AMINO-ACID-ANALYSIS BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - IMPROVED DERIVATIZATION AND DETECTION CONDITIONS WITH 9-FLUORENYLMETHYL CHLOROFORMATE

An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in berate buffer at pH 11.4 for 40 min at ambient tem perature. All amino acids resulted in stable derivatives. In particula r, improved derivatization was obtained with the troublesome amino aci ds His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivati ves show a higher fluorescence response than their disubstituted and m onosubstituted counterparts, respectively, resulting from other protoc ols. Under the new conditions, considerable less of the hydrolysis pro duct of FMOC-Cl is seen in the chromatograms. Baseline noise was subst antially reduced at a higher emission wavelength (630 nm instead of 31 3 or 340 nm). With simple precautions, extensive adsorption of the dis ubstituted derivatives (Lye, Hyl, and Tyr) on plastic or glass surface s could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid The detection Limits are in the femtomole range and the derivatives are stable for more than 48 h , thus permitting automated analysis of multiple samples. (C) 1996 Aca demic Press, Inc.