapic (2) and imazamethabenz-methyl (5). However, enantiomers of all imidazolinone herbicides except for imazamethabenzmethyl (5) were adequately resolved on Chiralcel OJ column in the normal-phase mode using n-hexane (0.1% FTA) with an alcohol modifier. Enantiomers of five methyl derivatives of imidazolinone herbicides were also resolved on Chiralcel OJ column. Imazamethabenz-methyl (5) and its derivatives (10) exhibited isomeric separation but no enantiomeric separation. The order of k values (which measures the “true selectivity”) followed imazapyr (1) > imazamox (4) > imazapic (2) > imazethapyr (3) > imazaquin (11). By comparison of k values a structure-enantioselectivity relationship was observed. The developed chiral separation methods may be used to evaluate enantioselectivity of these compounds during biodegradation, toxicity, and other biologically-mediated processes. Acknowledgments We thank P. Resketo and F. Ernst for their technical assistance. This study was supported by a USDA-National Research Initiatives grant No. 2005-35107-16189.
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High-performance liquid chromatographic separation of imidazolinone herbicide enantiomers and their methyl derivatives on polysaccharide-coated chiral stationary phases Wenjian Lao, Jay Gan ∗ Department of Environmental Sciences, University of California, Riverside, CA 92521, USA Received 5 December 2005; received in revised form 23 March 2006; accepted 24 March 2006 Available online 18 April 2006
Abstract Many chiral pesticides exhibit enantioselectivity in biotransformation and ecotoxicity in the environment. A significant class of chiral pesticides is imidazolinone herbicides, of which enantioselectivity has not been well studied. Development of efficient chiral separation methods is the first step for allowing characterization of enantioselectivity in environmental processes. In this study, we attempted to resolve enantiomers of imidazolinone herbicides using reversed-phase and normal-phase high-performance liquid chromatography with polysaccharide-type chiral columns. Enantiomers of imazethapyr, imazaquin, and imazamox were separated on a Chiralcel OD-R column using 50 mM phosphate buffer-acetonitrile as mobile phase. Enantiomers of imazapyr, imazapic, imazethapyr, imazamox and imazaquin were resolved on a Chiralcel OJ column using n-hexane (0.1% trifluoroacetic acid)-alcohol as mobile phase. The enantiomers of five methyl derivatives of imidazolinone herbicides were also resolved on the Chiralcel OJ column. The k values revealed a structure-enantioselectivity relationship for the separation behaviors of the enantiomers on the OJ column. The described method was successfully applied for chiral analysis of two imidazolinone herbicides (imazapyr and imazaquin) in spiked soil samples. © 2006 Elsevier B.V. All rights reserved. Keywords: Imidazolinone herbicides; Enantiomer separation; Chiral stationary phases; Chiral contaminants
1. Introduction High-performance liquid chromatography (HPLC) has become one of the most powerful techniques in the field of enantioselective separation. Although a large number of chiral compounds, including pharmaceuticals, food additives, natural products, synthetic intermediates, pollutants and pesticides have been separated by HPLC, separation of enantiomers is still one of the most challenging tasks. Up to 25% of pesticides are chiral compounds, and most of them are currently used as mixtures of stereoisomers or enantiomers. Enantioselectivity in degradation, toxicity and biological activity has been reported for various chiral pesticides, such as synthetic pyrethroids, organophosphate insecticides, and some chiral herbicides [1–3]. The need for characterizing enantioselective environmental behaviors of chiral pesticides has spurred the development of analytical methods for enantiomer separation. Gas chromatography (GC)
∗
Corresponding author. Tel.: +1 951 787 2712; fax: +1 951 787 3993. E-mail address: [email protected] (J. Gan).
0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.03.094
and capillary electrophoresis (CE) techniques have shown high efficiency for separating enantiomers of some chiral compounds [4–6]. However, capillary GC and CE have drawbacks for preparative separation due to the small injection quantity and concentrations [7–9]. In contrast, HPLC is currently the best option for analytical and small-scale preparative separation through which enantiomerically pure chemicals may be obtained and used for evaluation of environmental processes and toxicities. Chiral stationary phases (CSPs) play a key role in enantiomer separation by HPLC. The chiral selectors used in CSPs include polysaccharide derivatives, cyclodextrin derivatives, macrocyclic antibiotics, proteins, ligand exchange complex, crown ether, imprinted polymers and some low-molecular-mass selectors such as Pirkle-type compounds [10]. Polysaccharidetype CSPs developed by Okamoto’s group have been shown to be widely useful for chiral separation [11]. More than 80% of chiral drugs currently available on the market can be separated by three kinds of polysaccharide CSPs that have complementary characteristics. The commercial columns containing these CSPs include Chiralcel OD [cellu-
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Fig. 1. Chemical structures of imidazolinone herbicides and their methyl derivatives. (1) Imazapyr (R1 = H, R2 = R3 = H, R4 = N); (2) imazapic (R1 = –CH3 , R2 = R3 = H, R4 = N); (3) imazethapyr (R1 = –CH2 CH3 , R2 = R3 = H, R4 = N); (4) imazamox (R1 = –CH2 OCH3 , R2 = R3 = H, R4 = N); (5) imazamethabenz-methyl (R1 = –CH3 , R2 = CH3 , R3 = H, R4 = C); (6) imazapyr derivative (R1 = H, R2 = R3 = CH3 , R4 = N); (7) imazapic derivative (R1 = –CH3 , R2 = R3 = CH3 , R4 = N); (8) imazethapyr derivative (R1 = –CH2 CH3 , R2 = R3 = CH3 , R4 = N); (9) imazamox derivative (R1 = –CH2 OCH3 , R2 = R3 = CH3 , R4 = N); (10) imazamethabenz-methyl derivative (R1 = –CH3 , R2 = R3 = CH3 , R4 = C); (11) imazaquin (R5 = R6 = H); (12) imazaquin derivative (R5 = R6 = CH3 ).
