High‐performance liquid chromatographic separations of stereoisomers of chiral basic agrochemicals with polysaccharide‐based chiral columns and polar organic mobile phases

The separation of the stereoisomers of 23 chiral basic agrochemicals was studied on six different polysaccharide‐based chiral columns in high‐performance liquid chromatography with various polar organic mobile phases. Along with the successful separation of analyte stereoisomers, emphasis was placed on the effect of the chiral selector and mobile phase composition on the elution order of stereoisomers. The interesting phenomenon of reversal of enantiomer/stereoisomer elution order function of the polysaccharide backbone (cellulose or amylose), type of derivative (carbamate or benzoate), nature, and position of the substituent(s) in the phenylcarbamate moiety (methyl or chloro) and the nature of the mobile phase was observed. For several of the analytes containing two chiral centers all four stereoisomers were resolved with at least one chiral selector/mobile phase combination.     

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Enantioseparation of aldols by high‐performance liquid chromatography on polysaccharide‐based chiral stationary phases that bear chlorinated substituents

Two families of aldols, obtained from the condensation of aromatic aldehydes with cyclohexanone or acetone (ten examples in each group), were analyzed by high‐performance liquid chromatography in normal phase elution mode on three polysaccharide‐based chiral stationary phases of the Lux series, namely, Lux Cellulose‐2, Lux Cellulose‐4 and Lux Amylose‐2, which share the common feature of chlorinated substituents in the chiral selectors. Following simple optimization steps, the enantioseparation of all aldols derived from cyclohexanone was achieved and the highest values of separation factor (α, 1.32 < α < 2.20) and resolution (R_s, 4.5 < R_s <17.2) were observed on Lux Cellulose‐2, with the only exception of the 4‐nitro‐substituted derivative that was better resolved on Lux Cellulose‐4. On the contrary, Lux Amylose‐2 was the best choice for aldols derived from acetone and only specific analytes in this group were resolved on the cellulose‐based supports. A variable‐temperature study of selected compounds allowed us to determine thermodynamic parameters of the enantioseparation process, which was enthalpy‐controlled in all the cases except one.     

Monolithic silica columns functionalized with substituted polyproline‐derived chiral selectors as chiral stationary phases for high‐performance liquid chromatography

In this study, two polyproline‐derived chiral selectors are bonded to monolithic silica gel columns. In spite of high chiral selector coverage, the derivatization was found to have only a slight effect on the hydrodynamics of the mobile phase through the column. The enantioseparation ability of the resulting chiral monolithic columns was evaluated with a series of structurally diverse racemic test compounds. When compared to analogous bead‐based chiral stationary phases, higher enantioseparation and broader application domain were observed for monolithic columns. Moreover, the increase in flow rate produces a minor reduction of resolution, which permits to shorten analysis time. Additionally, increased loadability defines chiral polyproline derived monoliths as adequate for preparative chromatography.     

How mobile phase composition and column temperature affect enantiomer elution order of liquid crystals on amylose tris(3-chloro-5-methylphenylcarbamate) as chiral selector

A comprehensive study into the effects of mobile phase composition and column temperature on enantiomer elution order was conducted with a set of chiral rod-like liquid crystalline materials. The analytes were structurally similar and comprised variances such as length of terminal alkyl chain, presence of chlorine, number of phenyl rings, and type of chiral center. Experiments were carried out in polar organic and reversed-phase modes using amylose tris(3-chloro-5-methylphenylcarbamate) immobilized on silica gel as the chiral stationary phase. For all liquid crystals, reversal of elution order of enantiomers was observed based on type of used cosolvent and/or its content in the mobile phase; for some of the liquid crystals a temperature-induced reversal was also observed. Both linear and nonlinear dependencies of natural logarithm of enantioselectivity on temperature were found. Tested mobile phases comprised pure organic solvents and binary and tertiary mixtures of acetonitrile with organic solvents and/or water. Effect of acidic/basic mobile phase additives was also tested. Effect of structure of chiral selector is briefly discussed.     

