Sequential flow-injection spectrofluorimetric determination of coumarins using a double-injection single-line system

A simple and rapid flow-injection (FI) technique with spectrofluorimetric detection is presented for the sequential determination of two model compounds: 6-methoxy-7-hydroxycoumarin (scopoletine) and 7-hydroxycoumarin (umbelliferone) without prior separation. A double-injection valve with a single-line flow-injection system is used for the simultaneous injection of a sample and a phosphate buffer solution in aqueous 50% (vv) ethanol in order to measure the fluorescence signal of the analytes successively at pH ∗ 6.0 and 11.0. The optimum excitation and emission wavelengths are 350 nm and ≥ 418 nm, respectively. Using a calculation method based on differential fluorimetry it is possible to determine ≥ 1 μmol l−1 of scopoletine in the presence of ≥ 9-fold molar excess of umbelliferone or 1 μmol l−1 of umbelliferone in the presence of ≥ 23-fold molar excess of scopoletine. This technique can be used for sequential determination of various other fluorescent compounds (exhibiting pH-dependent excitation spectra) in two-component mixtures; it allows for a quick interchange of reagents within a single-line FIA system and the reagent consumption is decreased substantially.