Chiralpak IA Research Articles

Comparative studies of immobilized polysaccharide derivatives chiral stationary phases for enantioseparation of furanocoumarins and dihydroflavones and discussion on chiral recognition mechanism.

Enantiomeric separation of furanocoumarins and dihydroflavones compounds were systematically studied in the normal-phase mode using four different polysaccharide-type chiral stationary phases, namely, Chiralpak IA, Chiralpak IC, Chiralpak IG, and Chiralpak IK-3 by high-performance liquid chromatography. The effect of alcohol modifiers and alcohol content on enantiomeric separation was evaluated for the separation of furanocoumarins and dihydroflavones. All the eight compounds have achieved baseline separation with the resolutions ranging between 1.52 and 23.11. For a better insight into the enantiorecognition mechanisms, thermodynamic analysis was carried out. The mechanisms of chiral recognition have been discussed. Among four chiral columns, Chiralpak IG exhibited the most universal and the best enantioseparation ability toward furanocoumarins and dihydroflavones when used n-hexane-isopropanol and n-hexane-ethanol as mobile phase, respectively. The steric hindrance, hydrogen bonding, and π-π interaction played major roles in chiral recognition on Chiralpak IG. By comparing four chiral columns, this work systematically analyzed the separation methods of furanocoumarins and dihydroflavones for the first time and reported some active chiral ingredients of traditional Chinese medicine that have never been separated, which provided a further insight into the enantioseparation of furanocoumarins and dihydroflavones on chiral stationary phases.

Effect of mobile-phase modifiers on the enantioselective retention behavior of methyl mandelate with an amylose 3,5-dimethylphenylcarbamate chiral stationary phase under reversed-phase conditions.

In this study, methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone, and tert-butanol were used as organic modifiers in reversed-phase mode chiral liquid-chromatography to systematically investigate the effects of mobile phase components on the enantioselective retention behavior of methyl mandelate with immobilized amylose 3,5-dimethylphenylcarbamate-based sorbent called Chiralpak IA. A two-site enantioselective model was used to obtain information on the recognition mechanisms by observing the dependence of the enantioselectivity and retention factor difference on the modifier content. Similar enantioselective retention behaviors were observed for all modifiers, and characteristic modifier concentration points (PL , PM , and PH ) were identified. At modifier concentrations up to PM , the weakened hydrophobic environment resulted in polymer structural relaxation, which changed the recognition mechanisms. By contrast, at concentrations beyond PH , considerably different enantioselectivity behaviors were observed, indicating that the existence of dipole-dipole interaction, which was stronger at higher modifier concentrations, contributed to the retention mechanisms. The concentrations at which these characteristic points occurred were dependent on the carbon number of the modifier molecule. Modifiers with more carbon numbers facilitated the transition in the enantioselective behaviors. These results demonstrated that the proposed method can provide a physically consistent quantitative description of enantioselective retention behavior in reversed-phase mode.

Chiral separation of conazole pesticides using supercritical fluid chromatography

ABSTRACT Supercritical fluid chromatographic methods for enantiomer separation of six conazole-type antifungal pesticides (bromuconazole, cyproconazole, prothioconazole, tebuconazole, ipconazole, triticonazole) are presented in this work. Screening tests on six different chiral analytical columns (Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralpak AS, Chiracel OJ and Kromasil Cellucoat) were carried out using methanol as organic modifier. Cellulose-based stationary phases were found to be better for separation of each tested conazole, than the amylose-based columns. The influence of the most commonly used co-solvents (methanol, ethanol and 2-propanol) on the separation was also studied on selected columns. Suitable analytical methods were found for enantiomer separations for all studied conazoles; moreover, the methods are good starting points to semi-preparative separation. The importance of sample solvent selection in the semi-preparative separation of triticonazole isomers was proved. Acetonitrile as sample solvent resulted in a narrow, nice peaks compared to those obtained with methanol or dichloromethane. Twenty milligrams of each enantiomer of triticonazole was collected for ecotoxicity testing using the analytical size column.

Rapid chiral separation of potential antibiotics using supercritical fluid chromatography.

