Comparative investigation of anthracene derivatives as fluorescent pre-labeling agents for carboxylic acids

Abstract

Three anthracene derivatives {1-bromomethylanthracene (1-BrMA), 9-bromomethylanthracene (9-BrMA) and 9-chloroacetylanthracene (9-ClAA)} were investigated for their abilities as fluorescent pre-labeling agents for HPLC analysis of saturated aliphatic carboxylic acids. After various conditions such as reaction temperature, reaction time, and reagent concentration were examined, the derivatization procedures were established for ten saturated fatty acids (C₄, C₆, C₈, C₁₀, C₁₂, C₁₄, C₁₆₁₈ and C₂₀). In the next step, separation conditions of the derivatives were studied for a reversed phase column, WAKOSIL 5C-8, with gradient elution using 80100% (v/v) aqueous methanol. According to the conditions established, it was shown that all the fluorescent esters produced by the derivatization of the acids with the three reagents could be separated within ca. 25 min. The detection limits for caproic acid were 100folds lower by using 1-BrMA or 9-BrMA {100 fmol (S/N≥3)} than by 9-ClAA (10 pmol).