Direct HPLC separation of enantiomers of pantoprazole and other benzimidazole sulfoxides using cellulose-based chiral stationary phases in reversed-phase mode

A direct, isocratic, and simple reversed-phase HPLC method was described for the separation of enantiomers of the proton pump inhibitor, rac-pantoprazole (PAN) using cellulose-based chiral stationary phases (Chiralcel OD-R and Chiralcel OJ-R). Some structurally related chiral benzimidazole sulfoxides, rac-omeprazole (OME) and raclansoprazole (LAN), were also studied. Chiralcel OJ-R was successful in the resolution of enantiomers of rac-PAN and rac-OME, while Chiralcel OD-R was most suitable for resolving the enantiomers of rac-LAN. Highest enantioselectivity to rac-PAN and rac-OME was achieved on Chiralcel OJ-R by using acetonitrile as an organic modifier, whereas methanol afforded better resolution of rac-LAN on Chiralcel OD-R than acetonitrile. Increases in buffer concentration and column temperature decreased retention and did not improve the resolution of the enantiomers on both columns. Using a mixture of 50 mM sodium perchlorate solution and acetonitrile as a mobile phase at a flow rate of 0.5 ml/min, maximum separation factors of 1.26 and 1.13 were obtained for the enantiomers of rac-PAN and rac-OME using a Chiralcel OJ-R column, while maximum separation factor of 1.16 was obtained for the enantiomers of rac-LAN using a Chiralcel OD-R column. © 1995 Wiley-Liss, Inc.     

Separation of (R)- and (S)-tert-butyl 2-tert-butyl-4-methoxy-2,5-dihydro-1,3-imidazole-1-carboxylate (building block for amino acid synthesis) by preparative high performance liquid chromatography on a polysaccharide stationary phase

The preparative separation of the enantiomers of the title compound, a versatile chiral building block for the synthesis of unnatural amino acid esters, by high performance liquid chromatography on a chiral stationary phase (CSP), is reported for the first time. The CSP consists of amylose-(3,5-dimethylphenyl-carbamate), which has been coated onto the surface of macroporous aminopropyl-functionalized silica gel. The effect of mobile phase composition and the amount of amylose derivative on the silica gel has been thoroughly investigated. Using 2-propanol as organic modifier in hexane as mobile phase, on a semi-preparative column (200 mm × 40 mm ID, containing 192 g of stationary phase) about 200 mg of the racemate was separated per injection. Running the equipment under automatic conditions with repetitive injection mode allowed for the separation of 30 g per day. Both enantiomers were obtained with enantiopurities >99.75:0.25. Chirality 10:217–222, 1998. © 1998 Wiley-Liss, Inc.     

Enantiomeric Separations of Pyriproxyfen and its Six Chiral Metabolites by High‐Performance Liquid Chromatography

Pyriproxyfen is a chiral insecticide, and over 10 metabolites have been identified in the environment. In this work the separations of the enantiomers of pyriproxyfen and its six chiral metabolites were studied by high‐performance liquid chromatography (HPLC). Both normal phase and reverse phase were applied using the chiral columns Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralcel OD‐RH, Chiralpak AY‐H, Chiralpak AD‐H, Chiracel OJ‐H, (R,R)‐Whelk‐O 1, and Lux Cellulose‐3. The effects of the chromatographic parameters such as mobile phase composition and temperature on the separations were investigated and the enantiomers were identified with an optical rotation detector. The enantiomers of these targets could obtain complete separations (resolution factor Rs > 1.5) on Chiralpak IA, Chiralpak IB, Chiralcel OD, Chiralpak AY‐H, or Chiracel OJ‐H under normal conditions. Chiralcel OJ‐H showed the best chiral separation results with n‐hexane as mobile phase and isopropanol (IPA) as modifier. The simultaneous enantiomeric separation of pyriproxyfen and four chiral metabolites was achieved on Chiralcel OJ‐H under optimized condition: n‐hexane/isopropanol = 80/20, 15°C, flow rate of 0.8 ml/min, and UV detection at 230 nm. The enantiomers of pyriproxyfen and the metabolites A , C , and D obtained complete separations on Chiralpak IA, Chiralpak IC, and Lux Cellulose‐3 under reverse phase using acetonitrile/water as the mobile phase. The retention factors (k) and selectivity factors (α) decreased with increasing temperature, and the separations were better under low temperature in most cases. The work is of significance for the investigation of the environmental behaviors of pyriproxyfen on an enantiomeric level. Chirality 28:245–252, 2016. © 2016 Wiley Periodicals, Inc.     

