The use of reverse-phase high-performance liquid chromatography and precolumn derivatization with dansyl chloride for...

A rapid and accurate reverse-phase high-performance liquid chromatography procedure for amino acid analysis of connective tissue proteins has been optimized. The method is based on quantitative dansyl chloride precolumn derivatization of protein hydrolyzates and chromatographic separation of the dansyl derivatives on Ultrasphere ODS C18 column. High molar uv absorption of Pro and Hyp derivatives, quantitation of low Des and Ide amounts (60 pmol), and good separation of all the amino acid derivatives (CV mostly less than 0.1%) including Hyl overcome the difficulties of other methods in producing reliable single run amino acid analysis of collagen and elastin. The use of fluorescence detector and the possibility of concentrating derivatized samples would give even higher sensitivity to the system. The procedure appeared to be suitable for single run analysis of other proteins, particularly those having an unbalanced amino acid composition.