J. Phys. Chem. 1996, 100, 669-671
669
Role of Excited Singlet State in the Photooxidation of Carotenoids: A Time-Resolved Q-Band EPR Study A. S. Jeevarajan and L. D. Kispert* Department of Chemistry, The UniVersity of Alabama, Tuscaloosa, Alabama 35487
N. I. Avdievich and M. D. E. Forbes Venable and Kenan Laboratories, Department of Chemistry, CB#3290, UniVersity of North Carolina, Chapel Hill, North Carolina 27599 ReceiVed: July 31, 1995; In Final Form: October 3, 1995X
Spin-polarized 35 GHz time-resolved EPR (TREPR) spectra were obtained for the first time of the cation radicals of β-carotene (I), 15,15′-didehydro-β-carotene (II), 7′,7′-dicyano-7′-apo-β-carotene (III), and 7′-cyano7′-ethoxycarbonyl-7′-apo-β-carotene (IV) and the anion radical of the solvent which were formed by 308 nm photoexcitation of the carotenoids in carbon tetrachloride solution. Although the EPR spectra are weak in intensity due to the small dimensions of the 35 GHz quartz flat cell and have very broad line widths, it was possible to positively determine from the polarization pattern that the electron transfer to the solvent occurs from the excited singlet state of the carotenoids. The 35 GHz EPR spectra consist of two resolved EPR lines (one absorption and one emission) from which it has been possible to measure the g factor and ∆Hpp of the solvent-separated radical ion pair that was formed; measurements were not possible at 9 GHz.
Introduction
TABLE 1: EPR Parameters of Carotenoid Cation Radicals
Although the importance of carotenoids in photosynthesis as a photoprotector and an auxiliary antenna pigment is well established,1-3 the role of the carotenoid cation radical in the photosynthetic apparatus is not well understood.4 Further, to utilize the photoprotect characteristics of the carotenoids in artificial solar devices, the decomposition processes should be understood. Determining the properties of the carotenoid and related polyene radical cations and the reason for the strong lifetime dependence on the host matrix5-7 are important aspects for understanding the role of carotenoids in electron transport processes. It is known that acidic sites in the Nafion or silicaalumina matrix help to stabilize carotenoid cation radicals, while water in the matrix has the opposite effect. In the case of electrochemical experiments,8 the acidic and hydrophobic nature of the dichloromethane solvent help to stabilize the carotenoid cation radicals. The photophysics and photochemistry of β-carotene (I, Table 1) in a good electron-acceptor solvent such as carbon tetrachloride, reported previously9 at X-band frequency (9.5 GHz) are outlined in Scheme 1. The excited singlet-state lifetime of β-carotene (I) is about 10 ps,10 and this lifetime increases with decreasing chain length. Experiments with perdeuterated β-carotene have shown11 that radiationless decay to the ground state is the major relaxation path caused by strong vibronic coupling of the 1 1Ag- and 2 1Ag- states via the CdC stretch. The lifetime also decreases with increasing solvent polarity for carotenoids with polar substitutents.12 The quantum yield of fluorescence13 of I is about 10-5, and the quantum yield of triplet formation14 is about 10-3. Time-resolved X-band EPR (TREPR) spectra of β-carotene excited using 308 nm laser light consisted of a first derivative-like line thought to be composed of an absorptive low-field line and an emissive high-field line, separated by only 11 G. The TREPR spectra were tentatively assigned9 to the cation radical of the carotenoid (high-field emissive line) and the anion radical of the solvent (low-field * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, December 1, 1995.
0022-3654/96/20100-0669$12.00/0
a
References 19-21. b Parameters measured from the TREPR spectra.
SCHEME 1
absorptive line). Since the two broad lines overlapped, the resulting spectrum was too distorted that accurate values of the g factors and their characteristic line widths, which are essential for correct assignment of the radicals and the CIDEP mechanism, could not be established with any degree of certainty. It was expected that overlap of the two signals could be eliminated, or at least greatly reduced, by using a higher EPR frequency. © 1996 American Chemical Society
670 J. Phys. Chem., Vol. 100, No. 2, 1996
Jeevarajan et al.
Figure 1. TREPR signal (35 GHz) after 0.1 (top) and 0.5 µs (bottom) delay of 308 nm laser light photolysis of β-carotene (0.2 mM) in CCl4 purged with nitrogen.
