A new approach to the old problem: Inner filter effect type I and II in fluorescence.

The fluorescence technique is very popular and has been used in many fields of research. It is simple in its assumptions but not very easy to use. One of the main problems is the inner filter effect (IF) I and II which takes place in the cuvette. IF type I is permanently present, but IF type II occurs only when absorption and fluorescence spectra overlap. To avoid IF type I, absorbencies in the cuvette should be smaller than 0.05, which is however very difficult to obtain in many experiments. In this work we propose a new method to solve these problems in the case of a Cary Eclipse fluorimeter, having horizontally-oriented slits, based on old equations developed in the middle of the last century. This method can be applied for other instruments, even these with vertically-oriented beams, because we share scripts written in MATLAB and GRAMS/AI environment. Calculations in our method enable specifying beam geometry parameters in the cuvette, which is necessary to obtain the correct shape and fluorescence intensity of emission and excitation spectra. Such a specific fluorescence intensity dependence on absorbance can, in many cases, afford possibilities to determine the quantum yield (QY) using slopes of the straight-lines, which was demonstrated with the use of Tryptophan (Trp), Tyrosine (Tyr), and Rhodamine B (RhB) solutions. For example, assuming that QY=0.14 for Tyr, the QY determined for RhB reached QY=0.71±0.05, although the measurement for Tyr and RhB was performed at a completely different spectral range.