2
WILLIAMARNOLD AND HELEN SHERWOOD
West studies the formation of the latent image in silver bromide and the influence of impurities in the crystal lattice. Eggert summarizes the influence of radiation on the ignition and detonation of explosives, and the transfer of energy in solids. Fluorescence is a simpler phenomenon than photochemical reaction, and fundamental information about excited states can be obtained from its study. Bowen studies the effect of temperature on the fluorescence yields of substituted anthracenes in several solvents, particularly the activation energy required for quenching. Dubois proposes a mechanism for the gas-phase fluorescence of P-naphthylamine, using benzene vapor a t 150' as photosensitizer.
Vol. 63
Chlorophyll is of special significance because it absorbs such a large fraction of sunlight and because it is necessary in biological photosynthesis. Thomas reports induction phenomena in fluorescence of different types of chloroplasts and correlates them with photosynthetic enzymes. Arnold describes experiments on the thermoluminescence of dried and preilluminated chloroplast films which showed that five different activation energies may be involved. Coleman and Rabinowitch report evidence of the photoreduction of chlorophyll in vivo, derived from observations of changes in the absorption spectrum which cells undergo during illumination. R. M. Noyes presents general kinetic considerations applicable to reactions involved in the photochemical storage of energy.
ENERGY STORAGE I N CHLOROPLASTS BY WILLIAMARNOLD AND HELEN SHERWOOD Biology Division, Oak Ridge National Laboratory,' Oak Ridge, Tennessee Received Julg 11, I968
Dried chloroplast films that have been illuminated exhibit thermoluminescence. The glow curves have been analyzed to give the activation energies associated with this energy storage. The analysis shows that at least five different activation energies are involved: one a t 0.93 e.v. represents a little less than half of the stored energy, another at 0.69 e.v. is a minor part, between these two there are two or three unresolved levels that represent the major fraction, finally, unilluminated samples always give a small signal corresponding to an activation energy higher than 0.93 e.v.
The phenomenon of energy storage in dried chloroplast films when illuminated was reported in a previous paper.* When the films are heated this energy' is emitted as red light, showing that an activation energy is involved in the process. Evidence was given that not one but rather a distribution of activat,ion energies was needed to explain the experiments. The present paper is a more detailed study of this distribution of activation energies. Materials and Methods Chloroplasts were prepared and the films painted and dried on stainless steel disks as described in the previous paper.a The glow curves (that is, the emitted light intensity as a function of the temperature or time as the sample was heated) were measured as before. However, two modifications in procedure were introduced. First each sample was heated only once. This change was made in response to sharp criticism on the part of the biochemists to the repeated heating of biological samples to 150". Second, the rate of heating the sample was adjusted so as to make the reciprocal of the absolute temperature a linear function of time. A schedule of settings of the Variac, that controlled the heater, wa.s made by trial and error. Figure 1 shows the heating curve used. The exact mechanism whereby energy is stored in dried chloroplasts is not yet known. However two interpretations seem to be reasonable. (1) The chloroplasts act like a semi-conductor and the energy is stored as trapped electrons. On heating these electrons are released and produce the light. An activation energy E is needed to transfer an electron from the trap to the conduction band. ( 2 ) During illumination some high energy compound is
formed, that on heating decomposes by a first-order reaction, and emits light. E is the activation energy for the decomposition. Either case would be expected to follow the differential equation3 where
N
the number of trapped electrons or high energy molecules K = frequency factor k = Boltzmann's constant 5" = absolute temperature t = time It is assumed that the signal 8, given by the photomultiplier used to measure the emitted light, is proportional to - dN /dt . I n the ordinary method of making a glow curve the sample is heated at a constant rate p so that T = TO pt where To = the tern erature a t the beginning of the experiment and t = time start of experiment. Using this expression for the temperature equation 1 can be integrated and written as In N = - f Ke-E/k(To+tWdt (2) The integral on the right leads to a series in which a great many terms must be used, making the comparison with experimental results very clumsy. The equations describing glow curves made with the heating schedule shown in Fig. 1 take on a more tractable form using the relation 1_ - 1 T - C Y t Equation 1 can now be integrated to give N = NoeQ(l-eat) (3) where No = value of N a t start of experiment =
+
Born
a = -E Y
~
(1) Operated by Union Carbide Nuclear Company for the U. S. Atomic Energy Commission. (2) W, Arnold and H. K. Sherwood, Proc. Nat. Acad. Sci., 43, No. 1, 105 (1957).
k
~
(3) J. T. Randall and M. H. F. Wilkins, Proc. Rob. Soc. (London). 184,372 (1945).
