RP-LC with On-Line Recycled Mobile Phase for Analysis of Amino Acids Pre-Derivatized with o-Phthalaldehyde

Reversed-phase high-performance liquid chromatography (RPLC) of 17 amino acids and ammonium ion, pre-derivatized with o-phthalaldehyde and ethanethiol, has been investigated using on-line recycled mobile phase. The mobile phase was sodium phosphate buffer, pH 7.2, containing acetonitrile and 1 mM potassium peroxodisulfate. Fifty consecutive separations on an octadecyl silica gel column, kept at 35 °C, could be achieved by use of 50 mL mobile phase, at a flow rate of 2.0 mL min⁻¹, without significant changes of peak height and retention times of valine (Val), methionine (Met), and isoleucine (Ileu). RSD of peak heights and retention times were, respectively, 7.6 and 1.1% for Val, 9.6 and 0.9% for Met, and 4.9 and 0.8% for Ileu. The volume of mobile phase used was only one sixtieth that used for conventional RPLC. Analysis of Val, Met, and Ileu in beverages was achieved by use of this method. The results were in agreement with those obtained by conventional RPLC. This method could be also used for analysis of the other amino acids.