Acid-base dissociation and tautomerism of two aminofluorescein dyes in solution

The four-step dissociation of two fluorescein dyes bearing the amino group in 4'- and 5'-positions of the phthalic moiety was studied in water. The constants describing the equilibria H4R2+ reversible arrow H3R+ reversible arrow H2R reversible arrow HR- reversible arrow R2- were determined using the spectrophotometric method, at 25 degrees C. In water, four steps of aminofluoresceins dissociation occur within pH range of 0-9. On going to 50 mass % aqueous ethanol, the increase in the pK(a1), pK(a2) and decrease in the pK(a0), pK(a(-1)) values take place. The visible absorption spectra, referring to all the ionic (molecular) forms, were singled out. On the whole, the behavior of 4'-aminofluorescein is closer to that of the mother compound. The shift of the equilibria on introducing 50 mass % ethanol into the aqueous solution allowed better understanding the detailed scheme of prototropic equilibrium. The tautomeric equilibrium of the molecules shifts toward the colorless lactone. This effect is expressed stronger for the 5'-aminofluorescein. For this dye, even signs of such tautomers of the single-charged anion as anion-lactone and phenolate anion are observed in aqueous ethanol, which are atypical for fluorescein in water-based solvents. (C) 2016 Elsevier B.V. All rights reserved.