NOTES
absorbed, indicating the major product is polymer formation. The polymer was observed under all reaction conditions as evidenced by the decreased transmission of the cell after each run. Quantitative results were difficult to obtain, except a t high benzene pressures, because of the small amount of gaseous products formed. At these pressures of benzene, most of the light is absorbed in a region near the window of the reaction vessel and some of the observed products may represent secondary photolysis. Except for butene, the products formed a t high pressure, shown in Table I, are identical with the products formed a t low pressures. The higher acetylenes have been observed in the photolysis of acetylene’ and are probably the result of secondary photolysis of this compound. The present work shows that reactions 1 and 2 are not major reactions in the photolysis of benzene at 1470 A.
3347
more dimer is formed in alkaline solution. They suggested that the red form of the dye, extracted into benzene a t pH 11.2, is a dimer of MB. These observations contradict those of Lewis, et al.,“ who had found that MB does not dimerize in solvents of low dielectric constant (cf. also Rabinowitch and Epsteing). I
I
I
I
I
I
0.8
Changes in the Absorption Spectrum
of Methylene Blue with pH1
by G. S. Singhal and E. Rabinowitch Department of Botany, University of Illinois, Urbana, Illinois (Received March 7 , 1067)
Knowledge of the variations in the absorption spectrum of methylene blue (JIB) in different solvents and a t different pH is important in view of applications of this pigment in biology, clay mineralogy, and some basic photochemical experiments.2 The effects of pH, time, and temperature on the methylene blue spectrum in aqueous solutions had been studied Demethylation of tetramethyl thionine (methylene blue) to trimethyl thionine (TMT, or “methylene azure B”) and to asymmetric dimethyl thionine (“methylene azure A”) had been shown to O C C U ~ . ~Suggestion that T M T might exist in two tautomeric forms was made by Holmes and S n ~ d e r . ~ b T M T is present as a positive ion in acidic solution and as a neutral molecule in alkaline solution.8 The dimer MB2+ is formed in aqueous solution at pH 3.4 a t concentrations above 2.5 X 10-6.9 Palit and Saxena’O have found, a t pH 11.2, an increase in the shoulder-topeak ratio in the RIB absorption band, suggesting that
460
500
540
500
620
660
700
(nm)
Figure 1. Absorption spectra: 1, methylene blue in water; 2, TMT in water; 3, TMT in ether; 4, TMT in benzene.
(1) This research was supported by grants from the National Science Foundation (GB 3305) and the U. S. Atomic Energy Commission (AT(ll-1)-1502). (2) D. Frackowiak and E. Rabinowitch, J . Phys. Chem., 70, 3012 (1966). (3) W. J. hiacNeal and J. A. Killian, J . A m . Chem. SOC.,48, 740 (1926). (4) (a) W. C. Holmes, Stain Techn., 3, 45 (1928); (b) W. C. Holmes and E. F. Snyder, ibid., 4, 7 (1929). (5) R. D. Lillie, ibid., 18, 1 (1943). (6) Y. Koyama, hl. Masuda, and hl. Yoshikawa, Osaka Daigaku Igaku Zasshi. 10, 1193 (1958). (7) L. Michaelis, H. P. Schubert, and S. Granick, J . A m . Chem. Soc., 62, 204 (1940). (8) K. Bergmann and C. T. O’KonRki, J . P h y s . Chem., 67, 2169 (1963). (9) E. Rabinowitch and L. P. Epstein, J . Am. Chem. SOC.,63, 69 (1941). (10) S. R. Palit and G. K. Saxena, Nature, 209, 1127 (1966). (11) G. N. Lewis. 0 . Goldschmid, T. T. Magel, and J. Bigeleisen, J . A m . Chem. Soc., 65, 1150 (1943).
Volume 71.Number 10 September 1967
3348
I n the above-mentioned studies, spectrophotometric identification of the different forms of the dye was obtained either by visual examination of the spectrum, or by measuring the optical density in the absorption maximum, or the peak-to-shoulder ratio in the absorption band in J f B solutions of different pH, and comparing it with the corresponding values in pure water. We thought it might be of interest to know more about the different stages in the change of the absorption spectrum of MB with growing pH and to check whether the “red” form of 1 I B extracted into benzene, a t pH 11.2, really is a dimer. For this purpose, we extracted J I B into ether or benzene a t different pH’s and measured the absorption spectrum after transfer of the dye from the organic solvent back into water. The I\IB of Nerck and Co. was purified by crystallizing it once from 0.1 N HC1 and then twice from water. The resulting product had an absorption peak a t 663661 nm (Figure l), and a molar extinction coefficient in this peak equal to 9.0 X lo4. In the concentration used, its aqueous solution had a pH of 4.5. Concentrations used were below 1 X M . All of the absorption spectra were measured with a Bausch and Lonib spectrophotometer (Spectronic 505).