Enantiomer elution order reversal of fluorenylmethoxycarbonyl-isoleucine in high-performance liquid chromatography by changing the mobile phase temperature and composition

In this paper the elution order reversal of enantiomers of fluorenylmethoxycarbonyl- or FMOC-isoleucine is described depending on the separation temperature and composition of the mobile phase when using the polysaccharide-based chiral column Lux Cellulose-1 in HPLC with normal-phase eluent. Reversal of the enantiomer elution order (EEO) in HPLC depending on the column temperature and content of the polar modifier in the mobile phase has been reported before in the literature. However, EEO reversal by changing the content of acidic modifier in the mobile phase seems to be described for the first time in the present work.     

Structure‐enantioselectivity relationships for the study of chiral recognition in peptide enantiomer separation on cinchona alkaloid‐based chiral stationary phases by HPLC: Influence of the N‐terminal protecting group

Eleven different N‐terminal protecting groups (acetyl, benzoyl, FMOC, etc.) were employed for the HPLC separation of oligoalanine peptide enantiomers containing up to six amino acids. Isocratic HPLC separations were performed using a hydro‐organic buffered mobile phase and 4 mm ID columns containing three different chiral anion exchange stationary phases based on cinchona alkaloid‐derived chiral selectors. For most peptides successful separations could be obtained with all protecting groups, although those comprising aromatic moieties were found to yield higher enantioselectivities than those with aliphatic residues, since they are capable of undergoing favourable π‐π interactions with the selector. Systematic investigations concerning the presence or absence of structural features of related protecting groups showed that the use of protecting groups that are optimally adjusted to the binding pocket of the chiral selector effects a significant gain in enantioselectivity. At the same time these studies provided new insights into the chiral recognition mechanism.     

Liquid chromatographic enantiomer separations of novel quinazolone derivatives on quinine carbamate based chiral stationary phases using hydro-organic mobile phases

Quinine carbamate-type weak chiral anion-exchange selectors (SOs) and the respective chiral stationary phases (CSPs) have been used for the direct liquid chromatographic enantiomer separation of a wide range of chiral acids. In the present work, we demonstrate that these CSPs can also be extended to chiral discrimination of a set of neutral polar potential NMDA (N-methyl-D-aspartic acid) and/or AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) antagonist imidazo-quinazoline-dione derivatives (selectands, SAs) using acetonitrile and methanol containing hydro-organic and buffered mobile phases. The influence of mobile phase composition, column temperature and structure variation of the SAs and SOs on retention and enantioselectivity was systematically investigated to gain insight into the overall chiral recognition mechanism. As was expected for the reversed-phase mode, acetonitrile has a stronger eluotropic effect compared to methanol. Except for two analytes, the acetonitrile containing mobile phases provided baseline resolution (R(S)) of the enantiomers with R(S) values ranging between 1.68 and 2.76. Using methanol as the organic modifier enhanced the enantioselectivity. The enthalpic and entropic terms for the SO-SA association were calculated from the linear van't Hoff plots. Data reveal that the enantiomer separations are predominantly enthalpically driven.     

Study of the enantiomeric separation of the anticholinergic drugs on two immobilized polysaccharide‐based chiral stationary phases by HPLC and the possible chiral recognition mechanisms

In this work, the enantiomeric separation of ten anticholinergic drugs was first examined on two derivative polysaccharide chiral stationary phases (CSPs), i.e., Chiralpak ID and Chiralpak IA in the normal phase mode. Except for scopolamine hydrobromide, the remaining nine analytes could be completely separated with good resolutions using both columns under the optimized mobile phase conditions. And the enantiomeric discrimination ability of the studied CSPs towards nine analytes was in the order of Chiralpak ID > Chiralpak IA. The influences of organic modifier types, alcohol content, and base/acid additives on the enantiomeric separation were evaluated and optimized. According to the experimental results, the effect of the structures of analytes on enantiomeric separation was discussed. Additionally, the chiral recognition mechanisms were proposed based on the thermodynamic analysis of the experimental data.     

