T H E EFFECT OF ULTRA-VIOLET RAYS ON T H E ULTRAVIOLET ABSORPTION SPECTRUM OF AQUEOUS METHYLENE BLUE SOLUTIONS CARL E. NURNBERGER
AND
L. EARLE ARNOW
Laboratory of Biophysics, University of Minnesota, Minneapolis, Minnesota Received August 3, 1933
In 1912 Lasareff (7) observed that methylene blue was bleached by sunlight if it were spread on thin plates in the form of a gelatin solution. The bleaching of the visible spectrum of methylene blue has been reported also by Gebhard (1) and Krestownikoff (6). Iimori and Kitaoka (4) found that methylene blue was decolorized by exposure to the rays of the mercury arc. Hill (3) has used the fading of solutions of methylene blue in 30 per cent acetone to measure the ultra-violet intensity in the “physiologically active” region of the spectrum. Stenstrom and Lohmann (9), in an investigation of the fading action of Roentgen rays on various colored solutions, found that aqueous methylene blue solutions were faded by the rays. In this paper we shall report the changes in the ultra-violet absorption spectrum of aqueous methylene blue solutions following irradiation with ultra-violet rays. The accurate determination of absorption bands by the visual selection of points of equal density on a photographic plate is made more difficult by the uncontrollable variations in the intensity of the light source. Some of the errors of this method are eliminated by the use of the Judd Lewis (8) sector photometer, manufactured by Adam Hilger, London, since, by the use of this instrument, simultaneous and adjacent spectra of the light passing through both photometer and solution are obtained. In this laboratory, we use an under-water tungsten spark of the type recommended by the Bureau of Standards (2) as a source of light. The advantage of such a source lies in the fact that the resulting spectrum is continuous and relatively free from intense emission lines. Light from this source is split by the lens system of the apparatus into two parallel beams. The upper of the two beams passes through the solution whose absorption bands are being determined, while the lower one passes through a variable sector photometer and, if it is desired, through the pure solvent used in making the solution. (We have always taken the precaution of placing either the pure solvent or distilled water in the lower beam.) The two beams pass through the lens system of a quartz spectrograph and are 71
72
CARL E. NURNBERGER AND L . EARLE ARNOW
then brought together on a photographic plate, the image of the upper one lying just above that of the lower. The light source previously has been carefully adjusted so that the two images are equally intense when the photometer is opened completely and when no solution is placed in either beam. A series of photographs is then made on one plate. The customary method is to take the first exposure with the photometer set at 0.1 (on the lower of the two photometer scales), the second exposure with the photometer set a t 0.2, and so on, the last exposure being made when the photometer is set at 1.5. In order to keep constant the density of the spectrum on the photographic plate produced by the light passing through the sectors, it is necessary to use a different time of exposure for each sector setting. The time of each exposure is calculated conveniently by means of the formula, Exposure time = A
J’
J
where A is a constant and where J’/J is the ratio of the light intensity before the light enters the photometer t o that after it has passed through the photometer. The constant, A , is easily determined by experiment. The ratio, J’IJ, is the antilogarithm of thelower scale reading of the photometer scale. After the plate is developed (which process requires no special care, since the pairs of spectra are both exposed and developed simultaneously and under identical conditions), the points of equal density on each of the pairs of spectra are determined by using a suitable ocular and are marked with India ink. The extinction coefficients ( 5 ) can then be calculated by means of the relation,
where K is the extinction coefficient, L is the length (in centimeters) of the absorption chamher uaed, and log,, J’/J (slight corrections, found in the directions furnished by Hilger, must be applied), the logarithm of the ratio of the light intensity before the beam enters the photometer to that after it leaves it, is the lower reading of the photometer scale. A wave length scale is provided with the spectrograph; two images, one a t the bottom and one a t the top of the series of exposures, are recorded on the photographic plate by means of 15-second exposures with a 40-watt electric lamp. The wave length corresponding to each India ink dot is determined by placing a straight edge across the two wave length scales and moving it until the edge passes through the center of the dot. By plot,ting the extinction coefficient as a function of the wave length, the absorption bands can be illustrated in graphic form.
