Detection of a distance-dependent rate of quenching by frequency-domain fluorometry

The effect of the collisional quenching on the fluorescence intensity decays has been studied by frequency-domain fluorometry. We used an efficient (CBr₄) and/or inefficient (CCl ₄ quencher to quench the fluorescence of 1,2-benzanthracene (1,2-BA). The wide range of diffusion has been obtained by using propylene glycol at different temperatures (-40°C to 40°C). The measured intensity decays cannot be satisfactorily fitted either to the Smoluchowski or Collins-Kimball (RBC) model, except the case of inefficient quencher in the presence of high diffusion. In particular, we observed quenching in diffusionless conditions (-40°C). To describe the collisional quenching of the fluorescence more correctly we propose a new model which includes a distance-dependent quenching rate (DDQ model). The DDQ simulations show that the local concentration of quencher surrounding the excited fluorophore cannot be approximated by using the RBC model, except in the case of high diffusion and low quenching rate. The DDQ model describes well all measured intensity decays of 1,2-benzanthracene in the presence of CBr₄ and/or CCl₄. Also, the DDQ model more correctly predicts the curvature of Stern-Volmer plots and activation energies obtained from the temperature dependent rate of quenching.