Chromatographic Chiral Stationary Phase Research Articles

Liquid Chromatographic Chiral Separations by Crown Ether‐Based Chiral Stationary Phases

Liquid chromatographic chiral stationary phases (CSPs) based on chiral crown ethers have been successfully utilized in the separation of the enantiomers of various racemic compounds containing a primary amino group. Especially, CSPs based on chiral crown ethers incorporating chiral binaphthyl unit or tartaric acid unit have been most successful. In this review, the development of CSPs based on chiral crown ethers incorporating a chiral binaphthyl unit or a tartaric acid unit, their applications to the resolution of various primary and non‐primary amino compounds with the variation of the type and the content of organic, acidic, and inorganic modifiers in an aqueous mobile phase or with a non‐aqueous mobile phase and the efforts to improve the chiral recognition efficiency or the stability of the CSPs have been discussed.

Resolution of β-Amino Acids on a Chiral Stationary Phase Based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxilic Acid without Extra Free Aminopropyl Groups on Silica Surface

A liquid chromatographic chiral stationary phase (CSP) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxilic acid without extra free aminopropyl groups on silica surface has been demonstrated to be quite effective for the resolution of various /?-amino acids. The retention factors (k 1 ) for the resolution ofy β-amino acids on the CSP were quite large and the large retention factors might be quite attractive along with the reasonable separation factors (a) for preparative scale enantioselective chromatography. The large retention factors on the CSP were found to be reduced effectively by adding ammonium ion to mobile phase without sacrificing the chiral recognition efficiency of the CSP. Consequently, the CSP is also quite applicable for use in analytical enantioselective chromatography.

Preparation of a New Chiral Stationary Phase Based on (2S,3S)-O,O'-Bis-(10-undecenoyl)-N,N'-bis-(3,5-dinitrobenzoyl)-2,3-diamino-1,4-butandiol and Its Application

A new liquid chromatographic chiral stationary phase based on (2S,3S)-O,O'-bis-(10-undecenoyl)-N,N'-bis-(3,5-dinitrobenzoyl)-2,3-diamino-1,4-butandiol was prepared starting from (2R,3R)-1,4-bis(benzyloxy)-2,3-butanediol. The new chiral stationary phase was applied to the resolution of racemic anilide derivatives of N-acetyl-a-amino acids, 1,1'-bi-2-naphthol and 3,3'-diaryl-1,1'-bi-2-naphthols. The CSP was also applied to the resolution of some chiral drugs including a diuretic, bendroflumethiazide, and non-steroidal anti-inflammatory agents such naproxen and alminoprofen. In every case, the chiral recognition efficiency of the new CSP was quite excellent.ࡺ?⨀ЀĀ衺?⨀ЀĀऐ돀졽?⨀堘�⨀ꢐ?⨀돐좐?⨀��⨀킐?⨀뤏덐?⨀렏頏࢑?⨀਀࢑?⨀ᢑ?⨀؀ကᢑ?⨀?幺Ѐက䂑?⨀䂑?⨀

Effect of the residual silanol group protection on the liquid chromatographic resolution of racemic primary amino compounds on a chiral stationary phase based on optically active (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6

A liquid chromatographic chiral stationary phase (CSP) based on (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6, which has been utilized in the resolution of α-amino acids, amines and amino alcohols, was treated with excess of n-octyltriethoxysilane to prepare a new improved CSP. The residual silanol groups of the original CSP were protected by n-octyl groups in the new CSP. The chiral recognition ability of the new CSP was superior to that of the original CSP in the resolution of α-amino acids, amines and amino alcohols. Retention factors ( k 1) for the resolution of α-amino acids were lower on the new CSP than on the original CSP while those for the resolution of amines and amino alcohols were higher on the new CSP than on the original CSP. The improved chiral recognition ability of the new CSP and the retention behaviors of the two enantiomers on the new CSP have been rationalized to stem from the removal of the non-enantioselective interactions between the analytes and the residual silanol groups of the original CSP and the improved lipophilicity of the CSP.

Combining the Enantioselectivities of l-Valine Diamide and Permethylated β-Cyclodextrin in One Gas Chromatographic Chiral Stationary Phase

An L-valine diamide chiral selector was attached to a polysiloxane through a long hydrocarbon spacer giving rise to a chiral stationary phase (CSP), Chirasil-Val-C11. The enantioselective properties of this readily accessible diamide CSP under gas chromatographic conditions were found to be similar to that of the commercially available Chirasil-Val CSP prepared by a polymer-analogous route. A new binary CSP, Chirasil-DexVal-C11, was synthesized by means of simultaneous attachment of both the L-valine diamide and permethylated beta-cyclodextrin selectors to a polysiloxane using platinum-catalyzed hydrosilylation, thereby overcoming the immiscibility problem known for Chirasil-Val and Chirasil-Dex. This binary CSP retained both the enantioselectivity of Chirasil-Val-C11 toward alpha-amino acid derivatives and the unsurpassed enantioselectivity of Chirasil-Dex toward underivatized chiral alcohols, ketones, and hydrocarbons. Furthermore, it was shown that the presence of the cyclodextrin selector in Chirasil-Val-C11 significantly improved the enantioseparation of proline, which represented a problematic amino acid on diamide CSPs.

