The stepwise ionization (H(3)R(+) <==> H(2)R <==> HR(-) <==> R(2-)) of four fluorescein fluoro derivatives was studied by visible spectroscopy. The pK(a) values were determined in water, in 50 mass % aqueous ethanol, in oil-in-water microemulsions (benzene + CTAB + pentanol-1 in water with 1.0 M KCl; CTAB = cetyltrimethylammonium bromide), and in reversed ones (water + AOT in n-octane; AOT = bis-2-ethylhexylsulphosuccinate or Aerosol OT). The medium effects, DeltapK(a), i.e., changes in pK(a) of these dyes on going from water to some other solvent systems, were rationalized by considering the tautomerism, the values of microscopic ionization constants, and the charge types of the acid-base couples. An expressed shift of the tautomeric equilibria of H(2)R toward colorless lactone was registered on going from water to both aqueous ethanol and organized solutions. While the monoanions HR(-) of 3',4',5',6'-tetrafluoro- and 2,7,3',4',5',6'-hexafluorofluorescein exist in all the systems studied as a tautomer with ionized carboxylic and nonionized hydroxy groups, in the case of 2,4,5,7-tetrafluorofluorescein, the prevalence of another tautomer was observed (COOH and O(-) groups). For 2,7-difluorofluorescein (Oregon Green 488), the partial shift of the tautomeric equilibrium of HR(-) was registered from (COO(-) and OH) in water to (COOH and O(-)) in other solvent systems. The data for the dyes located in an AOT-based pseudophase indicate that the interior of the latter exerts essential differentiation of the acid strength of the dyes, probably caused by the peculiarity of dye species location in water pools. While the state of tautomeric equilibria resembles that in nonaqueous media, the absorption maxima of R(2-) species are close to those in water. Such nonuniform influence displayed by AOT-based water droplets should be taken into account when examining them by using different molecular probes.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.