The Separation of Dipeptides by High Performance Liquid Chromatography

Abstract

The separation of underivatized phenylalanine dipeptides by high performance liquid chromatography using a beta-cyclodextrin bonded silica gel column was evaluated. Parameters such as per cent organic modifier, pH, buffer type, and temperature were shown to have various effects on the dipeptides separation. The results revealed that the separation of dipeptides was possible using the beta-cyclodextrin column. Changing the per cent methanol in the mobile phase from 15 to 10 percent although improved the separation, did not affect it drastically. Changing the pH of the mobile phase affected the peak shape and retention times depending on the amino acid in the dipeptide. It was found that pH changes affected the retention times of the acidic dipeptides more than the uncharged amino acids. Two buffer systems were evaluated, triethylammonium acetate (0.1%, pH 4.01) and ammonium acetate (0.01 M, pH 4.5); both systems gave comparable separations. The effect of temperature on the separation of the phenylalanine dipeptides was also studied. The only observed effects in varying the temperature from ambient to 57 degrees centigrade were the decrease in retention times and the peaks were sharper at higher than lower temperatures. A comparison between using the beta-cyclodextrin column and the reversed-phase C-18 column for the separation of the phenylalanine dipeptides and other nonaromatic dipeptides indicated that both columns work equally well, with the C-18 requiring a higher concentration of methanol in the mobile phase. A comparison of the separation of a group of stereoisomers showed that the beta-cyclodextrin and C-l8 columns gave comparable results with the C-18 requiring different concentrations of methanol in the mobile phase. It was possible to separate a mixture of di-, tri-, and tetrapeptides using both columns.