1312
ROBERT LIVINQSTON
+ 3.78 X 14.21(0.7721 - 0.7035) 4- 0.155 X 89.63 (0.152 X 0.1030 + 3.15 X
(RaC) = 1.4 X 0.7035
= 1.0
+ 3.7 + 1.75 = 6.45 millicuries.
0.7721
- 3.30 X 0.7035)
REFEREXCES BATEMAN: Proc. Cambridge Phil. s o c . 16, 423 (1910). DUNWORTH: Kiature 144, 153 (1939). FEATHER ASD BRETSCHER: h o c . Roy. 8oc. (London) A166, 530 (1938). KOVARIK A K D ADAMS:Phys. Rev. 64, 413 (1938). (5) KIER: Phys. Rev. 66, 150 (1939). (6) PEREY:Compt. rend. 208, 97 (1939). (7) Report of International Radium Standards Commission: Rev. Modern Phys. 3, 427 (1931).
(1) (2) (3) (4)
TKE REVERSIBLE BLEACHIXG OF CHLOROPHYLL ROBERT LIVIXGSTOX
School of Chemistry, Institute of Technology, Cniversity of Minnesota, Minneapolis, Minnesota Rrceived M a y 10, 1941
It has been demonstrated by Porret, and Rabinowitsch (5) that chlorophyll in methanol solutions is reversibly bleached by exposure to visible light. Since this result is unique in the photochemistry of dyes' and has important bearing upon the interpretation of other photochemical reactions of chlorophyll, it was thought worthwhile to repeat and slightly extend these experiments. -4PPAFtATUS
Instead of using the elegant but rather difficult technique of Rabinow-itsch (6, 8), the measurements were made with simpler apparatus, which is illustrat'ed in figure 1. In this apparatus the chlorophyll solution was divided between t,wo plane-ended cylindrical glass cells (F and F') which were 10 cm. in length. These cells were supported in a blackened box (E) which was provided with the necessary windows. They were thermo1 The reversible bleaching of eosin and of thionine, observed by Weiss ( l l ) , occurs only in the presence of suitable reducing agents. I t appears to be more closely analogous t o the reversible photobleaching of methylene blue by phenylhydrazine (3) than t o the photobleaching of chlorophyll in the absence of a substrate.
REVERSIBLE BLEACHING OF CHLOROPHYLL
1313
stated by a steady flow of distilled water. One of the cells (F’) was so situated that it could be illuminated from the side. The illuminating system consisted of a 500-watt carbon arc (K), a shutter (L), a spherical condensing lens (M), a cylindrical lens (0),and a light filter (N). This light filter, which was made up of a saturated ferrous ammonium sulfate solution and a Corning glass filter H 348, transmitted the red end of the spectrum but absorbed the infrared as well as the blue and ultraviolet. The light which was used to determine the absorption of the solutions came from two 6-volt “headlight” lamps (.4and A’), which were run from the same storage battery. This light was rendered parallcl by the two
FIG.1 Thr apparatus
lenses (C and C’) and after being diaphragrned by the front windows of the box (E) passed through the cells (F and F‘). These two beams were then brought together upon the active surface of a photoelectric cell (J) by means of a lens (G). The beams passed through a Corning filter 352 (B), 5 cm. of 1.5 per cent copper sulfate aolution (H), and a Corning filter 241 (I). This filter combination transmits a narrow band with its rnaximum approximately a t 6500 A. The sector nheel (D) revolved in front of the box (E) and alternately interrupted the beams passing through the two cells (F and F’). The spacing of the sectors relative to the openings in the box (E) was so arranged that the intensity of the light falling upon
1314
ROBERT LIVIKGSTON
the photocell (J) did not fluctuate. Final adjustments were made by altering the position of the wheel relative to the box and by means of a resistance which was in series with one of the lamps. A change in the transmissivity of one of the cells resulted in an alternation in the intensity of the light falling upon the photocell. This alternating component was picked up by an A . C . amplifier (tuned to the frequency of the sector nheel) and was impressed upon a sensitive galvanometer by means of a rectifier. Changes in the transmission of the cell (F’) of 0.001 per cent could be detected by this device. The response of the amplifier-galvanometer system x a s calibrated by means of a thin test piece of glass, of known refractive index, which was immersed in the water of the thermostat (E). It \{as computed by Fresnel’s law that this piece of glass reduced the intensity of the transmitted beam by 0.8 per cent. MATERISLS
