High-performance liquid chromatography with photochemical fluorimetric detection of tryptophan based on 4-fluoro-7-nitrobenzo-2-oxa- 1,3-diazole : Total protein amino acid analysis |
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Authors: | Kazuhiro Imai Eiji Ueda Toshimasa Toyooka |
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Institution: | Branch Hospital Pharmacy, University of Tokyo, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112 Japan;Faculty of Pharmaceutical Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 Japan;Division of Foods Institute of Hygienic Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158 Japan |
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Abstract: | The simultaneous determination of amino acids including trytophan is described. The NBD- F forms a single adduct with tryptophan as with other amino acids, but the adduct lacks intrinsic fluorescence. After ultraviolet irradiation, the adduct fluoresces (pale-green); the fluorescence intensity increases with increasing irradiation time at pH 2-10, Under the same conditions, the other amino acid adducts are slowly decomposed. When the tryptophan adduct, separated on a Nucleosil ODS column (150×4.6 mm, 6 μm), is irradiated in an on-line photochemical reactor (310 nm), its fluorescence peak appears between those of the phenylalanine and lysine adducts. The detection limit for tryptophan by the proposed method is 3 pmol; the limits for other amino acids are 10–100 fmol. |
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