Modification and Evaluation of a Reverse Phase High Performance Liquid Chromatographic Method for Amino Acid Analysis

Abstract

A reverse phase high performance liquid chromatographic method was used to separate the o-phthaldialdehyde derivatives of of amino acids from food protein hydrolysates. Comparison of hydrolysis with hydrochloric, methanesulfonic and mercaptoethanesulfonic acids revealed that the latter could not be used because it interfered with derivatization: Methanesulfonic acid is preferable to hydrochloric acid since it protects the tryptophan in samples low in carbohydrates. Hydrolysates were prepurified with a Sep Pak Cl8 cartridge prior to derivatization. A linear gradient was used for combining the binary solvent system composed of acetonitrile and phosphate buffer. The derivatives were measured fluorometrically and histamine was used as an internal standard. Results obtained with this method were similar to those obtained with an ion exchange chromatography except that threonlne and glycine coeluted and cysteine, cystine and proline were not detected. INTRODUCTIONHIGH PERFORMANCE liquid chromatography (HPLC) has recently been applied to amino acid anysis since it is rapid and utilizes equipment that is more economical and versatile than a traditional ion exchange ammo acid analyzer. This research was undertaken to determine the feasibility of using the reverse phase HPLC procedure of Hill et al. (1979) for the analysis of amino acids in foods. Specifically the study was designed to determine the best acid hydrolysis and solvent elution procedures and to select a suitable internal standard.

Emprego de cromatografia líquida de alta eficiência hidrofílica na determinação dos aminoácidos de hidrolisados de caseína

“…Posteriormente, introduziu-se a cromatografia líquida de alta eficiência (HPLC), utilizando um equipamento mais econômico e versátil que um analisador de aminoácidos de troca iônica [11]. Assim, várias técnicas de cromatografia líquida de fase reversa (HPLC-RP), empregando gradiente de eluição, diferentes tipos de coluna e detectores, seguida da derivação das amostras com reagentes variados, estão sendo utilizadas para a determinação dos aminoácidos [1,5,7,21].…”