The chlorophyll used in these evperiments \vas a mixture of chlorophylls a and b, prepared by Professor Stoll and kindly put a t the author’s disposal by Professor James Franck. The methanol used in the preliminary experiments was of reagent grade and was redistilled before use. The other chemicals were of reagent grade and were used M ithout further purification. In the final series of experiments the methanol was refluxed with furfural (4, 12) and was finally distilled off from aluminum amalgam in an atmosphere of nitrogen through a 5-ft. column packed with glass spirals. The acetone was first precipitated as the sodium iodide complex (9) and was then distilled off from magnesium turnings in an atmosphere of nitrogen through the 5 f t . packed column. PROCEDURE
I n the preliminary experiments the solutions ivere made up in air (or in a n atmosphere of carbon dioxide), and they were then freed from dissolved gases by a method nhich was a slight modification of the technique* of Professor Thunberg (10). I n the final series of experiments approximately 0.020 mg. of chlorophyll \\as weighed directly into the celL3 The cell was then sealed to a vacuum line. The sol1 ent was freed from dissolved gases by successive low-temperature distillations, and 10 cc. of it was distilled into the cell. The cell v a s then sealed off under vacuum. EXPERIJIEXTAL RESULTS
I n several respects the results of the present investigation were qualitatively similar to those reported by Porret and Rabinowitsch ( 5 ) . In both 2 The author wishes t o express his indebtedness to Professor Thunberg for his kindness in demonstrating personally his simple and efficient technique. 3 The author is indebted t o N r . E. E. Renfren. of the School of Chemistry for performing this rather difficult manipulation.
REVERSIBLE BLEACHIXG O F CHLOROPHYLL
1315
series of experiments reversible bleaching was observed in air-free solutions, and this effect was suppressed and replaced by a slow irreversible bleaching by oxygen. On the other hand, in many of the present experiments the half-life of the bleached chlorophyll was greater than 10 sec., rather than less than 1 sec., as Porret and Rabinowitsch report. The results of three experiments are plotted in figure 2. These experiments were performed with an air-free solution in purified methanol. The form of these curves is typical of the experiments in which the half-life was relatively long, the regeneration of the chlorophyll following a secondorder law within the limits of experimental accuracy.
FIG.2 The results of three experiments on the reversible bleaching of chlorophyll
ii series of experiments were performed with varying intensity and time of illumination, with acetone as well as methanol as the solvent, and (in the preliminary experiments) with a variety of other substances added to the solutions. While the quantitative results of these several measuiements were not sufficiently reproducible to warrant their publication ,in detail, the general or qualitative effects appear to be significant. They are summarized by the following statements: When the half-life of the bleached product was relatively long, the bleaching produced by a brief exposure to light was directly proportional to the product of the duration and intensity of the illumination. The thermal rate of regeneration of the chlorophyll was proportional to the
1316
ROBERT LIVINGSTON
square of the concentration of the bleached form. The effect of this backreaction became noticeable when the illumination was prolonged, as is shown by curve A of figure 2. Although no determinations of the steadystate bleaching were made with such systems, the separately determined rate laws for the photochemical and thermal reactions are consistent with the proportionality between the steady-state bleaching and the square root of the intensity, which was observed with systems of short half-life. This latter result was reported by Porret and Rabinowitsch (4) and is confirmed by the present work. I n the present experiments the reversible bleaching appeared to be accompanied by a relatively slow irreversible bleaching. I n pure methanol solutions the irreversible effect was less than 10 per cent of the reversible one. However, in acetone and in some of the methanol solutions which contained added substances, i t appeared to be 30 or 40 per cent of the total bleaching. The slowest rates of regeneration were observed