Chiral Separation Of Amino Acids Research Articles

Adsorption liquid chromatography on silica for the chiral separation of amino acids and asymmetric amines derivatized with optically active N-α-9-fluorenylmethyloxycarbonyl-amino acid-N-carboxyanhydrides

Optically pure N-α-Fmoc-amino acid-N-carboxyanhydrides (Fmoc-AA-NCAs, Fmoc=9-fluorenylmethyloxycarbonyl) are proposed as precolumn reagents for the chiral analysis of asymmetric amines including α-amino acid alkyl esters. Separation of diastereomers arising from racemic amines is better achieved by liquid-solid adsorption chromatography on silica than by reversed-phase techniques. The sample preparation is easily performed while the properties of the Fmoc group allows high sensitivity by fluorescent detection. In this mode, picomolar limits of enantiomeric excess are detected.

Direct chiral separation of amino acids derivatized with 2-(9-anthryl)ethyl chloroformate by capillary electrophoresis using cyclodextrins as chiral selectors Effect of organic modifiers on resolution and enantiomeric elution order

Abstract Direct chiral separation of amino acids derivatized by 2-(9-anthryl)ethyl chloroformate (AEOC) has been performed by capillary electrophoresis in capillary zone electrophoretic (CZE) mode and micellar electrokinetic (MEKC) mode using cyclodextrins (CDs) as chiral selectors. The concentrations of chiral selector, surfactant and organic modifier were optimized. Application of β - and γ -CD as chiral selectors in MEKC, resulted in d/l separation of 12 and 13 amino acids, respectively. In CZE, chiral separation was obtained only with γ -CD, 8 amino acids were chirally separated in this mode. Separation in the MEKC mode resulted in higher resolutions of the enantiomers than in the CZE mode. Addition of 2-propanol to the buffer caused reversal of the enantiomeric elution order for some amino acids in MEKC mode.

Direct and indirect chiral separation of amino acids by capillary electrophoresis

Two approaches to the chiral separation of racemic mixtures of amino acids by means of capillary electrophoresis have been evaluated. These were indirect separation of diastereomers formed by derivatization with (+)- or (−)-1-(9-fluorenyl)ethyl chloroformate and direct chiral separation after derivatization with 9-fluorenylmethyl chloroformate. Separation conditions were optimized by the application of a full factorial design. For indirect separation, concentration of sodium dodecyl sulphate (SDS) and pH were the most important separation variables, and for direct separation, concentration of isopropanol (IPA), β-cyclodextrin and SDS were the most prominent factors affecting separation. The presence of IPA was a prerequisite for chiral recognition. With regard to selectivity, efficiency, analysis time and ease of method development the best results were provided by the indirect method. It should be noted, however, that the success of this approach is based on the availability of a derivatization reagent in high optical purity.

Gas chromatographic separation of enantiomeric amino acids and amines with α-methoxy-α-trifluoromethylpropionic acid as a chiral derivatizing agent

Enantiopure α-methoxy-α-trifluoromethylpropionic acid was prepared from 1,1,1-trifluoroacetone for the chiral separation of amino acids and amines by gas chromatography. Separation of the diastereomeric amide analytes was carried out on a capillary column coated with polyethylene glycol and showed good selectivity for these amino compounds at low column temperatures. A regularity was observed in the elution order, namely that a heterochiral diastereomer, i.e., the S, R-isomer, eluted faster than the homochiral S, S counterpart.

Enantiospecific drug analysis via the ortho-phthalaldehyde/homochiral thiol derivatization method

Pre-column derivatization with o-phthaldialdehyde and an optically active thiol has hitherto been used mainly for liquid-chromatographic chiral separation of amino acids. Chiral separation of non-amino-acid primary amines, especially of pharmaceuticals, via this approach has been largely ignored. We have therefore examined the applicability of the method to the chiral resolution of several pharmaceutical amines, o-Phthaldialdehyde and four commercially available homochiral thiols were used to study the separation of the enantiomers of amphetamine, p-hydroxyamphetamine, p-chloroamphetamine, 3-amino-1-phenylbutane, 3-amino-1-(4-hydroxyphenyl)-butane, mexiletine, tocainide, tranyleypromine and rimantidine. The resulting highly fluorescent isoindole derivatives were resolved on a Waters Nova-Pak C18 column using mobile phases consisting of mixtures of methanol, a sodium acetate buffer and acetonitrile, and the column effluent was monitored using fluorescence or UV detection. In some cases the fluorescence and/or the UV absorbance of the two diastereomers were unequal. It was found that the resolution of most of the amines could be optimized by varying the homochiral thiol in the derivatization step. This method of chiral separation may have wide applicability in enantiospecific drug analysis of non-amino-acid primary amines due to its simplicity and the high sensitivity it provides.

Systematic study on the resolution of derivatized amino acids enantiomers on different cyclodextrin-bonded stationary phases

The chiral separation of amino acids by liquid chromatography using cyclodextrin-bonded stationary phases was studied systematically. Six types of native and chemically modified β-cyclodextrin-bonded stationary phases have been used to separate enantiomers of some derivatized amino acids in the reversed-phase high-performance liquid chromatography mode. Chiral separations with ( R)- and ( S)-naphthyl-ethylcarbamate-βy̧clodextrin (NEC-β-CD) bonded phases were compared with similar separations with the native β-CD stationary phase. Racemic dansyl amino acids were separated best on β-CD column while 3,5-dinitro-2-pyridyl-, dabsyl-, and 3,5-dinitrobenzoyl-amino acids were resolved best on the ( R)- or ( S)NEC-β-CD column. The role of the mobile phase was studied. Effects of organic modifiers, ionic strength, and pH of the mobile phase on retention and enantioselectivity of the analytes were investigated on three stationary phases. The enantiomer elution order of all dansyl amino acids on native and NEC-β-CD bonded stationary phases was L before D enantiomer, while a reverse elution order for dabsyl amino acids was observed. Hence, elution order can be controlled by choosing the appropriate functional group. This provides an alternative to the current method of changing retention order by changing the stationary phase ( i.e., obtaining a different column). It appears that the NEC-β-CD bonded phases are highly effective multimodal chiral stationary phases. The chiral recognition model involving inclusion complexation and π-π interactions is discussed.

Application of micro high-performance liquid chromatography to the separation of chiral amino acids

Micro high-performance liquid chromatographic separation of dansyl- d,l-amino acids was examined using copper (II)- l-histidine or copper (II)- l-histidine methyl ester as eluent. Parameters that affect the retention and the resolution of dansyl- d,l-amino acids were examined. Gradient elution was used to separate many pairs of amino acids in a single chromatographic run.