lose tri(3,5-dimethylphenylcarbamate)], Chiralcel OJ [cellulose tri(4-methylbenzoate)], and Chiralpak AD [amylose tri(3,5dimethylphenylcarbamate)] [12]. Polysaccharide CSPs can be used in polar-organic, polar aqueous-organic and normal-phase modes [13]. The polysaccharide CSPs have relatively high loading capacities and are the most used CSPs for preparative separations [14]. A great number of pesticides, including organophosphorus pesticides [15], synthetic pyrethroids [16] and organochlorine compounds [17], have been enantiomerically resolved using columns coated with polysaccharide CSPs. Imidazolinone herbicides are widely used for broad-spectrum weed control. This class of herbicides currently consists of six commercially available members: imazapyr (1), imazapic (2), imazethapyr (3), imazamox (4), imazaquin (11) and imazamethabenz-methyl (5). The structures of the six imidazolinone herbicides and their methyl derivatives are shown in Fig. 1. The imidazolinone herbicides are chiral compounds in which a stereogenic center is located at the imidazolinone ring. Enantioselectivity in herbicidal activity was recognized for these herbicides in a previous study, in which the R enantiomer of imazethapyr was found to be 10-fold more inhibitory to acetohydroxy acid synthase (AHAS) than the S enantiomer [18]. However, so far only very limited research has been reported on the separation of imidazolinone enantiomers. Cyclodextrinmodified capillary zone electrophoresis was applied to separate enantiomers of imazaquin (11) and imazamethabenz-methyl (5) [19]. The enantioselective transformation of imazaquin in soil slurries was investigated using a CE method with dimethyl -cyclodextrin as the chiral selector [20]. In this study, we evaluated the use of HPLC with polysaccharide CSPs to resolve enantiomers of imidazolinone herbicides. Two different columns were tested in the reversed and normal-phase mode, respectively, and were shown to enantiomerically resolve these imidazolinone herbicides. The retention behavior and effect of mobile phase modifiers and compositions were investigated. Conditions for chiral separation of the methylation derivatives of these herbicides were also evaluated in the normal-phase mode, as methyl derivatization is often used prior to GC analysis of imidazolinone herbicide residues in environmental samples.
2. Experimental 2.1. Equipment Two HPLC systems were used. A modular Dionex HPLC system (Dionex, Sunnyvale, CA, USA) was used for the reversed-phase analysis. The Dionex system was equipped with a GP50 gradient pump, an AS50 thermal compartment, an AS50 autosampler, a PDA-100 photodiode array detector, and Chromeleon software for data acquisition and processing. An Agilent 1100 HPLC system (Agilent, Wilmington, DE, USA) equipped with a vacuum degasser, a quaternary pump, an autosampler, a thermostatic column compartment, and a multiple wavelength detector was used for the normal-phase analysis. An Agilent 6890N GC-5973 mass selective detector system with a DB-1701 column (30 m × 0.25 mm, film thickness: 0.25 m) was used for structural confirmation of methyl derivatives of imidazolinone herbicides. Two chiral columns were evaluated in this study. A Chiralcel OJ [cellulose tri(4-methylbenzoate)] column (250 mm × 4.6 mm I.D. with 10 m particle size) was used for normal-phase analysis, and a Chiralcel OD-R [cellulose tri(3,5-dimethylphenylcarbamate)] column (250 mm × 4.6 mm I.D. with 10 m particle size) was used for reversed-phase analysis. Both columns were purchased from Chiral Technologies (West Chester, PA, USA). 2.2. Chemicals and reagents Racemic standards of imazapyr (1) (99%) (Generic name: 2-(4-Isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid; trade names include Arsenal, Chopper, Contain), imazapic (2) (99%) (Generic name: 2-[4,5-dihydro-4-methyl-4(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-methyl-3-pyridinecarboxylic acid; trade names include Plateau, Cadre, Plateau Eco-Paks), imazethapyr (3) (99%) (Generic name: (2[4.5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-lH-imidazol2-yl]-5-ethyl-3-pyridinecarboxylic acid; trade names include Contour, Hammer, Overtop, Passport, Pivot, Pursuit, Pursuit
