The factors that influence the elution order for the resolution of amino acids on vancomycin phase using the polar‐organic mobile phases after their pre‐column derivatization with electrophilic reagents

Abstract

A variety of amino acids were enantioresolved on a vancomycin bonded chiral phase using the polar-organic mobile phases after their pre-column derivatization with electrophilic reagents in alkaline medium. The resolution was highly dependent on the analyte's structure and was enhanced as the aromatic side-chain group on the skeleton of analyte for pi-pi interaction with the chiral selector became available. The steric hindrance resulting from the bulky side-chain group on the analyte also affected the resolution. Elution reversal, not found on the teicoplanin phase under the same chromatographic conditions, was possible through altering the type of reagent used in the derivatizing reaction (e.g. 2,4-difluorophenylisothiocyanate to 2,4-difluorophenylisocyanate). It is believed that the steric hindrance, as a result of the bulky sulfur atom in reagents such as methylisothiocyanate and others examined in this study, was responsible for the reversed elution order. The bulkiness of the substituent on the aromatic ring of derivatizing reagents (i.e. 2,3- and 3,5-dichlorophenylisothiocyanate) was observed to affect the resolution and alter the elution order as well.

Reversal in elution order in liquid chromatography—compilation of experimental observations

“…The elution order of the separated amino acid enantiomeric peaks appeared to be varied on Chiralcel OD and Chiralcel OJ columns, suggesting the use of the two chiral columns in a complimentary manner (Lee et al, 2008). Chen (2005) made observations of elution order switches when amino acids were resolved on vancomycin bonded chiral phases after the analytes were subjected to pre-column derivatization with electrophilic reagents. Elution reversal was readily observed when the derivatization reagent was changed from 2,4-difluorophenylisocyanate to 2,4-difluorophenylisothiocyange (Chen, 2005).…”

“…Elution reversal was readily observed when the derivatization reagent was changed from 2,4-difluorophenylisocyanate to 2,4-difluorophenylisothiocyange (Chen, 2005). It was attributed to the bulkiness produced by the sulfur atoms in the reagent which resulted in steric hindrance phenomenom leading to reversal in elution order (Chen, 2005).…”

“…Elution reversal was readily observed when the derivatization reagent was changed from 2,4-difluorophenylisocyanate to 2,4-difluorophenylisothiocyange (Chen, 2005). It was attributed to the bulkiness produced by the sulfur atoms in the reagent which resulted in steric hindrance phenomenom leading to reversal in elution order (Chen, 2005). Dungelová et al (2004) reported an interesting work by chromatographing a number of enantiomers of alkoxy substituted esters of phenylcarbamic acid on serially connected Whelk-O 1 columns that displayed reversal enantioselectivities [i.e.…”

Chromatography of amino acids and short peptides. New advances

“…Analytes were detected at 260 and 270 nm. It was concluded from the results that amino acid enantiomers can be readily resolved under the experimental conditions outlined above and the structure of analyte exerts a considerable influence on the separation of enantiomers (Chen, 2005).…”