Predictability of Enantiomeric Chromatographic Behavior on Various Chiral Stationary Phases Using Typical Reversed Phase Modeling Software

Pharmaceutical companies worldwide tend to apply chiral chromatographic separation techniques in their mass production strategy rather than asymmetric synthesis. The present work aims to investigate the predictability of chromatographic behavior of enantiomers using DryLab HPLC method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. Three different types of chiral stationary phases were tested for predictability: macrocyclic antibiotics‐based columns (Chirobiotic V and T), polysaccharide‐based chiral column (Chiralpak AD‐RH), and protein‐based chiral column (Ultron ES‐OVM). Preliminary basic runs were implemented, then exported to DryLab after peak tracking was accomplished. Prediction of the effect of % organic mobile phase on separation was possible for separations on Chirobiotic V for several probes: racemic propranolol with 97.80% accuracy; mixture of racemates of propranolol and terbutaline sulphate, as well as, racemates of propranolol and salbutamol sulphate with average 90.46% accuracy for the effect of percent organic mobile phase and average 98.39% for the effect of pH; and racemic warfarin with 93.45% accuracy for the effect of percent organic mobile phase and average 99.64% for the effect of pH. It can be concluded that Chirobiotic V reversed phase retention mechanism follows the solvophobic theory. Chirality 25:506–513, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Separation of eight bedaquiline analogue diastereomers by HPLC on an immobilized polysaccharide‐based chiral stationary phase

The high‐performance liquid chromatography (HPLC) is a powerful method in the area of stereoisomer separation. In this study, separation of eight bedaquiline analogue diastereomers (compounds 1‐8) was first examined on a cellulose tris‐(3,5‐dichlorophenylcarbamate) chiral stationary phase, ie, Chiralpak IC in the normal phase mode. The influences of organic modifier types, alcohol content, and column temperature on diastereoseparation were evaluated. Under the optimum chromatographic conditions, all the analyte stereoisomers were successfully separated. The experimental results revealed the great influence of analytes' structures on the diastereoseparation with Chiralpak IC. In addition, thermodynamic parameters were calculated by Van't Hoff plots, and correlative chiral recognition mechanisms were discussed further.     

Enantioseparation of benzazoles and benzanilides on polysaccharide‐based chiral columns

The chiral recognition ability of the polysaccharide‐based chiral columns (Chiralpak AD‐RH, Chiralpak AS‐RJ, Chiralpak IC, Chiralcel OD‐RH, and Chiralcel OJ‐RH) for the benzazoles and the benzanilides was evaluated under reversed phase conditions. The columns showed the high chiral recognition ability for a wide range of benzazoles and benzanilides. Twenty‐one racemates were used for the evaluation, and 20 racemates were completely separated on at least one of the columns. In particular, AS‐RH and OJ‐RH showed the high chiral recognition ability for the benzazoles, and the AD‐RH, IC, and OJ‐RH were effective for the benzanilides. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Enantioseparation of racecadotril using polysaccharide‐type chiral stationary phases in polar organic mode

Enantioseparation of the antidiarrheal drug, racecadotril, was investigated by liquid chromatography using polysaccharide‐type chiral stationary phases in polar organic mode. The enantiodiscrimininating properties of 4 different chiral columns (Chiralpak AD, Chiralcel OD, Chiralpak AS, Chiralcel OJ) with 5 different solvents (methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile) at 5 different temperatures (5–40 °C) were investigated. Apart from Chiralpak AS column the other 3 columns showed significant enantioseparation capabilities. Among the tested mobile phases, alcohol type solvents were superior over acetonitrile, and significant differences in enantioselective performance of the selector were observed depending on the type of alcohol employed. Van't Hoff analysis was used for calculation of thermodynamic parameters which revealed that enantioseparation is mainly enthalpy controlled; however, enthropic control was also observed. Enantiopure standard was used to determine the enantiomer elution order, revealing chiral selector—and mobile‐phase dependent reversal of enantiomer elution order. Using the optimized method (Chiralcel OJ stationary phase, thermostated at 10 °C, 100% methanol, flow rate: 0.6 mL/min) baseline separation of racecadotril enantiomers (resolution = 3.00 ± 0.02) was achieved, with the R‐enantiomer eluting first. The method was validated according to the ICH guidelines, and its application was tested on capsule and granules containing the racemic mixture of the drug.     

