Liquid Chromatographic Chiral Stationary Phase Research Articles

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 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.

Experimental support differenciating two proposed chiral recognition models for the resolution of N-(3,5-Dinitrobenzoyl)-α-arylalkylamines on high-performance liquid chromatography chiral stationary phases

In rationalizing the odd chromatographic behavior for the separation of the enantiomers of N-(3,5-dinitrobenzoyl)-α-arylalkylamines on HPLC chiral stationary phases (CSPs) derived from α-(6,7-dimethyl-1-naphthyl)alkylamines, we initially suggested the occurrence of two competing, opposite sense chiral recognition processes termed the “dipole-stacking process” and the “hydrogen-bonding process”. A simplified “single mechanism” model was later suggested with the importance of face to edge π–π interaction between aromatic rings come to recognized. The initial and subsequent chiral recognition models can be differentiated by noting the chromatographic trends for the enantioseparation of a homologous series of N-(3,5-dinitrobenzoyl)-α-( p-alkylphenyl)ethylamines on the aforementioned CSPs. Data so obtained were consistent with the second “single mechanism” model but not with the first “two competing mechanism” model. From these results, it has been concluded that the “single mechanism” model is more plausible than the “two competing mechanism” model.

Resolution of Racemic N-Acyl-1-arylaminoalkanes on a (3,5-Dinitrobenzoyl)phenylglycinol-Derived High Performance Liquid Chromatography Chiral Stationary Phase

A chiral stationary phase (CSP 2) was prepared by connecting N-(3,5-dinitrobenzoyl)-(R)-phenylglycinol to silica gel through carbamate linkage. The CSP was applied in resolving racemic N-acyl-1-arylaminoalkanes and the chromatographic resolution results were compared with those on the previously reported CSP (CSP 1) derived from N-(3,5-dinitrobenzoyl)-(2S,3R)-norephedrine. From the comparison of the resolution results on CSP 1 and CSP 2, it was concluded that CSP 2 is more effective than CSP 1 in the resolution of racemic N-acyl-1-arylaminoalkanes.

Chitosan derivatives as chiral selectors bonded on allyl silica gel: preparation, characterisation and study of the resulting high-performance liquid chromatography chiral stationary phases

Several mixed 10-undecenoyl/phenylaminocarbonyl or benzoyl derivatives of chitosan, differently substituted in the aromatic ring, were prepared, characterized and immobilized on allylsilica gel. The resulting high-performance liquid chromatography (HPLC) chiral supports were tested chromatographically. The influence of the starting polysaccharidic material, as well as that of the solvents used as mobile phase modifiers, on the preparation and performance of the resulting chiral stationary phases (CSPs) is discussed.

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.

Evaluation of β-lactoglobulin as a stationary phase in high-performance liquid chromatography and as a buffer additive in capillary electrophoresis: observation of a surprising lack of stereoselectivity

Previous studies have reported that α 1-acid glycoprotein is quite similar in amino acid sequence and disulfide bond arrangements to members of a group of proteins which include β-lactoglobulin (BLG). Since generally homologous proteins retain some similarity in function at the molecular level, we decided to evaluate the enantioselective properties of BLG as an high-performance liquid chromatographic chiral stationary phase (HPLC-CSP), and as an additive in capillary electrophoresis (CE). Two columns with differences in internal diameter and method of immobilisation on epoxide silica were prepared. Chiral acidic, basic and uncharged drugs were chromatographed and mobile phase parameters, namely pH and type of organic modifier, were varied in order to test the column performance. The CE approach has some advantages in that there is no need for immobilisation and only a small amount of protein is required. BLG was therefore tested as a CE buffer additive, using the same analytes as in the HPLC study. Although one would expect that a protein would display some enantioselectivity, BLG did not show any enantioselectivity whatsoever in either system; the protein has fairly weak interaction with the majority of the test solutes, as indicated by both techniques.

