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Updated: May 16, 2026

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
Fully automated reagent peak-free liquid chromatography fluorescence analysis of highly polar carboxylic acids using
Kenichiro Todoroki1, Hiroki Hashimoto, Kazuyuki Machida
1Laboratory of Analytical and Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan. todoroki@u-shizuoka-ken.ac.jp
Abstract:
In this study, we combined a column-switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak-free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1-pyrenemethylamine in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxy-1H-benzotriazole. Residual excess of the unreacted reagent was tagged with 2-(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column-switching system placed in front of an analytical reversed-phase column. The signal of the fluorous-tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type-II model mice and their control.
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