Enantiomeric separation of prothioconazole and prothioconazole‐desthio on chiral stationary phases

Prothioconazole is a type of broad‐spectrum triazole thione fungicide developed by the Bayer Company. Prothioconazole‐desthio is the main metabolite of prothioconazole in the environment. In our study, enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on various chiral stationary phases (CSPs) by high‐performance liquid chromatography (HPLC). It was found that polysaccharide CSPs showed better ability than brushing CSPs in enantiomeric separation. The successful chiral separation of prothioconazole could be achieved on self‐made Chiralcel OD, commercialized Chiralcel OJ‐H and Lux Cellulose‐1. Chiralpak IA, Chiralpak IB, Chiralpak IC, Chiralcel OD, Chiralpak AY‐H, Chiralpak AZ‐H, and Lux Cellulose‐1 realized the baseline separation of prothioconazole‐desthio enantiomers. Simultaneous enantiomeric separation of prothioconazole and prothioconazole‐desthio was performed on Lux Cellulose‐1 using acetonitrile (ACN) and water as mobile phase. In most cases, low temperature favored the separation of two compounds. The influence of the mobile phase ratio or type was deeply discussed. We obtained larger Rs and longer analysis time with a smaller proportion of isopropanol (IPA) or ethanol and more water content at the same temperature. The ratio of ACN and water had influences on the outflow orders of prothioconazole‐desthio enantiomers. This work provides a new approach for chiral separation of prothioconazole and prothioconazole‐desthio with a discussion of chiral separation mechanism on different CSPs.     

Evaluation of the chiral recognition properties and the column performances of three chiral stationary phases based on cellulose for the enantioseparation of six dihydropyridines by high‐performance liquid chromatography

Separations of six dihydropyridine enantiomers on three commercially available cellulose‐based chiral stationary phases (Chiralcel OD‐RH, Chiralpak IB, and Chiralpak IC) were evaluated with high‐performance liquid chromatography (HPLC). The best enantioseparation of the six chiral drugs was obtained with a Chiralpak IC (250 × 4.6 mm i.d., 5 μm) column. Then the influence of the mobile phase including an alcohol‐modifying agent and alkaline additive on the enantioseparation were investigated and optimized. The optimal mobile phase conditions and maximum resolution for every analyte were as follows respectively: n‐hexane/isopropanol (85:15, v /v) for nimodipine (R  = 5.80) and cinildilpine (R  = 5.65); n‐hexane/isopropanol (92:8, v /v) for nicardipine (R  = 1.76) and nisoldipine (R  = 1.92); and n‐hexane/isopropanol/ethanol (97:2:1, v /v/v) for felodipine (R  = 1.84) and lercanidipine (R  = 1.47). Relative separation mechanisms are discussed based on the separation results, and indicate that the achiral parts in the analytes' structure showed an important influence on the separation of the chiral column.     

Chiral separation of neonicotinoid insecticides by polysaccharide‐type stationary phases using high‐performance liquid chromatography and supercritical fluid chromatography