Enantiomeric separation and recognition mechanism of 2-arylpropionic acid derivatives by high-performance liquid chromatography using a chiral column

An optical resolution of the amide derivatives of ibuprofen and the carbamate-alkylester derivatives of the trans-alcohol metabolite of loxoprofen and an analogous compound, CS-670, was studied by chiral high-performance liquid chromatography (HPLC). The chiral columns SUMIPAX OA-4000 and OA-4100 were used to investigate the enantiomeric separation behavior of these derivatives using both reversed and normal mobile phases. A better separation factor (α) of the amide and the carbamate ester derivatives was obtained in the normal mobile phase than in the reversed mobile phase HPLC. In addition, the recognition mechanisms of both amide and carbamate ester enantiomers were investigated by ¹H-nuclear magnetic resonance (NMR). It is suggested that the important driving forces for the enantiomeric separation are the formation of hydrogen bonding and the charge transfer complex between these derivatives and an active site of the chiral stationary phase. © 1995 Wiley-Liss, Inc.     

Experimental studies of competition between enantiomers in chiral liquid chromatography through their system peaks

Competition between the (+)- and (?) enantiomers of 2,2,2-trifluoro-1-(9-anthryl) ethanol as mobile phase additives was indicated by the chromatographic behavior of their system peaks. Two types of chiral stationary phases were used, one based on dinitrobenzoylphenylglycine and the other on dinitrobenzylphenylethylamine plus tartaric acid. The racemic mixture was used as the mobile phase additive and k′ of their system peaks was studied as a function of the mixture concentration in the mobile phase in both cases. A shift in k′ of the two system peaks was observed and considered as an indication that competition occurred. The areas of the two system peaks were also studied as a function of the concentration of the enantiomers in the samples, using two different compositions of the mobile phase. The dependency of system peaks' area on the sample composition indicated whether competition between the enantiomers occurred. One mobile phase contained 0.1 mM of the racemic mixture, where the area of the two retained system peaks behaved independently, i.e., only the peak corresponding to the enantiomer was affected by its presence in the sample. The other mobile phase contained 0.75 mM of the racemic mixture, and both peaks were affected by the injection of any one of the enantiomers. The interdependency of the system peaks' area on both the enantiomers indicated that their distribution in the chiral system was interrelated due to mutual interactions. A quantitative treatment of the interdependency and competition was excluded, due to the irreversible adsorption of the two enantiomers on the chiral stationary phase after using overloading concentrations. This irreversible adsorption was visualized by the appearance of two retained system peaks of the two residual enantiomers. These system peaks were detected only when the sample contained pure enantiomers due to competition between the enantiomer in the sample with the residual enantiomers in the stationary phase. © 1994 Wiley-Liss, Inc.     

Determination of panthenol enantiomers in cosmetic preparations using an achiral-chiral–coupled column HPLC system

A new high-performance liquid chromatography (HPLC) method for separation and determination of panthenol enantiomers in hair care products was developed. Two types of detectors, low-wavelength ultraviolet (UV) and polarimetric, were used. Optimized conditions consisted of coupled achiral, amino type, and chiral, amylose tris(3,5-dimethylphenylcarbamate), stationary phases, mixture of n-hexane/ethanol (60:40, v/v) as mobile phase under isocratic conditions and flow rate 0.8 cm³ min⁻¹. The effect of column temperature on retention and resolution of enantiomers was studied. The analysis runtime was 10 minutes, and the average retention times for d - and l -panthenol were 7.10 ±0.1 minutes and 8.21 ±0.2 minutes, respectively. The resolution of enantiomers on coupled achiral-chiral columns was R_s = 2.7. The solid-phase extraction method was employed for extraction and purification of analytes. The validated method was selective, accurate, and linear (R² > .998) over the concentration range of 0.001 to 1.0 mg cm⁻³ for both enantiomeric forms. The limits of detection (LOD) and quantitation (LOQ) of each enantiomer were 0.3 and 1.0 μg cm⁻³, respectively. The results demonstrated the occurrence of d -panthenol in hair care products.     

