Derivatization of Hydrophobic Amino Acids in Nonaqueous Media and Separation by Nonaqueous Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Abstract.

A method of nonaqueous capillary electrophoresis was established for the separation and determination of alanine, phenylalanine, isoleucine and valine after derivatization with 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazol in nonaqueous media. The derivatization and separation conditions were optimized. The optimum derivatization conditions were 70 min for the reaction time, 55 °C for the reaction temperature and 20 mM ammonium acetate in methanol for the derivatization buffer. The most suitable running buffer was composed of 60 mM ammonium acetate, 10% acetonitrile in methanol with a fused-silica capillary column (47 cm × 75 µm i.d.), 20 kV applied voltage and 20 °C capillary temperature. The correlation coefficients were better than 0.995 in the investigated concentration ranges. The relative standard deviation (R.S.D.) (n = 5) of the migration times and peak areas were 2.0–4.3% and 1.9–4.5%, respectively. The method was applied to the determination of four compounds in two compound amino acid injections, with the exception of phenylalanine, the recoveries for the other three compounds ranging between 88–116%.