Impact of substituents on the enantioseparation of racemic 2-amidotetralins on polysaccharide stationary phases. I. chiralcel OD

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and π-π interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R_S), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. Chirality 8:574–578, 1996. © 1997 Wiley-Liss, Inc.     

Benzoylcellulose derivatives as chiral stationary phases for open tubular column chromatography

The application of cellulose-based stationary phases for chiral separations has been extended to open tubular column chromatography. Efficient columns were obtained by coating the capillaries with mixtures of chiral cellulose materials and conventional achiral stationary phases for gas chromatography. In this study, various siloxane and polyethylene glycol polymers were used as achiral components and mixed with different substituted benzoylcellulose derivatives as chiral components. Systematic investigations were carried out to determine the optimal ratio for the components of the stationary phase. Depending on the chromatographic mode—gas chromatography (GC) or supercritical fluid chromatography (SFC)—the stationary phases were found to behave differently. The applicability of the technique was demonstrated by the resolution of various racemic compounds. © 1993 Wiley-Liss, Inc.     

Study on the HPLC‐based separation of some ezetimibe stereoisomers and the underlying stereorecognition process

The enantioseparation of ezetimibe stereoisomers by high‐performance liquid chromatography on different chiral stationary phases, ie, 3 polysaccharide‐based chiral columns, was studied. It was observed that cellulose‐based Chiralpak IC column exhibited the best resolving ability. After the optimization of mobile phase compositions in both normal and reversed phase modes, satisfactory separation could be obtained on Chiralpak IC column, especially in normal phase mode. The use of prohibited solvents as nonstandard mobile phase gave rise to better resolution than that of standard mobile phases (n‐hexane/alcohol system). In addition, the presence of ethanol in nonstandard mobile phase has played an important role in enhancing chromatographic efficiency and resolution between ezetimibe stereoisomers. Various attempts were made to comprehensively compare the chiral recognition capabilities of immobilized versus coated polysaccharide‐based chiral columns, amylose‐based versus cellulose‐based chiral stationary phases, reversed versus normal phase modes, and standard versus nonstandard mobile phases. Moreover, possible solute‐mobile phase‐stationary phase interactions were derived to explain how stationary and mobile phases affected the separation. Then the method validation with respect to selectivity, linearity, precision, accuracy, and robustness was carried out, which was demonstrated to be suitable and accurate for the quantitative determination of (RRS)‐ezetimibe impurity in ezetimibe bulk drug.     

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.     

Synthesis of polymer-type chiral stationary phases and their enantioseparation evaluation by high-performance liquid chromatography

Two new chiral polymers of different molecular weights were synthesized by the copolymerization of (1R,2R)-(+)-1,2-diphenylethylenediamine, phenyl diisocyanate and terephthaloyl chloride. The polymers were immobilized on aminated silica gel to afford two chiral stationary phases. The polymers and the corresponding chiral stationary phases were characterized by Fourier transform-IR, elemental analysis, 1H and 13C NMR. The surface coverages of chiral structural units on the chiral stationary phases were estimated as 0.27 and 0.39 mmol/g, respectively. The enantioseparation ability of these chiral stationary phases was evaluated with a variety of chiral compounds by high-performance liquid chromatography. The effects of the organic additives, the composition of mobile phases, and the injection amount of sample on enantioseparation were investigated. A comparison of enantioseparation ability between these two chiral stationary phases was made. It was believed that the chain length of polymeric chiral selector significantly affected the enantioseparation ability of corresponding chiral stationary phase.     

Enantiomer separation of amino acids in immunoaffinity micro LC-MS

Chiral immunoaffinity microbore columns were directly interfaced with MS detection, and the effect of column length and temperature on the enantiomer separation of a number of underivatized aromatic and aliphatic amino acids was investigated utilizing an antibody chiral stationary phase that had been prepared by immobilizing a monoclonal anti-D-amino acid antibody onto silica. The stronger affinity of the antibody towards aromatic and bulky amino acids allowed separation of such analytes in a 0.75 x 150 mm column, while an increase in column length enabled separation of more weakly bound compounds. The strength of interaction between chiral selector and analytes could be modulated conveniently by lowering the temperature. For the first time, simultaneous enantiomer separation of mixtures of amino acids was achieved on antibody-based chiral stationary phases using extracted ion chromatograms.     

Chromatographic properties of composite chiral stationary phases based on cellulose derivatives

Chiral stationary phases (CSPs) prepared by mixing together two different cellulose derivatives, before or after being coated on macroporous silica gel, were developed in order to determine the mutual influence of two different polymers on global chiral recognition capacity. The chromatographic properties of these CSPs were evaluated using a wide range of racemic test solutes. The mixing method does not significantly affect the enantioselectivities. The composite CSPs obtained by cocoating of two different cellulose derivatives on silica generally exhibit chiral recognition capacities intermediate between those of the two individual phases, and thus broadening the application range of a single column. These results indicate that the simultaneous coating of two different cellulose derivatives does not significantly alter the optical resolution power of each chiral material and are discussed in relationship with the supramolecular structure of the polymeric stationary phases. © 1995 Wiley-Liss, Inc.     

