AMINO-ACID-ANALYSIS BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - IMPROVED DERIVATIZATION AND DETECTION CONDITIONS WITH 9-FLUORENYLMETHYL CHLOROFORMATE
Ra. Bank et al., AMINO-ACID-ANALYSIS BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY - IMPROVED DERIVATIZATION AND DETECTION CONDITIONS WITH 9-FLUORENYLMETHYL CHLOROFORMATE, Analytical biochemistry, 240(2), 1996, pp. 167-176
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.