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Fluorescein is exclusively popular owing to bright fluorescence of its R2− dianion. While the structure of the latter and tautomerism of the H2R form are well established, only the species with COO− and OH groups has been supposed for HR− up to now. Using absorption, emission, and excitation at different pH in non-HBD solvents, DMSO, acetonitrile, and acetone, we reveal that 10–12% of HR− ions nevertheless exist as “phenolate” monoanion with nonionized COOH group.

The ionization constants of 2,7-dichlorofluorescein were determined and the molecular structures of the equilibrium forms were established in the ternary solvent system benzene-ethanol-water (mass ratio 47:47:6) by using visible spectroscopy. In this solvent mixture with relative dielectric constant e r ¼ 12.8 and normalized Reichardt's parameter E N T ¼ 0:587; an inversion of the stepwise ionization constants of the dye, K a1 and K a2 , occurs. The K a1 /K a2 ratio changes from 16 in water and 4.8 in 50 mass % aqueous ethanol to 0.1 in the ternary solvent system with ionic strength of 0.002 mol dm À3 , where pK a1 ¼ 9.23 AE 0.08 and pK a2 ¼ 8.22 AE 0.08 (25 8C). Such an extraordinary interrelation between the K a constants is in line with the molecular structure of ionic and nonionic species of 2,7-dichlorofluorescein deduced from their vis absorption spectra, as well as with the expressed salt effects and the probable ion association. The dianion R 2À (5 in Scheme 1) possesses a band with l max ¼ 512 nm and a molar absorptivity of E ¼ 84.3 Â 10 3 cm À1 mol À1 dm 3 . The tautomeric equilibrium of the neutral form, H 2 R, is strongly shifted toward the colorless lactone (2 in Scheme 1). The (very intensive) absorption band of the HR À species singled out from the vis spectra at different acidity, is red-shifted by Dl % 20 nm against the band of the dianion R 2À . Hence, the monoanion HR À is found to be completely converted into the 'phenolate' tautomer 4 with groups -COOH and -O À , while in aqueous solutions the 'carboxylate' tautomer 3 with groups -COO À and -OH predominates. In 50 mass % aqueous ethanol, the two tautomers 3 and 4 exist in commensurable concentrations. Emission and fluorescence excitation spectra and fluorescence quantum yield of the dianion R 2À in the ternary solvent mixture were determined.

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