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Plus, and Resolve), imazamox (4) (99%) (Generic name: (2[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol2-yl]-5-(methoxymethyl)-3-pyridinecarboxylic acid; trade name: Raptor), imazamethabenz-methyl (5) (99%) (reaction mixture of (RS)-6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin2-yl)-m-toluic acid and (RS)-2-(4-isopropyl-4-methyl-5-oxo2-imidazolin-2-yl)-p-toluic acid; trade name: Assert), and imazaquin (11) (99%) (Generic name: (2-[4,5-dihydro-4methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-y1]-3-quinolinecarboxylic; trade names include Ala-Scept, Scepter, Squadron, Tri-Scept, and Partner), were purchased from Chem Service (West Chester, PA, USA). Mobile phases used in this study were prepared from HPLC grade solvents. Acetonitrile, n-hexane, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butanol, acetone, H3 PO4 , Na2 HPO4 , and NaH2 PO4 were purchased from Fisher (Springfield, NJ, USA). Trifluoroacetic acid (TFA, an acidic mobile phase additive), 1,3,5-tri-tert-butylbenzene (a void time marker), iodomethane, and tetrabutylammonium hydroxide (1.0 M solution in methanol) were purchased from Sigma–Aldrich (Milwaukee, WI, USA). The water used in mobile phases was prepared with a Milli-Q water purification system. 2.3. Chromatographic conditions The herbicides were dissolved in acetonitrile or 2-propanol at 0.2 mg/ml, and 20 l was injected for analysis. The signals of HPLC UV detector were recorded at 254 nm. GC–MS analysis was performed with injection of 1 l in the splitless mode (260 ◦ C, 60 s splitless). The oven temperature program was as follows: 70 ◦ C held 1 min, ramped to 150 ◦ C at 10 ◦ C/min, held at 150 ◦ C for 5 min, ramped to 280 ◦ C at 10 ◦ C/min, and held at 280 ◦ C for 2 min. The transfer line temperature was 280 ◦ C, and the ion source temperature was 230 ◦ C. 2.4. Preparation and characterization of methylated derivatives of herbicides The derivatives of imidazolinone herbicides were prepared using a published method with some modifications [21]. The general procedure was as follows: racemic herbicide (5 mg) was added to 2 ml acetone in a 4.6 ml vial with a small magnetic stir bar. The vial was sealed by a screw cap and kept in a 40 ◦ C water bath. Tetrabutylammonium hydroxide (200 l, 1.0 M solution in methanol) and 400 l of iodomethane were added into the solution. The solution was stirred at 40 ◦ C for 1.5 h, and then blown to dryness under a gentle nitrogen stream. After water was added to the reaction mixture, the solution was extracted with ethyl ether-n-hexane (1:2, v/v, 30 ml) for three times. The extract was dried with sodium sulfate and concentrated on a rotary evaporator to dryness. The residue was recovered in 2propanol for HPLC normal-phase analysis or in n-hexane for GC–MS analysis. The derivatives of imidazolinone herbicides were characterized by GC–MS analysis with the following identifiers [21]: imazapyr derivative (6): retention time 24.53 min, and MS (EI) M+ m/z 289 (relative intensity 5.28%); imazapic deriva-
tive (7): retention time 25.64 min, and MS (EI) M+ m/z 303 (6.78%); imazethapyr derivative (8): retention time 25.26 min, and MS (EI) M+ m/z 317 (7.82%); imazamox derivative (9): retention time 27.31 min, and MS (EI) M+ m/z 333 (11.15%); imazamethabenz-methyl derivative (10): retention times 25.19 and 25.28 min, respectively, and MS (EI) M+ m/z 302 (1.68% and 1.72%, respectively); and imazaquin derivative (12): retention time 29.46 min, and MS (EI) M+ m/z 339 (9.02%). 2.5. Calculation The hold-up time (t0 ) was measured for every mobile phase with 1,3,5-tri-tert-butylbenzene as the tracer. The retention factor (k ) was calculated using the equation k = (tr − t0 )/t0 where tr is the retention time. The separation factor (α) was calculated as the ratio α = k2 /k1 , where k1 is the retention factor of the first eluted enantiomer and k2 is the retention factor of the second eluted enantiomer. Two different equations for resolution calculation were adopted due to the different softwares available on the two HPLC systems. The resolution factor (Rs ) for the reversed-phase mode was calculated using the equation Rs = 2 × (tr2 − tr1 )/(w1 + w2 ), where tr1 and tr2 are the retention times of the first and second eluted enantiomer, respectively, and w1 and w2 are the corresponding base peak widths. The resolution factor (Rs ) for the normal-phase mode was calculated using the equation Rs = 1.18 × (tr2 − tr1 )/((w1/2 )1 + (w1/2 )2 ), where (w1/2 )1 and (w1/2 )2 are the widths at half peak height. The structures of six imidazolinone herbicides and their methyl derivatives were calculated using Chem 3D Ultra version 7.0.0 (CambridgeSoft, Cambridge, MA, USA). The minimized energy of molecule was calculated by molecular mechanics using MM2 force field (minimum root-mean-square gradient was 0.01), following the semi-empirical AM1 (minimum rootmean-square gradient was 0.1). 2.6. Analysis of soil samples Soil samples (Arlington sandy loam with organic matter content of 0.92%) were collected from a field located at the Agricultural Experiment Station on the campus of University of California in Riverside, CA, USA. To prepare herbicide-spiked soil samples, 1.0 ml of stock solution of imazapyr (1) or imazaquin (11) in acetone-water was added to 30 g of soil in 200-ml flasks, which resulted in an initial herbicide concentration of 10.0 mg/kg. The flasks were allowed to sit for 2 h (to allow acetone to evaporate) and then were mixed thoroughly by rotation and shaking. The soil sample was then transferred into a 200ml propylene centrifuge bottle, and sodium hydroxide solution (0.5 M, 100 ml) was added. The mixture was shaken for 20 min on a mechanical shaker followed by centrifugation at 3000 rpm for 20 min. The supernatant was decanted into a 1000 ml separation funnel. The soil sample was extracted with fresh sodium hydroxide solution for two additional times, and the extracts were combined. The alkaline extract was adjusted to pH 2.8 with 6 M hydrochloric acid and centrifuged at 9000 rpm for 20 min. The supernatant was decanted into a 1000-ml separation funnel, and extracted with 60 ml of dichloromethane for three times. The