Interactions of chiral molecules with an (r)-n-(3,5-dinitrobenzoyl) phenylglycine HPLC stationary phase

Forty different chiral molecules were studied by liquid chromatography with a Pirkle-type, (R)-N-(3,5-dinitrobenzoyl) phenylglycine (DNBPG), chiral stationary phase column. The dramatic effect of a small molecular change on chiral recognition was demonstrated using DL-amino acid derivatives. The inductive effect on chiral recognition was also studied using trifluoro-, trichloro-, dichloro-, monochloroacetyl, and acetyl derivatives of four different chiral amines. The study of the enantiomer separation of 11 different crown ethers of 2,2′-binaphthyldiyl showed that the rigidity of the chiral center can be an additional parameter in chiral recognition for the DNBPG phase but not for a β-cyclodextrin bonded chiral phase. It is apparent from this study that steric effects, inductive effects, and molecular rigidity play important roles in chiral recognition with DNBPG chiral stationary phases.     

Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography

The chiral separation of amino acids (AA) derivatised with ethyl chloroformate by using comprehensive two-dimensional gas chromatography is reported. A commercially available enantioselective capillary column (Chirasil-l-Val) has been tested as first-dimension column. Two nonenantioselective stationary phases (BPX50 and BP1) with different column lengths were combined with the enantioselective column, which represent chiral/polar and chiral/low-polarity column sets, respectively. These column sets were evaluated to determine the most useful column combination to provide improved separation efficiency of enantioselective AA analysis. Separations of AA mixtures derivatised either as their N-trifluoroacetyl methyl esters or with methyl chloroformate, performed on a chiral/low-polarity column set, are also shown. The method was demonstrated for chiral analysis of AAs in different beer samples. The major AA in the beer samples was proline with amounts ranging from around 65-95% with minor contents of glycine and the l-enantiomers of alanine, valine, leucine, and isoleucine. Small amounts of d-alanine, at about 1, 1.5, and 15% were detected in the three samples.     

The covalently bonded cellulose tris(3,5-dimethylphenylcarbamate) on a silica monolithic capillary column for enantioseparation in capillary electrochromatography

A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-microm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.     

Functionalities tuned enantioselectivity of phenylcarbamate cyclodextrin clicked chiral stationary phases in HPLC

The mixed chloro‐ and methyl‐ functionalities can greatly modulate the enantioselectivities of phenylcarbamate cyclodextrin (CD) clicked chiral stationary phases (CSPs). A comparison study is herein reported for per(4‐chloro‐3‐methyl)phenylcarbamate and per(2‐chloro‐5‐methyl)phenylcarbamate β‐CD clicked CSPs (i.e., CCC4M3‐CSP and CCC2M5‐CSP). The enantioselectivity dependence on column temperature was studied in both normal‐phase and reversed‐phase mode high performance liquid chromatography (HPLC). The thermodynamic study revealed that the stronger intermolecular interactions can be formed between CCC4M3‐CSP and chiral solutes to drive the chiral separation. The higher enantioselectivities of CCC4M3‐CSP were further demonstrated with the enantioseparation of 17 model racemates in HPLC.     

Immobilization of (1S,2R)-(+)-2-amino-1,2-diphenylethanol derivates on aminated silica gel with different linkages as chiral stationary phases and their enantioseparation evaluation by HPLC

A chiral selector was prepared through the reaction between (1S,2R)-(+)-2-amino-1,2-diphenylethanol and phenyl isocyanate. This selector was immobilized on aminated silica gel, respectively, with bifunctional group linkers of 1,4-phenylene diisocyanate, methylene-di-p-phenyl diisocyanate, and terephthaloyl chloride to produce corresponding three chiral stationary phases. The prepared compounds and chiral stationary phases were characterized by FT-IR, elemental analysis, (1)H NMR, and solid-state (1)H NMR. The enantioseparation ability of these chiral stationary phases was evaluated with structurally various chiral compounds. The chiral stationary phase prepared with 1,4-phenylene diisocyanate as linker showed excellent enantioseparation ability. The influence of different linkages on the enantioseparation was discussed.     

Chiral separations in polar organic solvent chromatography: updating a screening strategy with new chlorine-containing polysaccharide-based selectors

The screening conditions of an existing column and mobile phase selection strategy for chiral drug substances in polar organic solvent liquid chromatography (POSC) were tested for their applicability on two new chlorine-containing polysaccharide-based stationary phases. The selectors of these phases are cellulose tris(3-chloro-4-methylphenylcarbamate) and amylose tris(5-chloro-2-methylphenylcarbamate). The enantioselectivity of these phases is compared to that of the four phases (Chiralpak AD-RH, Chiralcel OD-RH, Chiralpak AS-RH and Chiralcel OJ-RH) used in the earlier defined strategy. A test set of 62 structurally diverse chiral drug substances is analyzed using the screening conditions of the strategy on the six phases. The results confirm that the acetonitrile-based mobile phase provides a higher success rate and better resolutions than the methanol-based also on the new phases. However, the importance of the methanol-based mobile phase cannot be neglected for complementarity reasons; the two mobile phases insure enantioselectivity for different compounds. A third (ethanol-based) mobile phase, not part of the strategy, was also used to screen the two new phases. The joint results led to different possibilities to upgrade the current screening strategy so that improved success rates are obtained. The chlorine-containing chiral stationary phases demonstrated to have an added value to the screening process since they provided enantioresolution for compounds not resolved by non-chlorine-containing ones.     

