Preliminary Evaluation of the Effects of Quenching and Inner-Filter on the Ratio Deconvolution of Fluorescence Data

The theory for the use of quenching as an aid in the ratio deconvolution of multicomponent fluorescence data is discussed. The advantage of quenching is that apparent changes in the relative fluorescence intensity of the components can be accomplished without extensive sample preparation. The disadvantage is that most quenchers exhibit significant absorption in the range of excitation wavelengths used for studying many fluorescent analytes. This absorption produces an attenuation of the excitation beam through an “inner-filter” mechanism. The problems associated with inner-filter effects are discussed.