WHO is searching most active antibiotics due to the bacterial resistance problem. The activities of the racemic antibiotics may be augment by preparing optically active antibiotics by the chiral separation. Chiral separation of potential antibiotics such as cefotaxime and ofloxacin was studied using amylose-based packing chiral stationary phases (CSPs) such as Chiralpak IA and Chiralpak IG. Supercritical fluid chromatography (SFC) was employed to carry out this study. Both immobilized CSPs such as Chiralpak IA and IG have sown remarkable selectivity for the reported drugs by using SFC. The values of retention factor (k) for ofloxacin enantiomers were 9.63 and 11.81, followed by 2.94 and 5.96 for cefotaxime enantiomers. The values of separation factor (α) for both the reported drugs were 1.22 and 2.03, respectively Similarly, the values of resolution factor (Rs) for both the enantio-selective drugs were 1.49 and 2.06, separately and respectively. The chiral recognition mechanism was developed and it was observed that the π-π interactions are playing a major role. The developed method is effective, reproducible, eco-friendly, and may be used to discriminate the enantiomers of the reported drugs in any sample.

Enantiomeric Separation of (R)-5-(1-Hydroxyethyl)-2-(pyridine- 2-yl)-[1, 3, 4]-thiadiazole, an Intermediate of Nafithromycin on Immobilized Amylose Based Stationary Phase

Chiral high performance liquid chromatography method for the separation of (R)-5-(1- Hydroxyethyl)-2-(pyridine-2-yl)-[1, 3, 4]-thiadiazole (R-alcohol) an intermediate of new antibiotic Nafithromycin (NFT) has been developed and validated. NFT is a ketolide class of antibiotic currently under Phase III clinical trials in India to treat community acquired bacterial pneumonia. Chiral separation of the enantiomers with resolution >2 was accomplished on Chiralpak IA column using a mixture of n-hexane and isopropanol (IPA) in the ratio of (90:10; v/v) as an elution solvent, run at a flow rate of 1.2 mL.min-1. The column was maintained at 25°C. The analytes were detected at 254 nm. The developed method was extensively validated as per the international council for harmonisation of technical requirements for pharmaceuticals for human use guideline. Sensitivity of the method was adequate to control the passage of unwanted enantiomer to next step with limit of quantification (LOQ) of the undesired enantiomer (S)-5-(1-Hydroxyethyl)-2-(pyridine- 2-yl)-[1, 3, 4]-thiadiazole (S-alcohol) of 0.37 µgmL-1. Mean recovery of the S-alcohol was 98.9±8.7%. During development influence of column oven temperature on the chiral separation was assessed.

Application of Polysaccharide-Based Chiral High-Performance Liquid Chromatography Columns for the Separation of Regio-, E/Z-, and Enantio-Isomeric Mixtures of Allylic Compounds.

Daicel Chiralpak IA, IB, and IC, which are the polysaccharide-based chiral stationary phase (CSP) columns for high-performance liquid chromatography (HPLC), were applied in the separation of the non-enantiomeric isomeric mixtures obtained by the various allylation reactions and were highly effective in separating the regio- and (E)/(Z)-isomers in the allylation products. Due to the close structural similarity of the isomeric allylic compounds in the reaction mixtures, separations of the isomers are laborious and could not be accomplished by the conventional methods such as silica gel column chromatography, silica gel HPLC, preparative GPC, distillation, and so forth. This study has shown potential advantages of using the polysaccharide-based CSP columns in the separation of not only enantiomeric but also non-enantiomeric isomeric mixtures.

A comparative study of chiral separation of proton pump inhibitors by supercritical fluid chromatography and high-performance liquid chromatography.

A comparative study of chiral separation of pantoprazole and rabeprazole is carried out using supercritical fluid chromatography and high-performance liquid chromatography. The columns used were Chiralpak IA and Chiralpak IE. The best mobile phase in supercritical fluid chromatography was carbon dioxide-0.2% triethylamine in methanol (60:40) and 0.1% triethylamine in n-hexane-ethanol (50:50) in high-performance liquid chromatography. For supercritical fluid chromatography, values of the retention factor of pantoprazole enantiomers were 3.97 and 4.88. These values for rabeprazole enantiomers were 6.10 and 7.52. The values of separation and resolution factor for pantoprazole and rabeprazole were 1.23 and 1.23 and 2.20 and 3.36, respectively. Similarly, for high-performance liquid chromatography, the values of retention factor for enantiomers of pantoprazole were 4.02 and 7.32. These values for rabeprazole enantiomers were 5.32 and 7.88, respectively. The values of separation and resolution factor for pantoprazole and rabeprazole were 1.82 and 1.48 and 9.22 and 6.58, respectively. A comparison was carried out, which confirmed supercritical fluid chromatography as the best method due to its fastness, eco-friendly, and inexpensiveness. The reported methods are effective, efficient, and reproducible and may be used to separate and identify pantoprazole and rabeprazole in any unknown samples.

