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.     

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.     

Synthesis and HPLC chiral recognition of regioselectively carbamoylated cellulose derivatives

Four regioselective-carbamoylated cellulose derivatives having two different substituents at 2-, 3-, and 6-position were prepared and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. Investigations showed that the nature and arrangement of the substituents significantly influenced the chiral recognition abilities of the heterosubstituted cellulose derivatives and each derivative exhibited characteristic enantioseparation. Some racemates were better resolved on these derivatives than the corresponding homogeneously substituted cellulose derivatives including a commercial CSP, Chiralcel OD. Racemic compounds shown in this study were most effectively discriminated on cellulose 2,3-(3-chloro-4-methylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate) and 2,3-(3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate).     

Direct high-performance liquid chromatographic separation of the enantiomers of an aromatic amine and four aminoalcohols using polysaccharide chiral stationary phases and acidic additive

The HPLC enantiomeric separation of N-benzyl-alpha-methyl-benzylamine, phenylalaninol, tryptophanol, 2 (diphenylhydroxymethyl)pyrrolidine, and isoproterenol was accomplished in the normal-phase mode using two polysaccharide-derived chiral stationary phases (CSPs) and various n-hexane/2-propanol mobile phases with acidic (TFA) or basic (DEA) additive. The compounds were separated without any derivatization and separation factor range between 2.09 and 1.09 with resolution factor 3.4 and 0.4, respectively. The best separation of the enantiomers of the amine was achieved on amylose tris (3, 5-dimethylphenylcarbamate) CSP with TFA additive in the mobile phase; in acidic conditions, instead, the best enantioseparation of the aminoalcohols was achieved on cellulose tris (3, 5-dimethylphenilcarbamate). A long equilibration time of the CSP when switching from an undoped mobile phase to a doped one is required to obtain reproducible results.     

Enantioseparation of glycyl-dipeptides by CEC using particle-loaded monoliths prepared by ring-opening metathesis polymerization (ROMP)

Novel particle-loaded monolithic capillary electrochromatography (CEC) phases for chiral separations were prepared via ring-opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 microm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8,exo,endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens, RuCl2(PCy3)2(CHPh) as initiator and silica-based particles containing the chiral selector. By suspending silica particles bearing the chiral selector in the polymerization mixture, particle-based monoliths are easily prepared. This approach has several advantages compared to particle-based separation media: (i) the concept of particle-based monoliths is broadly applicable, as any silica-based chiral phase can be used; (ii) they are inexpensive to prepare; and (iii) the manufacturing process is very simple, no sophisticated packing procedures or the preparation of end frits are required. To show the usefulness of this concept for chiral CEC, the chiral separation performance of particle-loaded CEC monoliths bearing teicoplanin aglycone, chemically bonded to 3 microm silica gel, was investigated for a set of glycyl-dipeptides. Particle-loaded ROMP CEC monoliths showed good separation performance for glycyl-dipeptides.     

Preparation and evaluation of regioselectively substituted amylose derivatives for chiral separations

Six novel regioselectively substituted amylose derivatives with a benzoate at 2‐position and two different phenylcarbamates at 3‐ and 6‐positions were synthesized and their structures were characterized by ¹H nuclear magnetic resonance (NMR) spectroscopy. Their enantioseparation abilities were then examined as chiral stationary phases (CSPs) for high‐performance liquid chromatography (HPLC) after they were coated on 3‐aminopropyl silica gels. Investigations indicated that the substituents at the 3‐ and 6‐positions played an important role in chiral recognition of these amylose 2‐benzoate serial derivatives. The derivatives demonstrated characteristic enantioseparation and some racemates were better resolved on these derivatives than on Chiralpak AD, which is one of the most efficient CSPs, utilizing coated amylose tris(3,5‐dimethylphenylcarbamate) as the chiral selector. Among the derivatives prepared, amylose 2‐benzoate‐3‐(phenylcarbamate/4‐methylphenylcarbamate)‐6‐(3,5‐dimethylphenylcarbamate) exhibited chiral recognition abilities comparable to that of Chiralpak AD and may be useful CSPs in the future. The effect of mobile phase on chiral recognition was also studied. In general, with the decreased concentration of 2‐propanol, better resolutions were obtained with longer retention times. Moreover, when ethanol was used instead of 2‐propanol, poorer resolutions were often achieved. However, in some cases, improved enantioselectivity was achieved with ethanol rather than 2‐propanol as the mobile phase modifier.     

