An HPLC detection scheme for underivatized amino acids based on tryptophan fluorescence recovery Original Research Article

A simple, sensitive fluorescence detection scheme has been developed for detecting underivatized amino acids following HPLC separation. This detection is based on a displacement reaction between the eluted amino acids and a copper(II)–l-tryptophan (l-Trp) complex, Cu(l-Trp)2. In the form of the complex Cu(l-Trp)2, the fluorescence of l-Trp is approximately 95% quenched; with the addition of analytes with strong affinity for Cu(II) such as the natural amino acids, l-Trp is released from the complex and l-Trp fluorescence is recovered. Thus, the presence of the eluted analytes is inferred by the recovered fluorescence of displaced l-Trp. Twenty amino acids have been detected with the proposed detection method. Eluent pH has a strong effect on detection. The detection limit for l-cystine (l-Cys) is 3.8 pmol (S/N=3) using a 10 μl injection volume. Relative standard deviations for four injections of 50 and 250 pmol of l-Cys are 2.9 and 0.6%, respectively. Detection limits for most of the other amino acids tested are below 10 pmol with linearity up to the order of nmol.