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As in our previous work, most attempts to study the self-aggregation of methylene blue (MB) in water have been limited to the dimer. In the present work, we have analyzed the self-aggregation of MB in water beyond the dimeric form. For this purpose, the visible light absorption spectra of a large number of aqueous solutions of MB (1.1 × 10 -6 to 3.4 × 10 -3 M) and NaCl (0.0-0.15 M) at different temperatures (282-333 K) have been fed to a mathematical routine in order to determine the potential existence of a unique higher-order aggregate without any preconception about the aggregation order or about the need of counterions, such as chloride, for compensating the positive charge of the aggregates. Contrary to the common belief that the trimer is the dominant aggregate at high MB concentration, to our surprise we found that the tetramer acting alone, and without any counterion, is the higher-order aggregate that yields the best fitting to all the experimental absorbance spectra, with a very low average relative error of 0.04 ± 0.34%. Also contrary to previous assumptions, it has emerged quite evidently that this aggregate is present in the solution at MB concentrations below 3.4 × 10 -5 M (11 ppm), though to a rather low extent. This has brought the need for the recalculation of the visible light absorption spectrum and the thermodynamic parameters for the dimer, which along with those for the tetramer are the main contributions of the present work.

Visible Light Spectroscopic Analysis of Methylene Blue in Water; What Comes after Dimer?