High-Speed Separation of Dabsyl-Amino Acids by Ultra High-Performance Liquid Chromatography

The analysis of amino acids is becoming important in a variety of application fields ranging from food analysis to protein science. A number of separation and detection methods are currently used. Among them, combination of precolumn derivatization with dabsyl chloride and separation on a C18 column with UV/VIS detection is generally preferred due to simplicity, excellent stability, good reproducibility and high sensitivity of the method. Although OPA (orthophthalic aldehyde) is very popular derivatization reagent, it reacts with only primary amino acids. On the other a hand, dabsyl chloride forms derivatives for both primary and secondary amino acids. Conventional methods use a 4.6 mm I.D. × 150 – 250 mm column with 5 μm particle size packing material that requires approximately 45 min to complete the analysis. We evaluated the ability to shorten the analysis time by using an ultra high-performance liquid chromatography (UHPLC) system with a 3.0 mm I.D. × 50 mm column packed with 1.8 μm particle size packing material. Our results show a reduction of the total analysis time from 45 min to 7.5 min. We also report examination results of pre-column reactions as well as an outline of the separation conditions and several applications for this rapid separation method.