Amino Acid Analysis by Precolumn Derivatization with 1-Fluoro-2,4-Dinitrophenyl-5-I-Alanine Amide (Marfey’s Reagent)

Precolumn modification of amino acids and the subsequent resolution of their derivatives by reverse-phase high-performance liquid chromatography (RP-HPLC) is the preferred method for quantitative amino acid analysis. The derivatization step introduces covalently bound chromophores necessary not only for interactions with the apolar stationary phase for high resolution but also for photometric or fluorometric detection. Marfey’s reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide or (S)-2-[(5-fluoro-2,4-dinitrophenyl)-amino]propanamide, is used to separate and to determine enantiomeric amino acids (1). The reagent reacts stoichiometrically with the amino group of enantiomeric amino acids to produce stable diastere-omeric derivatives, which can readily be separated by reverse-phase HPLC (Fig. 1). The dinitrophenyl alanine amide moiety strongly absorbs at 340 nm (ε = 30,000 M−1 cm−1), allowing detection in the subnanomolar range. With smaller columns, the sensitivity will correspondly increase.