CHLOROPHYLL-POLY(VINYLPYRIDINE) COMPLEXES
1667
Chlorophyll-Poly(viny1pyridine) Complexes. 111. Photochemical Activity and Fluorescence Yield by G. R. Seely Charles F . Kettering Research Laboratory,‘ Yellow Springs,
Ohio 46587
(Received December 16, 1970)
Publication costs assisted by the Charles F. Kettering Research Laboratory
The fluorescence of denser aggregates of pyrochlorophyll bound to poly(viny1pyridine)is partia,lly quenched, probably by transfer of energy to “quenching centers,” consisting of interacting pairs of pigment molecules, The quantum yield for the photoreduction of p-dinitrobenzene by hydrazobenzene, sensitized by pyrochlorophyll in these aggregates, is almost directly proportional to the quantum yield of fluorescence. It is concluded that a reactive metastable state, such as the triplet, is not formed in the process of self-quenching of pyrochlorophyll fluorescence in this system.
Introduction I n the first paper of this series,2 the formation of complexes between chlorophyll or its derivatives and poly(4-~inylpyridine),and some of their absorption and fluorescence spectral properties, were described. I n the second paperj3the probability of nonradiative energy transfer was examined as a function of pigment density in the polymer ~ o m p l e x . ~ These systems are of interest as a novel approach to the synthesis of artificial aggregates of chlorophyll having properties like those of the natural photosynthetic unit of the green plant. One of the more notable properties of the poly(viny1pyridine) complexes is the self-quenching of chlorophyll fluorescence when the density of pigment molecules on the polymer is high. Sparsely distributed on the polymer, chlorophyll fluoresces with an intensity normal for true solutions. Densely distributed, the fluorescence is 90-95% quenched. Efficient quenching at chlorophyll coverages well below saturation was attributed to nonradiative transfer of excited state energy to quenching centers consisting of pairs of chlorophyll molecules, associated in some way on the polymer.2 The energy of interaction between such pairs of chlorophyll molecules was estimated to be about 1 kcal/mol. The quenching would then be a form of concentration quenching of the kind proposed by P e r ~ i ndiscussed ,~ in some detail by ForsterI6and examined experimentally by Levshin, et al., with several dyes in concentrated solution.’ The question examined in this paper is whether singlet excited chlorophyll is quenched by internal conversion to the ground state or by intersystem crossing to a reactive, metastable triplet state. The question is important to photosynthesis. The fluorescence yield of chlorophyll in the chloroplast is of similar magnitude to that in our dense aggregatesj8yet the photochemical efficiency is quite high. It is not known whether chlo-
rophyll reacts in its singlet excited or triplet excited state in photosynthesis, and it was hoped that the present work with a model system might shed some light on this question. Our approach is basically quite simple. The quantum yield of fluorescence is compared with the quantum yield of a reaction known to be sensitized by the triplel excited state. If the photoreaction persists at pigment densities great enough to quench fluorescence, it is evident that an appreciable number of singlet excited states are converted into triplets in the process of quenching. The photoreaction chosen was the sensitized reduction of p-dinitrobenzene (DNB) by hydrazobenzene, previously studied in ethanol-pyridine s o l ~ t i o n . ~Like many other reactions sensitized by chlorophyll, lo it is evidently initiated by electron transfer from the photoexcited pigment to DNB. As beforej3a9we used the more stable pyrochlorophyll (10-decarbomethoxychlorophyll), in place of chlorophyll, with reasonable confi(1) Contribution No. 420. (2) G. R. Seely, J . P h y s . Chem., 71, 2091 (1967). (3) G. R . Seely, {bid., 74, 219 (1970). (4) In our discussion of the value of the critical energy transfer distance Eo in the preceding reference, we failed to cite the values of A. G. Tweet, TV. D. Bellamy, and G. L. Gaines, Jr., J . Chem. P h y s . , 41, 2068 (1964), of ca. 54 for chlorophyll-chlorophyll transfer, and 40 A for chlorophyll-Cu pheophytin transfer, in dilute monolayers. Although the experimental conditions are somewhat different, these values should be comparable with our value of 42 b for pyrochlorophyll-pyrochlorophyll transfer. (5) F. Perrin, C. R. Acad. Sci., P a r i s , 192, 1727 (1931). (6) T . Forster, “Fluoresaenz Organischer Verbindungen,” Vandenhoeck and Ruprecht, Gottingen, 1951, Chapter 11. (7) V. L. Levshin, Izv. A k a d . Nauk SSSR, Ser. Fiz., 20, 397 (1956); V. L. Levshin and L. V. Krotova, Opt. Spektrosk., 13, 809 (1962) [Opt. Spectrosc., p 4671. (8) P. Latimer, T. T . Bannister, and E. I. Rabinowitch in “Research in Photosynthesis,” H. Gaffron, et al., Ed., Interscience Publishers, Inc., New York, N . Y . , 1957, p 107. (9) G. R. Seely, J . P h y s . Chem., 73, 117 (1969). (10) G. R. Seely, ibid., 69, 2779 (1965). T h e Journal of Physical Chemistry, Vol. 76, No. 11, 1071
C.R. SEELY dence that the results with chlorophyll would have been essentially the same.