Development and Validation of Teneligliptin Stereoisomers by HPLC Using Cellulose Based Immobilized Polysaccharide Chiral Stationary Phase

Background: There is no single chiral method for the quantitation of teneligliptin stereoisomers by high performance liquid chromatography (HPLC). Hence, there is a need for the quantification of teneligliptin (TNGP) and its stereoisomers. Objective: The main aim of the research work is to develop a novel simple, selective, precise and accurate HPLC method for the separation of TNGP and its stereoisomers. Methods: Different screening trials were executed by changing the mobile phase compositions to normal phase and polar mode and also by utilizing the different immobilized polysaccharide chiral columns like CHIRALPAK IA, IC, ID, IE, IF and IG. All the stereoisomers were eluted with high resolution, on CHIRALPAK IC-3 (4.6×250 mm), 3 μm chiral stationary phase (CSP) with a flow rate of 0.7 mL/min. The chromatographic system was processed with isocratic mode comprising ethanol: acetonitrile: ethanolamine in the proportion of 90:10:0.1% v/v/v with a column oven temperature of 15°C and detection wavelength of 250 nm. Results: The limit of detection (LOD) and limit of quantification (LOQ) values of TNGP(API), R,S-isomer, S,R-isomer and R,R-isomer were found to be 0.036/0.11, 0.029/0.09, 0.038/0.011 and 0.020/0.06 μg/mL, respectively. The method was found to be precise, accurate and linear (R2 > 0.999). Conclusion: The developed method was successfully applied for the quantification of bulk drug without any interference with the extraneous components. Hence, the method can be utilized successfully in the pharmaceutical organizations for the separation and quantification of TNGP isomers.

Direct and simultaneous analysis of lipase-catalyzed hydrolysis of high-oleic oil model by chiral stationary phase HPLC-ELSD

A novel HPLC-based method for direct separation of trioleoylglycerol (TOG), a major component in high-oleic oils, and its seven hydrolysis products (i.e., oleic acid, monooleoylglycerol (MOG) and dioleoylglycerol (DOG) isomers) was established using a chiral stationary phase column, Chiralpak IA. Within 20 min, all species including enantiomeric MOG (1-sn-MOG and 3-sn-MOG) and DOG (1,2-sn-DOG and 2,3-sn-DOG) were baseline-resolved with resolution factors over 1.5 between adjacent peaks. The established method was used for investigating the integral stereoselectivity, which is the selectivity concerning all hydrolysis steps, of lipase from Pseudomonas fluorescens (PFL) with TOG as substrate. The time-course of DOGs and MOGs indicated that PFL had selectivity for TOG hydrolysis in the order of sn-1, sn-2, and sn-3 position, while it preferred to hydrolyze 2,3-sn-DOG over 1,2-sn-DOG. Being rapid and accurate to evaluate integral stereoselectivity, this method could promote the development and application of lipases with target stereochemistry in the food industry.

Enantiomer Separation of Chiral Amines and Amino Acid Esters on Polysaccharide Phenylcarbamates Derived Chiral Stationary Phases

We have developed an efficient chiral HPLC method for enantiomeric resolution of some chiral amines and α- amino acid esters as nitrobenzoxadiazole (NBD) derivatives on amylose and cellulose phenylcarbamates derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence (FL) detection. The observed enantioselectivities were affected by the type of the analyte (amines or α-amino acid esters), backbone structure (cellulose or amylose) of the CSP used, and substituents of phenyl moiety on the chiral selector of CSP. Further, for same chiral selectors, coated type CSPs showed better enantiomeric separation than those observed for the covalently bonded type CSPs. In general, for the enantiomeric resolution of chiral amines, Chiralpak IE and Chiralcel OD-H columns showed the highest enantioselectivities. However, for resolution of α-amino acid esters, the performances of Chiralpak IA and Chiralpak AD-H (or Lux Amylose-1) columns were superior to those of other CSPs used. In general, the degrees of enantioselectivity for α-amino acid ethyl esters are greater than those for chiral amines. The developed analytical method was applied to determine the enantiomeric purity of two commercially available chiral amine and α-amino acid ester; the enantiomeric impurities were observed to be 0.35 and 0.05% for (S)-α-methylbenzylamine and (S)-leucine ethyl ester, respectively. The validation data obtained from intra- and inter-day accuracy assays for (S)-3,3-dimethyl-2-butylamine and (S)-leucine ethyl ester were 99.92-101.46 and 100.61-101.78%; and 100.05-102.12 and 101.25-102.73%, respectively; the intra- and interday precision assays in terms of relative standard deviation (% RSD) were found to be 0.96-1.57 and 0.81- 1.91%; and 0.24-1.01% and 0.65-1.35%, respectively. Owing to the high accuracy and precision results, the developed analytical method can prove to be useful for the enantiomeric separation of various chiral amines and amino acid esters as NBD derivatives using polysaccharide derived CSPs.