Enantioseparation and determination of flumequine enantiomers in multiple food matrices with chiral liquid chromatography coupled with tandem mass spectrometry

The present work firstly described the enantioseparation and determination of flumequine enantiomers in milk, yogurt, chicken, beef, egg, and honey samples by chiral liquid chromatography‐tandem mass spectrometry. The enantioseparation was performed under reversed‐phase conditions on a Chiralpak IC column at 20°C. The effects of chiral stationary phase, mobile phase components, and column temperature on the separation of flumequine enantiomers have been studied in detail. Target compounds were extracted from six different matrices with individual extraction procedure followed by cleanup using Cleanert C18 solid phase extraction cartridge. Good linearity (R²>0.9913) was obtained over the concentration range of 0.125 to 12.5 ng g^‐1 for each enantiomer in matrix‐matched standard calibration curves. The limits of detection and limits of quantification of two flumequine enantiomers were 0.015‐0.024 and 0.045‐0.063 ng g^‐1, respectively. The average recoveries of the targeted compounds varied from 82.3 to 110.5%, with relative standard deviation less than 11.7%. The method was successfully applied to the determination of flumequine enantiomers in multiple food matrices, providing a reliable method for evaluating the potential risk in animal productions.     

Enantioseparation of chiral pharmaceuticals by vancomycin‐bonded stationary phase and analysis of chiral recognition mechanism

The drug chirality is attracting increasing attention because of different biological activities, metabolic pathways, and toxicities of chiral enantiomers. The chiral separation has been a great challenge. Optimized high‐performance liquid chromatography (HPLC) methods based on vancomycin chiral stationary phase (CSP) were developed for the enantioseparation of propranolol, atenolol, metoprolol, venlafaxine, fluoxetine, and amlodipine. The retention and enantioseparation properties of these analytes were investigated in the variety of mobile phase additives, flow rate, and column temperature. As a result, the optimal chromatographic condition was achieved using methanol as a main mobile phase with triethylamine (TEA) and glacial acetic acid (HOAc) added as modifiers in a volume ratio of 0.01% at a flow rate of 0.3 mL/minute and at a column temperature of 5°C. The thermodynamic parameters (eg, ΔH, ΔΔH, and ΔΔS) from linear van 't Hoff plots revealed that the retention of investigated pharmaceuticals on vancomycin CSP was an exothermic process. The nonlinear behavior of lnk′ against 1/T for propranolol, atenolol, and metoprolol suggested the presence of multiple binding mechanisms for these analytes on CSP with variation of temperature. The simulated interaction processes between vancomycin and pharmaceutical enantiomers using molecular docking technique and binding energy calculations indicated that the calculated magnitudes of steady combination energy (ΔG) coincided with experimental elution order for most of these enantiomers.     

Chiral discrimination by albumin: A mechanistic study of liquid chromatographic optical resolution of nonaromatic carboxylic acids

In order to get further insight into the mechanism by which bovine serum albumin (BSA) discriminates between enantiomers of organic acids, some radioisotopically labeled, nonaromatic carboxylic acids were studied under varying mobile phase conditions. It was found for a series of N-alkanoyl-DL -[³H]leucines that the D -enantiomers were much more strongly retained and that the composition of the mobile phase could be adjusted to give very large (α > 20) enantiomeric separation factors. The elution order was consistent with what has been found earlier for other N-acyl derivatives as well as for N-arylcarbamoyl derivatives of simple aliphatic amino acids. A marked increase in the hydrophobic interaction of the D -enantiomers with the chiral phase was found upon a lowering of the mobile phase strength, conditions under which the L -form was only slightly influenced. These and other results are consistent with a chiral recognition model by which inclusion of the compound in a hydrophobic chiral cavity of BSA with simultaneous charge interaction is assumed to take place and whereby discrimination is determined by the steric bulk and orientation of the α-substituent. © 1993 Wiley-Liss, Inc.     

