ABSORPTION AND FLUORESCENCE PROPERTIES OF FLUORESCEIN

We have characterized the protolytic equilibria of fluorescein and determined the spectroscopic properties of its protolytic forms. The protolytic constants relating the chemical activities (which at low ionic strength equal concentrations) of the cation, neutral form, anion and dianion are pK(1) = 2.08, pK(2) = 3.31, and pK(3) = 6.43. All forms have rather high molar absorptivities being epsilon(437)(FH3+) = 53 000, epsilon(434)(FH2) = 11 000 epsilon(453)(FH-) = 29 000 (epsilon(472)(FH-) = 29 000) and epsilon(490)(F2-) = 76 900 M(-1) cm(-1) for the cation, neutral form, anion and dianion, respectively. The dianion has the most intense fluorescence with a quantum yield of 0.93 but also the anion shows considerable fluorescence with a quantum yield of 0.37. The neutral and cationic species are upon excitation converted into the anion and fluoresce with quantum yields of about 0.30 and 0.18, respectively.