Optical resolution of alpha-alkyl phenyl acetonitriles by HPLC on cellulose triacetate chiral stationary phases coated on underivatized silica gel

Microcrystalline celluloses from two sources were used to prepare cellulose triacetate chiral stationary phases (CSPs) coated on underivatized silica gel, which shows discriminating chiral recognition for enantiomers. The chiral separation of four alpha-alkyl phenyl acetonitriles was investigated on the prepared CSPs. It was observed that the concentration of the coating solvent of phenol in dichloromethane plays an important role in the resolution of the solutes. A series of primary alcohols, including secondary and tertiary alcohols, were used as mobile phase modifiers to investigate the effect of the structures of these modifiers on the capacity factors (k') and the separation factors (alpha). Also, the effect of the concentration of alcohol on the capacity factors and separation factors was examined. The chiral recognition mechanism of alpha-alkyl phenyl acetonitriles on the prepared CSPs is discussed. Copyright 2000 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.     

Enantioseparation and molecular modeling study of chiral amines as three naphthaldimine derivatives using amylose or cellulose trisphenylcarbamate chiral stationary phases

This study describes the enantioseparation of three chiral amines as naphthaldimine derivatives, using normal phase HPLC with amylose and cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (CSPs). Three chiral amines were derivatized using three structurally similar naphthaldehyde derivatizing agents, and the enantioselectivity of the CSPs toward the derivatives was examined. The degree of enantioseparation and resolution was affected by the amylose or cellulose-derived CSPs and aromatic moieties as well as a kind of chiral amine. Especially, efficient enantiomer separation was observed for 2-hydroxynapthaldimine derivatives on cellulose-derived CSPs. Molecular docking studies of three naphthaldimine derivatives of leucinol on cellulose tris(3,5-dimethylphenylcarbamate) were performed to estimate the binding energies and conformations of the CSP–analyte complexes. The obtained binding energies were in good agreement with the experimentally determined enantioseparation and elution order.     

Enantioseparation of amfepramone (rac-diethylpropion): preparative separation of the enantiomers and enantioselective analysis

The enantiomers of the anorectic drug amfepramone [rac-diethylpropion, rac-2-(diethylamino)-1-phenyl-1-propanone; rac-DEP] were separated in the preparative scale by crystallization. With enantiopure di-O-benzoyltartaric acid as salt-forming chiral selector, diastereoisomeric salts of DEP enantiomers with a final purity of more than 97.5% were obtained. Analytical liquid chromatographic and electrophoretic methods for the control of the enantiomeric purity and the stoichiometry of the salts were developed. The enantioseparation of rac-DEP by capillary electrophoresis (CE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral discriminator and phosphate buffer (pH 3.3) as run buffer led to good separations. HPLC methods were developed using polysaccharide chiral stationary phases (CSP). The separation of the two enantiomers and the two main degradation products (1-phenyl-1,2-propanedione and propiophenone), known from solid and liquid pharmaceutical preparations, was attained in one run on the silica-based CSP cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD). The conditions which might affect the enantioselectivity and the quality of the enantiomeric separation were investigated for Chiralcel OD and the related CSP amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD). Both CSPs showed very similar chromatographic properties. The separation factors could be influenced significantly by varying the polar organic modifier added to the mobile phase.     

Vancomycin as chiral selector for enantioselective separation of selected profen nonsteroidal anti-inflammatory drugs in capillary liquid chromatography

