[21] Fluorescence quenching studies: Analysis of nonlinear Stern-Volmer data

This chapter illustrates the way two of the situations, static quenching and multiple species, cause the nonlinear deviations. The chapter evaluates the ability of the commonly used Marquardt nonlinear least-squares algorithm to recover known parameters from synthetic data. The results of the study point out the experimental and analysis criteria that must be met to obtain optimal information from fluorescence quenching studies. In particular, the chapter demonstrates the need to perform time-resolved fluorescence quenching studies. In the same way, the dynamic quenching parameters are either confirmed or evaluated, the distinction between single and multiple species is verified, and the existence of static quenching, or other process, is established. Multiple chromophores can arise in several ways even in a chemically pure system. Unless steady-state quenching experiments are performed to sufficiently high quencher concentrations, an incorrect analysis can occur because data that appear linear are actually nonlinear. It is difficult to distinguish between the two static quenching models for both single species and multiple species systems. It is necessary to obtain the dynamic quenching parameters as well as the fractional intensities to enable an analysis of multiple species quenching data.