Protolytic fluorescein species evaluated using chemometry and DFT studies

Fluorescein (C.I. Solvent Yellow 94) contains three acid-base groups that lead to a protolytic equilibrium involving four species namely, dianionic, monoanionic, neutral and cationic. Since these species display superimposed bands in their electronic absorption spectra and close pKa values, the determination of pKa using traditional methods is complex. By applying chemometric tools, principal component and Q factor analysis followed by varimax and Imbrie oblique rotations, pKa values in water, pKa1 = 2.5, pKa2 = 3.8 and pKa3 = 6.1, were obtained. Geometric parameters secured using Density Functional Theory combined with a polarized continuum model that simulates the surrounding water molecules showed that the predominant neutral fluorescein structure is quinoid, while the monoanion is carboxylic. Time-Dependent Density Functional Theory predicted the origin of electronic transitions of each species, which agreed with the spectra generated using chemometrics.