mole ratio of azobenzene produced to DMB initially present was about 2.3 (cf. 2.21 in ethanol-pyridineg). Azobenzene in nitromethane has a band centered Experimental Section about 444 nm. To reduce interference from pyrochloroWe have previously described the preparation or phyll, the appearance of azobenzene is better meapurification of pyrochlorophyll,2 poly(4-vinylpyridine),3 sured a t 470 nm where the absorptivity is 334 M-l n i t r ~ m e t h a n ehydrazobenzene, ,~ and p-dinitr~benzene.~ cm-I. The absorption increase at 400 nm, due to The polymer sample was a fraction of molecular weight products of reduction of DKB, is larger than but lin440,000. early correlated with the increase a t 470 nm. Quantum As m previous yields could be calculated with greater precision from a portion of a stock solution of pyrochlorophyll in benzene and a small amount (0.005 the absorbance change at 400 nm and the slope of a rci) of purified a-methylnaphthalene were introduced plot of that change, Ap400, against the absorbance change Into x cuvette, the benzene was evaporated in a stream at 470 nm, AP470. The ratio Ap400/Ap~,owas determined of N s r and the soIution of pyrochlorophyl1 in a-methylfor each run and ranged from 3.74 at (py)/(chl) = naphthalene was diluted with ca. 4 ml of nitromethane. 534 to 2.89 at (py)/(chl) = 9. With 0.23 M 4-ethylThe fluorescence intensity was measured to confirm pyridine in place of polymer, the ratio was 6.50. The quantum yield declined linearly as the reaction the absence of large amounts of polar impurities.2 To h e cuvette were added about 5 mg of hydrazobenzene progressed, in such manner as to suggest product inand appropriate amounts of stock solutions of polyhibition of t,he later steps in the reduction of the nitro (vinylpyridine) and DNB in nitromethane, the concompound. Quantum yields during each run were tents were flushed thoroughly with X?,and the fluorestherefore extrapolated back to their initial values, and cence was determined again. Fluorescence was also only these are reported. In ethanol-pyridine solution, determined after the reaction and after the addition of the quantum yield was constant until the DYB was 0 1 ml of pyridine to dissociate pyrochlorophyll from almost consumed. the polymer. Fluorescence intensity after the reaction Measurements were made a t room temperature, ca. differed little from that before. Absorption spectra 24". A low relative humidity is necessary for successful upre also recorded on a Cary 14R spectrophotometer experimentation with this system; during these runs it was 535%. at these times. Fluorescence was measured, and the quantum yield Association between Components. Proper interpretacorrected for reabsorption and secondary fluorescence, tion of the data required that unintended associations between the reagents in nitromethane be absent. The in apparatus and by procedures described attachment of pyrochlorophyll to the polymer was Fiuorescence yields were calculated relative to the verified by the absorption spectrum2 and was not afvalue 0.228 already determined for pyrochlorophyll in fected by the presence of hydrazobenzene and DYB. the presence of excess p01ymer.~ Based on this value, The interaction, if any, between hydrazobenzene, DNB, the quantum yield of fluorescence in the presence of and poly(viny1pyridine) was investigated with a Varian 0.1 ml of pyridine in nitromethane was 0.197, and this A-60 nuclear magnetic resonance spectrometer. served as an internal standard for the determination of The methyl group of nitromethane absorbs at 6 the quantum yield of fluorescence in each run. 4.4 with respect to tetramethylsilane internal standard. JIeasurernents were made a t various ratios of the One of the C13 peaks flanking this band, at 6 5.67, afpolymer pyridine unit concentration to pyrochlorophyll fords a convenient reference point for the aromatic concentration, (py)/(chl), which is the primary indeproton resonance bands of the solutes. pendent variable of the system and which determines In 1 4 % solutions of poly(4-vinylpyridine) there are the quantum efficiency of fluorescence.2*3 two broad, equally intense bands at 63 and 163 He The photoreaction was activated by light from a below the C13 band. DNB, 0.06 M , has a single sharp tungsten projector lamp focused