Retention of Fluorescent Amino Acid Derivatives in Ion-pairing Reversed-phase Liquid Chromatography

Recent studies have shown that pillar array columns enable fast and quantitative analysis of amino acids. However, hydrophilic amino acids still cannot be retained on pillar array columns since they have limited retention ability. Ion pairing liquid chromatography is a promising means of increasing analyte retention. In this study, the effects of ion pairing reagents on the retention of eight hydrophilic amino acids (histidine, asparagine, glutamine, serine, arginine, aspartic acid, glycine, and glutamic acid) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) under reversed phase conditions on a conventional ODS column were studied. Among the ion-pairing reagents investigated, tetraheptylammonium bromide proved to be the most effective for increasing analyte retention. With a mobile phase consisting of 20 mM citrate buffer (pH 6.3)-acetonitrile (100:40, v/v) and 2 mM tetraheptylammonium bromide, the retention times of the eight NBD-amino acids except NBD-arginine were longer than 19.4 min, which was the retention time of NBD-valine when eluted without an ion-pairing reagent. As NBD-valine was well retained on pillar array columns, the chromatographic conditions may thus be applied in the analysis of hydrophilic amino acids using pillar array columns.