Phase fluorometric method for determination of standard lifetimes

Rayleigh scatterers have long been used as standards for fluorescence lifetime determinations, but they have many drawbacks, including the well-known “color effect ’. To avoid these problems, various fiuorophores have been used as standards. Unfortunately, the lifetimes of these compounds are not agreed upon to better than 5%, and the compounds cited in the literature do not fully cover the 250–850 nm band of common fluorescence emission. We describe a multifrequency phase fluorometric method for accurately determining the lifetimes of monoexponential fluorophores (standards) without reference to another standard. Results are shown for some widely used standard fluorophores and some recently developed compounds. An Independent test of the accuracy of the method based on quenching experiments is presented. © 1988, American Chemical Society. All rights reserved