Fluorescein excited-state proton exchange reactions: Nanosecond emission kinetics and correlation with steady-state fluorescence intensity

Fluorescein is a complex fluorophore that can exist in one or more of four different prototropic forms (cation, neutral, dianion, and monoanion) depending on pH. In the pH range 6-10, only the dianion and monanion forms are important. In a previous article, we showed by steady-state fluorescein measurements that an excited fluorescein molecule displays excited-state proton transfer reactions which interconvert the monoanion and dianion forms. However, we found that these reactions can occur only in the presence of a suitable proton donor-acceptor buffer such as phosphate buffer. Assuming that, at 1 M phosphate buffer concentration, the excited-state proton exchange reaction of fluorescein rapidly equilibrates during the lifetime of fluorescein, we were able to fit quantitatively steady-state fluorescence intensity vs pH titration graphs to a relatively simple reaction model. In this article, we use nanosecond emission (decay time) methods to study the excited-state proton reactions of fluorescein in the pH range 6-10 and in the presence of a phosphate buffer concentration. Fluorescein is a challenging fluorophore for the study of excited-state proton reactions because of the strong overlap of the absorption and emission spectra of the monoanion and dianion forms of fluorescein. However by recording nanosecond emission graphs and using methods of analysis of high precision, we have been able to test kinetic mechanisms and evaluate the specific rate constants for the excited-state proton reactions as well as the lifetimes of the monoanion and dianion. Using these values for lifetimes and rate constants, we discuss the process of equilibration in the excited-state and derive expressions which allow us to predict how quickly the excited-state reactions can reach equilibrium. Moreover, we use the above kinetic and spectral parameters to calculate steady-state fluorescence intensity Fs vs pH at 1 M phosphate buffer concentration and compare this theoretically calculated graph with the experimental graph.