RP-HPLC Determination of Amino Acids with Precolumn Derivatization

Solution of amino acid was mixed with the derivatizing reagent, 4-methoxybenzenesulfonyl chloride (MOBS-Cl), and derivatized for 35 min at 55 ℃ in a water bath. The solution containing the derivative of amino acid was used for RP-HPLC analysis, using ODS C18 column (4.6 mm×150 mm, 5.0 μm) as stationary phase and mixtures of 15 mmol·L-1 NaH2PO4 solution and acetonitrile in different ratios as mobile phase in the gradient elution. UV-detection at the wavelength of 235 nm was adopted in the determination. Linear relationships between values of peak area and concentration of the 17 amino acids were obtained in the same range from 0.01 to 5.0 mmol·L-1. Detection limits (3S/N) found for this method were in the range of 0.001 0-0.005 0 nmol·L-1. Precision of the method was tested with 5.0, 25.0, 50.0 g·L-1 of the standard amino acid solutions for 5 determinations including the precolumn derivatization process, values of RSD′s calculated from data of peak area were less than 5.0%.