in the solutions made up with highly purified methanol, the half-times being from 50 to 100 sec. Because of the accompanying irreversible fading, it is difficult to draw definite conclusions about the effect in acetone, but the half-time of regeneration seemed to be of the same order of magnitude. I n the preliminary experiments (using once-distilled, reagent grade methanol) halftimes of from 5 to 20 sec. were observed. The addition of either hydroquinone or allylthiourea (at concentrations of 0.01 M ) had no marked effect upon the bleaching. The substitution of isoamylamine for half of the methanol in these solutions likewise had little effect upon the course of the bleaching. On the other hand, as Rabinowitsch and Porret report, formic acid M ) has a marked effect, increasing the half-time threeor four-fold. THE QUANTUM YIELD AND THE ABSOLGTE VAI'UE OF THE RATE CONSTAKT
For those experiments where the half-life of the bleached chlorophyll was long compared to the period of the galvanometer (3 sec.), it is possible to separate the photochemical and thermal processes and to determine the quantum yield of the first and the bimolecular rate constant of the second. Before these computations could be made, it was necessary to determine the number of quanta absorbed per second and to calibrate the deflection of the galvanometer in terms of the change in the concentration of the chlorophyll. The calibration of the galvanometer deflection was made with the aid of a double absorption cell, 1.0 mni. thick. One compartment of this cell was filled with methanol and the other with the chlorophyll solution. The cell was so placed that either the solution or the solvent could be introduced into one of the scanning beams. In this way it was determined that,
REVERSIBLE BLEACHING OF CHLOROPHYLL
1317
when the apparatus was adjusted to give a deflection of 3.5 cm. for the test plate, a change of 0.10 per rent in the concentration of the chlorophyll produced a deflection of 1.2 cm. I n other words, a change in intensity of the transmitted beam of 0.27 per cent corresponded to 0.1 per cent change in the concentration of the chlorophyll.4 The total radiant energy from the arc (K of figure 1) which was incident upon the cell (F') was determined with a galvanometer and thermopyle, which had been calibrated a i t h a standard lamp. To forus the light upon the thermopyle, the cylindiical lens (0)was replaced with a spherical one of approximately the same focal length. The incident energy, determined in this way, was approximately 4.3 X lo5ergs per second. By combining a n approximate computation of the wave-length distribution of the light transmitted by the filter system (N) with the extinction curve of chlorophyll ( 7 ) , it ~ v t estimated s that between 2 and 8 per cent of the incident light was absorbed. Taking 4 per cent as a probable value, it may be computed that 4 X 10'6 quanta are absorbed per second. Taking experiment A of figure 2 as typical of the experiments performed in pure methanol solution, the quantum yield may be determined as follows: The decrease in chlorophyll concentration which occurred during the first 20 sec. of illumination may be computed from the corresponding galmoles per liter. The quantum yield \anometer deflection to be 6.1 X (q) for this process is given by the following expression, where the factor 10-2 is introduced, since the volume of the cell was 10 cc. : q =
6.1 X lo-' X lo-* X 6.0 x 10'' 4 x 10'6 x 2 x 10
-
Although this value is based upon crude measurements, it appears very probable that the correct value of q is between 2 X 10-4 and 2 x 10-3. It is certainly much less than unity.5 The dark rate of regeneration of chlorophyll (after a period of illuniination) call be represented by the usual bimolecular equation
where k is the rate constant, co is the inolarity of the bleached chlorophyll a t the beginning of the dark period, and c is its molarity after a lapse of While t,llese measurements were only rough approximations, they appear to be consistent with the extinction measurements of Zscheile (13) and of Rabinowitsch and Weiss (7). A computation based upon the data of t,he latter experimenters indicates that the change in intensity should be about 0.20 per cent rather than 0.27 per cent. This difference is within the limits of error and is immaterial to the present considerations. 5 Contrast reference 5 .
