Studies on fluorescein-VII The fluorescence of fluorescein as a function of pH.

The relative fluorescence of fluorescein over the pH range 3-12 has been measured at 516 nm, with excitation at 489 nm. The relative fluorescence is essentially zero at pH 3, increases slowly between pH 4 and 5, rises rapidly between pH 6 and 7, reaches a maximum at pH 8, and remains constant at above pH 8. The curve of relative fluorescence as a function of pH lies somewhat above the corresponding curve describing the fraction of fluorescein present as the doubly charged anion, Fl(2-), indicating much weaker fluorescence of the singly charged anion, HFl(-), and very much weaker fluorescence by the neutral species, H(2)Fl. The fluorescence data have been used to calculate a value for the third dissociation constant. Because of the complexity of the system, one unknown dissociation constant and three (relative) fluorescence constants, a series of three variable regressions on the data was made. The final values were K(HFl) = 4.36 x 10(-7) (mu = 0.10) for the third dissociation constant and K(H(2)Fl) = 0.8; kappa(HFl) = 5.7; kappa(Fl) = 100.0 for the relative fluorescence constants.

Studies on fluorescein-III The acid strengths of fluorescein as shown by potentiometric titration.

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).

Spectrophotometric determination of iron in process samples from the chemical cleaning of coal.

Iron is removed during the desulphurization and demineralization of coal by a chemical cleaning process utilizing a mixture of molten sodium hydroxide and potassium hydroxide. When 1,10-phenanthroline is used for spectrophotometric determination of the iron in the various caustic, aqueous and acidic process streams, organic materials leached from the coal by the molten caustic interfere with the colour-forming reaction. Pre-oxidation of the samples with potassium persulphate has proved to be an effective means of removing the interfering organic material before the iron determination.

Studies on fluorescein-II The solubility and acid dissociation constants of fluorescein in water solution.

The solubility of yellow fluorescein and of red fluorescein as a function of pH has been measured in water at ionic strength 0.10. The pH of minimum solubility is the same for both, 3.28. The intrinsic solubility, defined as the solubility of the undissociated species, H(2)Fl, and assumed to be constant and independent of pH, was calculated from the observed solubilities on the low-pH side of the minimum: S(i, yellow) = 3.80 x 10(-4)M: S(i, red) = 1.45 x 10(-4)M. The first dissociation constants were evaluated from the intrinsic solubilities and the observed solubilities on the low-pH side: both fluoresceins yielded the same value, pK(H3Fl) = 2.13. In using the observed solubilities on the high-pH side of the minimum to evaluate the intrinsic solubility and the second dissociation constant it was necessary to modify the existing theoretical approach by taking into account the presence of the fully dissociated anion. Appropriate mathematical treatments were devised to handle the more complex equations. Both fluoresceins yielded the same value for the second dissociation constant, pK(H2Fl) = 4.44. Both fluoresceins give the same yellow colour in saturated solution and the results just reported for the pH of minimum solubility and for the dissociation constants also indicate that for each of the three prototropic forms of fluorescein present in solution, H(3)Fl(+), H(2)Fl, and HFl(-), only one structure exists.

The infrared spectra and structures of the three solid forms of fluorescein and related compounds.

The three solid forms of fluorescein, yellow, red and (newly isolated) colourless, have been shown to differ distinctly in structure. By spectroscopic methods, principally infrared absorption, the colourless form has been assigned the lactone structure, the red solid the p-quinone structure, and the yellow solid a zwitterion structure with a positive charge distributed over the oxygen-bearing ring.