Enantiomeric Separation and Thermodynamic Investigation of (R)-N-Tert-Butoxy Carbonyl-Piperidine-3-Carboxylic Acid Hydrazide, a Key Starting Material of Zidebactam.

A new selective, accurate and precise chiral high-performance liquid chromatography method for the separation of (R)-N-tert-butoxy carbonyl-piperidine-3-carboxylic acid hydrazide (RE) and its enantiomer was developed. RE is a key starting material of novel β-lactam enhancer drug Zidebactam. Chiral resolution of more than 10 was achieved on Chiralpak IA column using mobile phase consisting of n-hexane, ethanol in the ratio of 70:30, v/v. The flow rate of the mobile phase was 1.0mLmin-1 and the column oven temperature was 30°C. Detection was carried out at 225nm. The developed method was validated as per the International Conference on Harmonization guideline. Limit of detection and limit of quantification of the enantiomeric impurity (S)-N-tert-butoxy carbonyl-piperidine-3-carboxylic acid hydrazide (SE) was 2.5 and 7.5μgmL-1, respectively. Mean recovery of the SE was 96.83±1.4%. The effect of thermodynamic parameters on the chiral separation was evaluated.

Enantioseparation of lysine derivatives on amylose tris (3, 5-dimethylphenylcarbamate) as chiral stationary phase with high separation factor

Enantioseparation of lysine derivatives by chiral high-performance liquid chromatography (HPLC) was accomplished using two chiral stationary phases (CSPs, Chiralpak IA and Chiralpak IC) based on polysaccharides under normal phase (NP) conditions. All analytes were completely separated. The impacts of polar modifiers, analyte structure and column temperature on chiral separation were discussed. Moreover, the relationship between structure and retention was investigated. The van't Hoff equation was employed to evaluate the thermodynamic parameters of the chiral separation process. The data suggest that the chiral separation process was enthalpy-driven. Surprisingly, two uncommon phenomena were observed: (1) high separation factors on Chiralpak IA and (2) different binding mechanisms with CSP and the two enantiomers.

Enantioselective LC-MS/MS method for the determination of cloperastine enantiomers in rat plasma and its pharmacokinetic application.

Cloperastine is a central antitussive used to reduce the frequency and intensity of coughing on a short-term basis. In this study, a reliable chiral LC-MS/MS technology has been developed for the quantification of cloperastine enantiomers in the rat plasma. Carbinoxamine was selected as the internal standard. The enantioseparation of cloperastine was performed on a Chiralpak IA column with a mobile phase composed of acetonitrile-water-ammonium hydroxide (80:20:0.1, v/v/v) at a flow rate of 0.6 mL/min. Cloperastine enantiomers were detected by mass spectrometry in multiple reaction monitoring mode with a positive electrospray ionization source. The method was validated over the linear concentration range of 0.05 to 10.0 ng/mL (5.0 × 10-4 ng to 0.10 ng) for both enantiomers. The lower limit of quantification (LLOQ) for each analyte was determined as 0.05 ng/mL. The relative standard deviations (RSDs) of intraday and interday precision was less than 13.9%, and the relative error (RE) of accuracy ranged from -5.4% to 6.1%, which were within the acceptance criteria. Finally, an application to the stereoselective pharmacokinetics of cloperastine in rats was successfully realized in our assay. The developed method on a commercially available Chiralpak IA column under isocratic mobile phase is advantageous to analyze cloperastine enantiomers in plasma samples collected for enantioselective metabolism or drug interaction studies.

