Protolytic Equilibrium of Excited States of Fluorescein

The protolytic equilibrium of the excited states of fluorescein depends not only on the equilibrium in the ground state, but also on the efficiency of photoexcitation, relaxation, and proton transfer in the excited state. Due to rate-competing radiative and non-radiative processes occurring in a mixture of protolytic forms, there is still a problem of identifying the fluorescent characteristics of individual forms of fluorescein. In this work, the steady-state distribution of the concentrations of the ground and excited states of the cation, a number of tautomers of the neutral form, monoanion, and dianion of fluorescein under continuous photoexcitation was simulated. The pH values of the predominant existence of individual protolytic forms in the excited states were determined, which were confirmed by steady-state fluorescence measurements. Possible reasons for the discrepancies in the published data on the fluorescent properties of individual protolytic forms of fluorescein are discussed and refined data on the spectral shapes and lifetimes of the quinoid and monoanion of fluorescein are obtained.