The enantiomeric separations of three neonicotinoid insecticides (identified as compounds 1 , 2 , and 3 ) were performed on three polysaccharide‐type chiral columns, that is, Chiralcel OD‐H, Chiralpak AD‐H, and Chiralpak IB, by high‐performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). Effects of the modifier percentage and column temperature on chiral recognitions of chiral stationary phases were also studied. Both 1 and 2 could be resolved on all three columns selected, with the highest R_s values obtained on Chiralpak AD‐H and Chiralcel OD‐H, respectively. However, satisfactory separation of the four stereoisomers of 3 was only achieved on Chiralcel OD‐H. Considering the effects of ethanol on the values of k, α, and R_s, we concluded that hydrogen bonding, π–π, and/or dipole–dipole interactions might be all responsible for the chiral separation. In comparison to HPLC, a shorter run time was achieved for 1 and 2 by SFC. However, 3 could not be stereoselectively resolved using SFC. On the basis of the calculated thermodynamic parameters, we found that the separation processes of enantiomers of 1 and 2 were entropy controlled and enthalpy controlled, respectively. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors,potential adrenergic ligands,by analytical and preparative liquid chromatography on amylose chiral stationary phases and characterization of the enantiomers

To obtain milligram amounts of the enantiomers of benzoxazolinone derivatives to be tested for binding to adrenergic sites, analytical HPLC methods using derivatized amylose chiral stationary phases were developed for the direct enantioseparation of benzoxazolinone aminoalcohols and their aminoketone precursors, derivatives with one or two chirals centers. The separations were made using normal phase methodology with a mobile phase of n‐hexane‐alcohol (ethanol, 1‐propanol, or 2‐propanol) in various proportions, and silica‐based amylose (tris‐3, 5‐dimethylphenylcarbamate) Chiralpak AD and (tris‐(S)‐1‐phenylethylcarbamate) Chiralpak AS columns. The effects of concentration of various aliphatic alcohols in the mobile phase were studied. The best separation was achieved on Chiralpak AS, so preparative HPLC was set up with this chiral stationary phase using a mobile phase consisting of n‐hexane‐alcohol using isocratic conditions and multiple repetitive injections. Physicochemicals properties of enantiomers were reported The effect of structural features of the solutes on discrimination between the enantiomers was examined. Limit of detection (LD) and limit of quantification (LQ) were determined using both ultra‐violet (UV) and evaporative light‐scattering detection (ELSD). Chirality, 2009. © 2008 Wiley‐Liss, Inc.     

Enantioselective potential of polysaccharide‐based chiral stationary phases in supercritical fluid chromatography

The enantioselective potential of two polysaccharide‐based chiral stationary phases for analysis of chiral structurally diverse biologically active compounds was evaluated in supercritical fluid chromatography using a set of 52 analytes. The chiral selectors immobilized on 2.5 μm silica particles were tris‐(3,5‐dimethylphenylcarmabate) derivatives of cellulose or amylose. The influence of the polysaccharide backbone, different organic modifiers, and different mobile phase additives on retention and enantioseparation was monitored. Conditions for fast baseline enantioseparation were found for the majority of the compounds. The success rate of baseline and partial enantioseparation with cellulose‐based chiral stationary phase was 51.9% and 15.4%, respectively. Using amylose‐based chiral stationary phase we obtained 76.9% of baseline enantioseparations and 9.6% of partial enantioseparations of the tested compounds. The best results on cellulose‐based chiral stationary phase were achieved particularly with propane‐2‐ol and a mixture of isopropylamine and trifluoroacetic acid as organic modifier and additive to CO₂, respectively. Methanol and basic additive isopropylamine were preferred on amylose‐based chiral stationary phase. The complementary enantioselectivity of the cellulose‐ and amylose‐based chiral stationary phases allows separation of the majority of the tested structurally different compounds. Separation systems were found to be directly applicable for analyses of biologically active compounds of interest.     

Chiral recognition ability and solvent versatility of bonded amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase: enantioselective liquid chromatographic resolution of racemic N-alkylated barbiturates and thalidomide analogs

