Factors Affecting Calibration for Phase-Modulation Fluorescence Lifetime Determinations

Abstract

The effects of several instrumental and experimental factors on phase-modulation fluorescence lifetime determinations of dimethyl POPOP, 9,10-diphenylanthracene, and benzo(a)pyrene were evaluated with the use of both a scattering solution and dimethylPOPOP as references for phase and modulation calibration. For the scattering solution reference, best results were obtained when the scatterer was measured within a few nanometers of the resonance peak corresponding to the excitation wavelength used for measuring the sample. Intensity matching of the reference (either scatterer or dimethylPOPOP) to the sample solution does not appear to be required as long as the sample solution exhibits lifetime homogeneity and the sample and reference are measured with the same voltage applied to the photomultiplier tube detector. Intensity matching to within 10–20% does appear to be indicated for samples with lifetime heterogeneity. When intensity matching is used, the intensities should be matched by adjustment of the reference solution concentration rather than by changing the wavelengths used to measure the reference.