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combined dichloromethane extract was transferred into a 200ml centrifuge bottle, and centrifuged at 9000 rpm for 20 min (to eliminate emulsion). The dichloromethane layer was dried with anhydrous sodium sulfate, and then concentrated on a rotary evaporator to near dryness. The residue was dissolved in 0.5 ml of 2-propanol and an aliquot (50 l) was injected into the HPLC for analysis. 3. Results and discussion Imidazolinone molecules with a free carboxylic acid group and an imine group exhibit ampholytic nature. As shown by the equilibrium of imazaquin (11) (Fig. 2), imidazolinone pesticides can be in the neutral form (II), in the deprotonated form (III) on the carboxyl, or in the protonated form (I) on the imidazolinone nitrogen. The pKa1 and pKa2 values of imazaquin (11) are 1.8 and 3.7. The corresponding values are 1.9 and 3.6 for imazapyr (1), 2.1 and 3.9 for imazethapyr (3), and 3.1 and 3.9 for imazapic (2). The pKa1 value of imazamethabenz-methyl (5) is 2.9 and that of imazamox (4) 3.3. Imidazolinones were reacted with methyl iodide in the presence of tetrabutylammonium hydroxide to obtain methylated derivatives. The structures of methyl derivatives of the six imidazolinone herbicides were confirmed by GC–MS analysis [21]. A large number of compounds were reported to be enantiomerically separated on tribenzoate and triphenylisocyanate derivatized polysaccharide CSPs. The interactions such as hydrogen bonding, – interaction and dipole–dipole stacking are believed to be responsible for the chiral discrimination. With
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these interactions, the degree of steric fit of the enantiomers in the “chiral cavity” plays a fundamental role in chiral separation. For normal-phase mode, alkane-alcohol solvents are mainly used as the mobile phase for polysaccharide CSPs. Acetic acid, TFA, ethanesulfonic acid [22,23], diethyl amine and triethylamine [24,25] are commonly used as mobile phase additives to minimize peak broadening arising from unwanted interactions between polar solutes and the stationary phase. Neutral compounds have been shown to be unaffected by the acidic or basic additives, and can be chromatographed with or without the additives. For the reversed-phase mode, aqueous-alcohol or aqueousacetonitrile are commonly used as eluents. Several studies have shown that the effect of pH of the aqueous mobile phase on the separation is significant for ionizable compounds [24,26]. Polysaccharide CSPs do not have any ionic interaction sites. Analytes should be kept neutral to achieve better separation. 3.1. Reversed-phase mode The phosphate buffer solutions (PBS) were selected as mobile phase components due to their demonstrated usefulness for separating ionizable compounds in the reversed-phase mode on Chiralcel OD-R column [26]. Methanol as organic modifier in PBS (50 mM) was first tested. No resolution was observed for any of the six imidazolinone herbicides at pH 2.0 or 3.0. The PBS (50 mM)-acetonitrile mobile phase systems were then tested at pH from 2.0 to 7.0. Enantiomers of imazethapyr (3), imazaquin (11) and imazamox (4) were resolved at pH 2.0 and 1.0 ml/min flow rate with 70/30 (v/v) PBS-acetonitrile as the
Fig. 2. The dissociation equilibrium of imazaquin (11). Table 1 Effect of mobile phase compositions and pH on chiral separation of imidazolinone herbicides on Chiralcel OD-R column pH 2.0
Imazethapyr (3)a Imazaquin (11)a Imazamox (4)a Imazapyr (1)b Imazethapyr (3)c Imazaquin (11)c Imazamox (4)c a b c
pH 3.0
pH 4.0
k1
α
Rs
k1
α
Rs
k1
α
Rs
8.50 26.37 4.20 37.85 16.25 55.68 7.92
1.10 1.25 1.13 1.09 1.12 1.25 1.16
0.58 2.1 0.49 0.27 1.01 2.44 0.89
4.77 11.38 2.25 9.38 14.75 36.34 3.65
1.0 1.23 1.0 1.0 1.12 1.22 1.12
0 1.49 0 0 0.34 1.87 0.31
2.25 11.17 1.12 5.85 14.68 36.37 1.9
1.0 1.24 1.0 1.26 1.12 1.20 1.0
0 1.49 0 0.49 0.35 1.92 0
Mobile phase A: 70/30 (v/v) (50 mM phosphate buffer)-acetonitrile, flow rate 1.0 ml/min. Mobile phase B: 80/20 (v/v) (50 mM phosphate buffer)-acetonitrile, flow rate 0.5 ml/min. Mobile phase C: 80/20 (v/v) (50 mM phosphate buffer)-acetonitrile, flow rate 1.0 ml/min.
