Chiralpak AD Column Research Articles

Enantiomeric Separation and Degradation of Benoxacor Enantiomers in Horticultural Soil by Normal-Phase and Reversed-Phase High Performance Liquid Chromatography.

The separation of benoxacor enantiomers on six commercial chiral columns was investigated by high-performance liquid chromatography (HPLC) under normal-phase and reversed-phase conditions. The mobile phases included hexane/ethanol, hexane/isopropanol, acetonitrile/water, and methanol/water. The effects of the chiral stationary phases (CSPs), temperature, and mobile phase composition and ratio on the separation of benoxacor enantiomers were examined. Under normal-phase conditions, the two benoxacor enantiomers were completely separated on Chiralpak AD, Chiralpak IC, Lux Cellulose-1, and Lux Cellulose-3 columns and partially separated on a Lux Cellulose-2 column. Under reversed-phase conditions, benoxacor enantiomers were completely separated on a Lux Cellulose-3 column and partially separated on Chiralpak IC and Lux Cellulose-1 columns. Normal-phase HPLC performed better than reversed-phase HPLC for the separation of benoxacor enantiomers. As the column temperature increased from 10 °C to 4 °C, the enthalpy (ΔH) and entropy (ΔS) results indicated that the resolution was strongly affected by the temperature and that the lowest temperature did not always produce the best resolution. An optimized separation method on the Lux Cellulose-3 column was used to investigate the stability of benoxacor enantiomers in solvents and the degradation of benoxacor enantiomers in three types of horticultural soil. Benoxacor enantiomers were stable, and degradation or racemization were not observed in methanol, ethanol, isopropanol, acetonitrile, hexane, or water (pH = 4.0, 7.0, and 9.0). In three horticultural soils, the degradation rate of S-benoxacor was faster than that of R-benoxacor, resulting in soil enrichment with R-benoxacor. The results of this study will help to improve the risk assessment of enantiomer levels of benoxacor in the environment.

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (Rs > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.

Chiral and achiral liquid chromatographic separation of palonosetron hydrochloride and its related impurities utilizing two different stationary phases: Polysaccharide and alkyl amide columns

In this work, two simple, sensitive and rapid liquid chromatographic (LC) methods were assessed; for the determination of palonosetron hydrochloride and its related impurities. Method A comprised stress study, chiral separation and quantification of palonosetron (PALO) and its six related impurities. The successful separation of PALO and its six related impurities was achieved using Chiralpak AD column (250 mm × 4.6 mm, 10 µm), in a polar organic (PO) mode. PALO was eluted using gradient mode, consisting of the mobile phase A: 0.1% diethylamine in acetonitrile, the mobile phase B: isopropanol. Method B is a green UPLC method to estimate PALO in the presence of three of its related impurities. Effective separation was carried out on Bonus RP column (100 mm × 4.6 mm, 1.8 µm), based on purified water- ethanol- triethylamine (75: 25: 0.1, v/v), pH 4.0 adjusted with 1 M acetic acid as the mobile phase. The validated methods showed excellent linearity (r2˃ 0.999) over the concentration ranges of 5.0–100.0 μg mL−1 and 0.1–10.0 μg mL−1 for method A and B, respectively. The values of the detection limit (LOD) and quantification limit (LOQ) of PALO and its six related impurities were found in the range of 0.014–0.032 μg mL−1 and 0.043–0.097 μg mL−1, respectively for method A, while for method B; they were found to be in the range of 0.08 and 0.1 μg mL−1, respectively. The optimized methods have been validated and found to be appropriate for quality control of PALO in its pharmaceutical dosage form.

Enantiomeric quality control of R-Tofisopam by HPLC using polysaccharide-type chiral stationary phases in polar organic mode.