Figure 2. TREPR signal (35 GHz) after 0.1 (top) and 0.5 µs (bottom) delay of 308 nm laser light photolysis of 15,15′-didehydro-β-carotene (0.5 mM) in CCl4 purged with nitrogen.
We report here the results obtained at 35 GHz when a flowing solution of the carotenoids I-IV (Table 1) in CCl4 was irradiated with 308 nm laser light.
signal from the sulfite anion radical. The very broad signal obtained in our experiments and the uncertainty in the value of the resonance frequency increased the normally acceptable error limits in the measurements of the g factors of the cation radical of carotenoids and the anion radical of the solvent. Values of ∆Hpp, given in Table 1, were calculated with the assumption that the absorption or emission TREPR signals obtained in our measurements are Lorentzian. The fact that the signal to noise ratio in Figure 1 is not as good as in the case of the X-band measurements is primarily due to different experimental constraints. The Q-band flat cell is smaller than the X-band flat cell, and the active volume in the Q-band cell is about 6 times smaller than that of the X-band cell. The yield of the carotenoid cation radical is low due to a number of competing reactions as depicted in Scheme 1. The TREPR spectrum of Car•+ and CCl4•- radical ions as components of a solvent separated radical ion pair would consist of a signal at low field (g ∼ 2.009) due to CCl4•- and a signal near g ) 2.002 for Car•+. The TREPR spectra (35 GHz) obtained at two different delay times during the photolysis of 15,15′-didehydro-β-carotene (II) are shown in Figure 2. The cation radical of II decayed much faster than the cation radical of I. It should be noted that the ∆Hpp of II is somewhat smaller than that of I, and this behavior is consistent with our simultaneous electrochemical and EPR measurements.19-21 TREPR spectra at 35 GHz of the two synthetic unsymmetrical carotenoids III and IV, which contain electron-withdrawing groups, are similar to those obtained for I and II. Values of ∆Hpp and the g factors (Table 1) obtained from the experimental spectra of III and IV are greater than those of I and II and are consistent with ENDOR results.7
Experimental Section β-Carotene (I) was obtained from Fluka and was purified by column chromatography, and 15,15′-didehydro-β-carotene (II) was a gift from Hoffmann-La Roche. The purity of I and II was checked by TLC and 1H NMR spectroscopy. The synthesis and characterization of 7′,7′-dicyano-7′-apo-β-carotene (III)15 and 7′-cyano-7′-ethoxycarbonyl-7′-apo-β-carotene (IV)16 are described elsewhere. Millimolar solutions of carotenoids were prepared in CCl4 and purged with dry nitrogen gas before and during the experiments. Samples were photolyzed at 300 K with the 308 nm output of a pulsed excimer laser (Lambda Physik LPX 100i, 200 mJ/pulse, 17 ns fwhm) while being pumped through a 0.2 mm path length flat cell in the center of a Varian Q-band TE103 optical transmission microwave cavity. The EPR spectrometer was a Varian instrument operating at 35.2 GHz in direct detection.17 The signals from the detector diode before and after the laser pulse were passed through a fast preamplifier and fed to both gates of a Stanford Research Systems 250 boxcar signal averager. Gate widths were typically 100 ns, and the repetition rate of the laser pulse was 60 Hz. It is estimated that at least 10% of the 180 mJ/pulse of laser light was entering the cavity. Delay times quoted are the times between the peak of the laser flash and the opening of the boxcar gate. The boxcar output was the difference between a gate before the laser pulse and a gate after (known delay) the laser pulse and was downloaded continuously to an external microcomputer (Dell 386) as the magnetic field was swept to collect a complete EPR spectrum at each delay time.