Preparation and application of HPLC chiral stationary phases based on (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid

Preparation of liquid chromatographic chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid and their application are reviewed. The various methods of connecting (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel covalently or dynamically are demonstrated. The CSPs based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid have been very successful for the resolution of various primary amino compounds with the use of an aqueous mobile phase containing organic and acidic modifiers. In addition, the resolution of secondary amino compounds including beta-blockers and N-(3,5-dinitrobenzoyl)-alpha-amino acids has been demonstrated on a CSP based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid with a non-aqueous mobile phase.

Liquid chromatographic enantioseparation of aryl?-amino ketones on a crown ether-based chiral stationary phase

A liquid chromatographic chiral stationary phase based on (3,3'-diphenyl-1,1'-binaphthyl)-20-crown-6 covalently bonded to silica gel was applied in the resolution of aryl alpha-amino ketones including cathinone, the main psychoactive alkaloid found in the leaves of the khat plant. The resolution was excellent, the separation factors ranging between 1.72 and 8.58 and the resolution factors (R(S)) ranging between 2.60 and 11.10. The chromatographic resolution behaviour was dependent on the type and the content of organic and acidic modifiers and the ammonium acetate concentration in aqueous mobile phase and the column temperature.

Chiral investigation of midodrine, a long-acting alpha-adrenergic stimulating agent.

Midodrine hydrochloride is a peripheral alpha(1)-adrenoreceptor agonist that induces venous and arterial vasoconstriction. Midodrine, after oral or intravenous administration, undergoes enzymatic hydrolysis and releases deglymidodrine, a pharmacologically active metabolite. Midodrine and deglymidodrine have a chiral carbon in the 2-position. To investigate the bioactivity of racemates and enantiomers of the drug and metabolite, three chromatographic chiral stationary phases, Chiralcel OD-H, Chiralcel OD-R, and alpha(1)-AGP, were evaluated for enantiomeric resolution. Good enantioseparation of midodrine racemate was obtained using the Chiralcel OD-H column. This stationary phase was then used to collect separately the midodrine enantiomers. By alkaline hydrolysis of rac-midodrine and each separated enantiomer, rac-deglymidodrine and its enantiomers were prepared. The control of the enantiomeric purity was carried out by alpha(1)-AGP stationary phase, while the hydrolysis of rac-midodrine and its enantiomers was controlled by capillary electrophoresis using trimethyl-beta-cyclodextrin as chiral selector. The pharmacological activity of the two racemates and the two enantiomeric pairs was tested in vitro on a strip of rabbit descending thoracic aorta. The tests continued that the activity of the drug and metabolite is due only to the (-)-enantiomer because neither of the (+)-enantiomers is active.

Effect of analyte lipophilicity on the resolution of α‐ and β‐amino acids on liquid chromatographic ligand exchange chiral stationary phases

AbstractSeparation of the two enantiomers of racemic α‐ and β‐amino acids on two ligand exchange chiral stationary phases (CSPs) prepared previously by covalently bonding sodium N‐((S)‐1‐hydroxymethy‐3‐methylbutyl)‐N‐undecylaminoacetate or sodium N‐((R)‐2‐hydroxy‐1‐phenylethyl)‐N‐undecylaminoacetate on silica gel was studied with variation of the organic modifier (methanol) concentration in the aqueous mobile phase. In particular, the variation of retention factors with changing organic modifier concentration in the aqueous mobile phase was found to be strongly dependent on both the analyte lipophilicity and the stationary phase lipophilicity. In general, the retention factors of relatively lipophilic analytes on relatively lipophilic CSPs tend to increase with increasing organic modifier concentration in the aqueous mobile phases while those of less lipophilic or hydrophilic analytes tend to increase. However, only highly lipophilic analytes show decreasing retention factors with increasing organic modifier concentration in the aqueous mobile phase on less lipophilic CSPs. The contrasting retention behaviors on the two CSPs were rationalized by the balance of the two competing interactions, viz. hydrophilic interaction of analytes with polar aqueous mobile phase and the lipophilic interaction of analytes with the stationary phase.

Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC–MS) has been developed and validated for the simultaneous determination of saliva concentrations of ( R)- and ( S)-methadone (Met) and ( R)- and ( S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC–MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized α 1-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10 mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min and a temperature of 25 °C. Under these conditions, enantioselective separations were observed for methadone ( α=1.30) and EDDP ( α=1.17) within 15 min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/ z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0–600.0 ng/ml, and for EDDP from 0.5 to 15.0 ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5 ng/ml and for EDDP 0.5 ng/ml. Acceptable intra- and inter-day precision of the method (CVs<4.0%) and accuracy (CVs<4.0%) were obtained. These findings demonstrate the accuracy and precision of the method used to successfully analyze saliva obtained from patients enrolled in a methadone-maintenance program.

Resolution of Tocainide and Its Analogues on Liquid Chromatographic Chiral Stationary Phases Based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxylic Acid

Two liquid chromatographic chiral stationary phases (CSPs) based on (+)-(18-crown-6)-2,3, 11,12-tetracarboxylic acid were successfully applied in the resolution of racemic tocainide and its analogues. In the resolution oftocainide, especially, the CSP containing N-CH 3 amide tethering groups was quite effective, showing clear baseline resolution (R s : 2.66) with reasonable enantioselectivity (a: 1.25). Consequently, the CSP containing N-CH 3 amide tethering groups is expected to be useful to monitor the enantiomeric composition of tocainide in clinical samples. In addition, the chromatographic behaviors for the resolution of tocainide and its analogues on the two CSPs were found controllable by varying the content and the type of organic and acidic modifiers in aqueous mobile phase.

Determination of the enantiomers of ketamine and norketamine in human plasma by enantioselective liquid chromatography–mass spectrometry

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection has been developed and validated for the simultaneous determination of plasma concentrations of ( R)- and ( S)-ketamine, and ( R)- and ( S)-norketamine. The compounds were extracted from human plasma using solid-phase extraction and then directly injected into the LC–MS system for detection and quantification. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase based upon immobilized α 1-acid glycoprotein (the Chiral AGP column). The separations were achieved using a mobile phase composed of 2-propanol–ammonium acetate buffer (10 m M, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml/min and a temperature of 25 °C. Under these conditions, the analysis time was 20 min. Detection of the ketamine, norketamine and bromoketamine (internal standard) enantiomers was achieved using selected ion monitoring at m/ z 238.1, 224.1 and 284.0, respectively. Extracted calibration curves were linear from 1 to 125 ng/ml per enantiomer for each analyte with correlation coefficients better than 0.9993 and intra- and inter-day RSDs of less than 8.0%. The method was applied to samples from a clinical study of ketamine in pain management.

Comparison of Chirasil-DEX CB as gas chromatographic and ULMO as liquid chromatographic chiral stationary phase for enantioseparation of aryl- and heteroarylcarbinols

For a broad spectrum of simple chiral alcohols, incorporating a (substituted) (het)aryl building block, enantiomer separation characteristics are reported for both gas chromatography on a Chirasil-DEX phase, and liquid chromatography on an ( S, S)-ULMO phase. On this chiral Pirkle-type phase, homochiral enantiomers (mostly R) are eluted first without exception. The elution order R before S appears conserved as a rule also for gas chromatographic separations on Chirasil-DEX, though with some remarkable exceptions indicating a change in the dominant discriminative mechanism. This was shown in the homologous series 1-phenylethanol to 1-phenylhexanol having the point of reversal at C4, while the o-methoxy analogues elute from C1 to C4 already in the reversed order.

Preparation and application of a new modified liquid chromatographic chiral stationary phase based on (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid

As an effort to improve the chiral recognition efficiency of a previously reported chiral stationary phase (CSP) based on (+)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid, a new CSP was prepared by simply replacing the amide N–H hydrogens of the tethering groups of the old CSP with methyl groups. The new CSP was superior to the old one in the resolution of racemic primary amines. However, in the resolution of α-amino acids and amino alcohols, the new and the old CSPs were complementary with each other. The elution orders on the new CSP were sometimes opposite to those on the old one. Consequently, the chiral recognition mechanism on the new CSP was presumed to be different from that on the old one. The chiral recognition behavior of the new CSP were investigated with four selected analytes and found to be dependent to some extent on the content of organic and acidic modifiers in aqueous mobile phase and the column temperature.

Alkylated and silylated β-cyclodextrin for gas chromatographic chiral stationary phases

Three new chiral stationary phases, 2,6-di-O-heptyl-3-O-trimethylsilyl-β-cyclodextrin (DHTBCD), 2,6-di-O-pentyl-3-O-trimethylsilyl-β-cyclodextrin (DPTBCD) and 2,6-di-O-butyl-3-O-trimethylsilyl-β-cyclodextrin (DBTBCD), were synthesized. Chromatographic properties such as column efficiency, thermal stability and column life span, were studied. The separations of enantiomers, such as ketone, esters, alcohols and olefines, were investigated on the alkylated and silylated β-cyclodextrin stationary phases. The influence of diluent on chiral separation was studied. The experimental results indicate that the stationary phases show good chromatographic properties in separating enantiomers. It is observed that inclusion complexation and hydrogen bonding interaction hardly play a role in separating enantiomers.