Results and Discussion When a buffered aqueous solution of the dye (pH 8.5) was shaken with ether, the ether layer became slightly pinkish. The absorption spectrum of the ether solution and that of the residue from evaporation of the ether extract, dissolved in water, are given in Figure 1. Also given in this figure is the spectrum of the same residue dissolved in benzene. Shaking under argon instead of air did not make any difference. The position of the absorption maximum (650 nm) in water suggests that this product is trimethyl thionine (TAIT).8 The addition of oxalic acid to the ethereal solution turned the red color into pinkish blue. When this solution was shaken with water, the ether layer gradually turned from pinkish to colorless and the water layer became increasingly blue in color. Even without the addition of any oxalic acid, when the ethereal solution of T l I T was shaken with water, the water layer became bluish. The absorption maximum of aqueous solution was in both cases a t 650 nm. This observation indicates that, presumably, T M T (red form) is extracted into ether or benzene as a neutral molecule, while the blue form of T l I T , present in neutral or acidic aqueous medium, is an ion. A break in the Eovs.pH curve a t pF1 12.2 confirms the existence of two forms of TNT. A similar transition point could not be observed in h3B in the same pH range. These observaThe Journal of Physical Chemistry
KOTES
0.8
0.7
0.6
0.5
0.4
d d
0.3
0.2
0.1
460
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540
580
620
660
700
X (nm) Figure 2 . Absorption spectra: 1, TMT in water a t p H 13.0; 2, ThIT solution (pH 13.0) adjusted to p H 3.0; 3, ether extract of TMT solution (pH 13.0) dissolved in water (pH -6.5); 4, ether extract of TMT solution (pH 13.0) dissolved in water (pH ~ 4 . 0 ) .
tions are in agreement with those of Bergmann and O’Konski8referred to earlier. To check the reversibility of the acid-base conversion, the following two T M T spectra in aqueous solution were compared; one mas measured with T M T solution having pH 13.0 (Figure 2 ) and the other with T M T first dissolved in an aqueous solution of pH 13.0 and then adjusted to pH 3.0 (Figure 2 ) . The blue shift of -635 nm) in the second case suggests the band (A,, that the acid-base equilibrium of TAIT is not completely reversible. This contention is further supported by spectra of the aqueous solution, prepared from TAIT extract in ether (obtained from aqueous TJIT solution at pH 13.0), adjusted to pH 4.0 and pH 6.5 (Figure 2). This incomplete reversibility probably indicates a further demethylation of TJIT, which seems to be base-catalyzed, and also suggests that it may be difficult to obtain pure ThIT because of further demethylations. Following the observations5 that the absorbancy changes of alkaline solutions are time dependent, the time course of these changes was studied. The absorption in the region of 610 nm, in an alkaline solution of
NOTES
3349
Table I: OD a t 610 nm/OD a t 663 nm as a Function of Time a t p H 10.7 and as a Function of p H (after 30 min) Time, hr-
7 -
OD a t 610 nm OD a t 663 nm
460
500
540
0
0.83
2.5
0.705
0.748
0.768
580
620
660
700
X (nm) Figure 3. Absorption spectra of differently demethylated methylene blue: I, dimethyl thionine, obtained from M B 1 hr; 2, monomethyl solution (pH ~ 1 2 . 0within ) thionine, obtained from a MB solution (pH -10.7) after about 60 hr; 3, completely demethylated MB-MB solution (pH ~ 1 2 . 0measured ) after 1 week.
M B (pH 10.7) when measured within 1 hr, was comparatively stronger than in pure water. With time, the over-all absorption decreased and the relative absorption around 610 nm increased (Table I). Also included in the table is the pH effect on the ratio of OD a t 610 and 663 nm. After about 60 hr, the pink form was extracted from RIB solution (pH -10.7) into benzene and dried under vacuum. Its aqueous solution 610 nm and a shoulder a t -570 nm (Figure 3, had A,, curve 2). In another series of experiments, extraction into benzene from an aqueous solution a t pH 12 was completed within 1 hr. Aqueous solution of the extracted material a t 619 nm, with a shoulder a t -578 nm had its A,, (Figure 2, curve 1). Fornianek12 found the absorption maxima of mono-, di-, tri-, and tetramethyl thionines a t 610, 622 (symmetrical), 652, and 667 nm, respectively. As reported above, we have observed the absorption maxima for the red forms, obtained a t different pH values and a t different times, a t 610, 619, and 650 nm. These are in
PH 11.0
13.0
0.695 0.732
1.19
. 7
18
0.788
io
90
0.878
0,905
6.i
7
good agreement with Formanek’s values for mono-, di-(symmetrical) and trimethyl thionines. This suggests that the red or pink forms, which we observed in alkaline solutions under different pH conditions and a t different times, were the different demethylated products of MB. Comparison of absorption maxima a t 589 nm (Figure 3, curve 3) of the solution (pH 12) measured after a week with that of thionin (A, 595 nm) leads to the conclusion that ultimately RfB may be completely demethylated, L e . , converted to thionine. Our results do not support the conclusion of Palit and Saxena’O that the red extract of M B contains a dimer. The spectrum of all red forms observed had the shoulder attributable to a vibrational sub-band (Figure 3) in the same relative position in which the absorption band of the dimer usually appears in dye. It seems that the red form is not an MB dimer, which has been reported to have two absorption peaks, a t 610 nm and a t 700 nm.* The spectrum of T M T in benzene or ether (Figure 1) gives support to this conclusion. The two forms of the demethylated dye can be expressed as
blue form of T M T present in acidic or neutral aqueous solution
red or pink form of T M T present in basic solutions, readily soluble in ether or benzene
Bergmann and O’Konski also suggested a similar acidbase transition in TMT.8 Summarizing the above results, it seems that in basic solutions methylene blue is stepwise demethylated, depending upon pH and time. The red form, observed in basic solutions, probably is the neutral form of the demethylated product. (12) J. Formanctk, “Untersuchung und Nachweis organischer Farbstoffe aud spektroskopischem Wege,” 2nd ed, Part 1, J. Springer, Berlin, 1908, pp 142-164.
Volume 71, Number 10
September 1967