Memory effect of diethylamine mobile phase additive on chiral separations on polysaccharide stationary phases

The existence of a memory effect for amine additives on polysaccharide chiral stationary phases has often been suggested, but not clearly demonstrated. Demonstration of this effect is made difficult by the uncertainty as to which analytes benefit from use of amine additives and, typically, an unclear history of column use. In this work, analytes were selected for differences in their behavior with and without additives. Columns were used with no prior history. A persistent memory effect was demonstrated on a CHIRALPAK AD-H column in hexane-based mobile phases. This effect was short-lived, with polar organic mobile phases. Memory was short-lived on a CHIRALCEL OJ-H column. Flushing with isopropanol was shown to remove most of the memory effect. Compounds expected to require amine additives on CHIRALCEL OD-H column did not. Acid treatment of the AD-H and OD-H columns changed their performance, which was subsequently restored by the incorporation of amine.     

Novel chiral ionic liquids stationary phases for the enantiomer separation of chiral acid by high‐performance liquid chromatography

Novel chiral ionic liquid stationary phases based on chiral imidazolium were prepared. The ionic liquid chiral selector was synthesized by ring opening of cyclohexene oxide with imidazole or 5,6‐dimethylbenzimidazole, and then chemically modified by different substitute groups. Chiral stationary phases were prepared by bonding to the surface of silica sphere through thioene “click” reaction. Their enantioselective separations of chiral acids were evaluated by high‐performance liquid chromatography. The retention of acid sample was related to the counterion concentration and showed a typical ion exchange process. The chiral separation abilities of chiral stationary phases were greatly influenced by the substituent group on the chiral selector as well as the mobile phase, which indicated that, besides ion exchange, other interactions such as steric hindrance, π‐π interaction, and hydrogen bonding are important for the enantioselectivity. In this report, the influence of bulk solvent components, the effects of varying concentration, and the type of the counterion as well as the proportion of acid and basic additives were investigated in detail.     

Vancomycin‐based chiral stationary phases for micro‐column liquid chromatography

Vancomycin selectively immobilized to silica via either one of its two amino groups has been investigated and compared with columns made from native vancomycin. The chemical modification of vancomycin prior to immobilization involved protection of one amino group as a 9‐fluorenylmethyl carbamate. The immobilization and the subsequent cleavage of the protecting group was performed on‐column. The types of compounds that can be separated with the vancomycin chiral stationary phases resemble those separated previously by capillary electrophoresis and thin‐layer chromatography. The protected chiral stationary phases were also investigated and in some cases very high enantioselectivity were obtained. One example of this is a separation of thalidomide with an α‐value as high as 5.4. The soft immobilization procedure preserves the structure of native vancomycin, in contrast to other approaches. Good repeatability and stability of the columns have also been obtained. Chirality 11:121–128, 1999. © 1999 Wiley‐Liss, Inc.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

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.     

Enantiomeric LC separation of calcium antagonists on protein-based chiral stationary phases

Three chiral calcium antagonist drugs, bepridil and two dihydropyridine derivatives (nicardipine and REC 15/2375), have been successfully separated within short retention times using either the α₁-acid glycoprotein chiral stationary phase (Chiral AGP) or the ovomucoid column (Ultron ES-OVM). Aqueous buffer at defined pH is modified by the addition of an organic component (propan-2-ol, acetonitrile, ethanol) in order to modulate the retention properties of each system. The influence of pH and percentage of organic modifier on retention, selectivity, resolution, and column performance are discussed for bepridil analyzed on Chiral AGP and for the two dihydropyridines (nicardipine and REC 15/2375) analyzed on Ultron ES-OVM stationary phases. © 1993 Wiley-Liss, Inc.     

Enantiomeric separation of mineralocorticoid receptor (hMR) antagonists using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems

This study demonstrates the increased versatility of the Chiralcel OJ-H stationary phase when using various alcohol/acetonitrile mobile phases. This chiral stationary phase has traditionally been employed in the normal phase mode and more recently with neat alcohols as eluents. Selected isomeric human mineralocorticoid receptor (hMR) antagonist pharmaceutical candidates and synthetic intermediates were separated using the Chiralcel OJ-H HPLC column with novel polar cosolvent eluent systems. The capacity factors, resolution, and selectivity of the chiral separations were assessed while varying the alcohol/acetonitrile composition and alcohol identity. The mixed polar eluents provide separations that are nearly always superior to both the traditional hexane-rich and single-alcohol "polar organic" eluents for the compounds tested in this article.