Separation of enantiomers of triazole chiral pesticides under reversed phase chromatography

　　The enantiomers of hexaconazole, diniconazole, diniconazole, flutriafol, triadimefon and tebuconazole were successfully separated under reversed phase chromatography using self-made cellulose trichiral stationary phase and amylose trichiral stationary phase.

　　Both stationary phases have strong separation ability. Under the optimized chromatographic conditions, hexaconazole and diniconazole can be separated on both stationary phases; triadimefon can only be separated on CDMPC-CSP; fluconazole, tebuconazole, and tebuconazole can only be separated on ADMPC-CSP. The increase of water content in the mobile phase will enhance the retention of enantiomers and increase the possibility of separation. In the temperature range of 0-40℃, the capacity factor k decreases with the increase of temperature. Except for tebuconazole and tebuconazole, the selectivity factor α of other chiral pesticides also decreases with the increase of temperature. There is no obvious regularity in the change of resolution Rs with temperature, and the best resolution does not always appear at low temperature. The elution order of enantiomers is determined by circular dichroism detector.

　　1 Introduction

　　In recent years, with the development of chiral technology, the research on optically active pesticides has become a hot topic in the field of pesticide chemistry. Among the commercialized pesticides, about 26% of them have chiral centers, most of which are sold and used in the form of racemates. When racemic chiral pesticides are applied to organisms, the differences between the enantiomers are not only manifested in biological activity, but also in toxicity to organisms, absorption, transfer, metabolism and elimination in the body. Therefore, it is of great significance to establish a high-resolution and high-sensitivity chiral pesticide separation and determination method. Common chiral separation methods include high-performance liquid chromatography, gas chromatography, molecular imprinting and capillary electrophoresis, among which high-performance liquid chromatography chiral stationary phase method is an important one. Among the many chiral stationary phases, polysaccharide (commonly cellulose and starch) derivatives CSPs show advantages such as strong separation ability and wide application range. Hexaconazole, diniconazole, diniconazole, fluconazole, triadimefon and tebuconazole are all triazole pesticides, which are an important class of broad-spectrum fungicides. The main methods for the separation of their enantiomers include gas chromatography, capillary electrophoresis, chiral mobile phase additives, chiral stationary phases and polysaccharide chiral stationary phases. The polysaccharide chiral stationary phase HPLC method is mainly used to separate triazole pesticides under normal phase conditions. In this study, the enantiomers of hexaconazole, diniconazole, diniconazole, flutriafol, triadimefon and tebuconazole were separated on a high performance liquid chromatograph using homemade cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (CDMPC-CSP) and amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (ADMPC-CSP) under reverse phase chromatography conditions with methanol/water or acetonitrile/water as mobile phases . The effects of stationary phase, mobile phase and temperature on chiral separation were compared, and the elution order of enantiomers was determined by circular dichroism detector.

　　2 Experimental part

　　2.1 Instruments and reagents 1100 high performance liquid chromatograph (Agilent), equipped with a diode array detector; JASCO2000 high performance liquid chromatograph (JASCO, Japan), equipped with a circular dichroism detector; liquid chromatography column packing machine (Beijing Fusiyuan Mechanical Processing Department); AT-930 refrigeration and heating dual-purpose chromatographic column oven (Tianjin Aotesense Instrument Co., Ltd.). 3,5-dimethylphenylisocyanate (Merck); amylose spheres (Sigma); micro-silica gel (Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences): spherical, particle size 5-7μm, specific surface area 110m2/g, average pore size 6.7nm; microcrystalline cellulose (Shanghai Reagent Factory No. 4); 3-aminopropyltriethoxysilane (KH-550, Gaixian Chemical Plant, Liaoning). All reagents used were analytically pure, and the mobile phase was used after re-distillation. Six triazole chiral pesticides were provided by the Pesticide Residue Analysis Group of China Agricultural University.

　　2.2 Synthesis of chiral stationary phases The chiral stationary phases CDMPC and ADMPC were synthesized according to the method in the literature and loaded into a stainless steel column at a pressure of 37 MPa.

　　2.3 Chromatographic conditions Chromatographic columns: CDMPC-CSP (250 mm × 4.6 mm), ADMPC-CSP (150 mm × 4.6 mm); mobile phase: methanol-water or acetonitrile-water; flow rate: 0.8 mL/min (CDMPC) and 0.5 mL/min (ADMPC); injection volume: 10 μL, detection wavelength: 230 nm. Capacity factor (k), selectivity factor (α) and resolution (Rs) were calculated according to the formulas k = (t-t0)/t0, α = k2/k1, and Rs = 2 (t2-t1)/(w1 w2), respectively.