EFFECT O F ULTRA-VIOLET RAYS ON METHYLENE BLUE
73
EXPERIMENTAL
A standard solution of methylene blue, containing 0.1 mg. of dye per cubic centimeter was made by dissolving recrystallized methylene blue in distilled water. From this stock solution, working solutions were prepared by dilution. By preliminary experiments, it was found that 0.008 mg. per cubic centimeter represented the correct concentration to show photographically the ultra-violet absorption bands when a 2-cm. absorption chamber was used. Accordingly this concentration was used in all our experiments. As a source of ultra-violet light for the irradiation of the methylene blue solutions, we used a Victor mercury arc lamp. All the solutions irradiated were placed at a distance of 30 cm. from the center of the lamp bulb. The 0.7
220 230 240 250 260 270 280 290 300 310 320 3% 540 350
Wave length (millimicrons) FIQ. 1. ULTRA-VIOLET ABSORPTION SPECTRA OF METHYLENE BLUEIN DISTILLED WATER 9 before irradiation; 0 after irradiation
operating voltage of the lamp varied between 65and 70 volts. In all cases except one (where distilled water was used as the solvent) the time of exposure was 2 hours. Figure 1 shows the absorption curve of methylene blue in distilled water, both before and after irradiation. A 20-cc. sample of the solution was placed in a small Petri dish (forming a layer of solution 0.8 cm. thick) and exposed to the rays. After a period of one hour, fading had become so marked that irradiation was stopped. The ultra-violet absorption curve shows that a very definite decrease in absorption had occurred in the ultra-violet region of the spectrum. The transmission at 670 millimicrons (point of maximum absorption of visible light by methylene blue (9)), as
74
CARL E . NURNBERQER AND L. EARLE ARNOW
0.7 h
0.6 -I-’
F’ 0.5 aJ
.A
3 0.4
2=
Q)
2
0.3
g
0.2
3 0.0
w
220 230 240 250 260 270 280 290 300 310 320 330
Wave
340 350
lenqt h (millimicrons)
FIQ.2. ULTRA-VIOLET ABSORPTION SPECTRA OF METHYLENE BLUEIN HYDROCHLORIC 0
ACID-POTASSIUM CHLORIDE SOLUTION (pH 2.19) before irradiation; 0 after irradiation through quartz
0.7
G 0.6 W
-w
.-5
0,5
$ 0.4 0
v
0.3
s .?
0.2
4-J U
.5 0.1
-w
U
0.0
220 230 240 250 260 270 280 290 300 310 320 330 340 350 Wave lenqth (millimicrons) FIQ. 3. ULTRA-VIOLET ABSORPTIONSPECTRA OF NETHYLENE BLUE IN PHOSPHATE (pH 6.69) SOLUTIONS 9 before irradiation; 0 after irradiation through quartz filter; o after irradiation through Blue-purple Corex A filter.
determined by a Bausch and Lomb spectrophotometer, increased from 0,0008t o 0.478 as a result of the irradiation. In order to be sure that the observed fading was caused by the ultra-
75
EFFECT O F ULTRA-VIOLET RAYS ON METHYLENE BLUE
violet rays and not by the heat produced by the lamp, two experiments were performed. Some of the same solution used above was irradiated under conditions identical with those described, except that it was protected by a thin sheet of ordinary window glass. No change in either visible or ultraviolet regions of the spectrum occurred. I n addition, some of the same solution was boiled for several minutes, the beaker being covered with a
Y
0.7
+ 0.6
'6 0.5 . 4
% F:
0.4
03
5 0.2 c
3 0.1 r(
w
0.0 220 230 240 250 260 270 280 290 300 310 320 330 340 350
Wave
length (mi[limicrons)
FIG.4. ULTRA-VIOLET ABSORPTIOS SPECTRA OF METHYLENE BLUEIN SODIUMBORATE SOLUTION(pH 9.08) 9 before irradiation; 0 after irradiation through quartz filter TABLE 1
-
J/J' (670 PH SOLT'EIVT
HCI-KC1 buffer Sodium borate buffer Phosphate buffer Phosphate buffer
FILTER
Quartz Quartz Quartz Blue-purple Corex A
Before irradiation
2.19 9 08 6.69 6.69
~
MILLIMICRONS)
After irradiation
Before irradiation
2.18 9.07 6.68 6.70
0.0007 0.0008 0.0008 0.0008
I
After irradiation
0.125 0.699 0.660 0.005
watch glass t o prevent evaporation. In this case, also, the methylene blue spectrum was not altered. The work of Stenstrom and Lohmann (10) has brought out the fact that pH apparently has little or no effect on the visible absorption spectrum of methylene blue solutions before exposure to Roentgen rays, but that during exposure greatest fading takes place in solutions of highest pH. Our ex-
