The visible spectra of methylene blue (MB) in aqueous-methanol solutions were used to determine the extent of hydration of MB aggregates. The investigation was carried out at MB concentrations of 1.000 X 10<sup>-4</sup> M and 1.000 X 10<sup>-3</sup> M. Abstract factor analysis (AFA) was used to determine the number of chemical species responsible for the spectral data. The species were identified as aqueous MB monomer, dimer, and trimer, and a methanoic MB monomer. The absorptivity spectra of the aqueous MB species obtained from the previous studies were used to extract their concentration profiles in methanol-water mixtures. With the addition of methanol to the aqueous solvent, the concentration of water could be varied from 0% to 100%. This variation was used to determine the extent of hydration and solvation of each of the four MB species, as well as their mutual equilibria. The species were identified as (1) MB<sup>+</sup>CH<sub>3</sub>OH, MB monomer monomethanol; (2) MB<sup>+</sup>H<sub>2</sub>O, MB monomer monohydrate; (3) (MB)<sub>2</sub><sup>++</sup>6H<sub>2</sub>O, MB dimer hexahydrate; and (4) (MB)<sub>3</sub>Cl<sup>++</sup>(H<sub>2</sub>O)<sub>13</sub>, MB trimer monochloro tridecahydrate. The dissociation constants for the hydrated monomer, dimer, and trimer were determined to be 0.48, 1.29 X 10<sup>3</sup> M<sup>5</sup> and 7.2 X 10<sup>6</sup> M<sup>13</sup>, respectively.
Zhouming Zhao and Edmund R. Malinowski, "Determination of the Hydration of Methylene Blue Aggregates and Their Dissociation Constants Using Visible Spectroscopy," Appl. Spectrosc. 53, 1567-1574 (1999)