Molecular recognition in cinchona alkaloid molecular imprinted polymer rods

A series of cinchona alkaloids has been employed as templates for the preparation of in situ molecularly imprinted polymer rods for use as liquid chromatographic chiral stationary phases. The utility of the functional monomers 2-(trifluoromethyl)acrylic acid and methacrylic acid has been compared. The observed differences in selectivity of the imprinted polymer have provided information regarding the nature of functional monomer–template interactions.

3,5-Dimethylphenylcarbamates of amylose, chitosan and cellulose bonded on silica gel: Comparison of their chiral recognition abilities as high-performance liquid chromatography chiral stationary phases

Mixed 10-undecenoyl/3,5-dimethylphenylaminocarbonyl derivatives of amylose and chitosan were prepared and immobilized on allylsilica gel. The enantioselectivities of the resulting supports were compared to those of an analogous cellulose-derived chiral stationary phase previously prepared.

Enantioselective determination of isradipine in human serum using chiral stationary phase liquid chromatography and gas chromatography with nitrogen selective detection.

rac-Isradipine is a dihydropyridine type calcium antagonist. Its calcium entry blocking effect is due primarily to the (+)-(S)-enantiomer. This study describes a sensitive enantioselective method for the determination of isradipine in human serum. Following alkaline extraction into hexane, the enantiomers of isradipine are separated quantitatively by high-performance liquid chromatography on a Chiralcel OJ column at 39 degrees C. The collected fractions were evaporated and assayed using capillary gas chromatography on a HP 50+ column with nitrogen selective detection. Using 2.0 ml of serum, 0.7 nmol/1 (0.26 ng/ml) of each enantiomer could be determined with acceptable precision. The method has successfully been used to measure (+)-(S)- and (-)-(R)-isradipine concentrations in samples from volunteers after intravenous and oral administration of isradipine.

Enantioselective determination of isradipine in human serum using chiral stationary phase liquid chromatography and gas chromatography with nitrogen selective detection

Enantioselective determination of isradipine in human serum using chiral stationary phase liquid chromatography and gas chromatography with nitrogen selective detection

Highly enantioselective HPLC separations using the covalently bonded macrocyclic antibiotic, ristocetin A, chiral stationary phase.

The macrocyclic glycopeptide, ristocetin A, was covalently bonded to a silica gel support and evaluated as a liquid chromatographic (LC) chiral stationary phase (CSP). Over 230 racemates were resolved in either the reversed-phase mode, the normal-phase mode, or the polar-organic mode. The retention behavior and selectivity of this CSP were examined in each mode. Optimization of separations on this column is discussed. The ristocetin A CSP appeared to be complimentary to other glycopeptide CSPs (i.e., vancomycin and teicoplanin). Column stability was excellent. The CSP was not irreversibly altered when going from one mobile phase to another.

Bonded cellulose-derived high-performance liquid chromatography chiral stationary phases: III. Effect of the reticulation of the cellulose derivative on performance

The influence of the reticulation of a mixed 10-undecenoyl/3,5-dimethylphenylaminocarbonyl derivative of cellulose on the enantioselectivity of several high-performance liquid chromatography (HPLC) chiral stationary phases (CSPs) was investigated. Three cellulose-derived chiral columns were obtained by reticulation of the same cellulose derivative on three end-capped silica gels of different pore size. The performance of the obtained supports was studied and compared with that of three bonded cellulose-derived CSPs prepared by fixation of the same cellulose derivative on allylsilica gel.

Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection

Enantiomers of warfarin and 7-hydroxywarfarin in human plasma and urine, respectively, were determined by high-performance liquid chromatography using a cellulose-derivative column with UV or fluorescent detection, and their absolute configuration was determined simultaneously by a circular dichroism spectropolarimeter connected in series. Enantiomers of warfarin and its major metabolites [i.e., ( R)-6-hydroxywarfarin, ( S)-7-hydroxywarfarin and ( RS)-warfarin alcohol] were well resolved. The method was precise and sensitive: within- and between-day coefficients of variation were <9.6% for warfarin enantiomers in plasma and <7.1% for 7-hydroxywarfarin enantiomers in urine, respectively, and the lower detection limits were 20 ng/ml for ( R)-warfarin, 40 ng/ml for ( S)-warfarin, 2.5 ng/ml ( R)-7-hydroxywarfin and 4.5 ng/ml for( S)-7-hydroxywarfarin in 0.5 ml of both plasma and urine. The ultrafiltration technique was used for determining unbound concentrations of warfarin enantiomers in plasma using [ 14C]warfarin enantiomers resolved by the present HPLC system. Clinical applicability of the method was evaluated by determining unbound concentrations of warfarin enantiomers in five consecutive plasma samples obtained from a patient exhibiting an unstable anticoagulant response to warfarin (4 mg/day, p.o.). Results indicated that the present method would be useful in clarifying factors responsible for a large intra- and inter-patient variability in warfarin effects with regard to unbound plasmaenantiomer pharmacokinetics.

Benzoates of cellulose bonded on silica gel: Chiral discrimination ability as high-performance liquid chromatographic chiral stationary phases

In order to generalize the recently described method for the insolubilization of polysaccharide derivatives to benzoates of cellulose, five mixed 10-undecenoate/benzoates of this polysaccharide have been prepared and linked to allyl silica gel by means of a radical reaction. The chiral recognition ability of the resulting materials when used as high-performance liquid chromatographic chiral stationary phases was evaluated using heptane/2-propanol and heptane/chloroform mixtures as mobile phases. Chirality 9:145–149, 1997. © 1997 Wiley-Liss, Inc.

Bonded cellulose-derived high-performance liquid chromatography chiral stationary phases I. Influence of the degree of fixation on selectivity

Four mixed 10-undecenoyl-3,5-dimethylphenylaminocarbonyl derivatives of cellulose, with an increasing proportion of alkenoyl groups, were synthesized and chemically bonded on allylsilica gel. The influence of the degree of fixation of the cellulose derivative on the matrix for the four resulting chiral stationary phases on their selectivity is discussed.

Bonded cellulose-derived high-performance liquid chromatography chiral stationary phases II. Influence of the porosity of the silica gel matrix on performance

Two cellulose derivatives bearing simultaneously 10-undecenoyl and 3,5-dimethylphenylaminocarbonyl groups in different ratios were chemically bonded on allylsilica gels of different porosity. The systematic study of the effect of the silica gel pore size on the bonding of the polysaccharide derivative and on the performance of the resulting chiral supports is presented.

Observations relevant to the differential intercalation of enantiomers between the strands of brush-type chiral stationary phases

Differential penetration of the enantiomers of certain analytes between the strands of “brush-type” liquid chromatographic chiral stationary phases (CSPs) has been invoked in several instances to explain the progressive change in separation factors as one proceeds, chromatographically, through a homologous series of these analytes. These “intercalative effects” are presumed to arise as a consequence of the semi-ordered, side-by-side bonding of selector strands in conventional brush-type CSPs. Thus, they are not expected to occur in the absence of the stationary phase. A recently developed proline-derived CSP affords large separation factors for the enantiomers of some esters, thioesters, and amides of N-acylated α-amino acids, the separation factor being dependent upon the length of the C-terminal substituent of the analyte. For example, the separation factors increase with the length of the linear thioalkyl substituent when N-(3,5-dinitrobenzoyl)leucine thioesters are chromatographed on the proline-derived CSP. However, the length of this substituent does not affect the enantiomeric purity attained when the enantiomers of these thioesters are stereochemically equilibrated in solution in the presence of the proline-derived selector employed in the CSP. These observations further support the concept of differential intercalation of enantiomers between the strands of brush-type CSPs and may have implications for asymmetric syntheses conducted with immobilized catalysts or reagents.

Evaluation of the loadability of an immobilised protein chiral stationary phase

High performance liquid chromatography chiral stationary phases based on immobilized proteins are well known and commonly used. This work reports on an evaluation of a Human Serum Albumin stationary phase in the preparative separation of benzodiazepines.