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imazamox (4). Imazapyr (1) could be resolved at a flow rate of 0.5 ml/min using 85/15 (v/v) PBS-acetonitrile as the mobile phase. Imazamethabenz-methyl (5) is a mixture of 5-methyl (meta) isomer and 4-methyl (para) isomer, and the complete resolution of which would result in four peaks. However, only initial isomeric separation was found for imazamethabenz-methyl (5) on OD-R column at pH 2.0 by decreasing the ratio of acetonitrile from 30 to 20% (α = 1.03, Rs = 0.06). Imazethapyr (3), imazaquin (11) and imazamox (4) were separated under the same mobile phase conditions without overlapping peaks in the reversed-phase mode. This suggests that the determination of the enantiomers of these three herbicides may be achieved in one sample matrix. 3.2. Normal-phase mode
Fig. 3. Chromatograms of chiral separation on Chiralcel OD-R column. Mobile phase, 80/20 (v/v) (50 mM phosphate buffer)-acetonitrile; flow rate, 1.0 ml/min.
mobile phase (Table 1). Under these conditions, baseline separation was achieved only for imazaquin. However, when the ratio of PBS and acetonitrile was changed to 80/20 (v/v), the resolution of imazethapyr (3) and imazamox (4) were improved but not to baseline separation (Fig. 3). Baseline separation of imazaquin (11) was preserved at pH 3.0 and 4.0 with slight changes in the separation factor as compared to pH 2.0. At pH 5.0, only initial separation (Rs = 0.13) was observed for imazaquin (11). At pH 6.0 and 7.0, imazaquin (11) lost its separation. Enantiomers of imazethapyr (3) and imazamox (4) were not resolved at pH 3.0 or 4.0 using 70/30 (v/v) PBS-acetonitrile as the mobile phase. Modifying chromatographic conditions to 80/20 (v/v) PBS-acetonitrile (pH 4.0) resulted in resolution of enantiomers of imazethapyr (3) and imazaquin (11), but not
3.2.1. Effect of molecular structures A n-hexane-2-propanol mixture was first used as the mobile phase on Chiralcel OJ column for the separation of imidazolinone herbicides and their methyl derivatives. Trifluoroacetic acid (0.1%, v/v) was added to n-hexane to allow elution of imidazolinone herbicides with free carboxyl group [13,27]. Enantiomers of all herbicides except imazamethabenz-methyl (5) were resolved on the OJ column using 75/25 (v/v) n-hexane (0.1%, TFA)-2-propanol (Table 2). Imazapyr (1) gave the highest separation factor, while imazethapyr (3), imazamox (4) and imazapic (2) were also resolved at the baseline (Fig. 4). In contrast, imazaquin (11) exhibited the lowest resolution with the smallest separation factor. No chiral separation of imazamethabenz-methyl (5) was achieved under these conditions. However, when the content of 2-propanol was reduced from 25 to 10% by volume, separation of imazamethabenzmethyl isomers was observed. Imazethapyr (3), imazamox (4) and imazapic (2) have different substituents at the 5 position of the pyridine moiety from imazapyr (1). Although this substituent is far away from the chiral center and may not change the spatial conformation, the substituent made obvious differences in retention and separation factors. The k2 and k1 of imazapic (2) and imazethapyr (3) were both smaller than imazapyr (1). Imazapic (2) was added
Table 2 The resolution (Rs ) of enantiomers of imidazolinone herbicides and their methyl derivatives on the Chiralcel OJ column in the normal-phase mode Alcohol modifier
Imazapyr (1)a Imazapic (2)a Imazethapyr (3)a Imazamox (4)a Imazaquin (11)a Imazapyr derivative (6)b Imazapic derivative (7)b Imazethapyr derivative (9)b Imazamox derivative (10)b Imazaquin derivative (12)b a b
Ethanol
1-Propanol
2-Propanol
1-Butanol
2-Butanol
t-Butanol
4.13 3.87 3.85 3.41 1.41 0.40 0.81 0.57 2.61 1.10
4.18 3.04 2.98 2.87 0.45 0.98 0.00 0.00 0.27 0.00
2.67 2.02 1.51 2.00 0.95 2.02 0.84 0.74 0.96 0.51
3.18 2.05 1.36 1.99 0.00 0.97 0 0 0 0
2.40 1.06 0.91 1.39 0.00 0.80 0.54 0 0 0
1.37 0.70 0.55 0.99 0.25 2.74 1.42 0 1.72 0.54
Mobile phase A: 75/25 (v/v) n-hexane (0.1% TFA)/alcohol modifier Mobile phase B: 90/10 (v/v) n-hexane (0.1% TFA)/alcohol modifier.
