SEQUENTIAL FLOW-INJECTION SPECTROFLUOROMETRIC DETERMINATION OF COUMARINS USING A DOUBLE-INJECTION SINGLE-LINE SYSTEM

A simple and rapid flow-injection (FI) technique with spectrofluorimet ric detection is presented for the sequential determination of two mod el compounds: 6-methoxy-7-hydroxycoumarin (scopoletine) and 7-hydroxyc oumarin (umbelliferone) without prior separation. A double-injection v alve with a single-line now-injection system is used for the simultane ous injection of a sample and a phosphate buffer solution in aqueous 5 0% (v/v) ethanol in order to measure the fluorescence signal of the an alytes successively at pH 6.0 and 11.0. The optimum excitation and em ission wavelengths are 350 nm and greater than or equal to 418 nm, res pectively. Using a calculation method based on differential fluorimetr y it is possible to determine greater than or equal to 1 mu mol l(-1) of scopoletine in the presence of less than or equal to 9-fold molar e xcess of umbelliferone or 1 mu mol l(-1) of umbelliferone in the prese nce of less than or equal to 23-fold molar excess of scopoletine. This technique can be used for sequential determination of various other f luorescent compounds (exhibiting pH-dependent excitation spectra) in t wo-component mixtures; it allows for a quick interchange of reagents w ithin a single-line FIA system and the reagent consumption is decrease d substantially.