　　3 Results and discussion

　　3.1 Effect of different stationary phases and mobile phase compositions on enantiomeric resolution Direct chiral resolution of triazole pesticides hexaconazole, diniconazole, tinconazole, flutriafol, tebuconazole and triadimefon was performed on CDMPC-CSP and ADMPC-CSP using methanol-water or acetonitrile-water as mobile phases. Under the optimized chromatographic conditions, the resolution of hexaconazole, diniconazole and triadimefon on CDMPC was 1.72, 1.31 and 1.55, respectively; the resolution of hexaconazole, diniconazole, flutriafol, tinconazole and tebuconazole on ADMPC was 1.28, 2.75, 1.05, 2.41 and 1.30, respectively. Different mobile phases on the same stationary phase have different effects on sample retention and stereoselectivity. For example, on ADMPC-CSP, diniconazole and flutriafol can only be resolved in methanol-water as mobile phase, while hexaconazole can only be resolved in acetonitrile-water as mobile phase. Methanol is both a proton acceptor and a proton donor; acetonitrile is only a proton acceptor. This shows that hydrogen bonding is not necessarily the main force for separation under reversed-phase conditions. As the water content in the mobile phase increases, the possibility of separation increases, but considering the life and efficiency of the column, the water content in this experiment is 40% and 60% for methanol and acetonitrile, respectively. Our research group has reported that tebuconazole and triadimefon can be separated on CDMPC under normal phase chromatography conditions. Hydrogen bonding, π-π, and dipole-dipole interactions are generally considered to be the separation chromatograms of triazole chiral pesticides on polysaccharide stationary phases under reversed-phase conditions on ADMPC-CSP.

　　Fig.2 Chromatograms showing the enantiomeric separation of triazole chiral pesticides a. Hexaconazole, acetonitrile/water (ACN/water) 45:55, V/V; b. Diniconazole, methanol/water (Methanol/water), 80:20, V/V; c. Flutriafol, methanol/water (Methanol/water) 70:30, V/V; d. Uniconazole, acetonitrile/water (ACN/water) 60:40, V/V; e. Tebuconazole, acetonitrile/water (ACN/water), 50:50, V/V; room temperature; flow rate: 0.5 mL/min. The effective force for separation under normal phase chromatography conditions; under reverse phase conditions, the highly polar mobile phase is more likely to form strong hydrogen bonds with the CSP, thereby weakening the hydrogen bonding between the solute molecules and the CSP, which may make the inclusion of the analyte in the helical chiral cavity of the stationary phase and "stericfit", hydrophobic interaction, π-π, and dipole-dipole interactions become the key factors for chiral recognition.

　　3.2 Effect of temperature on enantiomer resolution The effect of temperature on the enantiomer resolution of triazole chiral pesticides was investigated in the range of 0-40°C. The results show that the capacity factor k of the chiral pesticide enantiomers studied decreases with increasing temperature; there is no consistent rule for the selectivity factor α; and the best resolution of different pesticides occurs at different temperatures, rather than all at low temperatures.

　　By measuring the capacity factor k and chiral selectivity factor α at different temperatures, lnk and lnα are plotted against 1/T, and a straight line can be obtained for each. By examining the effect of temperature on chiral separation, the thermodynamic parameters in the chiral separation process can be calculated, thereby having a deeper understanding of the chiral separation process. Except for the fact that the natural logarithm of the selectivity factor of tebuconazole and the reciprocal of the absolute temperature are not linearly related on ADMPC when methanol is used as a modifier (Figure 3), the van′tHoff curves of other chiral pesticides are linear, and the calculated thermodynamic parameters are shown in Table 1. When methanol is used as a modifier, the chiral separation of tebuconazole on ADMPC is mainly controlled by entropy, while when acetonitrile is used as a modifier, it is mainly controlled by enthalpy; the chiral separation of other pesticides (except tebuconazole) is mainly controlled by enthalpy. Table 1 Van′tHoff equation and ΔΔH°, ΔΔS° of chiral pesticide enantiomers in the range of 0 to 40°C.

　　3.3 Elution order of enantiomers In recent years, HPLC circular dichroism detector has become a powerful tool for the analysis of chiral compounds. In this study, the elution order of enantiomers was determined by HPLC circular dichroism detector (see Table 2). The elution order of three triazole chiral pesticides was consistent under the same wavelength under two mobile phase conditions on CDMPC-CSP; while on ADMPC-CSP, the elution order of diniconazole and diniconazole was opposite, and the elution order of other chiral pesticides was consistent. The reason for this phenomenon is still unclear, but it is generally believed that the change in the mobile phase composition leads to a change in the stereo environment of the chiral cavity. Table 2 Elution order of triazole chiral pesticides under reversed phase conditions on CDMPC-CSP and ADMPC-CSP.