Stability of phenyl(thiocarbamoyl) amino acids and optimization of their separation by high-performance liquid chromatography

Abstract

The optimization of the conditions which influence the determination of amino acids by reversed-phase high-performance liquid chromatography is described, using phenyl isothiocyanate as a pre-column derivatizing agent. The influence of the pH of the mobile phase and the eluent composition on the gradient chromatography, the effect of temperature, the characteristics of the column packing and the stability of the phenylthiocarbamoyl (PTC) amino acids with time were studied. Optimum separation of 17 amino acids was achieved using a C₁₈ column, 25 cm long, with a particle size of 5 m and a 30 nm pore size; working at 40 °C, pH 6.5 and eluting with a mixture of solvents: ammonium acetate buffer, 50 mmol dm^–3; and ammonium acetate buffer, 100 mmol dm^–1(46% acetonitrile, 44% water and 10% methanol). The precision of the retention times of the PTC amino acids expressed as the percentage relative standard deviation are less than 0.5% for most of the amino acids. The PTC amino acids are relatively unstable at ambient temperature, with the stability varying widely depending on the particular amino acid. The PTC amino acids are stable at 0 °C.