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

摘要

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 acis 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 acis, 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 fo 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-cystien (L-Cys) is 3.8 pmol (S/N=3) using a 10 #mu# 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 acis tested are below 10 pmol with linearity up to the order of nmol.