through a 670-nm band 174 Hz below the CI3 band. Hydrazobenzene, interference filter. The light absorbed was measured by difference with an Eppley thermopile. Changes in 0.06 114, has a complex group of bands between 72 and 114 Hz below the CI3band and a small band a t 36 Hz. the visible absorption spectrum after intervals of illumSone of these bands was appreciably altered in position ination were followed with the Cary spectrophotomor shape by the presence of one of the other solutes at eter. these concentrations. The quantum yields reported are for the production A mixture of solutions concentrated in D S B and in of azobenzene from hydrazobenzene. The distribuhydrazobenzene has a yellow color owing to increased tion of products of reduction of DXB is not known in absorption in the violet. The method of continuous detail, but the principal one appears to be p-nitrophevariations, a t 0.27 M total concentration, indicated nylhydroxylamine, which has an absorption band in the violet near the beginning of nitromethane absorption. In reactions that were driven to completion, the (11) G. R. Seely, J. Phys. Chem., 73, 125 (1969). T h e Journal of Physical Chemistry, Vol. 76, hTo.11, 1971
1669
CHLOROPHYLL-POLY (VINYLPYRIDINE) COMPLEXES the formation of a 1:1 complex between the two compounds, probably of the charge-transfer type. There was no departure from spectral additivity a t 2.7 X 10-3 M total concentration. The nmr spectra at 0.27 M total concentration did not show appreciable deviations from additivity. I n summary, although there is evidence of a small degree of complex formation between DNB and hydrazobenzene at high concentration, there is no evidence of complex formation between any of the solutes at the concentrations used, except of course between pyrochlorophyll and the polymer.
0.20,
I
I
I
I
I
Results The results listed in Table I include a series in which (py)/(chl) is varied at constant DNB concentration and series in which the DNB concentration is varied at fixed high and low (py)/(chl) ratios. The DNB concentration in the first series is too low to cause appreciable quenching of fluorescence by itself, so that the photoreactions must be sensitized by the triplet state of pyrochlorophyll only. At 10-2 M DNB, fluorescence in sparse aggregates is about one third quenched and reaction sensitized by the singlet excited state of pyrochlorophyll is to be expected. Table I : Photoreduction of p-Dinitrobenzene by Hydrazobenzene, Sensitized by Pyrochlorop hyll-Poly( vinylpyridine) Complexes in Nitromethane. Comparison Initial of Quantum Yield of Reduction, % ( O ) , with Pyrochlorophyll Fluorescence Yield, at"
0
0.20
0.10
0.30
+f Figure 1. Plot of initial quantum yield, W0),of sensitized photoreduction of D N B by hydrazobenzene against quantum yield of fluorescence of pyrochlorophyll, Qlf, in the same solution. Data of Table I: 0, [DNB] < 10-$M; 0, [DNB] k% 10-2 M . Least-squares line correlates former data points.
(chl) = pyrochlorophyll concentration, (DNB) = p-dinitrobenzene concentration, (py)/(chl) = ratio of polymer pyridine units to pyrochlorophyll. Hydrazobenzene Concentration in the range 6.1-7.6 X 10-3 M . 4-Ethylpyridine, 0.23 M,in place of polymer.
sented by the linear correlation in Figure 1. That is, the initial quantum yield of reaction is almost directly proportional to the quantum yield of fluorescence. This clearly argues against the formation of any sort of reactive metastable state12as a result of self-quenching of fluorescence, with a lifetime much greater than that of the excited singlet state. When [DNB] = M , and (py)/(chl) = 334, the quantum yield for reaction is about the same as at lower DNB concentrations, though fluorescence is partly quenched. In previous work," the quantum yield for the reaction of nitro compounds with the singlet excited state of pyrochlorophyll was deduced to be somewhat less than that for reaction with the triplet excited state. The present result is consistent with this. When [DNB] S M , and (py)/(chl) = 22, the quantum yield observed is accounted for if (1) quenching by DNB is competitive with self-quenching, and (2) the quantum yield of reaction initiated by singlet pyrochlorophyll is about 0.17. It is not necessary to suppose that DNB reacts with the product of selfquenching.
