Fluorescence Technical Note FL-8

This technical note describes kinetic fluorescence as an analytical technique to quantify non-fluorescent species. The technique is applied to the determination of thiamine (vitamin B1) in solution. Fluorescence intensity is monitored as thiamine converts to fluorescent thiochrome, and a relationship is established between the rate of increase of intensity and the concentration of thiamine. The technique is shown to have good sensitivity and selectivity. Introduction Luminescent substances are quantified readily by sensitive fluorescence and phosphorescence techniques. Kinetic fluorescent methods extend the sensitivity advantage of fluorescence analysis to non-fluorescing samples by converting them chemically to fluorescent species. For example, the technique can be used to analyze pharmaceutical preparations to quantify non-fluorescing thiamine (vitamin B1), which oxidizes to fluorescent thiochrome. Kinetic fluorescence is borrowed from methods for studying reaction rates of chemical or physical transformations. In these studies, the increase or decrease in fluorescence intensity (corresponding to formation or degradation of a fluorescent species) is monitored during a reaction in order to understand the reaction mechanism. Kinetic fluorescence establishes a relationship between the change in fluorescence intensity during a reaction and the concentration of the non-fluorescing reactant. Kinetic fluorescence’s precision and specificity allow quantification of individual components in a multi-component solution without complicated separations. To illustrate the applicability of kinetic fluorescence for analytical work, we describe herein a reaction-rate method for the determination of thiamine, an experiment using commercial multiple-vitamin tablets, and a calibration procedure for thiamine determination with the Fluorolog modular spectrofluorometer system. Because thiamine is involved in numerous metabolic processes, proper maintenance of thiamine levels in the diet is essential. Thiamine deficiency can produce degenerative disorders such as edema, muscular atrophy, or even beriberi. In addition to foods containing thiamine (e.g., nuts, vegetables, pork, and liver), thiamine is also available in many commercial supplements, including vitamin-B complexes and antistress preparations. Fig. 1. Reaction of thiamine and Hg to form