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.     

Enantiomeric separation of imidazolinone herbicides using chiral high-performance liquid chromatography

Chiral high-performance liquid chromatography (HPLC) is one of the most powerful tools to prepare enantiopure standards of chiral compounds. In this study, the enantiomeric separation of imidazolinone herbicides, i.e., imazethapyr, imazapyr, and imazaquin, was investigated using chiral HPLC. The enantioselectivity of Chiralpak AS, Chiralpak AD, Chiralcel OD, and Chiralcel OJ columns for the three analytes was compared under similar chromatographic conditions. Chiralcel OJ column showed the best chiral resolving capacity among the test columns. The resolved enantiomers were distinguished by their signs of circular dichroism detected at 275 nm and their structures confirmed with LC-mass spectrometric analysis. Factors affecting the chiral separation of imidazolinones on Chiralcel OJ column were characterized. Ethanol acted as a better polar modifier than the other alcohols including 2-propanol, 1-butanol, and 1-pentanol. Although the acidic modifier in the mobile phase did not influence chiral recognition, it was necessary for reducing the retention time of enantiomers and suppressing their peak tailing. Thermodynamic evaluation suggests that enantiomeric separation of imidazolinones on Chiralcel OJ column is an enthalpy-driven process from 10 to 40 degrees C. This study also shows that small amounts of pure enantiomers of imidazolinones may be obtained by using the analytical chiral HPLC approach.     

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.     

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.     

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.     

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.     

Separation and aquatic toxicity of enantiomers of 1-(substituted phenoxyacetoxy)alkylphosphonate herbicides

A series of organophosphorous compounds (OPs), 1-(substituted phenoxyacetoxy)alkylphosphonates containing a chiral carbon atom, show notable herbicidal activities. In this study, the enantioselective separation and biological toxicity of all these compounds were investigated. The enantioselective separation on the columns of Chiralpak AD, Chiralpak AS, Chiralcel OD, and Chiralcel OJ were compared under various chromatographic conditions. All the analytes investigated obtained baseline resolution (R(s) > 1.5) on Chiralpak AD column, which showed best chiral separation capacity. Further investigation was carried out on Chiralpak AD to evaluate the influence of the mobile phase composition and column temperature. The effect of the structural features on discrimination was also examined. The resolved enantiomers were distinguished by their signs of circular dichroism. The acute aquatic toxicity of enantiomers and racemate to Daphnia magna (D. magna) were assessed. The in vivo assays showed that compound 3 was about 2-148.5 times more toxic than the other four analogues to D. magna. The racemates of compounds 3 and 5 showed intermediate toxicity compare to their enantiomers, while those of compounds 1, 2, and 4 showed synergistic or antagonistic effect. These results suggest that the biological toxicity of chiral OPs to nontarget organisms is enantioselective and therefore should be evaluated with their pure enantiomers.     

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.     

Direct HPLC enantioseparation of chiral aptazepine derivatives on coated and immobilized polysaccharide-based chiral stationary phases

The direct HPLC enantioseparation of Mianserin and a series of aptazepine derivatives is accomplished on polysaccharide-based chiral stationary phases (CSPs). The resolutions are performed on the coated-type Chiralcel OD and Chiralpak AD CSPs and on the first commercially available immobilized-type Chiralpak IA CSP, in normal-phase and polar-organic modes. The complete separation of enantiomers of all racemates investigated was successfully achieved under at least one of CSP/eluent combinations employed. Pure alcohols such ethanol or 2-propanol, with a fixed percentage of DEA added, serve as valuable alternatives to the more common n-hexane-based normal-phase eluents in resolution of Mianserin on the AD CSP. In order to study the chiroptical properties of aptazepine derivatives, chromatographic resolutions are carried out at semipreparative scale using Chiralpak AD and Chiralpak IA as CSPs. Nonconventional dichloromethane-based eluents have permitted to expand the chiral resolving ability of the immobilized Chiralpak IA CSP and to perform mg-scale enantioseparations with an analytical-size column. Assignment of the absolute configuration of the separated enantiomers is empirically established by comparing their chiroptical data with those of structurally related Mianserin.     