With the exception of runs in which [DNB] g loM2M , the quantum yield data are quite well repre-
(12) The metastable state need not be a triplet; the formation of the ion pair Chi+. Chl- as a result of quenching by two interacting pyrochlorophylls is entirely conceivable, but such an ion pair, if long-lived, should react with both hydrazobenzene and DNB."
lW(chl),
10*(DNB),
M
M
1.5 1.5 1.5 1.6 1.6 1.6 1.6 1.9 2.4 2.4 2.4 2.5 2.1 2.0 2.3 2.2 2.2 2.7
3.8 4.3 4.3 4.3 4.4 4.3 4.8 3.6 4.3 102 3.9 0.79 106 55.5 16.7 8.6 4.4 4.4
(PY)/ (chi)
534 237 118
111 60 30 28
9 80 334 365 324 22 22 20 21 22
b
%(O)
0.185 0.162 0.115 0.118 0.068 0.031 0.026 0.018 0.069 0.17 0.175 0.16 0.057 0.017 0.015 0.027 0.019 0.205
*f
(0.228) 0.213 0.1.54
,.. 0.125 0,055 0.052 0.048 0.101 0.142 0.224 0.223 0.040 0.020 0.023 0.045 0.027 0.197
a
The Journal of Physical Chemistry, Vol. 7 6 , N o . 11, 1071
G. R. SEELY
1670 Examination of Table I shows that the quantum yield of self-quenched preparations is not tightly correlated with the value of (py)/(chl). On the basis of previous experience with the ~ y s t e m ,it~ i,s~reasonable to conjecture that the actual fluorescence intensity is affected by minute amounts of polar impurities which serve to dissociate quenching pairs of pyrochlorophyll. This does not diminish but rather affirms the significance of the correlation between the two quantum yields.
Discussion Induced intersystem crossing to the triplet state is a well-supported mechanism for quenching of fluorescence by foreign molecules containing heavy atoms, such as bron~obenzene.~~ Self-quenching through formation of triplet states is indicated in many of the systems studied by Kautslip, et al., in their research on fluorescence, phosphorescence, and reactivity of dyes in solution and in aggregates.14 Formation of a long-lived excited state has been postulated for the self-quenching of eosin bound to poly(vinylpyrro1idone) l5 but in view of the complexity of the mechanisms of photoreaction of this dye,16the long-lived state may not be a triplet. The yield of triplet states on excitation of chlorophyll in dilute solution is high, but is low or zero in concentrated solutions or solid solutions in which fluorescence is q ~ e n c h e d . ' ~ No ~ ' ~triplet states could be detected in green leaves.'s~'g Cellarius and ;\IauzerallZ0reported that pheophgtin adsorbed onto polystyrene particles retained the ability to sensitize the reduction of an azo dye even when the coverage was such that fluorescence was almost entirely quenched. The intervention of a long-lived state is implied. The work of Neltrasov, et aL12'with chlorophyll adsorbed onto polymer particles points to the
T h e Journal of Physical Chemistry, Vol. 75, N o . 11, 1971
same relationship between fluorescence and photoreactivity. It is not clear why self-quenching of fluorescence should produce triplet or at least metastable states of chlorophyll and its derivatives in some cases and not in others. Perhaps attachment to a solid support is necessary to induce intersystem crossing or to stabilize a triplet state once formed. The pyrochlorophyllpoly(viny1pyridine) aggregate resembles concentrated solutions and green leaves in that quenching of fluorescence does not result in the formation of observable amounts of metastable states.
Acknowledgments. This work was supported in part by National Science Foundation Grant No. GB-7893. We appreciate the assistance of Dr. J. Corbin with the nmr spectroscopy and in the interpretation of the spectra, and the able technical assistance of Mr, T. H. Rleyer. (13) T. Medinger and F. Wilkinson, Trans. Faraday SOC.,61, 620 (1965); P.G. Bowers and G. Porter, Proc. Roy. SOC.,Ser. A , 299, 348 (1967). (14) H. Kautsky, A. Hirsch, and W. Flesch, Ber. Deut. Chem. Ges., 68, 152 (1935).
(15) J. S. Bellin and G . Oater, J . A m e r . Chem. Soc., 79. 2461 (1957). (16) T. Ohno, S. Kato, and hf. Koizumi, Bull. Chem. SOC.Jap., 39, 232 (1966). (17) P. G. Bowers and G.Porter, Proc. Roy. Soc., Ser. A , 296, 435 (1967). (18) G. Porter and G. Strauss, ibid., 295, 1 (1966). (19) G . T. Rikhireva, L. A . Sibel'dina, 2. P. Gribova, B. S. Marinov, L. P.Kayushin, and A. A. Krasnovskii, Dokl. Akad. Nauk SSSR, 181, 1485 (1968). (20) R. A. Cellarius and D. Mauzerall, Biochim. Bwphys. Acta, 112, 235 (1966). (21) R . Kapler and L. I. Nekrasov, Biojizika, 11, 420 (1966) (Biophysics, p 477) ; L. I. Nekrasov, A. N. Kiseleva, and N.I. Kobozev, Biofizika, 11, 977 (1966) ( B w p h p i c s , p 1118).
