Effect of Column Type and Experimental Parameters on the HPLC Separation of Dipeptides

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Abstract
The separation of underivatized phenylalanine dipeptides by high performance liquid chromatography using a beta-cyclodextrin bonded silica and a reversed-phase C-18 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 percent methanol in the mobile phase from 15 to 10 percent improved the separation only slightly. 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 there tention times of Phe-Asp, Phe-Clu, and Phe-Trp more than the other dipeptides analyzed. Two buffer systems were evalusted, triethylammonium acetate (O.l%, pH 4.0) and ammonium acetate (0.01 M, pH 4.5); both systems gave comparableseparations. The effect of temperature on the Separation of the phenylalanine dipeptides was also studied. The only observed effects in varying the temperature from ambient t o 57°C were the decrease in retention times and the peaks were shirperat higher than lower temperatures.