Novel Reducibility of Methylene Blue by Hot Titanium Trichloride Chao-Yang Tzung Department of Gynecology and Obstetrics, Kao-Hsiung Municipal Hospital, 68 Chung-Hwa 3 Road, Kao-Hsiung, Formosa
IN DILUTED AQUEOUS SOLUTIONS, the sulfoxide (5-oxide) structure (CIF,HI~N~SO)+ is preferred for methylene blue, rather than the structure (Cl&N3S)+, proposed by Bernthsen or Kehrmann for explanation of bi- and tetravalent reversible reduction decolorization, pH indicator properties of bivalent reduction products, molecular weight determination, and elemental analysis of methylene blue thiocyanate. In the preparation of methylene violet, it is found that methylene blue reacts in a characteristic fashion with KC1O3, an oxygen donor, and Na2S03, an oxygen acceptor (1). A study has been undertaken to determine the oxidation state of methylene blue by viewing from the standpoint that an organic sulfide generally has as its derivatives sulfoxide and sulfone. That the simplest stable-in-air oxidation product of phenothiazine is phenothiazine 5-oxide is an accepted concept(2-5). It is proposed that the behavior of methylene blue in a strongly diluted solution may be explained by assuming the dye is a 3,7-bis(dimethylamino) derivative of phenothiazine 5-oxide, as structure (A) :
EXPERIMENTAL
Methylene Blue Thiocyanate. Since the chloride is highly hygroscopic and difficult to standardize, the thiocyanate (13) is prepared after the contaminating lower homologs, such as azure B, azure A, or methylene violet are minimized from a commercial dye (14). The sample used is Biological Stain Commission certified methylene blue chloride, which loses 16.5 of its weight in a sulfuric acid desiccator. A 3.5gram sample of the dye is dissolved in 300 ml of borate buffer solution at pH 8.2, and filtered. Add successive portions of CHC13, 50 ml at a time, and extract. The CHC13 layer is rose red, but after several extractions the CHCl3 layer comes off colorless. Save the blue dye solution and add 5 ml of concentrated HCl, then a 650-mg sample of KSCN in 30 ml water. Purple crystals which turn to metallic green after a short time are formed (15). One drop of the dye is placed on a filter paper moistened by FeCI3 solution; that no excessive KSCN remains is indicated by the absence of a red-brown halo around a blue center. The crystal is filtered, washed with water, and while it is slushy it is poured onto layered filter papers on granular activated alumina in a refrigerator kept at 2 to 3" C. When the moisture is almost completely blotted, the crystal is dried to constant weight in a sulfuric (CH&N O m N I C H 3 ) z acid desiccator in an incubator at 36" C. The yield is 1.9 1 t 0 grams. Melting point, 219" C (corr). The melting point range (A) is 0.25-0.35" C, measured by a Beckmann type differential thermometer. A solution (Solution I) is prepared which This differs from the structure (B) proposed by Bernthsen consists of 20.1 mg of desiccated methylene blue thiocyanate, (6) or the structure (C) by Kehrmann (7, 8). 1680 mg of NaHC03 and 0.4N acetic acid in a total volume of 100.0 ml. The pH is determined to be 4.7 by a glass electrode. Five milliliters of Solution I is extracted by 2 ml of CHC13. No cherry red dye is extracted and the CHC13 layer remains colorless. This indicates that methylene violet contamination in the prepared methylene blue thiocyanate is not appreciable. Ten milliliters of Solution I is alkalinized to pH 8.0 by adding 260 mg of NaHC03 and 1 ml of 10% The elemental analysis and molecular weight determination of borax solution, and is extracted by 2 ml CHCI,. After being a dehydrated sample of methylene blue thiocyanate is accomvigorously shaken for ten seconds, the CHCI3 layer, after modated by the empirical formula (Cl7H17N4S2)20. At 3 to separation from the aqueous phase, is almost colorless. 8" C, 1 mole of (C16H18N3SO)+ is reduced to colorless C16H19The similar tests reveals, in CHC13,the rose red color of azure N3S0 by titrant, 2 N T P . But 4N Ti+3kept at 70 to 75" C B intentionally mixed with the prepared methylene blue reduces lmole of (C16H18N3SO)+ titrant to colorless C ~ ~ H N N ~ S . thiocyanate to a ratio 1 :150; at 1 :300 the result is ambiguous. In 28N H2SO41 mole of green (H+)(C16H18N3S)+3is reduced by Cold and Hot Reduction of Methylene Blue with Ti+3. 