Enantioselective separation of racemic secondary amines on a chiral crown ether-based liquid chromatography stationary phase

The first general enantioselective separation of racemic secondary amines on a crown ether-based liquid chromatography chiral stationary phase (CSP) is presented. The CSP is based on (+)- or (−)-(18-crown-6)-2,3,11,12-tetracarboxylic acid covalently bonded to silica gel. A mobile phase containing methanol, acetonitrile, triethylamine and acetic acid was employed in these separations of secondary amines with crown ether CSPs. The separation mechanism is believed to be the secondary amine forming a complex which includes crown ether coordination and electrostatic interaction of the positively charged amine with a carboxylate anion of the immobilized crown ether.

Note on the use of reciprocity of chiral recognition in designing liquid chromatographic chiral stationary phases

A new high-performance liquid chromatographic chiral stationary phase (CSP) was prepared from ( S)- N-(3,5-dimethylbenzoyl)leucine N-phenyl N-allyl amide. The new CSP was applied for the resolution of N-(3,5-dinitrobenzoyl)-α-amino amides and esters and the chromatographic resolution results were compared with those on another CSP derived from ( S)- N-(3,5-dimethoxylbenzoyl)leucine N-phenyl N-allyl amide. The new CSP was found to exert greater enantioselectivity than the other one. These results are contrary to what was expected from the reciprocity of chiral recognition. From these results it was concluded that the reciprocity of chiral recognition should be used with some degree of care in developing effective CSPs or in predicting chromatographic resolution behaviors.

Chiral resolution of flurbiprofen and ketoprofen enantiomers by HPLC on a glycopeptide-type column chiral stationary phase.

Vancomycin is an amphoteric, glycopeptide, macrocyclic antibiotic. When attached to 5 microspherical silica gel, vancomycin proved to be an effective chromatographic chiral stationary phase that could be used in the reversed-phase mode. In this study, a bonded vancomycin chiral stationary phase (Chirobiotic Vtrade mark) was investigated for the chiral liquid chromatography analysis of ketoprofen and flurbiprofen. The selectivity factor (alpha) and the chiral resolution factor (RS) of Chirobiotic Vtrade mark were evaluated first as a function of the buffer pH and molarity, and second as a function of organic modifier type and composition of the mobile phase. Four organic modifiers (tetrahydrofuran, 2-propanol, 1,4-dioxane and methanol) have been tested for their selectivity. Optimized conditions using 20% of tetrahydrofuran in ammonium nitrate (100 mM, pH 5) were selected for the enantioseparation of flurbiprofen and ketoprofen from their racemic forms. At pH 5, these acidic compounds are almost negatively charged, while the chiral selector possesses a positive charge allowing it to interact electrostatistically with the analytes. Using these chromatographic conditions, the column stability was excellent over several months of experiments.

Chiral selectors based on C2-symmetric dicarboxylic acids

Bis-allylamides of rigid C 2-symmetric dicarboxylic acids, useful as precursors in the synthesis of liquid chromatographic chiral stationary phases via hydrosilylation reactions, have been prepared by two different approaches. One involved resolution of the dicarboxylic acid followed by reaction with allylamine via the acid chloride or by a carbodiimide-assisted condensation. The other route involved acetalization of N,N′-diallyl- l-tartardiamide (DATD) with aromatic aldehydes. Moreover, transformation of the enantiopure dicarboxylic acid used in the first route into the corresponding diamine permitted the synthesis of selectors possessing a reversed amide functionality. The enantiomer-discriminating properties of some of these selectors were studied by NMR.

Liquid chromatographic resolution of racemic amino acids and their derivatives on a new chiral stationary phase based on crown ether

A new high-performance liquid chromatography chiral stationary phase (CSP) was prepared by bonding (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid to silica gel. The new CSP was employed in separating the two enantionmers of various natural and unnatural racemic α-amino acids and their derivatives. All natural and unnatural racemic α-amino acids tested were resolved with reasonable separation factors on the new CSP, except for proline, which does not contain a primary amino group. Racemic α-amino acid derivatives were also tested for their separability on the new CSP. In general, N-monoalkyl amides of α-amino acids were resolved better than the corresponding free α-amino acids. However, N, N-dialkyl amides and esters of α-amino acids were not resolved as well as the corresponding free α-amino acids, except for phenylglycine derivatives. In the case of phenylglycine, both N-monoalkyl and N, N-dialkyl amides of phenylglycine were resolved better than phenylglycine itself and its ester derivative.