Pulsed Amperometric Detection of Amino Acids Separated by Anion Exchange Chromatography

Abstract

Previous work has shown that the analysis of amino acids requires derivatization for spectrophotometric (UV-Vis) and fluorescence detection schemes. In this study, direct determination of both primary and secondary amino acids with pulsed amperometic detection (PAD) was achieved under alkaline conditions by oxidation of the amine and hydroxyl functional groups with a gold working electrode. PAD was found to be more sensitive (0.01 to 1.2 nM. mL⁻¹) than ninhydrin derivatization (4.5 to 55.0 nM mL⁻¹) with UV-Vis detection. Detection of amino acids by PAD was in good agreement with previous published work on the amino acid composition for the enzyme, ribonuciease A. High performance anion-exchange chromatography (HPAC) with PAD for amino acid analysis requires little to no sample preparation and was not subjected to matrix interferences previously noted with reverse phase separation and OPA or ninhydrin detection. Application of the developed methodology indicated that several wheat (Triticum aestivum L) varieties grown in sterile nutrient-sand media had a wide range in amino acid composition in their respective root mass and root exudates.