Manipulation of chiral resolution for isoxazoline-based IIb/IIIa receptor antagonists using various mobile phase additives in subcritical fluid chromatography

Subcritical fluid chromatography (SubFC) using a carbon dioxide-methanol mobile phase is used for the chiral resolution of IIb/IIIa receptor antagonist enantiomers. The chiral resolution of three analogs, each containing two chiral centers, is optimized using various mobile phase additives. The effects that acidic, basic, and neutral additives have on retention, efficiency, and resolution are examined. The additive that gives the best resolution was found to be dependent upon the functionality and charge of the chiral analyte. For charged analytes, additives that act as competing ions of the same charge as the chiral analyte dramatically improve efficiency and resolution. Resolution of neutral chiral analyte enantiomers is also greatly affected by the choice of mobile phase additive. Chirality 10:338–342, 1998. © 1998 Wiley-Liss, Inc.     

A New Chiral Residue Analysis Method for Triazole Fungicides in Water Using Dispersive Liquid‐Liquid Microextraction (DLLME)

A rapid, simple, reliable, and environment‐friendly method for the residue analysis of the enantiomers of four chiral fungicides including hexaconazole, triadimefon, tebuconazole, and penconazole in water samples was developed by dispersive liquid–liquid microextraction (DLLME) pretreatment followed by chiral high‐performance liquid chromatography (HPLC)‐DAD detection. The enantiomers were separated on a Chiralpak IC column by HPLC applying n‐hexane or petroleum ether as mobile phase and ethanol or isopropanol as modifier. The influences of mobile phase composition and temperature on the resolution were investigated and most of the enantiomers could be completely separated in 20 min under optimized conditions. The thermodynamic parameters indicated that the separation was enthalpy‐driven. The elution orders were detected by both circular dichroism detector (CD) and optical rotatory dispersion detector (ORD). Parameters affecting the DLLME performance for pretreatment of the chiral fungicides residue in water samples, such as the extraction and dispersive solvents and their volume, were studied and optimized. Under the optimum microextraction condition the enrichment factors were over 121 and the linearities were 30–1500 µg L^?1 with the correlation coefficients (R²) over 0.9988 and the recoveries were between 88.7% and 103.7% at the spiking levels of 0.5, 0.25, and 0.05 mg L^?1(for each enantiomer) with relative standard deviations varying from 1.38% to 6.70% (n = 6) The limits of detection (LODs) ranged from 8.5 to 29.0 µg L^?1(S/N = 3). Chirality 25:567‐574, 2013. © 2013 Wiley Periodicals, Inc.     

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose–based chiral stationary phase

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5‐dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n‐hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S‐enantiomer were found to be 0.45 and 1.5 μg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink′ _versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose–based chiral stationary phase     

Enantiomeric separation of type I and type II pyrethroid insecticides with different chiral stationary phases by reversed‐phase high‐performance liquid chromatography

The enantiomeric separation of type I (bifenthrin, BF) and type II (lambda‐cyhalothrin, LCT) pyrethroid insecticides on Lux Cellulose‐1, Lux Cellulose‐3, and Chiralpak IC chiral columns was investigated by reversed‐phase high‐performance liquid chromatography. Methanol/water or acetonitrile/water was used as mobile phase at a flow rate of 0.8 mL/min. The effects of chiral stationary phase, mobile phase composition, column temperature, and thermodynamic parameters on enantiomer separation were carefully studied. Bifenthrin got a partial separation on Lux Cellulose‐1 column and baseline separation on Lux Cellulose‐3 column, while LCT enantiomers could be completely separated on both Lux Cellulose‐1 and Lux Cellulose‐3 columns. Chiralpak IC provided no separation ability for both BF and LCT. Retention factor (k) and selectivity factor (α) decreased with the column temperature increasing from 10°C to 40°C for both BF and LCT enantiomers. Thermodynamic parameters including ?H and ?S were also calculated, and the maximum R_s were not always obtained at lowest temperature. Furthermore, the quantitative analysis methods for BF and LCT enantiomers in soil and water were also established. Such results provide a new approach for pyrethroid separation under reversed‐phase condition and contribute to environmental risk assessment of pyrethroids at enantiomer level.     

