Effect of solvent-water mixtures on the prototropic equilibria of fluorescein and on the spectral properties of the monoanion

A spectral resolution procedure was used to resolve the absorption, excitat ion and emission spectra of the fluorescein monoanion in a number of solven t-water mixtures. This permitted an analysis of the effect of the solvent e nvironment on the spectral properties of the monoanion and on the lactone/m onoanion/dianion transitions of fluorescein. The monoanion excitation and e mission spectra show relatively small changes with changing environment, a behavior that is related to the hydrogen-bonding environment of the solvent -water mixtures. There is also a general increase in the quantum yield of t he monoanion from 0.36 in water to values up to 0.49 in the solvent-water m ixtures. The presence of solvent also results in a general increase In the lactone content and in the monoanion:dianion and lactone:monoanion ratios. General polarity effects alone cannot account for the observed effects on t he prototropic transitions indicating that specific solute-solvent effects involving hydrogen bonding perturb the prototropic equilibria of fluorescei n.