Rapid and efficient chiral method development for lamivudine and tenofovir disoproxil fumarate fixed dose combination using ultra-high performance supercritical fluid chromatography: A design of experiment approach

Fixed dose combination (FDC) of tenofovir disoproxil fumarate (TDF) and lamivudine (3TC) is one of the most preferred FDC for the treatment of acquired immunodeficiency syndrome (AIDS)/human immunodeficiency virus (HIV) infection. To the best of authors’ knowledge there are no reported methods for chiral purity estimation of both drugs simultaneously from a FDC. The current study was focused on the development of a single chiral method uisng supercritical fluid chromatography (SFC) for separation of stereoisomers of TDF and 3TC combination employing design of experiment (DoE) approach. Method development was planned in three steps by using different experimental designs for each step. I-optimal, Taguchi orthogonal array and face-centred central composite designs (CCD) were employed for primary parameter selection, secondary parameter screening and final method optimization, respectively. All six stereoisomers were separated in a 10 minute run on Chiralpak IA column with carbon di-oxide /methanol (containing 0.5 % v/v n-butylamine) as mobile phase at 1.5 mL/min in gradient mode. The optimized method was verified for performance through establishing specificity, precision, linearity, accuracy, limit of quantification, and solution stability. Resolution between each isomeric pair was more than 1.5. The method was found to be linear from 1.5 µg/mL to 7.5 µg/mL for 3TC and 7.5 µg/mL to 37.5 µg/mL for TDF stereoisomers. The R2 values for all the linearity curves for undesired isomers were greater than 0.995. The method proved to be rapid, reproducible and efficient to quantify stereoisomers of both drugs in a single run.

Effects of the Sorbent Backbone and Side Chain on Retention Mechanisms Using Immobilized Polysaccharide-Based Stationary Phases in Normal Phase Mode

The effects of the side chain and polymer backbone on the retention mechanisms of four polysaccharide-based sorbents, namely amylose 3,5-dimethylphenylcarbamate (Chiralpak IA), cellulose 3,5-dimethylphenylcarbamate (Chiralpak IB), amylose 3,5-dichlorophenylcarbamate (Chiralpak IE), and cellulose 3,5-dichlorophenylcarbamate (Chiralpak IC), were investigated using simple achiral molecules with distinct functional groups as probes. The thermodynamic properties of solute retention behaviors were investigated using retention factor data, van’t Hoff plots, adsorption isotherms, and retention models. Frontal analysis was used to obtain the adsorption isotherms of acetone (AC) and isopropanol (IPA). The results revealed that the adsorptions of AC on the four sorbents followed Langmuir isotherms, whereas heterogeneous adsorption mechanisms more accurately fit the adsorptions of IPA. For solutes associated with hydrogen-bonding groups, the retention factors and enthalpy changes suggest that cellulose sorbents may have more binding sites available than the amylose sorbents. The van’t Hoff analysis indicated that sorbents with Cl-substituted phenyl groups enhanced the binding strength of sorbent NH groups with the solutes. For aromatic solutes, larger retention factors and stronger adsorption energies were found for the cellulose sorbents. The results suggested that side chains with Cl-substituted phenyl groups weakened the π interactions. The inferences obtained from these sorbents were used for the interpretation of the chiral recognition mechanisms of pantolactone enantiomers.

Separation and quantitation of notopterol enantiomers in notopterygii rhizoma et radix using solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

In this study, a specific, sensitive, and reliable high performance liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantitative determination of notopterol enantiomers in Notopterygii Rhizoma et Radix. Solid-phase extraction was used for the extraction of notopterol enantiomers from Notopterygii Rhizoma et Radix. Enantiomeric separation of notopterol was achieved on a Chiralpak IA column with the mobile phase consisting of acetonitrile-water (50:50, v/v) at a flow rate of 0.6 mL/min. Quantification was performed on a triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source in the positive ionization and multiple reaction monitoring mode. The lower limit of quantification and lower limit of detection were 0.09 and 0.04 mg/g for each enantiomer in NRR samples. And each enantiomer showed good linearity (R2≥0.999) in the range of 0.09–9.55 mg/g. The precision, repeatability, and stability were all within satisfaction. The average recoveries of (-)-notopterol and (+)-notopterol were demonstrated to be 99.3 % and 101.1 %, respectively, with the relative standard deviations less than 5.0 %. Finally, the validated method was successfully applied to the quantification of notopterol enantiomers in Notopterygii Rhizoma et Radix from different sources.

