Evaluation and optimization of high performance liquid chromatographic conditions for optical purity analysis of amino acid residues from a synthetic decapeptide using Marfey's reagent

An optimized liquid chromatographic system was developed to separate a mixture of Marfey's amino acid derivatives (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA). Alanine, glutamic acid, isoleucine, proline, and tyrosine were the five natural amino acid residues from the synthetic decapeptide, Suc-Tyr-Glu-ProIle-Pro-Glu-Glu-Ala-Cha-Glu-OH, which was analyzed for optical purity as part of this study. Excellent chromatographic resolution of the five pairs of natural amino acid derivatives, including D-alanine and D-proline, within a 70 minute gradient elution using an ODS (C-18) column, was obtained using acetonitrile as the organic modifier and sodium acetate buffer. The effects of the mobile phase pH with respect to peak retention factor (k) and separation factor (alpha) were studied in this chromatographic system.