76
CARL E. NURNBERGER A N D L. EARLE A R N O W
periments have shown this to be true also in the case of ultra-violet irradiation. The effect of pH is illustrated by figures 2, 3, and 4 and by table 1. The pH of the solutions was measured potentiometrically. I n an attempt to determine which wave lengths were most effective, several filters-including window glass, Vita glass, Clear Corex D, Bluepurple Corex A, and quartz-were used. In the case of all the filters except quartz, the following procedure was adopted: 20c c. of the methylene blue solution was placed in a small Petri dish, after which the dish was covered with the filter. Irradiation was carried out as described above. In order to eliminate any possible effects due t o ozone formation, irradiations in which the quartz filter was used were carried out by placing the
1.0 0.9
d
0.6
.-.I
0.5 3
E
ul
0.4 0.3
? 0.2 0.1 0.0 230 240 250 260 270 280 290 300 310 320 330
Wave
length
340 350
Imillimicrond
FIQ.5. ULTRA-VIOLET TRANSMISSION CURVESFOR FILTERS 0 quartz; o Blue-purple Corex A solution in a small, completely closed quartz vessel. Ultra-violet transmission curves of the filters were determined by means of a quartz spectrograph in combination with a microphotometer. The curves for the particular samples of Blue-purple Corex A and quartz used are given in figure 5 . The transmission curve for the quartz was taken through the quartz vessel mentioned above and represents, therefore, the transmission of a filter of twice the thickness actually used. Figure 4 and table 1 show that the decrease in absorption produced when the Blue-purple Corex A filter was used was slight as compared with that produced with the quartz filter. Since the transmissions of the other filters were less than that of the Bluepurple Corex A glass, their transmission curves are not included here. It appears that the wave lengths most active in decreasing the absorption bands of methylene blue are shorter than 270 millimicrons. For this
EFFECT O F ULTRA-VIOLET RAYS ON METHYLENE BLUE
77
reason we do not believe that the change in aqueous solutions of methylene blue produced by ultra-violet rays can be used for the purpose of measuring the radiation intensity between 290 and 310 millimicrons (the “physiologically active” region of the spectrum). SUMMARY
1. The method of using the Judd Lewis sector photometer in combination with the quartz spectrograph is briefly described. 2. The ultra-violet spectrum of methylene blue is included. Maximum absorption occurs at 292 millimicrons and at 246 millimicrons. 3. Exposure of methylene blue solutions to ultra-violet rays causes a decrease in the absorption bands in both visible and ultra-violet regions of the spectrum. 4. pH has no effect on the ultra-violet spectrum of methylene blue solutions, but irradiation with ultra-violet light causes greatest change in solutions which have highest pH. 5 . The wave lengths most active in decreasing the absorption bands of methylene blue solutions are shorter than 270 millimicrons.
The authors wish to express their gratitude to Dr. W. K. Stenstrom who suggested the problem and whose cooperation has made this investigation possible. REFERENCES (1) GEBHARD, K.: 2. physik. Chem. 79, 639 (1912); Phot. Korr. 60, 118 (1913). (2) GIBSON,K. S., MCNICHOLAS, H. J., TYNDALL, E. P. T., ANDFREHAFER, M. K.: Bur. Standards Sci. Paper No. 440, p. 129 (1922). (3) HILL, LEONARD: Strahlentherapie 34, 117 (1929). (4) IIMORI, SATOYASU, AND KITAOKA, KAORU:J. Chem. SOC.Japan 48, 479 (1927). (5) International Critical Tables, 1st edition, Volume V, p. 359. McGraw-Hill Book Co., New York (1929). ( 6 ) KRESTOWNKOFF, A. : Skand. Arch. Physiol. 62, 199 (1927). ( 7 ) LASAREFF, P.: Z. physik. Chem. 78,661 (1912); 79,638 (1912). J. Chem. SOC. 116, 312 (1919); Proc. Roy. SOC.London 93B, 178 (8) LEWIS,S. JUDD: (1922). (9) STENSTROM, WILHELM,AND LOHMANN, ANNE:Radiology 16,322 (1931). (10) STENSTROM, WILHELM, AND LOHMANN, ANNE: Radiology 21, 29 (1933).
