Comparison of Reverse-Phase High-Performance Liquid Chromatographic Methods for Precolumn-Derivatized Amino Acids

Abstract

A comparison was made among five precolumn derivatization techniques for amino acid analysis using reverse-phase high-performance liquid chromatography (HPLC). All chromatographic analyses were conducted using the same instrumentation and a C₁₈ Ultrasphere ODS column (5 μm, 250 × 4.6 mm). The precolumn derivatization methodologies studied included the formation of OPA (o-phthaldialdehyde), DANSYL (dimethylaminonaphthalenesulphonyl), DABSYL (dimethylaminoazobenzenesulphonyl), PTH (phenylthiohydantoin), and PTC (phenylthiocarbamyl) derivatives. The derivatization procedures were evaluated for simplicity, time required, and derivative stability. HPLC analyses of the amino acid derivatives were compared in terms of resolution, sensitivity, reproducibility, and time of analysis.