Technique for the determination of fluorescence quantum efficiencies: a method avoiding direct measurement of absorbance

Abstract

A method is described for the determination of the absorbance of a fluorescent solution by recording the fluorescence intensities at two points along the excitation path. An expression is developed that relates the ratio of these two fluorescence intensities to the absorbance of the solution. Thereby both the fluorescence and the absorbance of a solution can be obtained by recourse only to a spectrophotofluorimeter. This approach is extended to the determination of fluorescence quantum efficiences and an expression is presented that relates the fluorescence intensities at two points along the excitation path to the quantum efficiency of the fluorescent material. Quantum efficiences can therefore be determined without the need for an accurate separate measurement of the absorbances of solutions of standard and sample materials. A cuvette with a nitrogen deoxygenation facility is described for use in these and other related determinations.