The chiral selector vancomycin was used either as mobile phase additive or bound as a chiral stationary phase (CSP) for the stereoselective separation of seven racemic nonsteroidal anti-inflammatory drugs (NSAIDs), fenoprofen, carprofen, flurbiprofen, indoprofen, flobufen, ketoprofen, and suprofen, by capillary liquid chromatography. The effect of the type of stationary phase, the chiral column Chirobiotic V or the achiral stationary phases Nucleosil 100 C8 HD and Nucleosil 100 C18 HD, and the concentration of vancomycin in the mobile phase on separation of the drug enantiomers were evaluated. All the drugs, except flobufen, were successfully enantioseparated on Nucleosil 100 C8 HD with 4 mM vancomycin present in the mobile phase (composed of methanol and buffer) in the reversed phase mode. On the vancomycin-bonded chiral stationary phase, it was difficult to get enantioseparations of the profen NSAIDs. However, flobufen gave better enantioseparation on the vancomycin CSP. The better enantioresolution of the majority of profen derivatives on the achiral columns with vancomycin added to the mobile phase can be attributed in particular to the higher separation efficiency of this capillary chromatographic system. In addition, vancomycin dimers, formed in the mobile phase, seem to offer a better steric arrangement for stereoselective interaction to these analytes than the vancomycin bonded on the CSP. These substantial differences in the CS structure significantly influence the chiral discrimination mechanism.     

Separation of nicotine and nornicotine enantiomers via normal phase HPLC on derivatized cellulose chiral stationary phases

This paper describes the enantiorecognition of (±)nicotine and (±)nornicotine by high-performance liquid chromatography using two derivatized cellulose chiral stationary phases (CSPs) operated in the normal phase mode. It was found that different substituents linked to the cellulose backbone significantly influence the chiral selectivity of the derivatized CSP. The results showed that, in general, the tris(4-methylbenzoyl) cellulose CSP (Chiralcel OJ) surpasses tris(3,5-dimethylphenyl carbamoyl) cellulose CSP (Chiralcel OD). On the former column, the resolution (±)nicotine and (±)nornicotine enantiomers depended largely on mobile phase compositions. For the separation of the nicotine enantiomers, the addition of trifluoroacetic acid to a 95:5 hexane/alcohol mobile phase greatly improved the enantioresolution, probably due to enhanced hydrogen bonding interactions between the protonated analytes and the CSP. For (±)nornicotine separation, a reduction in the concentration of alcohol in the mobile phase was more effective than the addition of trifluoroacetic acid. Possible solute-mobile phase-stationary phase interactions are discussed to explain how different additives in the mobile phase and different substituents on the cellulose glucose units of the CSPs affect the separation of both pairs of enantiomers. Chirality 10:364–369, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

Proteins display interesting chiral discrimination properties owing to multiple possibilities of intermolecular interactions with chiral compounds. This review deals with proteins which have been used as immobilized chiral selectors for the enantioseparation of drugs in liquid chromatography and capillary electrophoresis. The main procedures allowing the immobilization of proteins onto matrices, such as silica and zirconia particles, membranes and capillaries are first presented. Then the factors affecting the enantioseparation of drugs in liquid chromatography, using various protein-based chiral stationary phases (CSPs), are reviewed and discussed. Last, chiral separations already achieved using immobilized protein selectors in affinity capillary electrochromatography (ACEC) are presented and compared in terms of efficiency, stability and reproducibility.     

Optical resolution of glycerin sulfides and glycerin selenides on the chiral stationary phase of cellulose‐tris(3,5‐dimethylphenylcarbamate)

Cellulose‐tris(3,5‐dimethylphenylcarbamate) was prepared after a reported method and was coated onto an aminopropylated mesopore spherical silica gel. The final product was used as a chiral stationary phase of high performance liquid chromatography for the enantioseparation of a series of glycerin sulfides and glycerin selenides. Mixtures of hexane and 2‐propanol were used as mobile phases. The effects of 2‐propanol concentration in the mobile phase on the retention and resolution were investigated. Some enantiomers of the glycerin monosulfides and monoselenides could be separated satisfactorily, but none of the disulfides could be separated. The structural features of the solutes that influence chiral separation were discussed. Chirality 11:598–601, 1999. © 1999 Wiley‐Liss, Inc.     

The use of multilayered capillaries for chiral separation by electrochromatography

Fused-silica capillaries were modified by the successively multiple ionic-polymer layer (SMIL) coating technique for a capillary electrochromatography (CEC) analysis of binaphthyl enantiomers. The SMIL coating capillaries consisting of three different polymers (A(+)-B(-)-C(+) coating) were prepared by the alternative deposition of positively charged chiral or achiral polymers and negatively charged DNA. Previous studies have indicated that DNA-cationic polypeptide or synthetic polymer complexes immobilized onto the inner surface of the capillary worked as the chiral stationary phases for 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNP). In this study, to investigate the chiral recognition mechanism and optimize the CEC separation condition in the DNA-cationic polymer coating, effects of the chirality of the polymer unit, the strand of DNA, and the number of layer pairs on the separation were investigated. It should be noted that, since single stranded DNA (ssDNA) was more suitable to immobilize cationic polymers than double stranded DNA, the ssDNA-cationic polymer immobilized capillaries gave a stable electroosmotic flow and reproducible CEC analyses. As a result, a poly(ethyleneimine)-ssDNA-protamine (Prt) coating provided the best chiral separation of BNP. The high separation performance of the prepared capillary is discussed in terms of DNA/polycations interaction.     

