Homogeneous liquid–liquid extraction method for the selective spectrofluorimetric determination of trace amounts of tryptophan

Abstract

Twenty-one amino acids were derivatized with fluorescamine (FLA) under basic conditions (pH 9) and the extraction of the amino acid–FLA derivatives was investigated using a homogeneous liquid–liquid extraction with perfluorooctanoic acid (HPFOA) based on phase separation under strongly acidic conditions. Under the optimum concentration conditions for the reagents ([PFOA]_T = 3 × 10⁻³ mol dm⁻³, [acetone]_T = 3 vol.%, [HCl]_T = 1.8 mol dm⁻³), the concentration factor was approximately 1000-fold (i.e., 30 μl of the sedimented liquid phase was produced from 33 ml of the homogeneous aqueous solution). The percentage extraction (E) was determined for the 21 amino acid–FLA derivatives; the value for the tryptophan (Trp)–FLA derivative was 80.9%, whereas the other derivatives were not almost extracted (E < 0.4%). The Trp–FLA derivative was selective for the extraction using the homogeneous liquid–liquid extraction method with HPFOA. After the sedimented liquid phase containing Trp–FLA has been placed on a polytetrafluoroethylene filter-paper, the fluorescence intensity was determined using a spectrofluorimeter with filter-paper as the solid-sample holder. The calibration graph of Trp was linear over the range 1.0 × 10⁻⁸–1.5 × 10⁻⁶ mol dm⁻³. The relative standard deviation for the central value of the calibration graph was 4.5% (five determinations) and the detection limit (S/N=3) was 8.9 × 10⁻⁹ mol dm⁻³. When the proposed method was applied to the highly sensitive spectrofluorimetric determination of Trp in animalin-L syrup, the results were satisfactory.