RESUMEN
Potentiometric back-titration of yellow solid fluorescein (H(2)Fl) and of red solid fluorescein in alkali with acid yielded titration curves that were practically identical in shape and position. The end-points at pH 8.5, 5.40 and 3.3 corresponded, respectively, to titration of the excess of standard alkali, and the successive protonations Fl(2-) + H(+) = HFl(-) and HFl(-) + H(+) = H(2)Fl. The pH at the mid-point of the first protonation yielded a value of 6.36 for pK(HFl) (ionic strength 0.10). Because of precipitation of yellow fluorescein during the second protonation step, a value for pK(H(2)Fl) could not be obtained. The total concentration of fluorescein at the first appearance of the precipitate fell on the curve for the solubility of yellow fluorescein as a function of pH. The titrations and the pK values found for the three acid groups of protonated fluorescein (H(3)Fl(+)) have been interpreted on the basis that in water fluorescein exists in only one structural form the yellow zwitterion. Similar back-titrations of alkalinized solutions of yellow or red fluorescein in 50% aqueous ethanol showed that in this medium fluorescein is present in only one form, presumably the quinonoid structure, with much weaker apparent acid functions, pK'(1) = 6.38 and PK'(2) = 7.16 (ionic strength 0.10).