Comparative studies between covalently immobilized and coated chiral stationary phases based on polysaccharide derivatives for enantiomer separation of N‐protected α‐amino acids and their ester derivatives

Liquid chromatographic enantiomer separation of several N‐benzyloxycarbonyl (CBZ) and N‐tert‐butoxycarbonyl (BOC) α‐amino acids and their corresponding ethyl esters was performed on covalently immobilized chiral stationary phases (CSPs) (Chiralpak IA and Chiralpak IB) and coated‐type CSPs (Chiralpak AD and Chiralcel OD) based on polysaccharide derivatives. The solvent versatility of the covalently immobilized CSPs in enantiomer separation of N‐CBZ and BOC‐α‐amino acids and their ester derivatives was shown and the chromatographic parameters of their enantioselectivities and resolution factors were greatly influenced by the nature of the mobile phase. In general, standard mobile phases using 2‐propanol and hexane on Chiralpak IA showed fairly good enantioselectivities for resolution of N‐CBZ and BOC‐α‐amino acids and their esters. However, 50% MTBE/hexane (v/v) for resolution of N‐CBZ‐α‐amino acids ethyl esters and 20% THF/hexane (v/v) for resolution of N‐BOC‐α‐amino acids ethyl esters afforded the greatest enantioselectivities on Chiralpak IA. Also, liquid chromatographic comparisons of the enantiomer resolution of these analytes were made on amylose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IA and Chiralpak AD) and cellulose tris(3,5‐dimethylphenylcarbamate)‐derived CSPs (Chiralpak IB and Chiralcel OD). Chiralpak AD and/or Chiralcel OD showed the highest enantioselectivities for resolution of N‐CBZ‐α‐amino acids and esters, while Chiralpak AD or Chiralpak IA showed the highest resolution of N‐BOC‐α‐amino acids and esters. Chirality 2009. © 2008 Wiley‐Liss, 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.     

Enantiomers of C(5)-chiral 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives: Analytical and semipreparative HPLC separation, chiroptical properties, absolute configuration, and inhibitory activity against monoamine oxidase

The HPLC enantiomer separation of a novel series of C(5)-chiral 1-acetyl-3-(4-hydroxy- and 2,4-dihydroxyphenyl)-5-phenyl-4,5-dihydro-(1H)-pyrazole derivatives, with inhibitory activity against monoamine oxidases (MAO) type A and B, was accomplished using polysaccharide-based chiral stationary phases (CSPs: Chiralpak AD, Chiralcel OD, and Chiralcel OJ). Pure alcohols, such as ethanol and 2-propanol, and typical normal-phase binary mixtures, such as n-hexane and alcohol modifier, were used as mobile phases. Single enantiomers of several analytes examined were isolated on a semipreparative scale, and their chiroptical properties were measured. The assignment of the absolute configuration was established for one compound by single-crystal X-ray diffraction method and for the other three by CD spectroscopy. The inhibitory activity against MAO of racemic samples and single enantiomers were evaluated in vitro.     

Enantioselective chromatography and absolute configuration of N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines: potential sigma1 ligands

We describe the preparation of racemic N,N-dimethyl-3-(naphthalen-2-yl)-butan-1-amines, potential sigma1 ligands, and their resolution via chiral HPLC. In order to obtain enantiopure compounds, direct chromatographic methods of separation using chiral stationary phases were investigated. Different methods suitable for both analytical and semipreparative purposes are proposed. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD and OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). On the basis of the preliminary chromatographic results, the resolution of compound 1 was transferred onto a Chiralcel OD semipreparative column. The enantiomers were obtained in high enantiomeric excess. The configurational assignment was performed by circular dichroism. Computational analysis was used to explore the enantioselective recognition process of compound 1 with the Chiralcel OD stationary phase.     

Chiral separation of some 4a-methyl-1,2,3,4,4a,9a-hexahydro-fluoren-9-one derivatives as a probe for difference in solvation by 2-propanol of carbamate moiety in chiralcel OD-H,chiralpak AD,and chiralpak AS chiral stationary phases

Chromatographic resolution of 12 derivatives in the 4a-methyl-2,3,4,4a-tetrahydro-1H-fluorene and 4a-methyl-1,2,3,4,4a,9a-hexahydrofluoren-9-one series differing by the framework around position 9 and substitution in position 6, are reported on Chiralcel OD, Chiralpak AD, and Chiralpak AS under two elution conditions and according to the two classes of enantiomers. Results from principal component analysis (PCA) as well as hierarchical clustering show a clustering of the actual compounds depending on properties around position 9, the effect of the substituent in position 6 (methyl, chloro or fluoro) not being strong enough to intermesh the data. Carbamate phases show very different properties when they are used in the separation of a series of ketones C and α-chloroketones D , which differ in basicity and the steric requirement around the carbonyl. Analysis of the effect of 2-PrOH content in hexane on the retention is consistent with a large difference in solvation of the carbamate moiety by 2-PrOH, in the order Chiralcel OD > Chiralpak AD > Chiralpak AS. 4a-Methyl-2,3,4,4a-tetrahydro-1H-fluorene derivatives, which lack hydrogen bonding sites, are not discriminated on these CSPs and show identical k′ responses to 2-PrOH content changes on the three CSPs. Chirality 10:770–777, 1998. © 1998 Wiley-Liss, Inc.     

