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Updated: Jun 2, 2026

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
Automated pre-column derivatization of thiolic fruit-antibrowning agents by sequential injection coupled to
Theano D Karakosta1, Paraskevas D Tzanavaras, Demetrius G Themelis
1Department of Chemistry, Laboratory of Analytical Chemistry, Aristotelian University of Thessaloniki, Thessaloniki, Greece.
Abstract:
The present study reports the very first application of ethyl propiolate (EP) as an advantageous pre-column derivatization reagent for the determination of thiols by liquid chromatography (LC). Cysteine (CYS), glutathione (GSH) and N-acetylcysteine (NAC) were derivatized online under stopped-flow conditions in a sequential injection (SI) system coupled to HPLC. The formed derivatives were separated isocratically with a monolithic stationary phase (100×4.6 mm id) and UV detected at 285 nm. Critical parameters that affected the online pre-column derivatization reaction (e.g. the reaction time and the amount concentration of EP) and the separation (e.g. pH and the composition of the mobile phase) were investigated. The developed analytical scheme offers a total analysis time of less than 10 min, limits of detection in the range of 0.24-0.35 μmol/L and satisfactory linearity up to 200 μmol/L for all analytes. The proposed method was applied to the analysis of the selected thiols--that are often employed as antibrowning agents--in fresh fruit samples.
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