The chiral recognition ability and solvent versatility of a new chiral stationary phase containing amylose 3,5-dimethylphenylcarabamate immobilized onto silica gel (CHIRALPAK IA) is investigated. Thus, the direct enantioselective separation of a set of racemic N-alkylated barbiturates and 3-alkylated analogs of thalidomide was conducted using different nonstandard solvents as eluent and diluent, respectively in high-performance liquid chromatography (HPLC). The separation, resolution, and elution order of the investigated compounds were compared on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) using a mixture of n-hexane/2-propanol (90:10 v/v) as mobile phase with different flow-rates and fixed UV detection at 254 nm. The effect of the immobilization of the amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted as the bonded phase (Chiralpak IA) was superior in chiral recognition and possesses a higher resolving power in most of the reported cases than the coated one (Chiralpak AD). A few racemates were not or poorly resolved on the immobilized Chiralpak IA or the coated Chiralpak AD when using standard solvents were most efficiently resolved on the immobilized Chiralpak IA upon using nonstandard solvents. Furthermore, the immobilized phase withstands the nonstandard (prohibited) HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, methyl-tert-butyl ether, and others when used as eluents or as a dissolving agent for the analyte itself. The direct analysis of a real sample extracted from plasma using DCM on Chiralpak IA is also shown.     

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.     

Green chiral HPLC enantiomeric separations using high temperature liquid chromatography and subcritical water on Chiralcel OD and Chiralpak AD

We report here the application of subcritical water in chiral separations on two popular polysaccharide chiral stationary phases (CSPs): Chiralpak AD and Chiralcel OD. The behavior of these two CSPs was studied under reversed phase conditions at room temperature to discover the maximum percentage of water in the mobile phase, which provided the separation of enantiomers of flavanone and benzoin, respectively, in a reasonable time (i.e., less than 1 h). Then, the stability of Chiralpak AD and Chiralcel OD versus temperature was investigated and discussed. Chiralcel OD separation of flavanone racemate was obtained at 120 °C with water and 2-propanol (80/20) as the mobile phase, while benzoin racemate was separated in pure water at 160 °C. Separations of several racemates were also presented, and advantages and limitations of the technique were discussed.     

Chiral separation of novel iminonaringenin derivatives

A series of 4‐iminonaringenin derivatives 2 ‐ 6 have been prepared in good overall yields from a condensation reaction between naringenin and primary amines. The structures of all products were confirmed by ultraviolet, infrared, proton nuclear magnetic resonance, and carbon‐13 nuclear magnetic resonance spectroscopic techniques. These derivatives were analyzed by high‐performance liquid chromatography using polysaccharide‐based chiral stationary phases, namely, Chiralpak IB and Chiralcel OD, using various mobile phases. 2‐Propanol showed a high enantioselectivity for naringin and its derivatives using achiral column containing immobilized polysaccharides (Chiralpak IB).     

Normal phase chiral HPLC of methylphenidate: comparison of different polysaccharide-based chiral stationary phases.

A comparison of the enantiomeric resolution of (+/-)-threo-methylphenidate (MPH) (Ritalin) was achieved on different polysaccharide based chiral stationary phases. The mobile phase used was hexane-ethanol-methanol-trifluoroacetic acid (480:9.75:9.75:0.5, v/v/v/v). Benzoic acid and phenol were used as the mobile phase additives for the enantiomeric resolution of MPH on Chiralcel OB column only. The alpha values for the resolved enantiomers were 1.34, 1.29, 1.30, and 1.24 on Chiralpak AD, Chiralcel OD, Chiralcel OB (containing 0.2 mM benzoic acid in mobile phase), and Chiralcel OB (containing 0.2 mM phenol in mobile phase) columns, respectively. The R(s) values were 1.82, 1.53, 1.19, and 1.10 on Chiralpak AD, Chiralcel OD, Chiralcel OB (containing 0.2 mM benzoic acid in mobile phase), and Chiralcel OB (containing 0.2 mM phenol in mobile phase), respectively. The role of benzoic acid and phenol as mobile phase additives is discussed.     

