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

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.