1318
ROBERT LIVINGSTON
t sec. The values of k obtained in this way differ widely for the several solutions. Even for a single solution k appears to vary with the previous treatment of the solution. For example, the value of k corresponding to the smooth curves drawn for experiments A, B, and C of figure 2 are 6.5 X 106, 5.9 X lo6, and 8.3 X lo6, respectively. During illumination the rate of fading should be the difference between the photochemical bleaching and the bimolecular regeneration
where I is the intensity of the absorbed light and K is a constant proportional to the quantum yield, p. The integrated fon.1 of this equation can be put in the following convenient form by substituting CY = ( K l / k ) * . In CY+c = 2akt CY-c The smooth curve corresponding to the light interval of experiment A is a plot of this function with k = 6.5 x 105 li. per mole per second and CY = 1.66 X lo-* moles per li. per second. GENERAL DISCUSSIOS OF THE RESULTS
While the experimental evidence presented here, like the results of Porret and Rabinowitsch ( 5 ) , appears to be too uncertain to justify any detailed theoretical conclusions, it may be of interest to compare these findings with the generalized mechanism of dye photochemistry which was proposed by Franck and Livingston (1). They suggested that the reversible fading of chlorophyll in the absence of a substrate could be represented by the following mechanisms
(1) .
GH
+ hv
-+
GH*
(2)
GH* -+ G H
(3)
GH* -+ HG
(5)
HG
(6)
G
+ GH
-+
(absorption)
+ hv,
GH2
+ GH, + 2GH
(fluorescence) (internal conversion accompanied by tautomerization)
+G
(the bleaching reaction) (regeneration of the chlorophyll)
The special symbols have the following significance: GH, normal chlorophyll; GH*, electronically excited chlorophyll; HG, a reaction tautomer of chlorophyll; GH,, the partly reduced, bleached form of chlorophyll; G, the partly oxidized form of chlorophyll. Both G and GH2 are radicals. Since the fluorescence yield is relatirely small (* 0.08), this mechanism leads to the conclusion that the quantum yield of the photochemical
REVERSIBLE BLEACHING OF CHLOROPHYLL
1319
bleaching should be nearly unity. Since one of the present findings is that the quantum yield6 is less than 2 X l O W , it is apparent that the mechanism must be modified. This modification can be made most simply by introducing a new step, the spontaneous reversion of the tautomer (HG) to the 'normal molecule (GH).
HG + G H
(-1)
It is a consequence of this assumption that the quantum yield of the bleaching process should increase with increasing concentration of chlorophyll. If, as the present work seems to indicate, the half-life of the bleached chlorophyll is longest in the most carefully purified solutions, the impurities must catalyze the regeneration of the chlorophyll. Furthermore, any mechanism which may be suggested for this catalysis must be consistent with the fact that the steady-state bleaching is proportional to the square root of the light intensity even when the bleaching is relatively small. These conditions are satisfied by the assumption that the two following reactions occur simultaneously with the six steps already postulated':
(7) (8)
+ HG -+ BH + G BH + G GH + B B
+
The symbols B and B H denote, respectively, an oxidizing impurity and its reduced form. The fact that the rate constant for the bimolecular regeneration in pure solution is about fold less than predicted by the simple collision theory (assuming zero heat of activation and an arbitrary collision diameter of 3 X lo-* cm.) is consistent with this explanation. If the foregoing general explanation is assumed to be correct, the halflife of the reactive tautomer, in respect to its spontaneous rearrangement to form a nornial chlorophyll molecule, can be estimated from the available data as follows: If we assume, in the usual way, that the rates of change of the concentrations of the unstable intermediates, GH* and HG, are negligibly small, it follows that
The possibility that the low quantum yield is due to a process of internal conversion followed by degrading collisions with the solvent is rendered highly improbable b y Gaffron's (2) observation of quantum yields approaching unity for photooxidations sensitized by chlorophyll. ' The explanation suggested by Franck and Livingston (1) for the inhibition by ferrous ion cannot be extended to the present case, since it leads to the conclusion t h a t in the presence of a moderate amount of the impurity the (residual) bleaching would be directly proportional to the intensity of the absorbed light. The present mechanism is analogous to their explanation of the inhibiting action of oxygen.
