High-pressure liquid chromatography of amino acids and dipeptides on a tripeptide bonded stationary phase

A bonded tripeptide (L-valyl-L-phenylalanyl-L-valine) phase has been used as a stationary phase in liquid chromatography. Unique retention orders and retention variations are shown for phenylthiohydantoin (PTH) derivatives of amino acids and isomeric dipeptides using acidic and basic mobile phases. It is shown that the 25 PTH-amino acids studied here have different capacity ratios when 1% citric acid in water (ca. pH 2.5) is used as the mobile phase. The analysis time for these amino acid derivatives could be shortened without a loss in the resolution by adding 5% methanol to the above mobile phase. Of particular importance is the fact that the basic amino acids are eluted first. Isomeric dipeptides have been resolved by using deionized water (pH 5.5), and 1% sodium citrate in water (pH 7.9), as mobile phases. The data show that the nature of the mobile phases, the pH, and the ionic strength are the important factors affecting the selectivity and efficiency of the separations of amino acids and dipeptides.