2N Ti+3 to yellow (H+)3(C16HlsN3S)+.This yellow dye beSolution 11 is diluted Tic13 in 1N HCl. While fresh, its comes colorless (H+)3(CI6Hl9N3SO)if the acidity is lower than reducing potency is found to be N = 1.04 X lo-? as TP3/ Ti0+2 by both Fe2(NH4)2(S04)4. 24H20 with KSCN indicator 20N HzS04; if the acidity of the colorless solution is raised and KMn04-oxalic acid methods. A cold reduction is to higher than 28N H2SO4,the yellow color appears again. This chill 10.0 ml of Solution I in a beaker to 3" C, and to titrate yellow form was postulated as the semiquinone free radical by dropwise addition of 1.11 ml of Solution I1 to decolorizaof methylene blue by Michaelis et al. in 1940 (9-12). tion as the end point, while sun light is avoided. The terminal temperature is about 8" C and the pH is 4.2. The newly devised hot reduction is to take 2.20 ml of Solution I1 in a beaker heated to 70-75°C during titration. After 9.96 (1) C. Y . Tzung, Stain Tech., 39, 351 (1964). ml of Solution I is added dropwise, the reagent is colored (2) S . Smiles, J. Chem. SOC.,95,1253 (1909). faintly blue. The calculated amount of Solution I1 required (3) Zbid.,97, 186 (1910). to decolorize 10.0 ml of Solution I, is 2.21 ml. White crystals (4) Zbid.,p. 1559. (5) Zbid.,101,2294 (1913). (6) A. Bernthsen, Liebig's Ann., 230, 73 (1885). (7) F. Kehrmann, Ber., 34, 4170 (1901). (8) Zbid.,39(II), 1403 (1906). (9) L. Michaelis, M. P. Schubert, and S. Granick, J. Am. Chem. SOC., 62,204 (1940). (10) Zbid.,62, 1802 (1940). (11) Zbid., 63, 351 (1941). (12) Zbid., p. 1636.
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ANALYTICAL CHEMISTRY
(13) H. J. Conn, "Biological Stains," 6th ed., Biotech Publications, Geneva, N. Y.,1953. (14) R. D. Lillie, Public Health Reports, Supplement No. 178, 17 ( 1944). (15) H. D. K. Drew and F. S. H. Head, J. Chem. SOC.,1933, p. 248.
precipitate during a hot reduction, but no H2S is appreciated in the reagent. After six hours of air oxidation, the reagent is centrifuged, and the blue supernatent is collected as completely as possible. The sediment is isolated, and identified as hydrated titanium dioxide (16). Methylene blue is identified (13) discoloring the collected supernatent. By a hot reduction, 2.2 ml of Scllution I1 is calculated as necessary to decolorize the recovered methylene blue. pH Response of Cold Reduction Products. Solution I11 consists of 20 mg of desiccated methylene blue thiocyanate in 100 ml of 28N H2S04. Solution I V is a Ti+3solution diluted in 28N H2S04; its reducing potency, determined as for Solution 11, is N = 1.02 :< lo-*, as Ti+3/Ti0+2. Solution V is 20N H & 0 4 for which the redox potential is poised by Ti+3 and methylene blue. To 50 ml of 20N H2S04 2.0 ml of Solution I11 is added. Then Solution IV is dropped m until the critical decolorization of green dye, and 1.0 ml of Solution 111 is added. The completed Solution V is faintly green colored. Ten rnilliliters of Solution I11 and 1.3 ml of Solution IV are mixcd. The green color (absorption maximum 400 to 440 mp, and 680 mp) turns to yellow (absorption maximum at 435 mp with a hump at 480 mp). Five-tenth ml of the yellow solution and 5 ml of Solution V are mixed. The yellow is decolorized (decolorization is not due to reduction, because the redox potential is indicated by green methylene blue in it). Upon addition of 10 ml of 9 6 z H2S04,the yellow color is again produced. This is not due to oxidation, as supplemental addition of 0.5 ml of Solution IV does not decolorize the yellow color produced. The green dye in 28N or higher H2S04is invariably completely decolorized if the acidity is made lower than 20N H2S04,provided the diluting solution does not contain an oxidizing agent. If an oxidizing agent is present, green, steel blue, or blue color are produced according to the terminal acidity. Anal. Calcd for (