Enantioseparation of chiral perfluorooctane sulfonate (PFOS) by supercritical fluid chromatography (SFC): Effects of the chromatographic conditions and separation mechanism

Perfluorooctane sulfonate (PFOS) is one of the most frequently detected perfluoroalkyl substances in environmental and human samples. Previous studies have shown that nonracemic PFOS in biological samples can be used as a marker of PFOS exposure sources. In recent years, supercritical fluid chromatography (SFC) has emerged as a powerful method to separate chiral compounds. In this study, a method of perfluoro‐1‐methylheptane sulfonate (1 m‐PFOS) enantioseparation by SFC was established. The optimal separation was obtained using a Chiralpak QN‐AX column with CO₂/2‐propanol (70/30, v/v) as the mobile phase with a flow rate of 1 mL/min, column temperature was 32°C, and BPR pressure was 1800 psi. The resolution (Rs) and retention time were 0.88 and 130 minutes, respectively. This method is more economic and greener than HPLC. Modifier pH and column temperature were determined to be significant factors of SFC chiral separation. Modifier pH is negatively correlated with the retention factors and Rs. Adsorption thermodynamics were used to explain the influence of temperature change, and it was concluded that the transfer of two enantiomers from the mobile phase to the stationary phase is enthalpy‐driven. Enantioseparation of 1 m‐PFOS by SFC follows the same rules of ion exchange as those for the chiral separation by HPLC.     

Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl‐Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation

Rapid and simple isocratic high‐performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of α_l‐acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (?) and the stereochemical resolution factor (R_s) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S‐hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1–10 µg mL^‐1 and 0.5–5 µg mL^‐1 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process. Chirality 28:49–57, 2016. © 2015 Wiley Periodicals, Inc.     

High-performance liquid chromatographic separation of a complex mixture of diuretics using a micellar mobile phase of sodium dodecyl sulphate: Application to human urine samples

A systematic optimization of the HPLC separation of a complex mixture containing 19 diuretics by micellar liquid chromatography using sodium dodecyl sulphate (SDS), a Hypersil (150 mm×3.0 mm I.D., 5 μm) C₁₈ column, a flow-rate of 0.5 ml min⁻¹ and UV absorbance detection has been carried out. Several mobile phases consisting of SDS and organic modifiers such as acetonitrile, tetrahydrofuran, propanol, butanol or pentanol, and the pH adjusted to 3.2, were tested. The effect of the organic modifier and SDS concentration on the retention behavior and separation of the diuretics was investigated. A mobile phase containing 40 mM SDS and 4% tetrahydrofuran was finally selected. Under these conditions, 14 out of 19 diuretics were separated in about 31 min. A bivariant optimization method for the mobile phase SDS–tetrahydrofuran corroborated the above results. The effect of temperature on the retention was also studied, and 50°C was selected. The optimized method was applied to human urine samples of subjects administered Diurex^® (tablets containing 20 mg of the active ingredient xipamide) without sample preparation.     

Solvent selectivity in chiral chromatography using a β-cyclodextrin-bonded phase

A β-cyclodextrin-bonded phase has been used to investigate the separation of the enantiomers of atenolol, oxprenolol, celiprolol, tertatolol, terbutaline, fluoxetine, norfluoxetine, and zopiclone, focusing on the importance of solvent selectivity. With cyclodextrin (CD)-bonded phases, chiral discrimination occurs because the two enantiomers of a racemate form inclusion complexes of different strengths within the CD cavity. The organic modifier molecules tend to compete with solutes for a definite number of adsorption sites on the stationary phase. Moreover, the ternary complex formation may play an important role in chiral recognition. In this study, it was of interest to estimate the influence of mobile phase modifiers with respect to solvent type (i.e., ACN, MeOH, EtOH, THF, i-PrOH, PrOH and t-BuOH), size and shape, and concentration. Solvent selectivity has been investigated by using different organic modifiers in mobile phases with the same polarity, and relationships were established between the logarithm of solvent partition coefficient (log P_s) and the three most important chromatographic parameters: retention time (t), resolution (R), and enantioselectivity (α). Thus, it seems that the hydrophobicity of the organic modifier becomes one of the dominant factors affecting the inclusion process phenomena. Further, the apparent partition coefficients of the compounds under study have been determined and a comparison has been attempted regarding the degree of their enantiomeric resolution. © 1995 Wiley-Liss, Inc.     