A novel, fast and economic chiral HPLC method was developed and validated for the resolution of the four isomers of tofisopam. The separation capacity of eleven different chiral columns: six polysaccharide-type including three amylose-based (Chiralpak AD, Chiralpak AD-RH and Chiralpak AS) and three cellulose-based (Chiralcel OD, Chiralcel OJ and Lux Cellulose-4); three cyclodextrin- (Quest-BC, Quest-C2 and Quest-CM) and two macrocyclic glycopeptide antibiotic-type (Chirobiotic T and Chirobiotic TAG) were screened using polar organic or reversed-phase mode. Chiralpak AD, based on amylose tris(3,5-dimethylphenylcarbamate) as chiral selector with neat methanol was identified as the most promising system. In order to improve resolution, an orthogonal experimental design was employed, altering the concentration of 2-propanol, column temperature, and flow rate in a multivariate manner. Using the optimized method (85/15 v/v methanol/2-propanol, 40°C, flow rate: 0.7mL/min) we were not only able to separate the four isomers but also detect 0.1% S-enantiomer as chiral impurity in R-tofisopam. This is important since the latter is under development as a single enantiomeric agent. Thermodynamic investigation revealed an unusual entropy and enthalpy-entropy co-driven controlled enantioseparation on Chiralcel OJ and on Chiralpak AD column, respectively. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested on a pharmaceutical formulation containing the racemic mixture of the drug. As a further novelty, a separate circular dichroism method was applied for the investigation of the interconversion kinetics of tofisopam conformers, which proved to be crucial for sample preparation and method validation.

Determination of AR-42 enantiomeric purity by HPLC on chiral stationary phase

A sensitive and accurate liquid chromatographic method for the determination of AR-42 enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 1.9 was accomplished within 10 min using a CHIRALPAK AD column (250 mm × 4.6 mm; particle size 5 μm) and n-hexane/2-propanol/diethylamine (75:25:0.1 v/v/v) as mobile phase at a flow rate of 1 mL min−1. Eluted analytes were monitored by UV absorption at 260 nm. The effects of mobile phase components, temperature and flow rate on enantiomeric selectivity and resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.001 and 0.5 mg mL−1 (n = 10), and the recoveries between 98.23 and 101.87% were obtained, with relative standard deviation lower than 1.31%. Limit of detection and limit of quantitation for AR-42 were 0.39 and 1.28 μg mL−1 and for its enantiomer were 0.36 and 1.19 μg mL−1, respectively. It was demonstrated that the developed method was accurate, robust and sensitive for the determination of enantiomeric purity of AR-42, especially for the analysis of bulk samples.

HPLC Determination of Enantiomeric Purity of Letermovir Based on CHIRALPAK AD.

A precise and sensitive LC method was developed and further validated for the determination of enantiomeric purity of {(4S)-8-Fluoro-2-[4-(3-methoxyphenyl)-1-piperazinyl]-3-[2-methoxy-5-(trifluoromethyl)phenyl]-3,4-dihydro-4-quinazolinyl} acetic acid (Letermovir). Baseline separation with a resolution >2.8 was accomplished within 10 min using a CHIRALPAK AD (250 mm × 4.6 mm; particle size 5 μm) column, with n-hexane/2-propanol (80:20 v/v) as mobile phase at a flow rate of 1 mL min-1. The eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity as well as resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within the concentration range between 0.003 and 1 mg mL-1 (n = 14), and the recoveries between 98.24 and 101.43% were obtained, with relative standard deviation <1.29%. The limit of detection (LOD) and limit of quantitation (LOQ) for Letermovir were 0.96 and 3.15 μg mL-1; those for its enantiomer were 1.01 and 3.39 μg mL-1, respectively. The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of Letermovir, especially for the analysis of bulk samples.

Competitive adsorption equilibrium model with continuous temperature dependent parameters for naringenin enantiomers on Chiralpak AD column

Determination of competitive adsorption equilibrium model with continuous temperature dependent parameters is important for the design and optimization of a chromatographic separation process operated under non-isothermal conditions. In this study, linear pulse experiments were first carried to determine the parameters of transport-dispersive model and their temperature dependences in the range of 283–313K. Overloaded band profiles of naringenin enantiomers on a Chiralpak AD column were acquired under various temperatures. Three of them were first separately fitted using Langmuir, linear-Langmuir and bi-Langmuir isotherm models substituted into the transport-dispersive column model. The comparison showed that bi-Langmuir model captures more details of the experimental results. This model was then extended with three extra parameters accounting for adsorption heat effects and used to simultaneously fit the band profiles at three temperatures.

