High-Speed RP-HPLC/FL Analysis of Amino Acids After Automated Two-Step Derivatization with o-Phthaldialdehyde/3-mercaptopropionic Acid and 9-Fluorenylmethyl Chloroformate

Abstract

This paper describes an automated high-speed RP-HPLC/FL method, including two-step precolumn derivatization with o-phthaldialdehyde/3-mercaptopropionic acid (OPA/3-MPA) for primary amino acids and with 9-fluorenylmethyl chloroformate (FMOC-Cl) for secondary amino acids. The OPA/3-MPA derivatives eluted within 6.5 min, while the retention time of the last eluted amino acid derivative (FMOC-Pro) was 8.0 min. The total analysis time, including precolumn derivatization, separation, column washing and reequilibration cycles, was only 18 min. Therefore, 75–80 samples per day can be analysed during unattended operation. The efficiency of the method was demonstrated by the separation of amino acids extracted from potato tubers.