METHYLENE VIOLET (BERNTHSEN), BY ZINC-ALKALI-CHLORATE HYDROLYSIS OF METHYLENE BLUE.

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Though Bernthsen's methylene violet (MV) is a common constituent of polychrome methylene blue, the hydrolytic oxydation of methylene blue to yield azure-free MV has been considered a difficult chemical reaction since the time of Bernthsen, who used Ag2O in the hydrolysis. MV is qualitatively distinguished from azures by Bernthsen's criteria and the author's new tests: (1) light-excited isomeric change, (2) reactivity to acidity, (3) reaction with KCIO, and (4) reaction with Na2SO3 of azures in CHCI3, while MV gives none. But MV shows (5) indicator properties at pH 4, while azures do not. For practical hydrolysis, treat methylene blue (10 parts by weight) and KCIO3 (1 part) with 1-2 N NaOH to convert methylene blue to a mixture of MV and azures. Then dilute the solution, add a Zn salt and NaHCO3 in excess of the amount needed to convert the NaOH to Na2CO3. Boil the solution gently for 1-2 hr. The end point of the reaction is found by pipetting a drop of reactant into 3% acetic acid in a test tube, adding CHC13 and extracting. The acetic layer should then be almost colorless while the CHC13 is colored intensely cherry red. After cooling, the precipitated dye is filtered and dried. This procedure gives good yields of a dye which meets the criteria given by Bernhsen. The peak of the absorption curve in solution, pH 4-11, is at 624 mμ (Bernthsen 625 mμ) and in acid solution, pH 0-4, 588 mμ (Biological Stains, 1953; 580 μ). The dye contains so little azures, that purification of the MV fraction obtained from the reaction mixture is unnecessary when it is used in the Wright-type Romanowsky stain. The remarkable staining effect of MV is its power to bring out red azurophil granules of monocytes and lymphocytes when used with eosinated thiazins in Wright's stain.