Discussion Results The Q-band (35 GHz) TREPR signals obtained at two different delay times during photolysis of β-carotene (I) in carbon tetrachloride are shown in Figure 1. The spectra consist of a broad absorption line in the low-field side and a broad emission line in the high-field side, reminiscent of the X-band results.9 The arrows indicate the location of the center of the emissive and absorption peaks based on the digital analysis of the EPR line shape. The two broad EPR lines were separated by about 45 G at 35 GHz but significantly overlapped at 9 GHz. The g factors (Table 1) were calculated with reference to the sulfite anion radical (2.003 16)18 and the two lines differed in g values by 0.007. The resonance frequency of the spectrometer was not measured with a frequency counter but was deduced from the value of the magnetic field and the known reference
Carotenoids play a role of antenna function in photosynthesis. It is important to understand the role of the excited states of the carotenoids. The photophysics and photochemistry of the excited state of carotenoids are shown in Scheme 1. In addition to the radiative and nonradiative decays of the excited state of the carotenoid, an electron-transfer process also occurs in the presence of carbon tetrachloride solvent. The polarized Q-band EPR signal can shed light on the reactive state of the carotenoid in the electron-transfer reaction. The cation radical and anion radical formed as a result of the electron transfer process can also be characterized from the polarized Q-band EPR spectra. The polarized TREPR spectra of I-IV are attributed to an anion radical of the solvent (CCl4•-) which gives rise to an absorption line at low field and the carotenoid cation radical which exhibits an emission line at high field. The possibility
The Photooxidation of Carotenoids that the absorption line is due to the •CCl3 radical, which could be formed by loss of Cl- from CCl4•-, is unlikely because of the following. It has been shown22 in a steady-state EPR experiment that the •CCl3 radical gives rise to 10 lines with a coupling constant of 6.25 G (35Cl). None of our transient TREPR spectra showed any evidence of such coupling nor a line width large enough to have resulted from 9 separations of 6.25 G. On the other hand, it has been established that during photoionization of p-cresol in carbon tetrachloride, the anion radical CCl4•- is formed by electron transfer from the excited cresol.9 Photolysis of neat CCl4 does not give rise to any transient EPR signal, although optical measurements obtained during radiolysis of CCl4 indicated the formation of the CCl4•species.23 TREPR spectra of the photolyzed cresol/CCl4 system showed a transient species with a life time of about 0.5 µs and, moreover, its g factor (∼2.009) was similar to that of the absorption line observed in the carotenoid/CCl4 TREPR experiments. The low-field peak in the latter is therefore assigned to the chlorinated solvent anion radical, one part of the solvent separated radical ion pair that was formed. The observed absorption/emission (A/E) unresolved TREPR signals indicate that a net effect geminate radical pair mechanism (RPM) is involved in the chemically induced dynamic electron polarization (CIDEP) of the radicals present. For this mechanism, if the product of the signs of the parameters in the expression µJ∆g is positive for one of the species, then an absorption spectrum is observed; if it is negative, an emission spectrum results.24 The electron transfer to the solvent occurs from the excited singlet state of the carotenoids; therefore µ, the nature of the electronic state of the geminate radical pair, is negative. The singlet level of the radical ion pair is below the triplet level; therefore, J, the sign of exchange energy, is negative.9 For the anion radical of CCl4, ∆g, the difference between the g factors of the anion radical of CCl4 and the cation radical of carotenoid, is positive; thus an absorption pattern (µJ∆g is positive) is predicted.24 Since for the carotenoid cation radical all three parameters are negative, µJ∆g is negative and emission is predicted, as is observed. The decay of the polarized TREPR signals (Figures 1 and 2) of the carotenoid cation radical and the anion radical of the solvent depends on the relaxation time and the chemical lifetime of the radicals. The fact that in steady-state EPR experiments of I high microwave power (100 mW) was required to saturate the carotenoid cation radical indicates that its spin-lattice relaxation time (estimated as less than 1 µs) is smaller than its chemical lifetime. Thus, the TREPR signal decay of the cation radicals of carotenoids is dominated by the relaxation time. It is also known that the relaxation times at the