High-performance liquid chromatography separation and quantitation of ofloxacin enantiomers in rat microsomes

A sensitive, simple and accurate method for determination of enantiomers of ofloxacin in microsomal incubates was developed by chiral ligand-exchange RP-HPLC with fluorescence detection to examine stereoselective metabolism of ofloxacin in the glucuronidation process. The C₁₈ stationary phase was used as analytical column. The solution of chiral mobile phase additive was made up of 6 mM l-phenylalamine mixed with 3 mM CuSO₄ in water. Mobile phase consisted of the solution of chiral mobile phase additive–methanol (86:14).The fluorescence detector was operated at λ_ex 330 nm and λ_em 505 nm. The flow-rate of mobile phase was set at 1.0 ml/min. The achiral ODS column offers good separation of the two enantiomers in less than 25 min. The recovery of the assay was 97.9±6.1% (n=10) for S-ofloxacin and 99.6±6.0% (n=10) for R-ofloxacin. The method provides a high sensitivity and good precision (RSD<10%). The LOD was 0.6 μM for both enantiomers and the LOQ was 5.70±0.45 μM (n=8) for S-ofloxacin and 5.66±0.47 μM (n=8) for R-ofloxacin. The standard curves showed excellent linearity over the concentration range 5.5–2078 μM for S-(−)-ofloxacin and R-(+)-ofloxacin. The enantioselective method developed has been applied to determine the stereoselectivity of glucuronidation metabolism of ofloxacin optical isomers in rat liver microsomes.     

Direct chiral separations of the enantiomers of phenylpyrazole pesticides and the metabolites by HPLC

The enantiomeric separation of the enantiomers of three phenylpyrazole pesticides (fipronil, flufiprole, ethiprole) and two fipronil metabolites (amide‐fipronil and acid‐fipronil) were investigated by high‐performance liquid chromatography (HPLC) with a CHIRALPAK^® IB chiral column. The mobile phase was n‐ hexane or petroleum ether with 2‐propanol or ethanol as modifier at a flow rate of 1.0 mL/min. The influences of mobile phase composition and column temperature between 15 and 35°C on the separations were studied. All the analytes except ethiprole obtained complete enantiomeric separation after chromatographic condition optimization. Fipronil, flufiprole, amide‐fipronil, and acid‐fipronil obtained complete separation with the best resolution factors of 2.40, 3.40, 1.67, and 16.82, respectively, but ethiprole showed no enantioselectivity under the optimized conditions. In general, n‐ hexane with 2‐propanol gave better separations in most cases. The results showed decreasing temperature and content of modifier in the mobile phase resulted in better separation and longer analysis time as well. The thermodynamic parameters calculated according to linear the Van't Hoff equation indicated the chiral separations in the study were enthalpy‐driven. Fipronil and its two chiral hydrolyzed metabolites obtained baseline separation simultaneously under optimized conditions.     

Effect of Chromatographic Conditions on Enantioseparation of Bovine Serum Albumin Chiral Stationary Phase in HPLC and Thermodynamic Studies

Twelve chiral compounds were enantiomerically resolved on bovine serum albumin chiral stationary phase (BSA‐CSP) by high‐performance liquid chromatography (HPLC) in reversed‐phase modes. Chromatographic conditions such as mobile phase pH, the percentage of organic modifier, and concentration of analyte were optimized for separation of enantiomers. For N‐(2, 4‐dinitrophenyl)‐serine (DNP‐ser), the retention factors (k) greatly increase from 0.81 to 6.23 as the pH decreasing from 7.21 to 5.14, and the resolution factor (R_s) exhibited a similar increasing trend (from 0 to 1.34). More interestingly, the retention factors for N‐(2, 4‐dinitrophenyl)‐proline (DNP‐pro) decrease along with increasing 1‐propanol in mobile phase (3%, 5%, 7% and 9% by volume), whereas the resolution factor shows an upward trend (from 0.96 to 2.04). Moreover, chiral recognition mechanisms for chiral analytes were further investigated through thermodynamic methods. Chirality 25:487–492, 2013. © 2013 Wiley Periodicals, Inc.