Determination of the Enantiomeric Purity of Trelagliptin by Pre-Column Derivatization and Liquid Chromatography on a Chiral Stationary Phase

A precise and sensitive LC method for determination of enantiomeric purity of trelagliptin has been developed and validated. Pre-column derivatization was performed before separation. Baseline separation with a resolution factor >2.5 was accomplished within 10 min by use of a Chiralpak AD column (250 × 4.6 mm; particle size 5 µm) and n-hexane–2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min−1. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity and on resolution of enantiomers were thoroughly investigated. Calibration curves were plotted within the concentration range 0.005–2 mg mL−1 (n = 12), and recoveries between 98.23 and 101.34 % were obtained, with relative standard deviation (RSD) <1.39 %. LOD and LOQ for the trelagliptin derivative were 1.51 and 5.03 µg mL−1; those for its enantiomer were 1.49 and 4.94 µg mL−1, respectively. The method was evaluated and validated by analysis of bulk samples of trelagliptin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

HPLC Determination of Enantiomeric Purity of PF-04136309 Based on a Chiral Stationary Phase

A precise and sensitive LC method was developed and further validated for the determination of enantiomeric purity of (S)-N-[2-(3-{trans-4-hydroxy-4-[5-(pyrimidin-2-yl)pyridin-2-yl] cyclohexylamino} pyrrolidin-1-yl)-2-oxoethyl]-3-(trifluoromethyl) benzamide (PF-04136309). Baseline separation with a resolution higher than 1.8 was accomplished within 40 min using a CHIRALPAK AD (250 × 4.6 mm; particle size 5 μm) column, with n-hexane:2-propanol (70:30 v/v) as mobile phase at a flow rate of 1 mL min−1. The eluted analytes were subsequently detected with a UV detector at 260 nm. The effects of mobile phase components and temperature on enantiomeric selectivity as well as the resolution of enantiomers were thoroughly investigated. The calibration curves were plotted within a concentration range between 0.01 and 1 mg mL−1 (n = 9), and recoveries between 98.17 and 101.28 % were obtained, with relative standard deviation (RSD) lower than 1.44 %. The LOD and LOQ for PF-04136309 were 3.59 and 11.54 μg mL−1 and for its enantiomer were 3.39 and 11.28 μg mL−1, respectively. The developed method was demonstrated to be accurate, robust and sensitive for the determination of enantiomeric purity of PF-04136309, especially for the analysis of bulk samples.

Enantioseparation of 3α-acyloxy-6β-acetoxyltropane compounds with Chiralpak AD and Chiralcel OD-H chiral stationary phases

Six 3α-acyloxy-6β-acetoxyltropane compounds were enantioseparated by high performance liquid chromatography with amylose-based chiral stationary phase Chiralpak AD and cellulose-based chiral stationary phase Chiralcel OD-H in the normal phase mode, using various mixtures of n-hexane-isopropanol as mobile phases. The enantiomers 6 were completely separated on a Chiralpak AD column. While the enantiomers 1, 4 and 3 got complete, baseline and basic separation respectively on a Chiralcel OD-H column. However, the enantiomers 6 were partially separated on the Chiralcel OD-H column and enantiomers 1 could not be separated on the Chiralpak AD column. This indicated that the cave structure of chiral stationary phase exerted great effect on the resolutions. The enantiomers 5 could not be separated on both of the chiral stationary phases. The main possible mechanism of chiral resolution involves in spatial adaptability and molecular interactions between chiral stationary phases and compounds. The substituents in C-3α position of 3α-acyloxy-6β-acetoxyltropane compounds play an important role in spatial adaptability. And it was suggested that the steric hindrance effect of the substituent in C-3α position was the key factor of determining the selective recognition of chiral stationary phase to the enantiomers of 3α-acyloxy-6β-acetoxyltropane compounds. Besides, the molecular interaction, such as π-π interaction, also exerts great influence to the chiral resolution. This study provides a reference for the enantioseparation of many other tropane derivatives.

Determination of Henry Constants of Azelnidipine Enantiomers on Chiralpak AD Column at Different Temperatures

Simulated moving bed (SMB) chromatographic separation has become a highly promising method for chiral separation due to the less solvent consumption and efficient utilization of stationary phase. As reported, Azelnidipine exists two isomeric forms, it is necessary to product azelnidipine single enantiomer for medical safety and drug developmen. However, the separation of azelnidipine enantiomers on SMB is complex, requiring many operating parameters. And the SMB processes with temperature gradient (non-isotherm) mode may have significant advantages over the traditional isothermal mode. In this article, the Henry constants of azelnidipine enantiomers were determined with pulse experiments on Chiralpak AD column at different temperatures. It provided important references for the production of azelnidipine single enantiomer with non-isotherm SMB.