Q-band frequency are longer than those at X-band frequency.25 Assuming that the relaxation times of the cation radical of I and II are similar, the cation radical of II (high-field line in Figure 2) chemically decayed much faster than the anion radical of the solvent (lowfield line) and the cation radical of I. Values of the g factor and ∆Hpp of the cation radicals of I-IV in dichloromethane determined by simultaneous electrochemical EPR (SEEPR) spectroscopy19-21 differed considerably from the values estimated from the TREPR spectra obtained previously at the X-band frequency.9 This discrepancy is now known to be due to the line-shape distortion resulting from extensive overlap of the two polarized TREPR radical signals. In the Q-band experiments, overlap of the two radical signals is reduced so that better evaluation of the g factors and ∆Hpp values was possible. The line widths and g factors observed in the TREPR spectra of the photochemically generated cation radicals of I-IV are nearly equal to those obtained in the SEEPR
J. Phys. Chem., Vol. 100, No. 2, 1996 671 experiments. Our results show that Q-band TREPR spectroscopy can play a significant role in unequivocally assigning the transient radicals with different g factors and large line widths. Conclusions Photoexcitation by 308 nm laser light of millimolar solutions of carotenoids in carbon tetrachloride produces a solvent separated radical ion pair between CCl4•- and carotenoid cation radicals. The transient EPR spectra are spin polarized, and a radical pair mechanism of CIDEP is involved in the polarization. The counter radical is the CCl4•- species formed by electron transfer to CCl4. From the polarization pattern, it is concluded that the electron transfer occurs from the excited singlet state of the neutral carotenoids. The yield of the cation radical formed from the excited singlet state is very low owing to a number of competing reactions. The EPR parameters of photochemically prepared carotenoid cation radicals are consistent within error limits with those of electrochemically prepared carotenoid cation radicals. Acknowledgments. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research of the U.S. Department of Energy under Grant No. DE-FG05-86ER13465. We thank Dr. Elli Hand for critically reading the manuscript. We thank Hoffmann-La Roche of Basel, Switzerland, and Roche Vitamins and Fine Chemicals of Nutley, NJ, for providing samples of II and 8′apo-β-caroten-8′-al, a precursor for III and IV. References and Notes (1) Mathis, P.; Schenck, C. C. In Carotenoid Chemistry and Biochemistry; Britton, G., Goodwin, T. W., Eds.; Pergamon Press: New York, 1981; pp 339-351. (2) Koyama, Y. J. Photochem. Photobiol. 1991, B9, 265 and references therein. (3) Frank, H. A.; Violette, C. A.; Trautman, J. K.; Shreve, A. P.; Owens, T. G.; Albrecht, A. C. Pure Appl. Chem. 1991, 63, 109. (4) Riva´s, J. O. L.; Telfer, A.; Barber, J. Biochim. Biophys. Acta 1993, 1142, 155 and references therein. (5) Piekara-Sady, L.; Jeevarajan, A. S.; Kispert, L. D. Chem. Phys. Lett. 1993, 207, 173. (6) Jeevarajan, A. S.; Kispert, L. D.; Piekara-Sady, L. Chem. Phys. Lett. 1993, 209, 269. (7) Piekara-Sady, L.; Jeevarajan, A. S.; Kispert, L. D.; Bradford, E. G.; Plato, M. J. Chem. Soc., Faraday Trans. 1995, 2881. (8) Khaled, M.; Hadjipetrou, A.; Kispert, L. D.; Allendoerfer, R. D. J. Phys. Chem. 1991, 95, 2438. (9) Jeevarajan, A. S.; Khaled, M.; Forbes, M. D. E.; Kispert, L. D. Z. Phys. Chem. 1993, 82, 51. (10) Wasielewski, M. R.; Kispert, L. D. Chem. Phys. Lett. 1986, 128, 238. (11) Wasielewski, M. R.; Johnson, D. G.; Bradford, E. G.; Kispert, L. D. J. Chem. Phys. 1989, 91, 6691. (12) O’Neil, M. P.; Wasielewski, M. R.; Khaled, M.; Kispert, L. D. J. Chem. Phys. 1991, 95, 7212. (13) Shreve, A. P.; Trautman, J. K.; Owens, T. G.; Albrecht, A. C. Chem. Phys. Lett. 1991, 178, 89. (14) Hashimoto, H.; Koyama, Y.; Hirata, Y.; Mataga, N. J. Phys. Chem. 1991, 95, 3072. (15) Hand, E.; Belmore, K.; Kispert, L. D. HelV. Chim. Acta 1993, 76, 1939. (16) Hand, E.; Belmore, K.; Kispert, L. D., manuscript in preparation. (17) Forbes, M. D. E. ReV. Sci. Instrum. 1993, 64, 397. (18) Jeevarajan, A. S.; Fessenden, R. W. J. Phys. Chem. 1989, 93, 3511. (19) Jeevarajan, A. S.; Khaled, M.; Kispert, L. D. J. Phys. Chem. 1994, 98, 7777. (20) Jeevarajan, A. S.; Khaled, M.; Kispert, L. D. Chem. Phys. Lett. 1994, 225, 340. (21) Jeevarajan, J. A.; Kispert, L. D. J. Electroanal. Chem., submitted for publication. (22) Hudson A.; Hussain, H. A. Mol. Phys. 1969, 16, 199. (23) Klassen, N. V.; Ross, C. K. J. Phys. Chem. 1987, 91, 3668. (24) Adrian, F. J. Res. Chem. Intermed. 1990, 16, 99. (25) Forbes, M. D. E.; Ruberu, S. R. J. Phys. Chem. 1993, 97, 13223.
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