Direct Quenching Fluorescence

In some cases it is possible to obtain a measure of the association constant for intercalation directly from fluorescence quenching data. This method is applicable when the dynamic quenching of the hydrocarbon fluorescence by DNA is small and when the intercalated hydrocarbon has a negligible fluorescence quantum yield compared to that of the free hydrocarbon. If these conditions are met, the association constant for intercalation, Kq, is equal to the Stern-Volmer quenching constant Kgy (76) and is given by Equation 1. 

The riboflavin binding protein that occurs in eggs has been exploited for the radio-ligand binding assay of riboflavin. Because binding to the protein quenches the native fluorescence of riboflavin, it can be exploited for a direct titrimetric fluorescence assay of the vitamin in urine and other biological samples (Kodentsova et al., 1995). 

However, the direct determination of absorption at the wavelength of maximum absorption is more sensitive (or in the worst case at least as sensitive) as the indirect measurement of absorption by fluorescence or phosphorescence quenching. 

The LIF technique is extremely versatile. The determination of absolute intermediate species concentrations, however, needs either an independent calibration or knowledge of the fluorescence quantum yield, i.e., the ratio of radiative events (detectable fluorescence light) over the sum of all decay processes from the excited quantum state—including predissociation, col-lisional quenching, and energy transfer. This fraction may be quite small (some tenths of a percent, e.g., for the detection of the OH radical in a flame at ambient pressure) and will depend on the local flame composition, pressure, and temperature as well as on the excited electronic state and ro-vibronic level. Short-pulse techniques with picosecond lasers enable direct determination of the quantum yield [14] and permit study of the relevant energy transfer processes [17-20]. 

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