Development and Validation of an New High Performance Liquid Chromatography Method for Determination of Apixaban Isomers

The purpose of this research study is to develop a novel, simple, precise, accurate and economical method for determination of Apixaban API isomers. Apixaban API has three isomer as ortho, meta and para. In reverse phase and normal phase chromatography it was very difficult to separate these isomer; hence new chiral technique was adopted. This chromatographic method was developed on chiralpak IA column (250×4.6×5µm) with isocritic technique. The detection of isomeric impurities were observed at wavelength 290nm. This analytical method was validated as per ICH guideline and regression analysis showed R value (correlation coefficient) > 0.999 for Apixaban API and its isomeric impurities. A solution of Apixaban in dichloromethane was found stable up to 48 hrs. The degradation study was done within the given guidelines prescribed by ICH. The method is validated for Linearity, Accuracy and Precision.

Enantiomeric separation and enantioselective determination of some representive non-steroidal anti-inflammatory drug enantiomers in fish tissues by using chiral liquid chromatography coupled with tandem mass spectrometry

In this work, enantiomeric separation of nine non-steroidal anti-inflammatory drugs including ibuprofen, indoprofen, pranoprofen, flurbiprofen, ketoprofen, carprofen, naproxen, loxoprofen and etodolac was investigated by HPLC combining with four polysaccharide based chiral stationary phases (Chiralpak IA, Chiralpak IB, Chiralpak IC and Chiralpak ID). The effect of various types of the mobile phase, acidic additive types, as well as mobile phase pH on enantiomeric separation was evaluated and optimized in detail. Chiralpak ID showed the best enantioselectivity towards seven of the above nine chiral drugs. Additionally, a sensitive and simple method for simultaneous enantioselective determination of indoprofen, flurbiprofen, carprofen and naproxen enantiomers in fish tissues based on C18 solid phase extraction (SPE) with chiral liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) on Chiralpak ID was established. Method validation showed that good linearity (R2 > 0.99) for each enantiomer with the limit of quantification in the range of 2–10 ng g−1 was obtained. Recoveries of the studied profens enantiomers in fish tissues were 82.6% - 106.7% with RSD less than 8.2% at three concentration levels. The proposed method was applied to the real fish samples analysis, providing a reference to fully evaluate the risks of these drug enantiomers in food or environmental samples.

Enantiomeric Separation and Thermodynamic investigation of (R)-5-[1-(4-Nitrobenzylsulfonyloxy)-ethyl]-5-(pyridine-2-yl)- [1,3,4]-thiadiazole, a Key Intermediate of Nafithromycin

A selective and precise chiral high performance liquid chromatography method for the separation of (R)-5-[1-(4-Nitrobenzylsulfonyloxy)-ethyl]-5-(pyridine-2-yl)-[1, 3, 4]-thiadiazole (RE) has been developed and validated. It is an intermediate of a novel antibiotic nafithromycin. Nafithromycin is a ketolide class antibiotic being developed to treat community acquired bacterial pneumonia. Enantiomeric separation was achieved on Chiralpak IA column using mobile phase consisting of n-hexane and ethanol in the ratio of (15:85; v/v) with the resolution of more than 3. Separation was achieved at flow rate of 1.0 mL min−1 and column temperature 30°C. The eluent was monitored at 254 nm. The developed method was validated as per the guideline. The method was found to be selective, precise, accurate and linear. Limit of detection and limit of quantification of the undesired enantiomer (S)-5-[1-(4-Nitrobenzylsulfonyloxy)-ethyl]-5-(pyridine-2-yl)- [1, 3, 4]-thiadiazole (SE) was 0.11 and 0.35 μgmL−1 respectively. Recovery of the SE was found to be in the range of 104.52 % and 105.83 %. The impact of thermodynamic parameters on the chiral separation was evaluated.

Liquid chromatographic chiral recognition of phytoalexins on immobilized polysaccharides chiral stationary phases. Unusual temperature behavior

Three immobilized polysaccharide chiral stationary phases, Chiralpak IA, Chiralpak IB and Chiralpak IC, were used for the study of enantioseparation of 36 derivatives of natural indole phytoalexins, in most cases bioactive, including racemic spirobrassinin, 1-methoxyspirobrassinin and 1-methoxyspirobrassinol methyl ether. Almost all analytes were baseline resolved at least on two different polysaccharide columns in normal phase mode. The effects of mobile phase composition, the analyte structure and the column temperature on the retention and enantioseparation were investigated. Evaluation of the corresponding thermodynamic parameters using van´t Hoff plots (ln k versus 1/T) in the temperature range -15 to 50 °C indicated that separations were enthalpy controlled in most cases, but some entropy controlled separations were also observed. Moreover, unusual phenomenon, an increase retention with increasing temperature accompanied with increased resolution was observed on the Chiralpak IC column. The elution order of enantiomers was determined in some cases and reversed elution order was also observed.