Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids

Abstract

The principle of sequential injection (SI) was exploited to develop a fully automated pre-column derivatization procedure combined on-line to liquid chromatography (LC). Using SI–LC derivatization 14 amino acids were determined fluorimetrically in pharmaceuticals with o-phthaldialdehyde (OPA) as the derivatization reagent. The SI system was used for the handling of samples and reagents, on-line mixing and introduction to the LC injection system. Chemical (pH and reagents concentrations) and instrumental variables (sample and reagent volumes, reaction time and flow rate) were optimized to attain the highest reaction yield and detector signal. Reversed phase chromatographic resolution of 14 amino acids was achieved within 35 min using gradient elution. The automated operation of the coupled SI–LC system resulted in very satisfactory performance. The method was applied for the simultaneous determination of amino acids in pharmaceutical formulations.