Jan. , 1950
ENERGY STORAGE IN CHLOROPLASTS Q=-
Ke -E/ k TO
1.o
E = 0.926 BY K = 2.45 x 1 0 ~ ~ ~ - '
01
Since the signal S (the intensity of the emitted light) is proportional to -dN/dt we can write S = - A dN - = Ah~,,~&e(Q[1-eQt1+~t) dt where A = proportionality constant. S , the maximum value of the signal will occur at the time t , when dS d t -- aS(1 - &eat) = 0
0.8
?
.
0.4 -
0.2 . 0
1
-
pO.6 -
so t h a t tm is given by &eat, =
(4)
1
S, = AaNoe(Q-1) The ratio of the signal a t any time to the maximum signal
5
= 6(1+a[t-tml
- ea[t-tm~)
(5)
can be used to give the activation energy E and the frequency factor K . The experimental data are plotted using S / S , as the ordinate and time as the abscissa. The time of maximum signal Im is determined by inspection. Equation 5 is then used to determine the value of a that gives the best fit to the data. Knowing a and t m e uation 4 gives the value of Q. E and K then are calculated %om the values of CY and Q.
,
,
3 '4 5
and
Sm
3
*x/;
,
,
,
,
,
6 7 8 , 9 10 11 12 13 14 Time (min.). Fig. 2.
3
1.2
B
iii
1
0
Time (min.). Fig. 3.
1 2
3
4 5 6 7 8 Time (min.). Fig. 1.
9 1 0 1 1 1 2 13
1.5 -
ILLUMINATED AT -4O'C HEATING RATE= 13.5°/m~n
4 1.0 -
Results .Y m Glow curves made on dried chloroplasts that have not been illuminated always show a small 0.5 signal a t the higher temperatures. The magnitude of this background signal is different from one batch of chloroplasts to another, but no treatment is known that reduces it to zero. In the following -50 0 +50 +lo0 +150 it is assumed that the effect of illumination is given Temperature ("C.). by the difference between the glow curve made with Fig. 4. an illuminated sample and one made on a sample The glow curve for that part of the stored energy held a t the same temperature in the dark. Glow curves made on samples of dried chloro- that is removed by annealing is shown in Fig. 3 as plasts illuminated in air by neon light for 12 hours the dashed curve with a peak a t 10 minutes. The a t 20" and corrected for background do not cor- curve is the difference between an illuminated respond to equation 5. The experimental curve sample and a sample illuminated and annealed. is much wider than the calculated one, showing The curve can only be fitted to equation 5 by using again that there are several activation energies two or three different activation energies, all someinvolved. Partial heating before the experiment what smaller than the 0.93 e.v. found before. will anneal out tha,t part of the stored energy that Figure 3 also gives as the solid line the data from has the lower activation energy. Figure 2 gives Fig. 2 and the background signal from a dark data for an experiment made after both the il- sample. All three curves are plotted on the same luminated and dark sample had been heated to scale. As can be seen the activation energy a t 88" and slowly cooled down to 20". The points 0.93 e.v. represents nearly one-half of all the light are the experimental results and the solid line emitted. has been calculated by equation 5. The activation Figure 4 gives a glow curve, made by heating energy E is 0.93 e.v. and the frequency factor a t a constant rate, for a sample illuminated in dry K is 2.5 X lo9set.-'. air a t -40". The curve shows there is a small
4
E. J. BOWEN AND J. SAHU
peak a t 40" that precedes the main light emission. The equations of Randall and Wilkins3 with the value of K as 2.5 X lo9 set.-' give the activation energy for this peak as 0.69 e.v. Unless the sample is cooled this peak is largely annealed out during the illumination. During the course of these experiments a number of qualitative observations were made that bear on the mechanism whereby dried chloroplasts store energy. They are summarized in the following statements. (1) The composition of the atmosphere surrounding the sample during the heating seems to have no effect on the glow curve. (2) Samples illuminated in air or 0 2 become charged, that is, store energy. (3) Samples illuminated in N2 or SO2 do not become charged. (4) Neither water vapor or COz seem to have any effect on the charging. ( 5 ) Air and 0 2 illuminated with short wave length ultraviolet light can charge the dried chloroplasts by flowing over the surface in the dark, ( 6 ) Nitrogen illuminated by ultraviolet does not charge the samples. (7) Air illuminated by ultraviolet and passed through a strong electric field to remove ions will still charge the samples. Discussion The two most reasonable explanations of the storage of energy in dried chloroplasts are the trap-
Vol. G3