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.     

Evaluation of dalbavancin as chiral selector for HPLC and comparison with teicoplanin‐based chiral stationary phases

Dalbavancin is a new compound of the macrocyclic glycopeptide family. It was covalently linked to 5 μm silica particles using two different binding chemistries. Approximately 250 racemates including (a) heterocyclic compounds, (b) chiral acids, (c) chiral amines, (d) chiral alcohols, (e) chiral sulfoxides and sulfilimines, (f) amino acids and amino acid derivatives, and (g) other chiral compounds were tested on the two new chiral stationary phases (CSPs) using three different mobile phases. As dalbavancin is structurally related to teicoplanin, the same set of chiral compounds was screened on two commercially available teicoplanin CSPs for comparison. The dalbavancin CSPs were able to separate some enantiomers that were not separated by the teicoplanin CSPs and also showed improved separations for many racemates. However, there were other compounds only separated or better separated on teicoplanin CSPs. Therefore, the dalbavancin CSPs are complementary to the teicoplanin CSPs. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Recent progresses in protein-based chiral stationary phases for enantioseparations in liquid chromatography

Chiral stationary phases (CSPs) based on proteins orglycoproteins have been developed for the enantioseparations of various compounds. In 2001, a review article [J. Haginaka, J. Chromatogr. A, 906 (2001) 253] dealing with CSPs based on proteins and glycoproteins was published. After that serum albumin from other species, penicillin G-acylase, antibodies, fatty acid binding protein and streptavidin were newly introduced as the chiral selectors in CSPs. This review article deals with recent progresses in CSPs based on protein or glycoproteins in LC after 2001, focusing on their enantioselective properties and chiral recognition mechanisms.     

Liquid Chromatographic Resolution of Amino Acid Esters of Acyclovir Including Racemic Valacyclovir on Crown Ether‐Based Chiral Stationary Phases

Valacyclovir, a potential prodrug for the treatment of patients with herpes simplex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs) based on (3,3’‐diphenyl‐1,1’‐binaphthyl)‐20‐crown‐6 covalently bonded to silica gel. In order to find out an appropriate mobile phase condition, various mobile phases consisting of various organic modifiers in water containing various acidic modifiers were applied to the resolution of valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved quite well on the two CSPs with the separation factors (α) in the range of 2.49 ~ 6.35 and resolutions (R_S) in the range of 2.95 ~ 12.21. Between the two CSPs, the CSP containing residual silanol protecting n‐octyl groups on the silica surface was found to be better than the CSP containing residual silanol groups. Chirality 27:268–273, 2015. © 2015 Wiley Periodicals, Inc.     

Enantiomeric discrimination of pyrethroic acid esters on polysaccharide derived chiral stationary phases

Enantiomeric separation of pyrethroic acid methyl and ethyl esters was examined on cellulose-based chiral stationary phases (CSPs): chiralcel OD (cellulose tris(3,5-dimethylphenyl carbamate)) and chiralcel OF (cellulose tris(4-chlorophenyl carbamate)). The good resolution of pyrethroic acid esters was achieved on chiralcel OD and OF. Separation factors ranged from 1.19-5.12 for Chiralcel OD and 1.00-1.59 for chiralcel OF. Hexane/2-propanol (100:0.15, v/v %) was used as the eluent. The resolution capability of CSPs was greater chiralcel OD than chiralcel OF in the case of the pyrethroic acid esters. The flow rate was 0.8 ml/min and detection was set at 230 nm. The results of the chromatographic data and molecular mechanics suggest that steric effect was a major factor in the enantioseparation. Furthermore, the hydrogen bond between analytes and CSP played an important role in the chiral recognition.