Chiral separation of phospine‐containing α‐amino acid derivatives using two complementary cellulosic stationary phases in supercritical fluid chromatography

Enantiomeric separations of six amino‐acid derivatives have been studied using packed‐column supercritical fluid chromatography with two polysaccharide‐based enantioselective stationary phases: cellulose tris(3,5‐dimethylphenylcarbamate) and cellulose tris(3‐chloro‐4‐methylphenylcarbamate) (Lux Cellulose‐1 and ‐2). The effect of analyte structure on retention and separation was studied. Varied mobile phase compositions were investigated: alcohol modifier percentage was increased from 3 to 40% but smaller amounts were most effective in separating these compounds. Besides, ethanol was preferred to methanol or isopropanol as it proved to be a good compromise to achieve sufficient resolution in a reasonable analysis time. Moreover, a carbon dioxide‐ethanol mixture allows performing analyses in safe and green conditions. The effect of temperature at constant mobile phase composition was explored between 10 and 40°C. In most cases, increasing the temperature improved the chiral separation, up to an optimum temperature. The results are discussed in line with the structure variation of the racemic derivatives analyzed and the two columns are compared. The two columns were shown to provide complementary selectivities for the investigated solutes: whereas Lux 1 provided separation for five of the six racemates, Lux 2 could resolve the last racemic mixture. Finally, optimized conditions of separation are defined. Chirality, 2010. © 2009 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.     

Access to Enantiomerically Pure cis‐ and trans‐β‐Phenylproline by High‐Performance Liquid Chromatography Resolution

The preparation of all four stereoisomers of the proline analog that bears a phenyl group attached to the β carbon either cis or trans to the carboxylic acid (cis‐ and trans‐β‐phenylproline, respectively) has been addressed. The methodology developed allows access to multigram quantities of the target amino acids in enantiomerically pure form and suitably protected for use in peptide synthesis. Racemic precursors of cis‐β‐phenylproline and trans‐β‐phenylproline were prepared from easily available starting materials and subjected to high‐performance liquid chromatography enantioseparation. Semipreparative columns (250 × 20 mm) containing chiral stationary phases based on amylose (Chiralpak IA) (Daicel‐Chiral Technologies Europe, Illkirch, France) or cellulose (Chiralpak IC) were used respectively for the resolution of the cis‐ and trans‐β‐phenylproline precursors. Chirality, 24:1082‐1091, 2012. © 2012 Wiley Periodicals, Inc.     

Studies on the effect of alcohols on the chiral discrimination mechanisms of amylose stationary phase on the enantioseparation of nebivolol by HPLC

The chiral recognition mechanism of amylose CSPs has been described by achieving the enantiomeric resolution of (+/-)-nebivolol on Chiralpak AD and Chiralpak AD-RH columns with methanol, ethanol, 1-propanol, 2-propanol, 1-butanol as mobile phases at different flow rates. The energies of interactions of methanol, ethanol, 1-propanol, 2-propanol and 1-butanol with both phases were calculated. The (+)-RRRS enantiomer eluted first when using methanol, ethanol and 1-propanol, while the elution order was reversed when using 2-propanol and 1-butanol as the mobile phases. It has been concluded that the reversal elution order observed was due in part to the chiral cavities on the amylose CSP which were responsible for the bondings of different magnitude between chiral stationary phase and enantiomers, which are influenced with the type of alcohol used as mobile phase on the conformation of the 3,5-dimethyl phenyl carbamate moiety on the pyranose ring system of the amylose.     

Enantioseparation and plant virucidal bioactivity of new quinazoline derivatives with α-aminophosphonate moiety

Enantiomers of some new quinazoline derivatives bearing α-aminophosphonate moiety were separated under normal-phase conditions on two immobilized polysaccharide-based chiral stationary phases (Chiralpak IA and Chiralpak IC). The role of two chiral stationary phases (CSPs), polar modifier and column temperature on retention time and separation factor was studied. Apparent thermodynamic parameters were deduced from Van’t Hoff plots and plausible mechanism of chiral recognition has been discussed. The semi-preparative separation of some compounds was executed successfully in n-hexane/isopropyl alcohol (IPA) on the Chiralpak IA column. The preliminary bioassay showed that both the enantiomers of the investigated series of compounds possessed similar anti-tobacco mosaic virus (TMV) activities.