USE OF CHROMOGENIC AND FLUORESCENT OXYCARBONYL CHLORIDES AS REAGENTS FOR AMINO-ACID-ANALYSIS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The aryloxycarbonyl chlorides 4-phenylazobenzyloxycarbonyl chloride (PAZ-CI), p-nitrobenzyloxycarbonyl chloride (PNZ-Cl), o-nitrobenzyloxycarbonyl chloride (ONZ-Cl), 2-naphthoxycarbonyl chloride (NOC-Q) and 2-(naphthylmethyl)oxycarbonyl chloride (NMOC-Cl) were used for the precolumn derivatization of amino acids. The separations of the derivatives thus formed by reversed-phase (C-18 or C-8) high-performance liquid chromatography (HPLC) were investigated and compared with those with the established reagent 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). A robotic autosampler enabled the fully automated mixing of samples with alkaline berate buffers, addition of excess of reagents (and of the scavenger 1-aminoadamantane if required), the final dilution of aliquots of the reaction mixtures with sodium acetate buffers and solvents and injection on to the HPLC column. Derivatives were eluted from the columns at 45 degrees C using gradients made up from sodium acetate buffers and acetonitrile. Derivatives were detected by their UV absorbance (PAZ-, PNZ- and ONZ-amino acids) or by their fluorescence (NOC-, NMOC- and FMOC-amino acids). Hydrolysates of equine myoglobin, bovine beta-lactoglobulin A and bovine serum albumin were quantitatively analysed with the reagents NOC-Cl and PNZ-Cl and the results were found to be in good agreement with those obtained by derivatization with FMOC-CL and with the calculated amino acid composition of the proteins.