ping of electrons or the formation of some high energy chemical compound. The trapping of electrons is suggested by the following: (1) at least five different activation energies are involved in the glow curve. A multiplicity of electron trap levels is common in the storage of energy in crystals. (2) The spikes in electrical conduction shown in Fig. 4 and 5 of the previous paper2 would seem to imply the freeing of electrons. (3) The value of the frequency factor K of 2.5 >( lo9set.-' is near to the l O S - l O g set.-' suggested by Randall and WiJkins3 as appropriate for the freeing of electrons. On the other hand the chemical storage is suggested by: (1) oxygen is needed in order for the energy to be stored. If all that oxygen did was to trap an electron then SO2 would be expected to serve as well, and it does not. (2) The fact that air irradiated with ultraviolet light can be used to charge a chloroplast sample would indicate a chemical reaction. Finally it might be argued that the storage represents a mixture of electron trapping and chemical compound formation. The occurrence of the spike in electrical conduction2 at a temperature considerably below the temperature of maximum light emission may indicate that these two effects are different.
THE EFFECT OF TEMPERATURE O N FLUORESCENCE OF SOLUTIONS BY E. J. BOWEN AND J. SAHU Physical Chemistry Laboratory, Oxford University,England Received M a y 10,1068
Measurements have been made of the effect of temperature on the fluorescence yields of substituted anthracenes dissolved in several solvents. 9-Substituted anthracenes show high yields and steep temperature dependencies, while side-substituted derivatives have low yields and small temperature variations. The results are interpreted in terms of the following concepts. There appear t o be two processes of energy degradation of the excited molecules, a substantially temperatureindependent one which probably is associated with the singlet-triplet conversion, and a temperature-dependent one having a heat of activation of degradat'ion and a dependence on solvent viscosity which may be associated with a direct transition from excited to the ground state. Values also are given of fluorescence quenching constants of dissolved oxygen, bromobenzene and carbon tetrachloride for these anthracene derivatives.
Measurement of true temperature effects of the fluorescence of solutions is rendered difficult by the smallness of the changes and of the complications inherent in the estimation of the total light emitted. Previous attempts to obtain reliable results have been subject to uncertainties of magnitudes approaching those of the quantities measured. Apart from experimental errors due to inconstancies of the apparatus, changes in the spatial distribution of the fluorescence caused by refractive index and polarization effects can occasion misleading interpretations of the results. 1.2 The correction to be applied for the refractive index effect can be assessed only roughly, as it depends on the detailed geometry of the apparatus, and it is sufficiently large in some instances to reverse the sign of the change of fluorescence with temperature. Measurements are here given which were made with a new form of apparatus designed par( I ) , E. J. Bowen and K. West, J . Chem. SOC.,4394 (1955). (2) E. J. Bowen and D. M. Stebbens, ibid., 360 (1957).
ticularly to minimize refractive index and polarization errors.
Experimental Figure 1 shows the arrangement in section and in plan. A 5-liter glass flask was fitted with a tube A for evacuation, and with a tube B, reaching to the center, to contain the solution. The flask was lightly smoked internally with esium oxide and painted over the external surface %,f?barium sulphate-Cellofas B mixture, except for holes to admit the exciting li h t and to observe the fluorescence. After evacuation the h s k was thus both a Dewar vessel and a0n integrating sphere. A beam of filtered mercury 3660 A. light was focussed by lens C on to the tube B, and the fluorescence collected from the flask wall a t E allowed to fall on a spectrometer slit a t D. The concentrations of the solutions were such as to give practically total light absorption in tube B, and the liquid level was above the top of the light beam to eliminate any effect from thermal expansion. This arrangement avoids errors due to changes in the spatial distribution of the fluorescence emitted from the solution, whether due to refractive index or to polarization effects. The collected fluorescence light waa dispersed by a Hilger D 246 spectrometer, with glass prism, to .the exit slit of which was attached a 13 stage photo-multiplier connected
h
