An HPLC detection scheme for underivatized amino acids based on tryptophan fluorescence recovery
2000; Elsevier BV; Volume: 409; Issue: 1-2 Linguagem: Inglês
10.1016/s0003-2670(99)00882-x
ISSN1873-4324
AutoresMin Yang, Sterling A. Tomellini,
Tópico(s)Analytical Chemistry and Chromatography
ResumoA 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.
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