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

Abstract

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. Ionpairing liquid chromatography is a promising means of increasing analyte retention. In this study, the effects of ionpairing 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 reversedphase 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.

Ultralow-Temperature HPLC Using Low-Molecular-Weight Hydrocarbons as Mobile Phases

“…Chromatography is not only useful for the separation of mixtures, it can also be used to study interactions between the stationary phase and compounds [9][10][11]. The development of chromatography under cryogenic conditions will allow to investigate interactions between compounds at ultralow-temperatures, and will contribute to understanding the chemistry at cryogenic conditions.…”

Liquid Chromatography

“…Kanao et al studied the isotope effect on hydrogen bonding and CH/CD-π interaction by LC. 17 Retention behaviors of fluorescent amino acid derivatives in ion-pairing reversed-phase LC 18 and inorganic anions in hydrophilic interaction chromatography 19 were also investigated. Recently, tube radial distribution chromatography (TRDC), a unique LC without a distinct stationary phase, was developed, and TRDC using a commercially available HPLC system was reported.…”