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Fig. 4. Chromatograms of chiral separation on Chiralcel OJ column. Mobile phase for imidazolinone herbicides: 75/25 (v/v) n-hexane (0.1%TFA)/alcohol modifier; and mobile phase for imidazolinone herbicides methyl derivatives: 90/10 (v/v) n-hexane (0.1%TFA)/alcohol modifier.
a methylene group to become imazethapyr (3) which exhibited a decrement in the retention factor and enantioselectivity. However, in imazamox (4) with an alkoxyl group replacing the methyl group of imazethapyr (3), the retention and separation factor were both enhanced. These results suggest that the alkoxyl group likely increased the dipole–dipole interaction with CSP. Wainer et al. [28] demonstrated that steric factor of an enantiomer played an important role in its fitting into chiral cavities of cellulose CSPs. The aromatic portion of imidazolinone herbicides may fit into the chiral cavity to form complex with CSP [29]. The bulkiness of imazaquin (11) may have resulted in a
lower degree of steric fit in the chiral cavities and consequently the poor resolution. To better understand this assumption, the optimization structures of imidazolinone herbicides and their derivatives were calculated by using the AM1 semi-empirical quantum mechanics method [30]. The values of 1-2-2 -1 dihedral angles of aromatic ring and imidazol ring reflect differences in structural characteristics (Table 3). The values of dihedral angles were negative for all compounds, indicating that the twisting directions of aromatic rings were on the same side of the imidazolinone ring. From the values of the 1-2-2 -1 dihedral angle, imidazolinone compounds could be divided into
Table 3 Calculated dihedral angles of 1-2-2 -1 of imidazolinone herbicides and their methyl derivatives Dihedral angel of 1-2-2 -1 (degree) Imazapyr (1)
Imazapic (2)
Imazethapyr (3)
Imazamox (4)
Imazaquin (11)
Imazamethabenz-methyl (5) meta isomer
Derivative
para isomer
−44.9
−43.8
−43.4
−45.1
−26.0
−63.6
−68.8
−58.9
−58.3
−58.6
−59.1
−5.8
−65.7
−84.2
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three groups. Imazapyr (1), imazapic (2), imazethapyr (3) and imazamox (4) are in the first group with almost identical 1-2-2 1 dihedral angles. Imazaquin (11) and imazamethabenz-methyl (5) are in the second and third group, respectively. The absolute values of the dihedral angle for the first group are greater than the absolute value for imazaquin (11). A higher absolute value of the dihedral angle implies that the twisting of the aromatic ring out of the plane of the imidazol ring is greater. It is therefore possible that one of the reasons for the lower separation factor of imazaquin (11) stems from its particular conformation. All of the methyl derivatives of imidazolinone compounds except imazamethabenz-methyl (10) were enantiomerically resolved using 90/10 (v/v) n-hexane-2-propanol as the mobile phase (Table 2). These derivatives evidently do not offer any hydrogen bonding interaction with the OJ column. Okamoto et al. confirmed that hydrophobic interactions could be a major force in the chiral separation on OJ column [13]. The hydrophobic interactions between the enantiomers of these derivatives and CSP seem to be of importance for the formation of the solute-
CSP complex and may contribute to proper steric fit to the cavity of CSP. The enantiomers of imazamethabenz-methyl derivatives (10) were not separated using n-hexane-2-propanol 90/10 (v/v) as the mobile phase, although isomeric separation was obtained. From the structures of imazapic (6) and imazamethabenz-methyl derivatives (10), it is likely that the nitrogen of the pyridine ring is beneficial for the chiral separation through dipole–dipole interactions. 3.2.2. Effect of alcohol modifier The effect of polar alcohol modifiers on enantiomer separation was investigated, where ethanol, 1-propanol, 2propanol, 1-butanol, 2-butanol or t-butanol was tested as the polar modifier. For all the alcohol modifiers, k1 consistently decreased in the order imazaquin (11) > imazamox (4) > imazapyr (1) > imazapic (2) > imazethapyr (3), while the order for k2 followed imazamox (4) > imazapyr (1) > imazaquin (11) > imazapic (2) > imazethapyr (3). Therefore, only the position of imazaquin (11) changed in the two orders. The reten-
Fig. 5. Influence of alcohol modifier on retention factor of imidazolinone herbicides and their methyl derivatives. (a) The first eluted enantiomer of imidazolinone herbicide; (b) the second eluted enantiomer of imidazolinone herbicide; (c) the first eluted enantiomer of imidazolinone herbicide methyl derivative; (d) the second eluted enantiomer of weimidazolinone herbicide methyl derivative. Chromatographic conditions were the same as given in Fig. 4.