Rapid Supercritical Fluid Chromatography Method for Separation of Chlorthalidone Enantiomers

Supercritical fluid chromatography employing chiral stationary phases is a popular separation technique to perform enantioselective separations. The main advantages of supercritical fluid chromatography are low analysis time, low consumption of organic modifiers, and therefore lower costs and higher environmental friendliness. A novel method for the separation of chlorthalidone enantiomers, widely used diuretic drug, is reported that clearly demonstrates the advantages of supercritical fluid chromatography. The effects of the amount and type of organic modifiers, temperature, and back pressure on enantioselectivity and resolution of the enantiomers were evaluated. The baseline separation was achieved in less than 2.5 min in the optimized system composed of Chiralpak AD column, mobile phase CO2/MeOH 50/50 (v/v), temperature 40°C, a flow rate of 4.0 mL/min, and 120 bar back pressure. Moreover, enantiomers of chlorthalidone were determined in two commercially available pharmaceuticals. The proposed method may be easily transferred to a semi-preparative scale.

Effects of organic modifier and temperature on the enantiomeric separation of several azole drugs using supercritical fluid chromatography and the Chiralpak AD column

The effects of organic modifier and temperature on the enantioseparation of 10 triazoles and eight imidazoles, using supercritical fluid chromatography with the Chiralpak AD column, have been investigated in this work. For this purpose four different organic modifiers (methanol, ethanol, 2-propanol and acetonitrile) were evaluated. Only in the case of two compounds could the enantiomeric separation not be achieved with any of the modifiers tested; the rest of compounds were baseline or partially resolved with at least one of the modifiers. The alcohol-type modifiers provided the best results in terms of retention time and resolution. In general, retention increased in the order methanol < ethanol < 2-propanol; moreover it was possible to establish a relationship between the retention and the number of aromatic rings and dioxolane groups in the molecule, that is, the higher the number is, the higher the retention time. From the study of the temperature effect, the enthalpy-entropy compensation was demonstrated for all the compounds, except for bifonazole using methanol and miconazole using acetonitrile. This suggested that both analytes are enantiomerically resolved through different mechanisms.

Enantiomeric separation of citalopram base by supercritical fluid chromatography

The chiral separation of citalopram base by supercritical fluid chromatography on a semipreparative Chiralpak AD column was studied with the use of three alcohol-type modifiers (methanol, ethanol, and 2-propanol) with different volume percentages (5, 10, and 15%). The best separation was achieved when 10% 2-propanol was used in the presence of 0.1% diethylamine as additive. Under these conditions, the resolution reached 2.15 and the selectivity was 1.388. In addition, other parameters that affected the retention and separation properties, i.e. temperature, pressure, and density, were studied in detail. At the same pressure, a decrease in the temperature improved the enantioselectivity as the experimental temperature range was below the isoelution temperature. However, the temperature dependence of the retention factor was complicated. As a rule, the retention factor decreased when the temperature increased at the same density. A satisfactory regression of the logarithm of the retention factor versus density and temperature was obtained using a simplified lattice-fluid model. Surprisingly, the relationship between the Henry constant and density can be accurately correlated by using the same quadratic equation.

Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

Determination of competitive adsorption isotherm of enantiomers on preparative chromatographic columns using inverse method

Precise determination of competitive adsorption isotherm is of great importance for model-based optimization and scale-up of chromatographic separation processes, particularly for the simulated moving bed (SMB) systems due to its intrinsic complexity. In this study, five parameters of a bi-Langmuir model for adsorption of ketoprofen enantiomers on a preparative Chiralpak AD column at 25°C were determined by the application of inverse method (IM). Transport-dispersive (TD) model parameters, axial dispersion and mass transfer coefficients were independently estimated first by tuning a series of linear pulse experiments. Competitive adsorption isotherm model parameters were then determined independently by fitting the experimental data. A new numerical scheme that couples non-dominated sorting genetic algorithm (NSGA) and Levenberg–Marquardt algorithm (LMA) was proposed for the data fitting. NSGA was capable of rapidly locating the optimal solution in the parametric space while LGA allowed speedy convergence given a good initial guess. Independently measured band profiles and simulated moving bed (SMB) experiments were later used to validate the model parameters. The experimental results were in good agreement with the model predictions.