Use of native and derivatized cyclodextrin chiral stationary phases for the enantioseparation of aromatic and aliphatic sulfoxides by high performance liquid chromatography

Summary The enantioselectivity of native and derivatized cyclodextrin stationary phases for aromatic and aliphatic chiral sulfoxides was evaluated using high performance liquid chromatography (HPLC). Many sulfoxide enantiomers could be baseline resolved using the derivatized cyclodextrin stationary phases in the reverse phase mode. The most important factor influencing enantioselectivity is the presence of steric bulk alpha to the chiral center. However, substituents on an aromatic ring bonded to the sulfoxide have less pronounced effects on enantioselectivity. The 2,3-dimethyl β-cyclodextrin exhibits the broadest anantioselectivity for neutral chiral sulfoxides. Native cyclodextrins and hydroxypropyl-β-cyclodextrins were much less effective in separating this class of molecules. The hydrogen bonding ability of the organic modifier does not significantly affect enantioselectivity.     

Determination of enantiomeric purity of S‐amlodipine by chiral LC with emphasis on reversal of enantiomer elution order

An LC method was developed and prevalidated for the enantiomeric purity determination of S‐amlodipine in polar organic solvent chromatography using a chlorine‐containing cellulose‐based chiral stationary phase (CSP). The concentration of formic acid (FA) (0.01–0.2%) in the mobile phase containing acetonitrile as the main solvent was found to influence the elution order of amlodipine enantiomers as well as the enantioresolution. A reversal of the enantiomer elution order of amlodipine was only observed with chiral stationary phases with both electron‐withdrawing (chloro) and electron‐donating groups (methyl) on the phenyl moieties of the chiral selector, namely cellulose tris(3‐chloro‐4‐methylphenylcarbamate) and cellulose tris(4‐chloro‐3‐methylphenylcarbamate). The highest enantioresolution (R_s: 4.1) value was obtained at the lowest FA concentration (0.01%) using cellulose tris(4‐chloro‐3‐methylphenylcarbamate) as the chiral selector and the enantiomeric impurity, R‐amlodipine, eluted first under these conditions. Therefore, the mobile phase selected for the prevalidation of the method consisted of ACN/0.1% DEA/0.01% FA and the temperature was set at 25°C. The method was prevalidated by means of the strategy based on the total measurement error and the accuracy profile. The method was found to be selective and the limit of quantification was found to be about 0.05% for R‐amlodipine, while the limit of detection was close to 0.02%.     

Analytical and semipreparative chiral separation of cis‐itraconazole on cellulose stationary phases by high‐performance liquid chromatography

cis‐Itraconazole is a chiral antifungal drug administered as a racemate. The knowledge of properties of individual cis‐itraconazole stereoisomers is vital information for medicine and biosciences as different stereoisomers of cis‐itraconazole may possess different affinity to certain biological pathways in the human body. For this purpose, either chiral synthesis of enantiomers or chiral separation of racemate can be used. This paper presents a two‐step high‐performance liquid chromatography approach for the semipreparative isolation of four stereoisomers (two enantiomeric pairs) of itraconazole using polysaccharide stationary phases and volatile organic mobile phases without additives in isocratic mode. The approach used involves the separation of the racemate into three fractions (i.e. two pure stereoisomers and one mixed fraction containing the remaining two stereoisomers) in the first run and consequent separation of the collected mixed fraction in the second one. For this purpose, combination of cellulose tris‐(4‐methylbenzoate) and cellulose tris‐(3,5‐dimehylphenylcarbamate) columns with complementary selectivity for cis‐itraconazole provided full separation of all four stereoisomers (with purity of each isomer > 97%). The stereoisomers were collected, their optical rotation determined and their identity confirmed based on the results of a previously published study. Pure separated stereoisomers are subjected to further biological studies.     

Chiral separation of lenalidomide by liquid chromatography on polysaccharide‐type stationary phases and by capillary electrophoresis using cyclodextrin selectors

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

Enantiomeric resolution of new aromatase inhibitors by liquid chromatography on cellulose chiral stationary phases

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct enantioseparation of substituted [1-(imidazo-1-yl)-1-phenylmethyl)]-benzothiazolinone and benzoxazolinone derivatives with one chiral center. Those analogues of fadrozole constitute new potent nonsteroidal inhibitors of aromatase (P450 arom). The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes along with the temperature of the column on the discrimination between the enantiomers were examined. Baseline separation (Rs > 1.5) was easily obtained in many cases.     