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tion factors increased generally with increasing length of carbon chain of linear or branched alcohols (Fig. 5a and b). The change in the separation factor for imazapyr (1) was small with ethanol or 2-butanol as modifier instead of 2-propanol. However, with 1-propanol or 1-butanol as modifier, the separation factor of imazapyr (1) increased, but the magnitude of enhancement arisen from 1-butanol was smaller than with 1-propanol. The use of t-butanol as modifier deteriorated enantioselectivity of imazapyr (1). The separation factors of imazapic (2), imazethapyr (3) and imazamox (4) consistently decreased with the order of alcohol modifiers ethanol > 1-propanol > 2propanol > 1-butanol > 2-butanol > t-butanol. The enantioselectivity of imazaquin (11) on the OJ column was different from the other imidazolinone herbicides. First, the effect on separation factors of imazapyr (1) and imazaquin (11) was reversed when 1-propanol replaced 2-propanol as modifier. Second, using 1butanol or 2-butanol as modifier, the enantiomers of imazaquin (11) lost chiral separation. Third, when t-butanol was used as the modifier, enantioselectivity was recovered to a certain degree.
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It is likely that the imazaquin (11) enantiomers carrying a large quinoline group were in unfavorable situation to compete with the linear alcohol molecules, but with t-butanol as modifier, the enantiomers may access chiral interaction sites more easily. The combination of higher separation factor and lower retention factor indicated that ethanol was a more favorable mobile phase modifier for the separation of imidazolinone compounds on cellulose ester-type CSP [29]. However, with consideration of both enantioselective effect and cost, 2-propanol should be a good selection as the modifier. The alcohol modifiers showed different effects on retention factors and separation factors of imidazolinone herbicides and their methyl derivatives (Fig. 5c and d). The t-butanol as modifier gave the largest retention factors to enantiomers of imidazolinone herbicides and their methyl derivatives. However, retention factors with 2-propanol as modifier were larger than from ethanol, 1-propanol, 1-butanol and 2-butanol. Only imazapyr (6) methyl derivative showed enantiomeric separation with all of the alcohol modifiers, whereas the results on the other
Fig. 6. Influence of alcohol modifier on separation factor and k of imidazolinone herbicides and their methyl derivatives. (a) Separation factor of imidazolinone herbicide; (b) k of imidazolinone herbicide; (c) separation factor of imidazolinone herbicide methyl derivatives; (d) k of imidazolinone herbicide methyl derivatives. The x-axis for (a) and (b) are: imazapyr 1, imazapic 2, imazethapyr 3, imazamox 4 and imazaquin 5. The x-axis for (c) and (d) are: methyl derivative of imazapyr 1, imazapic 2, imazethapyr 3, imazamox 4 and imazaquin 5. Chromatographic conditions were the same as in Fig. 5.
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methyl derivatives were inconsistent. Enantiomers of methyl derivatives of imazapyr (6) and imazamox (9) were resolved with 1-propanol as modifier. With 1-butanol as the modifier, however, only methyl derivative of imazapyr (6) showed enantiomeric separation. Enantiomers of methyl derivatives of imazamox (9), imazethapyr (6) and imazaquin (12) were not resolved with 2-butanol as modifier. With the exception of imazethapyr derivative (8), the methyl derivatives gave the largest separation factors when t-butanol was used as the modifier. The separation factors of imidazolinone herbicides and their methyl derivatives measured by using different alcohol modifiers are shown in Fig. 6a and c. The structure-enantioselectivity relationships were different among the different alcohol modifiers. This suggests that comparing apparent separation factors alone may not be enough to gain a better understanding of trends in the structure-enantioselectivity relationship. Therefore, the k values were further considered for the evaluation. Schurig et al. first suggested the importance to differentiate contributions to α by physical (achiral) and chemical (chiral) interactions between a chiral compound and stationary phases in complexation GC [31]. Guiochon et al. also observed that k consisted of contributions from non-enantioselective and enantioselective interactions [32]. The conventional separation factor α is an apparent separation factor aapp : αapp =
+ k kns2 s2 + k kns1 s1
(1)
and k are the contributions of non-enantioselective where kns1 ns2 interaction of the less and the more retained enantiomer, respec and k the contributions of enantioselective interactively; ks1 s2 tion of the less and the more retained enantiomer, respectively. Since the non-enantioselective interaction is identical for both enantiomers, the difference between the retention factors of two enantiomers, k , is caused by the enantioselective interaction:
k = k2 − k1
Fig. 7. Effect of mobile phase compositions on the separation factor. Chromatographic conditions were the same as in Fig. 5, except for the volume fraction of alcohol modifier.
