Amino Acid Analysis With Postcolumn Fluorescent Derivatization

Abstract

Amino acid analysis may be considered to be a fundamental technique for all aspects of protein chemistry. It is used not only for obtaining new data, but also for confirming the results from other types of analyses. Most current commercial analyzers are essentially modernized versions of the original autoanalyzer of Spackman, Stein, and Moore (1), designed some 30 yr ago. However, other methodologies for amino acid analysis are now becoming more popular. They may be classified into two groups, i.e., precolumn and postcolumn derivatization. In the former approach the mixture is reacted with a reagent, and the resultant amino acid derivatives are resolved on a column. In the latter approach, which is the theme of this chapter, the mixture is first resolved, and the amino acids are derivatized as they elute from the column. Within each approach there are a number of variations. The authors’ primary method utilizes separation of the amino acids on a cation-exchange column, followed by postcolumn reaction with fluorescamine (2–4). This methodology will be presented in this article, but it will be placed in perspective with respect to the other approaches for amino acid analysis.