The Separation of 24 OPA-AA Of Natural Origin and Quantitative Analysis of Tyrosine by Means of HPLC

Abstract

The separation of a mixture of 24 amino-acids (AA) of natural origin is carried out by column HPLC μ. Bondapak C18 10 μm, following pre-column derivatization using orthophthalaldehyde (OPA). Because of the large numbers of AA to separate, the use of a linear gradient making use of the diversity of the AA polarities ensures the resolution of the mixture in less than 45 min.

The gradient is established through the progressive addition of acetonitrile to monopotassic phosphate solution (pH 6.80). The thioisoindol derivatives, which are thus formed and wich absorb greatly at 340 nm, are detected with a spectrophotometer. Quantitative analysis of tyrosine is carried out by means of the internal calibration method with two calibration substances: glutathion and 6amino caproic acid.

This analysis, which is directly applicable in industrial surroundings to the separation of primary AA, is both reproducible and sensitive.