imazamox (9) gave the highest “true selectivity”. The k values of the methyl derivatives illustrated that the t-butanol modifier led to the highest “true selectivity” in spite of no resolution of enantiomers of imazethapyr methyl derivatives (8). Ethanol as modifier showed average lower “true selectivity” than 2propanol. 1-propanol, 1-butanol and 2-butanol as mobile phase modifiers gave lower k values and did not favor the separation of imazaquin (12) and imazethapyr derivatives (8) (Fig. 6d). 3.2.3. Effect of mobile phase composition As the 2-propanol volume fraction in the n-hexane-2propanol mobile phase was increased from 20 to 35%, the
(2)
The higher k values imply that the “true selectivity” is larger. Linear chromatography does not provide enough information to distinguish the nonselective and enantioselective interactions of each enantiomer. However, some trends can still be observed from the k values (Fig. 6b). The measured k followed the order imazapyr (1) > imazamox (4) > imazapic (2) > imazethapyr (3) > imazaquin (11). This sequence revealed clearly a structure-enantioselectivity relationship: with increasing chain length of the substituent on the 5 position of pyridine ring (H < CH3 < CH2 CH3 ), the “true selectivity” decreased. However, if this substituent provides extra interaction other than pure hydrophobic interactions, such as the alkoxyl group in imazamox (4), the “true selectivity” increased. On the other hand, the bulkiness of a quinoline group appeared to result in deterioration of chiral separation. The different alcohol modifier did not affect the order of k . Therefore, this study showed that changes of separation factors for structurally related compounds may be manifested by comparison of k values. The k values further revealed a trend of chiral separation for the methyl derivatives. Methyl derivatives of imazapyr (6) and
Fig. 8. Typical chromatograms of imazapyr (1) and imazaquin (11) extracted from a sandy loam soil. Mobile phase for imazapyr: 75/25 (v/v) n-hexane (0.1%TFA)/2-propanol; and mobile phase for imazaquin: 80/20 (v/v) n-hexane (0.1%TFA)/2-propanol.
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k and Rs decreased and the enantioselectivity was essentially unchanged (Fig. 7). However, when the volume fraction of 2propanol was further increased from 35 to 40%, a more rapid decrease in separation factor was observed. As the fraction of 2-propanol was increased, it is likely that the strength of hydrogen bonds between imidazolinone compounds and CSP became weaker and the solubility of the solute increased. These results suggest that the hydrogen bonding interaction not only influenced the retention but also the enantiomeric selectivity [33]. 3.3. Applications Chromatograms on the OJ column of imazapyr (1) and imazaquin (11) extracted from spiked soil samples are shown in Fig. 8a and b, respectively. Enantiomers of imazapyr (1) were baseline separated (Fig. 8a), while enantiomers of imazaquin (11) were also adequately resolved (Fig. 8b). These results suggest that the developed method may be applied for chiral separation of imidazolinone herbicides in environmental matrices such as soil or sediment samples, and may be useful for enantioselectivity evaluation in environmental processes. 4. Conclusions Enantiomers of imazethapyr (3), imazaquin (11), and imazamox (4) were separated on Chiralcel OD-R column using PBS-acetonitrile as the mobile phase. Imazapyr (1) only showed faint enantioselectivity on this column in the reversed-phase mode, while no chiral separation was observed for imazapic (2) and imazamethabenz-methyl (5). However, enantiomers of all imidazolinone herbicides except for imazamethabenzmethyl (5) were adequately resolved on Chiralcel OJ column in the normal-phase mode using n-hexane (0.1% FTA) with an alcohol modifier. Enantiomers of five methyl derivatives of imidazolinone herbicides were also resolved on Chiralcel OJ column. Imazamethabenz-methyl (5) and its derivatives (10) exhibited isomeric separation but no enantiomeric separation. The order of k values (which measures the “true selectivity”) followed imazapyr (1) > imazamox (4) > imazapic (2) > imazethapyr (3) > imazaquin (11). By comparison of k values a structure-enantioselectivity relationship was observed. The developed chiral separation methods may be used to evaluate enantioselectivity of these compounds during biodegradation, toxicity, and other biologically-mediated processes. Acknowledgments We thank P. Resketo and F. Ernst for their technical assistance. This study was supported by a USDA-National Research Initiatives grant No. 2005-35107-16189.
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