1320
ROBERT LIVINGSTON
Since the fluorescentyieldls relatively small, kz > ks(GH) for the present conditions where (GH) = 2 X 1 P M . Therefore
Since it is very improbable that kg is larger than k6, we may write
Values of 'p = 5 X and ka = 5 X lo8 correspond to a (natural) halflife of the tautomer equal to or greater than 4 X le6 sec. SUMMARY
As was previously reported by Porret and Rabinowitsch ( 5 ) , air-free chlorophyll solutions are reversibly bleached by light. This reversible bleaching is inhibited by oxygen. The quantum yield of the photochemical bleaching is not unity, as previously suggested, but is approximately 5 X The regeneration of the bleached chlorophyll follows a secondorder law. The smallest rate constant which was observed for this regeneration reaction was 7 X l o 5 li. per mole per second. Allylthiourea and similar easily oxidizable reducing agents have, a t moderately low concentrations, little or no effect upon the bleaching. These several results are compared with a general theory of dye photochemistry which was suggested recently by Franck and Livingston (1). This study is an outgrowth of discussions of related problems with Professor James Franck, to whom the author is deeply indebted for many valuable suggestions. He also wishes to express his gratitude to Dr. Foster Rieke, who designed and supervised the construction of the differential photometer. REFERENCES (1) FRANCK AND LIVINGSTON: J. Chem. Phys. 9, 184 (1911). (2) GAFFRON: Ber. 60B,755, 2229 (1927). (3) HOLST:Z. physik. Chem. A179, 172 (1937); A182, 321 (1938); Dissertation, Lund, 1938. (4) MORTON AND MARCH: Ind. Eng. Chem. 6, 151 (1934). (5) PORRET AND RABINOWITSCH: Nature 140, 321 (1937). (6)RABINOWITSCA AND LEHMANK: Trans. Faraday SOC.31,689 (1935). (7) RABINOWITSCH AND WEISS:Proc. Roy. SOC. (London) A160,251 (1937). (8) RABINOWITSCH AND WOOD:J . Chem. Phys. 4,497 (1936). (9) SHIPSEY AND WERNER: J. Chem. SOC.103, 1255 (1913). (10) THUNBERQ: Handbuch der biologischen Arbeilsrnethoden, Teil I, Heft. 7. Urban and Schwarzenberg, Berlin (1920). (11) WEISS:Trans. Faraday SOC.36, 48 (1939). (12) WEISSBERGER AND PROSKAUER: Organic Solvents. The Clarendon Press, Oxford (1935). (13) ZSCHEILE: Botan. Gaz. 96, 529 (1934).
1321
ADSORPTION AT INTERFACES
ADSORPTION AT WATER-AIR AND WATER-ORGANIC LIQUID INTERFACESlv2 F. E. BARTELL
AND
JAMES K. DAVIS
Department of Chemistry, University of Michigan, Ann Arbor, Michigan Received May 3, 1841
The efficacy of a surface-active material in lowering the boundary tension a t an interface is indicative of the extent of adsorption of the material a t that boundary. The dependence of the extent of adsorption of any material upon its ability to lower the bouiidary tension a t constant temperature is stated quantitatively by the well-known equation of Gibbs ( 5 ) : -dS = rldpl
+ ridp2..
e .
*
- + rib,
(1)
where S is the boundary tension, I' is the surface excess per unit surface area, I.( is the chemical potential, and the subscripts 1,2, . . i refer to the components of the system. The extents to which a given surface-active solute lowers the boundary tensions a t the liquid-air and a t the liquid-liquid interfaces are a measure of the relative adsorptions of the solute a t the two boundaries concerned. I t has been generally considered that materials which are effective in lowering the boundary tension of water against air also tend to lower the interfacial tension between water and an immiscible organic liquid, but up to the present time only a few attempts have been made either to compare the extent of adsorption of a chemical compound at the various interfaces or to compare its effect on the boundary tensions a t those interfaces. Traube, Weber, and Guirini (16) have found that the aqueous solutions of a number of substances exhibit tensions a t the interface against oleic acid and against liquid paraffin which parallel in value the surfare tensions of the aqueous solutions of these substances. Other investigators have, in isolated instances, compared the adsorption of an organic solute between water and air and between water and an organic liquid. Tlius, Harkins and King (8) found that the maximum adsorption of butyric acid between water and benzene is the same as that between water and air, but that for equal concentrations of butyric acid in the aqueous phase, I
* Presented a t the Seventeenth Colloid Symposium, held a t Ann Arbor, Michigan, June 6-8, 1940. The material presented in this paper is from a dissertation submitted by James K. Davis t o the Horace H Rackham School of Graduate Studies of the University of Michigan in partial fulfillment of the requirements for the degree of Doctor of Science, 1940.
