Spectrophotometric estimation of the p Ka values, absorption spectra and structural formulas of molecular species in aqueous solutions of fluorescein and sulfonefluorescein

Abstract

The absorption spectra of 5×10-6M solutions of fluorescein (F) or sulfonefluorescein (SF) in 0.2 M phosphate buffers of integral pH numbers ranging from 0 to 10 were measured. The spectra of SF demonstrated three molecular species: H2SF, HSF and SF2. At the wavelength of the second isosbestic point, the pH at which the absorbance of SF became average expressed pK a1 = 3.10 when the acid dissociation constant of HA was defined as Ka = aH+ [A-] /[HA]. Similarly pKa2 = 6.25 was estimated based on the absorbance at the wavelength of the first isosbestic point. By using these values and computer graphics, identical spectra of HSF were obtained from both spectra of SF at pH 4 and 5. From these results, the structures of the three species were estimated to have quinoid forms. The spectra of F demonstrated four molecular species: H3F +, H2F, HF-and F2. At the wavelength common to both isosbestic points, the pH at which the absorbance of F became average was expressed pKa2 = 4.20. Succesively, by computer graphics and using trial and error, the coincident spectra of HF were obtained, together with the value of pKa3 = 6.39, from both spectra of F at pH 6 and 7 where the absorbance of H2F was negligible. The spectrum of H2F was similarly obtained together with the value of pKal = 2.24. By adding 1/5 of the H3F+ spectrum to 2/15 of the HF spectrum, an identical spectrum as that of H2F was obtained; hence, H2F is thought to be a mixture comprising a lactone and two quinonoid species, while H3F +, HF-and F2-are quinonoid forms. © 1994, The Japan Society for Analytical Chemistry. All rights reserved.