Enantiomeric analysis of simendan on polysaccharide‐based stationary phases by polar organic solvent chromatography

Herein, the enantiomeric separation of simendan by high‐performance liquid chromatography with ultraviolet detection using polysaccharide‐based chiral stationary phases in polar organic mode is described. Three chiral columns (Chiralpak AD‐H, Chiralcel OD‐H, and Chiralpak AS) were screened using pure methanol and acetonitrile without additives under isocratic conditions. A reversed elution order was observed on the Chiralpak AD‐H column when the methanol content in the mobile phase (methanol–acetonitrile mixtures) was above 10%, whereby levosimendan eluted prior to dextrosimendan. Further, it was found that increasing temperature effectively improved the enantioresolution on the Chiralpak AD‐H column. Van't Hoff analysis was performed to evaluate the contribution of enthalpy and entropy to the chiral discrimination process. The best enantioseparation (α = 3.00, Rs = 12.85) was obtained on the Chiralpak AD‐H column with methanol as the mobile phase at 40°C. Thus, a quantitative method for the resolution of dextrosimendan was established and validated, which could be used as a reference for the determination of dextrosimendan in levosimendan products.     

A new application of stopped-flow chiral HPLC: inversion of enantiomer elution order

A newly developed procedure to reverse the enantiomer elution order of compounds resolved on chiral stationary phases (CSPs) for HPLC is presented. The optimized analytical protocol is based on the effect of temperature on enantioselectivity and does not involve any changing in mobile phase composition or type of CSP. In essence, the approach entails variable temperature chromatography at two temperatures. The enantiomer separation is performed at a low column temperature, with stopping the flow prior to elution of the less retained enantiomer. Then, the column temperature is changed with the peaks trapped inside the column, followed by elution with the same mobile phase in reverse direction. Under these conditions, the more pronounced loss in free energy of binding for the more strongly bound enantiomer results in an inversion of the elution order. This procedure may be applied to each enantiomer pair that is separated by chiral HPLC under an appreciable enthalpy-control.     

Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

A unique reversal of elution order during direct enantiomeric resolution of amide derivatives of 1-phenyl-2-aminopropane by high performance liquid chromatography on chiral stationary phases

Enantiomeric amide derivatives of (S)- and (R)-1-phenyl-2-aminopropane (dextro- and levoamphetamine, respectively) were resolved by high performance liquid chromatography on commercially available ionically and covalently bonded chiral stationary phases ((R)-N-(3,5-dinitrobenzoyl)phenylglycine). Ten enantiomeric amide pairs were synthesized and chromatographed on the columns by using a mobile phase of hexane-isopropanol (97 : 3), a flow rate of 2 ml/min, and a column temperature of 20°C. The (R)-isomer of all 10 amides eluted first on the covalent column as did the (R)-isomer of nine derivatives on the ionic column. however, the 3,5-dinitrobenzoyl amide of (S)- amphetamine eluted before the (R)-isomer on the ionic column. This reversal in enantiomeric elution order reveals the complexity of the interactions occurring on these columns and emphasizes the hazards of relying on observed elution order as an a priori indication of absolute configuration.     

Improved preparation of chiral stationary phases via immobilization of polysaccharide derivative‐based selectors using diisocyanates

The classical method for the preparation of immobilized polysaccharide‐based chiral stationary phases (CSPs) with a diisocyanate was improved. Cellulose or amylose was directly coated onto 3‐aminopropyl silica gel after it was dissolved in a mixture of N,N‐dimethylacetamide, LiCl, and pyridine, then immobilized onto silica gel with a diisocyanate, and finally allowed to react with an excess of corresponding isocyanate. Four polysaccharide derivatives, 3,5‐dimethylphenylcarbamate and 3,5‐dichlorophenylcarbamate of cellulose, and 3,5‐dimethylphenylcarbamate and 5‐chloro‐2‐methylphenylcarbamate of amylose, were immobilized onto silica gel utilizing this method. Compared with the classical diisocyanate method, the improved procedure avoided the derivatization and regeneration of 6‐hydroxyl groups of cellulose and amylose, and thus showed an advantage for simple and economical preparation. The relationships among the amount of diisocyanate used, immobilization efficiency, and enantioseparation on the cellulose‐based CSPs were investigated. Also, the solvent durability of the obtained CSPs was examined with eluents containing chloroform or THF. By utilizing these eluents, the chiral recognition abilities of the obtained CSPs for some of the tested racemates were improved.