Enantioselective analysis of zopiclone in rat brain by liquid chromatography tandem mass spectrometry

A new high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection was developed and validated for the quantification of zopiclone enantiomers in rat brain samples. Zopiclone enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile/ethanol/methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL min(-1). Moclobemide was used as internal standard. The sample treatment procedure was carried out employing solid-phase extraction, yielding mean absolute recoveries of 89.6 and 91.7% for each zopiclone enantiomer. The validated method showed linearity in the range of 0.29-344.8 ng g(-1), with quantification limits of 0.29 ng g(-1) for both enantiomers. Precision and accuracy were within acceptable levels of confidence (<15%). The method was applied in a pilot study of zopiclone kinetic disposition in rats. It could be observed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-(R)-zopiclone, confirming the stereoselective disposition of zopiclone.

Stereoselective Determination of Metoprolol and its Metabolite α‐Hydroxymetoprolol in Plasma by LC‐MS/MS: Application to Pharmacokinetics during Pregnancy

Metoprolol is available for clinical use as a racemic mixture. The S-(-)-metoprolol enantiomer is the one expressing higher activity in the blockade of the β(1)-adrenergic receptor. The α-hydroxymetoprolol metabolite also has activity in the blockade of the β(1)-adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α-hydroxymetoprolol in plasma using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). 1-ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α-hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC-MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α-hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100-mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)(0-∞) values for S-(-)-metoprolol (AUC(S-(-)) /AUC(R-(+)) = 1.81) and the favoring of the formation of the new chiral center 1'R of α-hydroxymetoprolol (AUC(0-∞) (1'R/1'S) = 2.78).

Enantiomeric Separation and Toxicity of an Organophosporus Insecticide, Pyraclofos

Despite the fact that the biological processes of chiral pesticides are enantioselective, knowledge of the toxicities of pyraclofos due to enantiospecificity is scarce. In this study, the optical isomers of pyraclofos were separated and their toxicities to butyrylcholinesterase (BChE) and Daphnia magna were assessed. Baseline resolution of the enantiomers was obtained on both Chiralcel OD and Chiralpak AD columns. The effect of the mobile phase composition and column temperature were then discussed. The resolved enantiomers were characterized by their optical rotation and circular dichroism signs. The anti-BChE tests demonstrated that (-)-pyraclofos was about 15 times more potent than its (+)-form. However, acute aquatic assays suggested that (+)-pyraclofos was about 6 times more toxic than its antipode. Moreover, the joint toxicity of pyraclofos enantiomers to D. magna was found to be an additive effect. These results demonstrated that the overall toxicity of pyraclofos should be assessed using the individual enantiomers.

Enantiomeric Resolution and Growth-Retardant Activity in Rice Seedlings of Uniconazole

The increasing application of chiral pesticides has enhanced interest in their enantioselectivity. However, little relevant information is currently available for enantioselective activity of chiral plant growth regulators. In an attempt to screen active enantiomers of uniconazole, this work investigated enantiomeric separation and the enantioselective effect of uniconazole on the growth of rice seedlings and cyanobacteria. Baseline resolution of uniconazole enantiomers was achieved on a Chiralpak AD column by chiral high-performance liquid chromatography (HPLC). The relationship among circular dichroism (CD), optical rotation (OR), and absolute configuration was successfully established by coupling of CD and OR detection. The t test at the 95% level of confidence indicated significant differences between the enantiomers in their retardant activity toward growth of rice seedlings and stimulation effect on growth of cyanobacteria, the natural biofertilizers in rice paddy fields. The S-(+)-enantiomer was more active than the R-(-)-enantiomer in retarding growth of rice seedlings and stimulating growth of Microcystis aeruginosa . This special enantiomeric selectivity was further elucidated by probing the binding mode of enantiomers to gibberellin (GA) 20-oxidase by molecular docking. The S-(+)-enantiomer was found to bind tightly with GA 20-oxidase. The results suggested that the S-(+)-enantiomer instead of a racemate of uniconazole should be used to improve rice seedling quality.