Chiral HPLC separation and CD spectra of the C-2 diastereomers of naringin in grapefruit during maturation

Naringin is the chief flavanone glycoside of grapefruit (Citrus paradisi). It is responsible for part of the bitter taste of the fruit and can cause the inhibition of some cytochrome P450s. The direct separation of (2R)- and (2S)-naringin in the albedo of grapefruits was obtained in normal phase HPLC mode using Chiralcel OD as chiral stationary phase and n-hexane/ethanol with 0.1% of TFA as mobile phase. Chiralpak AD was almost ineffective in the separation. This procedure was used to evaluate the stereochemistry at C-2 during maturation of the grapefruit. The CD curves of (2R)- and (2S)-naringin isolated by semipreparative chiral HPLC were determined and the elution order of the chromatographic peaks was related to the absolute C-2 configuration. Partial resolution of the C-2 diastereomers of narirutin was obtained on Chiralpak AD.     

Chiral HPLC analysis of milnacipran and its FMOC-derivative on cellulose-based stationary phases

The HPLC enantioseparation of the last generation antidepressive drug milnacipran (+/-)-1 was investigated on different cellulose-based chiral stationary phases (CSPs). On carbamate-type columns, Chiralcel OD and OD-H (+/-)-1 could be separated with alpha value about 1.20 but the resolution was quite low because of the tailing of the peaks. Direct determination of (+/-)-1 with high selectivity and resolution was obtained on Chiralcel OJ in normal phase mode elution. Precolumn derivatization of milnacipran with Fmoc-Cl gave compound (+/-)-2 which was enantioseparated on all the investigated CSPs and allowed enhanced UV or fluorimetric detection. The Chiralpak IB, that could be considered the immobilized version of Chiralcel OD-H, was found completely ineffective in the chiral recognition of (+/-)-1 and moderately efficient in the separation of (+/-)-2.     

Direct high-performance liquid chromatography (HPLC) separation of etodolac enantiomers using chiral stationary phases

The enantiomers of the antiinflammatory drug Etodolac were separated without derivatization on Chiralcel OD and Pirkle (R)-DNBPG columns. Enantiomeric purity can be determined in less than 10 min. Optimization of separation was evaluated using various concentrations of 2-propanol (doped with TFA) in hexane as the mobile phase. © 1993 Wiley-Liss, Inc.     

Chromatographic resolution of chiral intermediates in β-adrenergic blocker synthesis on chiral stationary phases

The enantiomeric purities of optically active intermediates for β-adrenergic blocking agents prepared via enzyme-assisted processes can be determined rapidly and with high accuracy using HPLC on commercially available columns with chiral supports [Chiralcel OD, OB; Chiralpak OT(+)]. The dependence of the resolution parameters on the substitution pattern of both hydroxy compounds and their esters is reported. © 1993 Wiley-Liss, Inc.     

Chiral HPLC separation and CD spectra of the enantiomers of the alkaloid tacamonine and related compounds.

The HPLC enantiomeric separation of racemic indole alkaloids tacamonine, 17 alpha-hydroxytacamonine, deethyleburnamonine, and vindeburnol was accomplished using Chiralpak AD and Chiralcel OD as chiral stationary phases. Small structural differences affect the enantioselectivity ability of these phases. Single enantiomers of tacamonine and vindeburnol were isolated by semipreparative HPLC and their CD spectra and optical rotations were measured.     

Chiral Recognition of N‐Phthaloyl,N‐Tetrachlorophthaloyl,and N‐Naphthaloyl α‐Amino Acids and Their Esters on Polysaccharide‐Derived Chiral Stationary Phases

Enantiomeric separations of N‐phthaloyl (N‐PHT), N‐tetrachlorophthaloyl (N‐TCPHT), and N‐naphthaloyl (N‐NPHT) α‐amino acids and their esters were examined on several kinds of polysaccharide‐derived chiral stationary phases (CSPs). Resolution capability of CSPs was greater Chiralcel OF than the others for N‐PHT and N‐NPHT α‐amino acids and their esters. In N‐TCPHT α‐amino acids and their esters, good enantioselectivities showed Chiralcel OG for N‐TCPHT α‐amino acids, Chiralpak AD for N‐TCPHT α‐amino acid methyl esters, and Chiralcel OD for N‐TCPHT α‐amino acid ethyl esters, respectively. From the results of liquid chromatography and computational chemistry, it is concluded that l ‐form is preferred and more retained with electrostatic interaction in case of interaction between N‐PHT α‐amino acid derivatives and Chiralcel OF, N‐TCPHT α‐amino acid derivatives and Chiralcel OD, and N‐NPHT α‐amino acid derivatives and Chiracel OF. On the other hand, d ‐form is preferred and more retained with van der Waals interaction in case of interaction between N‐TCPHT α‐amino acid ester derivatives and Chiralcel OG and Chiralpak AD. Chirality 24:1037–1046, 2012. © 2012 Wiley Periodicals, Inc.